CN101541345A - Stabilized antibody formulations and uses thereof - Google Patents

Abstract

The present invention provides methods for optimizing certain stable liquid formulations of antibodies that immunospecifically bind an antigen of interest. Such formulations are suitable for parenteral administration to a subject with improved stability, low to undetectable levels of aggregation, low to undetectable levels of antibody fragmentation/degradation, and little to no loss of antibody biological activity, even with prolonged storage in this way. Compared to formulations produced by non-optimized methods, the formulations provided by the methods of the present invention have several advantages, including less stringent or more achievable shipping and storage conditions, less frequent dosing and/or less frequent administration in therapeutic, prophylactic or diagnostic applications. or in smaller doses. The invention also provides methods for identifying antibodies having certain phase properties for formulating the antibodies using the methods of the invention. Prophylactic, therapeutic and diagnostic applications of such antibody formulations are also provided.

Description

Stabilized antibody formulations and applications thereof

1 Introduction

The present invention provides methods for optimizing certain formulations of antibodies that immunospecifically bind an antigen of interest. Such formulations are suitable for parenteral administration to a subject with improved stability, low to undetectable levels of aggregation, low to undetectable levels of antibody fragmentation/degradation, and little to no loss of antibody biological activity, even with prolonged storage in this way. Compared to formulations produced by non-optimized methods, the formulations provided by the methods of the present invention have several advantages, including less stringent or more achievable shipping and storage conditions, less frequent dosing and/or less frequent administration in therapeutic, prophylactic or diagnostic applications. or in smaller doses. The invention also provides methods for identifying antibodies having certain phase behaviors for formulating the antibodies using the methods of the invention. Prophylactic, therapeutic and diagnostic applications of such antibody formulations are also provided.

2. Background of the invention

Protein instability is a major obstacle in the development of the protein drug market. In particular, protein aggregation, often caused by instability, is one of the major obstacles in all stages of drug development. Therefore, the presence of transient populations of partially unfolded intermediates and the conditions under which they form are important in predicting and understanding protein aggregation. This is particularly important for multidomain proteins such as monoclonal antibodies (mAbs), which are very popular drug candidates due to their high binding affinity and specificity, easy targeting of specific antigens, and overall anti-aggregation ability. Because of the high sequence identity of antibodies produced in the same manner, they are often assumed to have similar phase properties and stability during processing and storage.

Currently, many antibodies are offered as lyophilized formulations. Lyophilized formulations of antibodies have a number of limitations, including lengthy lyophilization processes and high manufacturing costs. In addition, lyophilized formulations must be precisely reconstituted under sterile conditions by healthcare workers before administration to patients. Accordingly, there is a need for liquid antibody formulations that are at concentrations comparable to or higher than reconstituted lyophilized formulations so that reconstitution of the formulation prior to administration is no longer required. Thus, such formulations allow health care workers to administer antibodies to patients more quickly and easily.

Moreover, some existing liquid antibody formulations have a short shelf life and may lose the biological activity of the antibody due to chemical and physical instability during storage. Chemical instability can be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination, or disulfide bond exchange, and physical instability can be caused by antibody denaturation, aggregation, precipitation, or adsorption. Among them, aggregation, deamidation and oxidation are known to be the most common causes of antibody degradation (Wang et al., 1988, J.of Parenteral Science & Technology 42 (Supplement): S4-S26; Cleland et al., 1993, Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377). Therefore, there is a need for a stable liquid formulation of an antibody that binds to an antigen of interest that exhibits high stability, low to undetectable levels of aggregation, and low to undetectable levels of aggregation, even during long-term storage. Antibody fragmentation/degradation level and the biological activity of the antibody decreased little or even not.

3. Contents of the invention

_The present invention provides stable antibody liquid antibody formulations containing non-zwitterionic buffers such as phosphate (eg _Na3PO4 ), tris, citrate, succinate and acetate buffers. In a specific embodiment, the invention provides stable liquid formulations containing high concentrations of antibodies. In other embodiments, antibody formulations of the invention are formulated in a form suitable for parenteral administration to a subject (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous). In another specific embodiment, the subject is a human.

The present invention provides stable antibody liquid formulations comprising phosphate in a concentration range of about 10 mM to 100 mM or greater, a pH range of about 4.0 to 8.0; NaCl in a concentration range of about 0 mM to 200 mM; and a concentration of about 10 mg /ml or more of an antibody of interest, eg, an antibody (including antibody fragments thereof) that immunospecifically binds IL-9 polypeptide, eg, 7F3com-2H2. The stable liquid formulations of the invention may also contain one or more excipients such as sugars, surfactants and polyols. In particular embodiments, the liquid formulations of the invention comprise a surfactant (such as Tween-20 or Tween-80) at a concentration ranging from about 0% to 0.1%; sucrose at a concentration ranging from about 0% to 10%; and and/or trehalose in a concentration range of about 0% to 10%. In certain embodiments, the stable liquid formulation is for subcutaneous delivery and comprises phosphate in a concentration range of about 25 mM to 75 mM (e.g., a concentration of about 50 mM), a pH range of about 6.0 to 6.5; a concentration range of about 100 mM NaCl to 200mM (such as a concentration of about 150mM); and an antibody of interest in a concentration range of about 50mg/ml to 150mg/ml (such as about 100mg/ml), such as an antibody that immunospecifically binds to an IL-9 polypeptide (including antibodies thereof) fragment), such as 7F3com-2H2.

The antibody preparation of the present invention is stored at room temperature or 4°C for a longer period of time (such as but not limited to 6 months, 1 year, 2 years, 3 years or 5 years) or at an elevated temperature such as 38-42°C for a period of time (for example, After, but not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year), the improved aggregation properties are preferably maintained. Such formulations have a pH in the range of 4.0 to 8.0, such as pH 6.2.

Stable liquid formulations of any type of antibody can be concentrated and produced using the methods of the invention. The antibodies used in the method of the present invention can be therapeutic or preventive antibodies, and can be used to treat and/or control various diseases, including but not limited to: diseases related to or characterized by abnormal expression and/or activity of IL-9 polypeptides Disease or disorder, disease or disorder associated with or characterized by aberrant expression and/or activity of the IL-9 receptor ("IL-9R") or one or more subunits thereof, autoimmune disease, inflammatory Disease, proliferative disease or infection (eg, respiratory infection), or one or more symptoms thereof (eg, wheezing). Examples of autoimmune diseases include, but are not limited to: diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, anemia vera, multiple sclerosis, rheumatoid carditis, systemic lupus erythematosus, rheumatoid arthritis , chronic inflammation, Sjögren's syndrome, polymyositis, dermatomyositis, and scleroderma. Examples of inflammatory diseases include, but are not limited to: asthma and allergies (types I-IV). Examples of respiratory tract infections include, but are not limited to, infections of the upper and lower respiratory tracts, including viral, bacterial and/or fungal infections. Examples of viral infections include parainfluenza virus infection, influenza virus infection, post pneumonia virus infection or respiratory syncytial virus (RSV) infection. Antibody formulations of the invention may also be used to treat subjects who have or have had bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis, or acquired or congenital immunodeficiency.

)；特异性结合RSV抗原的抗体，如

(帕丽珠单抗)、MEDI-524(莫维珠单抗；

)；特异性结合CD2的抗体，如MEDI-507(西利珠单抗)；结合CD19的抗体，如MT-103；结合EphA2的抗体，如EA2、EA5、B233(包括其人和人源化形式)；和结合EphA4的抗体，如EA44(包括其人和人源化形式)。可配制并用于本发明方法的其它治疗性和预防性抗体也参见2006年6月23日提交的美国申请号11/473,537，题为“具有优化聚集和片段化特性的抗体制剂”(Antibody Formulations Having Optimized Aggregationand Fragmentation Profiles)，通过引用将其全文纳入本文。Non-limiting examples of therapeutic or prophylactic antibodies that can be formulated and used in the methods of the invention are listed in Section 5.1.1 below, and include antibodies or fragments thereof that immunospecifically bind IL-9 polypeptides, e.g., 4D4, 4D4 H2- 1D11 _, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4; antibodies that specifically bind _αvβ3 integrin, such as MEDI-522 (dimensional Tasin

); antibodies that specifically bind to RSV antigens, such as

(Palivizumab), MEDI-524 (Motavizumab;

); antibodies that specifically bind CD2, such as MEDI-507 (cilizumab); antibodies that bind CD19, such as MT-103; antibodies that bind EphA2, such as EA2, EA5, B233 (including their human and humanized forms ); and antibodies that bind EphA4, such as EA44 (including human and humanized forms thereof). Other therapeutic and prophylactic antibodies that may be formulated and used in the methods of the invention are also described in U.S. Application No. 11/473,537, filed June 23, 2006, entitled "Antibody Formulations Having Optimized Aggregation and Fragmentation Properties." Optimized Aggregation and Fragmentation Profiles), which is incorporated herein by reference in its entirety.

In a specific embodiment, the present invention provides stable liquid formulations of antibodies (eg, monoclonal antibodies) that, during production, preparation, shipping, and storage, as determined by, for example, high performance size exclusion chromatography (HPSEC), It has high stability, the fragmentation and/or aggregation level of the antibody is so low that it cannot be detected, and the biological activity of the antibody (including its antibody fragments) has little or no decrease. In a specific embodiment, the stable liquid formulation of the present invention comprises an antibody, eg, a monoclonal antibody (eg, a monoclonal antibody that immunospecifically binds to an IL-9 polypeptide, such as 7F3com-2H2). In other embodiments, the stable liquid formulation of the present invention facilitates the administration of antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides (such as 7F3com-2H2) to prevent, treat and/or control IL-9 polypeptides Diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9 receptor ("IL-9R") or one or more subunits thereof A disease or disorder characterized by an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof (such as wheezing). Examples of autoimmune diseases include, but are not limited to: diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, anemia vera, multiple sclerosis, rheumatoid carditis, systemic lupus erythematosus, rheumatoid arthritis , chronic inflammation, Sjögren's syndrome, polymyositis, dermatomyositis, and scleroderma. Examples of inflammatory diseases include, but are not limited to: asthma and allergies (types I-IV). Examples of respiratory tract infections include, but are not limited to, infections of the upper and lower respiratory tracts, including viral, bacterial and/or fungal infections. Examples of viral infections include parainfluenza virus infection, influenza virus infection, post pneumonia virus infection or respiratory syncytial virus (RSV) infection. Antibody formulations of the invention may also be used to treat subjects who have or have had bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis, or acquired or congenital immunodeficiency. In particular, the stable liquid formulations of the present invention enable healthcare professionals to rapidly administer sterile doses of antibodies (including antibody fragments thereof) without the need for precise and aseptic reconstitution of the antibodies (including antibody fragments thereof) prior to administration.

In other embodiments, the present invention includes stable liquid formulations of antibodies that immunospecifically bind IL-9 polypeptides, including but not limited to 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 (see U.S. Application Serial No. 11/823,253 filed April 12, 2004 and published as U.S. Patent Publication No. US2005/0002934 A1 for amino acid sequence, via Incorporated herein by reference in its entirety), said preparations exhibit undetectably low levels of antibody fragmentation and/or aggregation during production, preparation, transportation, and long-term storage, and 4D4, 4D4H2-1D11, 4D4com-XF- 9. The biological activity of 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 decreased little or even not. The invention also includes stable liquid formulations of antibodies that immunospecifically bind IL-9 polypeptides and that have prolonged in vivo half-lives compared to known antibodies, including, for example, 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com- 2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4, the antibody fragmentation and/or aggregation level of the preparation is so low as to be undetectable, and the biological There is little or no decline in academic activity. The present invention also includes stable liquid preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies (including antibody fragments thereof) comprising heavy chain variable regions (VH) and/or light chain variable regions (VL ), which has the amino acid sequence of the VH and/or VL region of 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4, The antibody aggregation and/or fragmentation level of the preparation is so low as to be undetectable, and the biological activity of the antibody (including antibody fragments thereof) decreases little or even not. The invention also includes stable liquid formulations of antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides, said antibodies (including antibody fragments thereof) comprising one or more VH complementarity determining regions (CDRs) and /or one or more VL CDRs, which have the amino acid sequences of one or more VH CDRs and/or VL CDRs listed in Table 1 below, and the antibody aggregation and/or fragmentation level of the preparation is so low that it cannot be detected , and the biological activity of the antibody (including its antibody fragment) decreases little or even not. In specific embodiments, the invention does not include stable 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 antibodies liquid formulations.

The invention includes stable liquid formulations of antibodies (including antibody fragments thereof). Compared with antibodies formulated with zwitterionic buffers (such as histidine buffers) at the same pH and in the presence of the same concentration of salt, non-zwitterionic buffers (for example, phosphate (such as Na ₃ PO ₄ ), tris, lemon salt, succinate, or acetate buffers), certain properties of antibodies formulated at pH below the pI of the antibody and in the presence of salts (e.g., NaCl) (e.g., formation of unfolded intermediates, colloidal instability stability, solute binding or precipitation) may decrease or decrease, and the formulation is stable at 38-42°C as determined by high performance size exclusion chromatography (HPSEC). Also using static light scattering (SLS, static light scattering) technology, Fourier transform infrared spectroscopy (FTIR) technology, circular dichroism (CD) technology, urea-induced protein unfolding technology, tryptophan intrinsic fluorescence technology, Differential Scanning Calorimetry and/or ANS protein binding techniques to assess phase properties, other physical properties and stability of molecules. Assessed by HPSEC, the liquid formulation of the present invention has a stability of at least 15 days but not more than 25 days at a temperature of 38-42° C.; has a stability of at least 6 months but not more than 1.5 years at a temperature of 20-24° C. Stability; stability of at least 1.5 years, at least 2 years, at least 2.5 years or at least 3 years at a temperature of 2-8°C, especially 4°C. The invention also encompasses liquid formulations of antibodies (including antibody fragments thereof) that immunospecifically bind an antigen of interest (such as an IL-9 polypeptide) that have low to undetectable levels of antibody aggregation as determined by HPSEC . Also using static light scattering (SLS) techniques, Fourier transform infrared spectroscopy (FTIR) techniques, circular dichroism (CD) techniques, urea-induced protein unfolding techniques, tryptophan intrinsic fluorescence techniques, differential scanning volume Thermal and/or ANS protein binding techniques to assess phase properties, other physical properties and stability of molecules. In one embodiment, the liquid formulation of the present invention has a stability of at least 15 days at a temperature of 38-42° C. and a low to undetectable level of antibody aggregation as determined by HPSEC, and as determined by an antibody binding assay, such as ELISA. , compared with the reference antibody, the biological activity of the preparation antibody (including its antibody fragment) has little or no decrease.

The present invention provides methods for identifying antibodies, particularly therapeutic and prophylactic antibodies, using non-zwitterionic Buffers (e.g., phosphate (such as _Na3PO4 ), tris, citrate, succinate, or acetate buffers), formulated at a pH below the pI of _the antibody and in the presence of salts (such as NaCl) Certain properties of the antibody (eg, formation of unfolded intermediates, colloidal instability, soluble binding or precipitation) may be diminished or reduced, making them amenable to formulation using the methods of the invention. These phase properties may contribute to the stability of the antibody formulation. The stability of the antibody formulation can be determined, for example, by high performance size exclusion chromatography (HPSEC). Also using static light scattering (SLS) techniques, Fourier transform infrared spectroscopy (FTIR) techniques, circular dichroism (CD) techniques, urea-induced protein unfolding techniques, tryptophan intrinsic fluorescence techniques, differential scanning volume Thermal and/or ANS protein binding techniques to assess phase properties, other physical properties and stability of antibody molecules.

The present invention provides methods for preparing stable liquid formulations of antibodies (including antibody fragments thereof) that immunospecifically bind to an antigen of interest (e.g. IL-9 polypeptide) comprising the use of appropriate molecular weight (MW) cutoffs (e.g. , 30kD cutoff for intact antibody molecules and F(ab') ₂ fragments; and 10kD cutoff for antibody fragments such as Fab fragments) semipermeable membranes concentrate purified antibody-containing fractions to a final antibody concentration of about 1 mg/ ml, about 5mg/ml, about 10mg/ml, about 15mg/ml, about 20mg/ml, about 30mg/ml, about 40mg/ml, about 50mg/ml, about 60mg/ml, about 70mg/ml, about 80mg/ml ml, about 90 mg/ml, about 100 mg/ml, about 150 mg/ml, about 175 mg/ml, or about 200 mg/ml, and the concentrated antibody fraction is diafiltered into the formulation buffer using the same membrane. The formulation buffer of the present invention comprises phosphate, tris, citrate, succinate or acetate in a concentration range of about 1 mM to 100 mM, about 10 mM to 100 mM, about 5 mM to 50 mM, about 10 mM to 25 mM, or about 25 to 75 mM . The formulation buffers of the present invention also comprise NaCl at a concentration ranging from about 0 mM to 200 mM, from about 10 mM to 200 mM, from about 50 to 200 mM, from about 100 to 150 mM, or from about 100 mM to 200 mM. The formulation may have a pH in the range of about 4.0-8.0, such as about 6.0-6.5.

The liquid formulation of the present invention can be sterilized by filtration with a 0.2 μ filter. The sterilized liquid formulation of the present invention can be administered to a subject to prevent, treat and/or manage a disease or disorder, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, and A disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory tract infection), or one or more of its symptoms. The liquid formulations of the present invention can be administered in combination with other treatments (eg, prophylactic or therapeutic drugs other than antibodies that immunospecifically bind IL-9 polypeptides, such as anti-inflammatory agents, immunomodulators, and anticancer agents).

In a specific embodiment, the invention provides an antibody formulation comprising an aqueous carrier, phosphate, and an antibody or antibody fragment at a concentration of 50 mg/ml or greater, said antibody formulation formulated for administration to a human subject. In another specific embodiment, the present invention provides an antibody preparation comprising an aqueous carrier, phosphate, and an antibody or antibody fragment at a concentration of 10 mg/ml or higher, wherein the antibody or antibody fragment is used at the same pH and in the presence of the same concentration of salt. One or more lower phase properties of the antibody or antibody fragment are reduced when formulated using phosphate buffer at a pH below the pI of the antibody and in the presence of salt compared to the antibody formulated in a histidine buffer: (a) formation of an unfolded intermediate; (b) colloidal instability; (c) soluble association of the antibody molecule; or (d) precipitation of the antibody molecule; wherein said at least one or more phase characteristics are determined by selecting As determined by techniques from the following group: high performance size exclusion chromatography (HPSEC) technique, tangential flow filtration (TFF) technique, static light scattering (SLS) technique, Fourier transform infrared spectroscopy (FTIR) technique, circular dichroism (CD) technique, urea-induced protein unfolding technique, tryptophan intrinsic fluorescence technique, differential scanning calorimetry (DSC) and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding technique.

The invention also provides kits for use by healthcare professionals comprising stable liquid formulations of antibodies (including antibody fragments thereof) that immunospecifically bind an antigen of interest (eg, IL-9 polypeptide). In other embodiments, kits comprising stable formulations of the invention are formulated in a form suitable for parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous) to a human subject. The present invention also provides methods for preventing, treating and/or controlling diseases or disorders, including, for example, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9 polypeptides, Diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative diseases or infections (such as respiratory infections), or one or more of its symptoms. The stable liquid formulations of the present invention can be administered to a subject (such as a human subject) via parenteral (such as intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), oral or intranasal routes for the prophylaxis, treatment and/or Controlling a disease or disorder, e.g., a disease or disorder associated with or characterized by aberrant expression and/or activity of an IL-9 polypeptide, associated with aberrant expression and/or activity of IL-9R or one or more subunits thereof or a disease or disorder characterized thereby, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof. The stable liquid formulations of the invention can also be used to diagnose, detect or monitor diseases or disorders, for example, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9 polypeptides, IL-9R or one of its A disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory tract infection) related to or characterized by abnormal expression and/or activity of one or more subunits, or one or more of them symptoms.

3.1. Terminology

All formulations of antibodies and/or antibody fragments that immunospecifically bind to an antigen of interest (such as the IL-9 polypeptide described herein), collectively referred to herein as "liquid formulations of the invention", "highly concentrated stable liquid formulations of the invention ”, “antibody liquid preparation of the present invention” or “antibody preparation of the present invention”.

The term "abnormal" as used herein refers to a deviation from normal, eg, an average healthy subject and/or a population of average healthy subjects. The term "abnormal expression" as used herein refers to abnormal expression of a gene product (such as ribonucleic acid, protein, polypeptide or peptide) in a cell or subject compared to normal healthy cells or subject and/or a normal population of healthy cells or subjects. This abnormal expression may be a consequence of gene amplification. In a specific embodiment, the term "abnormal expression" refers to the gene product expression of a normal healthy cell or subject and/or a normal healthy cell or subject population, IL-9 and/or IL-9R or IL-9R of a cell or subject Abnormal expression of its subunits, including expression of IL-9 and/or IL-9R or its subunit gene products at unusual locations in cells or subjects, IL-9 and/or IL-9R or their subunit gene products in Altered expression levels in the cell or subject, expression of mutated IL-9 and/or IL-9R or subunit gene products thereof, or combinations thereof. The term "abnormal activity" as used herein refers to an altered level of a gene product, increased activity of a gene product, or decreased activity of a gene product in a cell or subject relative to normal healthy cells or subjects and/or a population of normal healthy cells or subjects. In a particular embodiment, the term "abnormal activity" refers to IL-9 and/or IL-9R or subunit activity thereof that deviates from the activity found in normal healthy cells or subjects and/or normal healthy cells or subject populations (such as , the affinity of IL-9 and IL-9R is improved). Examples of IL-9 activity include, but are not limited to: phosphorylation of IL-9R, Jak3 activation, MEK activation, STAT-1 activation, and STAT-3 activation.

As used herein, the term "about" when a value or range is given means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

As used herein, the term "analogue" when referring to proteinaceous substances (such as proteins, polypeptides, peptides, and antibodies) means similar or identical in function to a second proteinaceous substance, but does not necessarily include A similar or identical amino acid sequence, or a proteinaceous substance that is structurally similar or identical to a second proteinaceous substance. A proteinaceous substance having a similar amino acid sequence refers to a second proteinaceous substance that satisfies at least one of the following conditions: (a) has at least 30%, at least 35%, at least 40%, at least 45% of the amino acid sequence of the second proteinaceous substance; %, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the same amino acid sequence proteinaceous substance; (b) at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues encoding a second proteinaceous substance under stringent conditions group, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, A proteinaceous substance encoded by a nucleotide sequence hybridized to a nucleotide sequence of at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues; and ( c) at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% identical to the nucleotide sequence encoding the second proteinaceous substance A proteinaceous substance encoded by a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. A proteinaceous substance that is structurally similar to a second proteinaceous substance refers to a proteinaceous substance that has a secondary, tertiary or quaternary structure similar to that of the second proteinaceous substance. The structure of a proteinaceous material can be determined by methods known to those skilled in the art including, but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced into a first amino acid sequence or nucleic acid sequences for optimal alignment). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When the amino acid or nucleotide residue at a position in the first sequence is identical to the amino acid or nucleotide residue at the corresponding position in the second sequence, then the two molecules are identical at that position. The percent identity of two sequences is related to the number of identical positions shared by the two sequences (ie percent identity = number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are equal in length.

A mathematical algorithm can also be used to determine the percent identity of two sequences. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877 was modified. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol., 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter set, such as score=100, wordlength=12, to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program parameter set, eg, score-50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. For gapped alignments (for comparison purposes), Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Alternatively, an iterative search can be performed using PSI-BLAST, which is used to detect near and far relationships (Id.) between molecules. When utilizing the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used (see NCBI website). Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such algorithms are integrated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, which allow or disallow gaps. In the calculation of percent identity, generally only exact matches are counted.

As used herein, the term "analog" when referring to a non-proteinaceous analog refers to a second organic or inorganic molecule that is functionally similar or identical to a first organic or inorganic molecule and that is structurally similar to the first organic or inorganic molecule.

The term "antagonist" as used herein refers to any protein, polypeptide, peptide, peptidomimetic, glycoprotein, antibody, antibody fragment, sugar, Nucleic acids, organic molecules, inorganic molecules, macromolecules or small molecules. In various embodiments, the antagonist reduces the function, activity and/or expression of another molecule by at least 10%, at least 15%, at least 20%, at least 25%, compared to a control such as phosphate buffered saline (PBS). At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 95%, or at least 99%.

The term "antibody fragment" as used herein refers to an antibody fragment that immunospecifically binds an antigen of interest (eg, IL-9 polypeptide). Antibody fragments can be produced by any technique known to those of skill in the art. For example, Fab and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to yield Fab fragments) or pepsin (to yield F(ab')2 fragments). The F(ab') ₂ fragment contains the entire light chain, as well as the variable, CH1 and hinge regions of the heavy chain. Antibody fragments can also be produced by recombinant DNA techniques. An antibody fragment can be one or more complementarity determining regions (CDRs) of an antibody, or one or more antigen-binding fragments of an antibody.

The term "antibody" as used herein refers to a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a camelised antibody, a chimeric antibody, a single chain Fv (scFv), a single chain antibody, a single domain antibody , Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv) and anti-idiotypic (anti-Id) antibodies (including anti-Id antibodies such as antibodies of the invention), intrabodies and their Epitope binding fragments. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (such as IgG, IgE, IgM, IgD, IgA, and IgY), class (such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 ), or subclass.

The term "antibody or antibody fragment that immunospecifically binds an antigen of interest" and similar expressions as used herein refers to an antibody or antibody fragment that specifically binds an antigen of interest or a fragment thereof, and does not specifically bind other antigens or fragments thereof. As used herein, the term "antibody or antibody fragment that immunospecifically binds to an IL-9 polypeptide" and similar expressions refers to an antibody or antibody fragment that specifically binds to an IL-9 polypeptide or fragment of an IL-9 polypeptide, and does not specifically bind to other polypeptides. Preferably, an antibody or antibody fragment that immunospecifically binds to an IL-9 polypeptide has a higher affinity for the IL-9 polypeptide or IL-9 polypeptide fragment than for other polypeptides or other polypeptide fragments. Antibody affinity is a measure of its binding to a specific antigen at an antigen-antibody site, and is essentially the sum of all attractive and repulsive forces present in the interaction between the antigen-binding site of an antibody and a specific epitope. The affinity of an antibody to a specific antigen (such as an IL-9 polypeptide or a fragment of an IL-9 polypeptide) can be expressed as an equilibrium constant K, which is defined as K=[Ag Ab]/[Ag][Ab], which is the antibody-binding site The affinity of the spot, where [Ag] is the concentration of free antigen, [Ab] is the concentration of free antibody, and [Ag Ab] is the concentration of the antigen-antibody complex. If there is a strong reaction between antigen and antibody, then the amount of free antigen or free antibody will be very small, so the equilibrium constant or antibody affinity is high. Discovery of high-affinity antibodies with good fit between antigen and antibody (see Sigal and Ron eds., 1994, Immunology and Inflammation-Basic Mechanisms and Clinical Consequences for discussion of antibody affinity, MH Corporation, New York ( McGraw-Hill, Inc. New York), pp. 56-57; and Seymour et al., 1995, Immunology-An Introduction for the Health Sciences, McGraw-Hill Book Company, Australia ), pp. 31-32). Preferably, an antibody or antibody fragment that immunospecifically binds an IL-9 polypeptide or fragment thereof does not cross-react with other antigens. That is, the antibody or antibody fragment immunospecifically binds to the IL-9 polypeptide or fragment thereof with higher energy than other polypeptides or fragments of other polypeptides (for a discussion of antibody specificity see, e.g., Paul ed., 1989, Basic Immunology (Fundamental Immunology, 2nd ed., Raven Press, New York, pp. 332-336). Antibodies or antibody fragments that immunospecifically bind IL-9 polypeptides can be identified by, for example, immunoassays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and BIAcore assay (see Section 5.7 below) or Other techniques known to those skilled in the art (see, e.g., Seymour et al., 1995, Immunology-An Introduction for the Health Sciences for a discussion of various assays for assaying antibody-antigen interactions in vivo , McGraw-Hill Book Company, Australia, pp. 33-41). Antibodies or antibody fragments that immunospecifically bind IL-9 polypeptides or fragments thereof only antagonize IL-9 polypeptides and do not significantly antagonize other activities.

The term "control IgG antibody" as used herein refers to an IgG antibody or other "control antibody" that does not immunospecifically bind to an antigen of interest (eg, IL-9 polypeptide) and preferably does not cross-react with the antigen of interest (eg, IL-9 polypeptide).

The term "cytokine receptor modulator" as used herein refers to a substance that modulates phosphorylation of cytokine receptors, activation of signal transduction pathways associated with cytokine receptors, and/or expression of specific proteins such as cytokines. Such substances can directly or indirectly modulate the phosphorylation of cytokine receptors, the activation of signal transduction pathways associated with cytokine receptors and/or the expression of specific proteins such as cytokines. Thus, examples of cytokine receptor modulators include, but are not limited to, cytokines, cytokine fragments, fusion proteins, and antibodies or antibody fragments that immunospecifically bind to cytokine receptors or cytokine receptor fragments. In addition, examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides (such as soluble cytokine receptors), fusion proteins, and antibodies that immunospecifically bind cytokines or fragments thereof.

The term "derivative" as used herein in reference to proteinaceous substances such as proteins, polypeptides, peptides and antibodies refers to proteinaceous substances comprising amino acid sequences altered by introducing substitutions, deletions and/or additions of amino acid residues. The term "derivative" as used herein also refers to a proteinaceous substance which is modified by covalently linking a molecule of any type to the proteinaceous substance. For example, but not limited to, antibodies can be glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, attached to cellular ligands or other proteins, etc. The method is modified. Derivatives of proteinaceous substances can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. In addition, derivatives of proteinaceous substances may contain one or more non-classical amino acids. A derivative of a proteinaceous substance has similar or identical functions to the proteinaceous substance from which it was derived.

As used herein, the term "derivative" when referring to non-proteinaceous derivatives refers to a second organic or inorganic molecule formed from the structure of a first organic or inorganic molecule. Derivatives of organic molecules include, but are not limited to, molecules modified by, for example, the addition or deletion of hydroxyl, methyl, ethyl, carboxyl, nitro, or amine groups. Organic molecules can also be esterified, alkylated and/or phosphorylated.

As used herein, the terms "disorder" and "disease" are used interchangeably to refer to a condition in which a subject differs from healthy, non-diseased subjects. In particular, the term "autoimmune disease" is used interchangeably with the term "autoimmune disorder" and refers to a condition characterized by an immune response to the subject's own cells, tissues and/or organs Injury symptoms. The term "inflammatory disease" is used interchangeably with the term "inflammatory disorder" and refers to a subject condition characterized by inflammation, eg chronic inflammation. Autoimmune diseases may or may not be associated with inflammation. Also, inflammation may or may not be caused by autoimmune disease. Certain conditions may be identified as more than one disease. For example, certain conditions may be identified as autoimmune and inflammatory diseases.

As used herein, the term "effective amount" refers to a disease or disorder sufficient to reduce and/or alleviate, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, IL-9R or one of its A disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory tract infection) related to or characterized by abnormal expression and/or activity of one or more subunits, or one or more of them the severity and/or duration of a symptom, prevent the progression of the disease or disorder, cause regression of the disease or disorder, prevent the recurrence, development or appearance of one or more symptoms associated with the disease or disorder, or An amount of a treatment (eg, prophylactic or therapeutic agent) that enhances or improves the prophylactic or therapeutic effect of another treatment (eg, prophylactic or therapeutic agent).

The term "epitope" as used herein refers to a fragment of a polypeptide or protein that has antigenic or immunogenic activity in animals, preferably mammals, most preferably humans. An epitope having immunogenic activity is a fragment of a polypeptide or protein that is capable of eliciting an antibody response in an animal. An epitope with antigenic activity is a fragment of a polypeptide or protein that immunospecifically binds to an antibody as determined by methods well known to those skilled in the art, such as immunoassay. An antigenic epitope is not necessarily immunogenic.

The term "excipient" as used herein refers to a diluent, carrier, preservative, binder or stabilizer commonly used in pharmaceuticals to impart beneficial physical properties to the formulation, such as increased protein stability, increased protein solubility and reduced viscosity inert substances. Examples of excipients include, but are not limited to: proteins (such as serum albumin), amino acids (such as aspartic acid, glutamic acid, lysine, arginine, glycine), surfactants (such as SDS, Tween 20, Tween 80, polysorbate and non-ionic surfactants), sugars (such as glucose, sucrose, maltose and trehalose), polyols (such as mannitol and sorbitol), fatty acids and phospholipids (such as alkylsulfonate salt (alkyl sulfonate) and caprylate (caprylate)). Additional information on excipients is found in Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), pp. The full text is included in this article.

The term "fragment" as used herein refers to a fragment comprising at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues in the amino acid sequence of a second different polypeptide or protein , at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 A peptide or polypeptide of a sequence of amino acids contiguous with amino acid residues. In another embodiment, a fragment of a protein or polypeptide retains at least one function of the protein or polypeptide. In another embodiment, a fragment of a protein or polypeptide retains at least two, three, four or five functions of the protein or polypeptide. For example, a fragment of an antibody that immunospecifically binds IL-9 polypeptide retains the ability to immunospecifically bind IL-9 polypeptide. A "functional fragment" is a fragment that retains at least one function of a protein or polypeptide.

The term "fusion protein" as used herein refers to an amino acid sequence comprising a first polypeptide or protein or fragment, analog or derivative thereof, and a heterologous polypeptide or protein (i.e., different from the first polypeptide or protein or fragment, analog or A polypeptide or protein of the amino acid sequence of a second polypeptide or protein or a fragment, analog or derivative thereof). In one embodiment, the fusion protein comprises a prophylactic or therapeutic agent fused to a heterologous protein, polypeptide or peptide. According to this embodiment, the heterologous protein, polypeptide or peptide may or may not be a different type of prophylactic or therapeutic agent. For example, two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein. In one embodiment, the fusion protein maintains or increases the activity compared to the activity of the original polypeptide or protein prior to fusion to the heterologous protein, polypeptide or peptide.

The terms "high concentration" and "concentrated antibody" as used herein refer to an antibody (including antibody fragments thereof) that immunospecifically binds to an antigen of interest (such as IL-9 polypeptide) in an antibody preparation at a concentration of 50 mg/ml or higher, or 95 mg/ml ml or higher.

The term "host cell" as used herein includes a particular subject cell transfected or transformed with a nucleic acid molecule, as well as the progeny or potential progeny of such cells. The progeny of such cells may not be identical to the parent cell transfected with the nucleic acid molecule due to possible mutations and environmental influences in subsequent generations or integration of the nucleic acid molecule into the host cell genome.

As used herein, the term "human child" or "child" or variations thereof refers to a human being between 24 months and 18 years of age.

As used herein, the terms "elderly", "elderly" or variants thereof refer to persons 65 years of age or older, or 70 years of age or older.

The term "human infant" or "infant" or variants thereof as used herein refers to a human being less than 24 months, less than 12 months, less than 6 months, less than 3 months, less than 2 months or less than 1 month old.

As used herein, the terms "preterm human infant", "preterm infant" or "premature infant" or variants thereof refer to those born at less than 40 weeks of gestational age, less than 35 weeks, less than 6 months, less than 3 months, less than 2 months, or anyone younger than 1 month old.

The term "hybridizes under stringent conditions" as used herein describes such that at least 30% (at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Nucleotide sequences that are 90%, 95%, or 98%) identical generally maintain hybridization and washing conditions that hybridize to each other. Such stringent conditions are known to those skilled in the art, see, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and updated), 6.3.1 -6.3.6.

Generally, stringent conditions are selected to be about 5-10°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength, pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the target-complementary probe hybridizes to the target sequence in equilibrium (excess of target sequence, 50% of the probes are equilibrated at Tm). Stringent conditions are a salt concentration of less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salt), pH 7.0 to 8.3, and temperature for short probes (e.g., 10-50 nucleotides). Conditions of at least about 30°C, for long probes (eg, greater than 50 nucleotides), at least 60°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In selective or specific hybridization, a positive signal is at least two times, preferably 10 times, the background hybridization signal.

In one non-limiting example, stringent hybridization conditions are hybridization with 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes with 0.1X SSC, 0.2% SDS at about 68°C. In one non-limiting example, stringent hybridization conditions are hybridization with 6XSSC at about 45°C, followed by one or more washes at 50-65°C with 0.2X SSC, 0.1% SDS (i.e., at 50°C, 55°C, 60 °C or 65 °C for one or more washes).

The term "IL-polypeptide" as used herein refers to IL-9, its analogs, derivatives or fragments, including mature and immature forms of IL-9 (see Van Snick et al., 1989, J Exp. Med. 169:363-68 and Yang et al., 1989, Blood 74:1880-84, both incorporated herein by reference in their entirety), or fusion proteins containing IL-9, analogs, derivatives or fragments thereof. IL-9 polypeptides may be from any species. The nucleotide and/or amino acid sequence of the IL-9 polypeptide can be found in literature or public databases, or the nucleotide and/or amino acid sequence can be determined by cloning and sequencing techniques known to those skilled in the art. For example, the nucleotide sequence of human IL-9 can be found in the GenBank database (see eg, accession number NM_000590; Figure 12). The amino acid sequence of human IL-9 can be found in the GenBank database (see, e.g., Accession Nos. A60480, NP_000584, and AAC17735; Figure 13) and U.S. Patent Application No. 10/412,703, filed April 11, 2003, published November 27, 2003 is US 2003/0219439 A1, entitled Recombinant Anti-Interleukin-9 Antibodies (the amino acid sequence of human IL-9 on page 5 is specifically incorporated herein by reference). In one embodiment, the IL-9 polypeptide is human IL-9, an analog, derivative or fragment thereof.

The terms "IL-9 receptor" and "IL-9R" as used herein refer to IL-9 receptor or an analog, derivative or fragment thereof, or a fusion comprising IL-9 receptor, analog, derivative or fragment thereof protein. When referring to IL-9R, the terms "subunit or subunits" and "subunit" as used herein refer to the IL-9R ligand-specific alpha subunit of a functional IL-9R or an analog, derivative or fragment thereof. unit ("IL-9Rα") and/or the consensus _γc chain (also present in IL-2R, IL-4R, IL-7R and IL-15R complexes). In one embodiment, functional IL-9R mediates the proliferative response (e.g., [ ³ H]-thymidine incorporation, assay or hexosaminidase assay) (see for example, Renauld et al., 1992, Proc.Natl.Acad.Sci.USA, 89: 5690-94 and Bauer et al., 1998, JBiol. Chem. incorporated in its entirety here). Preferably, T cell lines expressing functional IL-9R (such as TS1RA3 cells expressing human and murine IL-9Rα (R&D Systems, Inc. (R&D Systems))), leading to a dose-dependent increase in T cell proliferation (see Renauld et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 5690-94 and Bauer et al., 1998, J Biol. Chem. -60). In another preferred embodiment, a functional IL-9R comprising _γc and IL-9Rα chains initiates a signal transduction cascade through Janus kinases JAK1 and JAK3, thereby activating signal transducers and activators of transcription (STAT) factors Homodimers and heterodimers of STAT-1, STAT-3 and STAT-5 (see Bauer et al., 1998, J Biol. Chem. 273:9255-60). In another preferred embodiment, functional IL-9R can prevent apoptosis through mechanisms including STAT-3 and STAT-5, as determined by apoptosis assays known to those skilled in the art (see Bauer et al., 1998, J Biol . Chem. 273:9255-60). IL-9R or one or more subunits thereof may be from any species. The nucleotide and/or amino acid sequences of IL-9R and its subunits can be found in literature or public databases, or the nucleotide and/or amino acid sequences can be determined by cloning and sequencing techniques known to those skilled in the art. For example, the nucleotide sequence of human IL-9R can be found in the GenBank database (see eg, accession numbers NM_002186, NM_176786, and NM_000206; Figure 14). The amino acid sequence of human IL9R can be found in the GenBank database (see, e.g., Accession Nos. NP_002177, NP_789743, and NP_000197; FIG. 15 ) and in U.S. Patent Application No. 10/412,703, filed April 11, 2003, published November 27, 2003 as US 2003/0219439A1, entitled Recombinant Anti-Interleukin-9 Antibodies (specifically the amino acid sequence of human IL-9R on pages 6-7 is incorporated herein by reference). In one embodiment, the IL-9R or one or more subunits thereof is human IL-9R or one or more subunits thereof, analogs, derivatives or fragments thereof.

As used herein, the term "immune modulator" and its variants include, but are not limited to: immunomodulators, immune modulators or immunomodulators, which refer to substances that modulate the immune system of a host. In a specific embodiment, an immunomodulator is a substance that alters some aspect of a subject's immune response. In certain embodiments, an immunomodulator is a substance that suppresses or lowers a subject's immune system (ie, an immunosuppressant). In certain other embodiments, an immunomodulator is a substance that activates or enhances a subject's immune system (ie, an immunostimulator). According to the invention, the immunomodulators used in the combination therapy of the invention do not include the antibodies of the invention. Immunomodulators include, but are not limited to: small molecules, peptides, polypeptides, proteins, nucleic acids (such as RNA and DNA nucleotides, including but not limited to: antisense nucleotide sequences, triple helices, RNAi, and encoding biologically active proteins, nucleotide sequences of polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetics and synthetic or natural organic molecules.

The term "combination" as used herein refers to the use of more than one treatment (eg, prophylactic and/or therapeutic agents). The term "combined" is not limited to patients with diseases or disorders (such as, diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or one or more of them) A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection), or one or more symptoms thereof, associated with or characterized by aberrant expression and/or activity of the subunit The sequence in which subjects are administered treatments (eg, prophylactic and/or therapeutic agents). It can be administered to patients with a disease or disorder (such as a disease or disorder related to or characterized by abnormal expression and/or activity of IL-9 polypeptide, abnormal expression of IL-9R or one or more subunits thereof) and/or activity associated with or characterized by a disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or one or more symptoms thereof) second treatment (eg, prophylactic or therapeutic) before (eg, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), at the same time or after (such as 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after the first treatment (such as prophylactic or therapeutic agents).

As used herein, the term "immunospecifically binds an antigen" and like terms refer to peptides, polypeptides, proteins, fusion proteins and antibodies (including antibody fragments thereof) that specifically bind an antigen or fragment and do not specifically bind other antigens. A peptide, polypeptide, protein or antibody that immunospecifically binds an antigen may bind other peptides, polypeptides or proteins with lower affinity as determined, for example, by immunoassays, BIAcore, or other assays known in the art. Antibodies (including antibody fragments thereof) that immunospecifically bind an antigen may cross-react with related antigens. Preferably, antibodies (including antibody fragments thereof) that immunospecifically bind an antigen do not significantly cross-react (ie, are not detectable in routine immunization assays) with other antigens. An antibody is said to specifically bind to an antigen when it is determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), to bind to an antigen with a higher affinity than any cross-reactive antigen. See, eg, Paul ed., 1989, Fundamental Immunology, 2nd ed., Raven Press, New York, pp. 332-336 for a discussion of antibody specificity.

The term "combination" as used herein refers to the use of more than one treatment (eg, more than one prophylactic and/or therapeutic agent). Use of the term "in combination" does not limit the order in which treatments (eg, prophylactic and/or therapeutic agents) are administered to a subject with a respiratory disorder. Before (eg, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks ago), at the same time or after (such as 5 minutes, 15 minutes, 30 minutes , 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks later) to administer the first (eg, first prophylactic or therapeutic agent).

The term "inorganic salt" as used herein refers to any compound containing no carbon in which some or all of the acid hydrogens or acids are replaced by metal or metal-like groups, which are often used as tonicity-modulating compounds in pharmaceutical compositions and biomaterial formulations. The most common inorganic salts are NaCl, KCl, NaH ₂ PO ₄ and so on.

As used herein, the term "isolated" when referring to organic or inorganic molecules (small or large), as opposed to proteinaceous substances or nucleic acid molecules, refers to organic or inorganic molecules that are substantially free of different organic or inorganic molecules. Preferably, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% are organic or inorganic molecules other than a second different organic or inorganic molecule. In one embodiment, the organic and/or inorganic molecules are isolated.

As used herein, the term "isolated" when referring to a proteinaceous material (such as a peptide, polypeptide, fusion protein, or antibody) refers to a proteinaceous material that is substantially free of cellular material or contaminating proteins produced by its cell or tissue of The case is substantially free of chemical precursors or other chemical substances. The term "substantially free of cellular material" includes preparations of proteinaceous material in which the proteinaceous material is separated from the cellular components of the cell from which it was isolated or recombinantly produced. Thus, proteinaceous material that is substantially free of cellular material includes substances that contain less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins, polypeptides, peptides, or antibodies (also referred to as "contaminating proteins"). preparations of proteinaceous substances. Where the proteinaceous substance is produced recombinantly, it may also be substantially free of culture medium, ie the volume of culture medium in the preparation of the proteinaceous substance is less than 20%, 10% or 5%. Where the proteinaceous substance is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, ie, it is separated from chemical precursors or precursor chemicals involved in the synthesis of the proteinaceous substance. Thus, such proteinaceous substance preparations contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the proteinaceous substance of interest. In a specific embodiment, the proteinaceous material described herein is isolated. In one embodiment, an antibody of the invention is isolated. In a specific embodiment, an "isolated" antibody is purified by a multi-step purification process comprising three chromatography steps (cation exchange, protein A, and anion exchange), a nanofiltration step, and a low pH treatment step (detailed in See section 6) below.

As used herein, the term "isolated" when referring to a nucleic acid molecule refers to a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or precursor chemicals when chemically synthesized. In a specific embodiment, a nucleic acid molecule is isolated; however, "isolated" does not include members of a population of clonal libraries, such as cDNA libraries.

As used herein, the term "low to undetectable aggregation level" refers to the analysis by high performance size exclusion chromatography (HPSEC), static light scattering (SLS) technique, Fourier transform infrared spectroscopy (FTIR) technique, circular dichroism (CD ) technique, urea-induced protein unfolding technique, tryptophan intrinsic fluorescence technique, differential scanning calorimetry and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding assay, by protein weight, containing Samples with not more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1% and not more than 0.5% aggregates.

The term "low to undetectable levels of fragmentation" as used herein means greater than or equal to 80%, 85%, 90%, 95%, 98% or 99% of the total protein within, for example, a single peak as determined by HPSEC, or Represents non-degraded antibody or non-degraded fragments by reduced capillary gel electrophoresis (rCGE) within two peaks (e.g., heavy and light chains) (or the same number of peaks as the number of subunits) and contains no greater than total protein 5%, 4%, 3%, 2%, 1%, or 0.5% of other unimodal samples. The term "reducing capillary gel electrophoresis" as used herein refers to capillary gel electrophoresis performed under reducing conditions sufficient to reduce the disulfide bonds of antibodies.

As used herein, the term "control" refers to the beneficial effect that a subject obtains from a treatment (eg, a prophylactic or therapeutic agent) that does not result in cure of the disease. In certain embodiments, one or more treatments (eg, one or more prophylactic or therapeutic agents) are administered to a subject to "manage" a disease such that progression or worsening of the disease is prevented.

The term "mast cell modulator" as used herein refers to a substance that modulates mast cell activation, mast cell degranulation and/or expression of specific proteins such as cytokines. Such substances may directly or indirectly modulate mast cell activation, mast cell degranulation and/or expression of specific proteins such as cytokines. Non-limiting examples of mast cell modulators include, but are not limited to, inhibition and/or reduction of stem cell factors, mast cell proteases, cytokines (such as IL-3, IL-4, and IL-9), cytokine receptors (such as IL -3R, IL-4R and IL-9R) and stem cell receptor expression, function and/or activity of small molecules, peptides, polypeptides, proteins, nucleic acids (such as DNA and RNA nucleotides, including but not limited to: antisense Nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides, or peptides), fusion proteins, antibodies, synthetic or natural inorganic molecules, synthetic or natural organic molecules, or mimetics. Other non-limiting examples of mast cell modulators include, but are not limited to: small molecules, peptides, polypeptides, proteins, nucleic acids (such as DNA and RNA nucleotides, including But not limited to: antisense nucleotide sequences, triple helix, RNAi and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), fusion proteins, antibodies, synthetic or natural inorganic molecules, synthetic or natural organic molecules or Simulator. In certain embodiments, a mast cell modulator is a substance that prevents or reduces activation of additional mast cells following mast cell degranulation. In other embodiments, a mast cell modulator is a substance that inhibits or reduces mast cell degranulation.

The terms "non-responsive" and "refractory" as used herein refer to patients who are not clinically relieved of one or more symptoms associated with their disease with currently available treatments (such as prophylactic or therapeutic agents) Diseases or disorders include, for example, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9 polypeptides, abnormal expression and/or activity of IL-9R or one or more subunits thereof A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease, or infection (eg, a respiratory infection) associated with or characterized thereby, or one or more symptoms thereof. Typically, such patients have severe ongoing active disease and require additional treatment to relieve symptoms associated with the disease.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government, or listed in the US Pharmacopoeia, European Pharmacopoeia or other generally accepted pharmacopoeia for use in animals, and more particularly in humans.

The term "polyol" as used herein refers to sugars containing many -OH groups compared to normal sugars.

The term "prevention" as used herein refers to the suppression of diseases or disorders, such as abnormal expression of IL-9 polypeptide and/or Diseases or disorders associated with or characterized by IL-9R or its activity, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory The development or onset of a disease, proliferative disease or infection (such as a respiratory infection) or one or more symptoms thereof, or the prevention of the recurrence, occurrence or development of one or more symptoms of a respiratory disease.

As used herein, the term "preventive agent" refers to a disease or disorder that can be used to prevent and prevent a disease or disorder, for example, a disease or disorder that is related to or characterized by abnormal expression and/or activity of IL-9 polypeptide, and IL-9R or one or more of it. Diseases or disorders, autoimmune diseases, inflammatory diseases, proliferative diseases or infections (such as respiratory infections) associated with or characterized by abnormal expression and/or activity of subunits, or the pathogenesis of one or more symptoms thereof , recurrence or development of any substance. In certain embodiments, the term "preventive agent" refers to an antibody that immunospecifically binds an IL-9 polypeptide. In certain other embodiments, the term "prophylactic agent" refers to a substance other than an antibody that immunospecifically binds an IL-9 polypeptide. Preferably, the preventive agent is known to be useful or has been used or is being used to prevent or prevent diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or its A disease or disorder associated with or characterized by aberrant expression and/or activity of one or more subunits, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection) or one or more thereof Onset, development, progression and/or severity of symptoms. Prophylactic agents can be identified into different classes based on one or more actions of the agent in vitro and/or in vivo. For example, mast cell modulators can also be identified as immunomodulators.

As used herein, the term "prophylactically effective amount" means sufficient to prevent a disease or disorder, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, IL-9R or one or more thereof A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection) associated with or characterized by aberrant expression and/or activity of the subunit, or the development of one or more symptoms thereof, An amount of a treatment (eg, a prophylactic) that relapses or morbidity, or enhances or improves the prophylactic effect of another treatment (eg, a prophylactic).

The term "prophylactic regimen" as used herein refers to a dosing and timing regimen for administering one or more treatments (eg, one or more prophylactic agents) that have a prophylactic effect.

The term "regimen" as used herein includes dosing schedules and dosing regimens. The regimens described herein are methods of use, including prophylactic and therapeutic regimens.

The term "sugar" as used herein refers to a class of molecules that are derivatives of polyols. Sugars are commonly referred to as carbohydrates and can contain varying amounts of sugar units such as monosaccharides, disaccharides and polysaccharides.

As used herein, the term "side effect" includes unwanted unwanted effects of a prophylactic or therapeutic agent. Side effects are always redundant, but redundant effects are not necessarily bad effects. An adverse effect of a treatment (eg, a prophylactic or therapeutic agent) may be a harmful, unpleasant, or risky effect. Adverse effects experienced by many patients are known in the art. Many of these effects are described in the Physicians' Desk Reference (60th Edition, 2006).

The term "small molecule" and similar terms as used herein include, but are not limited to: peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, molecular weight less than about 10,000 g/mole of organic or inorganic substances (i.e. including heterogeneous organic and organometallic compounds), organic or inorganic substances of molecular weight less than about 5,000 g/mole, organic or inorganic substances of molecular weight less than about 1,000 g/mole, molecular weight of less than about 500 g/mole of organic or inorganic substances, as well as salts, esters and other pharmaceutically acceptable forms of such drugs.

When referring to a liquid preparation containing an antibody (including antibody fragments thereof) that immunospecifically binds an antigen of interest (such as an IL-9 polypeptide), the terms "stability" and "stable" as used herein refer to a given production, preparation, Resistance of antibodies or (including antibody fragments thereof) in formulations against aggregation, degradation or fragmentation under shipping and storage conditions. A "stable" formulation of the invention retains biological activity under given manufacturing, manufacturing, shipping and storage conditions. Through HPSEC, static light scattering (SLS) technology, Fourier transform infrared spectroscopy (FTIR) technology, circular dichroism (CD) technology, urea-induced protein unfolding technology, tryptophan intrinsic fluorescence technology, differential The stability of the antibody (including antibody fragments thereof) is assessed by scanning calorimetry and/or ANS binding techniques to determine the degree of aggregation, degradation or fragmentation of the formulation compared to a reference formulation. For example, the reference preparation can be a reference standard frozen at -70°C, 10 mg/ml antibody (including its antibody fragments) prepared in phosphate buffer containing 150 mM NaCl (such as 4D4, 4D4 H2-1 D11, 4D4com- XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4), pH 6.0-6.5 composition, the reference preparation usually produces a single monomer peak in HPSEC (≥97 %area). Alternatively, the reference preparation can be a reference standard frozen at -70°C, 10 mg/ml antibody (including its antibody fragments) prepared in phosphate buffer (such as 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com -2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4), pH 6.0-6.5 composition, the reference preparation usually produces a single monomer peak (≥97% area) in HPSEC. The overall stability of formulations containing antibodies (including antibody fragments thereof) can be assessed by a variety of immunoassays including, for example, ELISA and radioimmunoassays with isolated antigen molecules or cells expressing it.

As used herein, the terms "subject" and "patient" are used interchangeably. The term "subject" as used herein refers to an animal, preferably a mammal, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, such as macaques, chimpanzees, and people), more preferably people. In certain embodiments, the subject is suffering from a disease or disorder associated with or characterized by aberrant expression and/or activity of an IL-9 polypeptide, an aberration with IL-9R or one or more subunits thereof A mammal, preferably a human, associated with or characterized by a disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof, in which the expression and/or activity is associated or characterized . In another embodiment, the subject is suffering from a disease or disorder associated with or characterized by aberrant expression and/or activity of an IL-9 polypeptide, an aberration with IL-9R or one or more subunits thereof Expression and/or activity associated with or characterized by diseases or disorders, autoimmune diseases, inflammatory diseases, proliferative diseases or infections (such as respiratory tract infections), or livestock (such as horses, pigs or cows) or pets (such as dogs or cats). In another embodiment, the subject is suffering from a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, and IL-9R or one or more subunits thereof. Aberrant expression and/or activity associated with or characterized by a disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or risk of one or more symptoms thereof Animals, preferably humans (eg, immunocompromised or immunosuppressed mammals). In another embodiment, the subject is not an immunocompromised or immunosuppressed mammal, preferably a human. In another embodiment, the subject is a mammal, preferably a human, having a lymphocyte count of not less than about 500 cells/mm3. In another embodiment, the subject is a human infant or a premature human infant. In another embodiment, the subject is a human child or adult. In another embodiment, the subject is a human child with bronchopulmonary dysplasia, congenital heart disease, or cystic fibrosis. In another embodiment, the subject is an elderly person. In yet another embodiment, the subject is a human institution or group home, such as, but not limited to, a person in a nursing home.

As used herein, the term "synergy" refers to a combined treatment (eg, a prophylactic or therapeutic agent) that is more effective than the sum of the effects of any two or more individual treatments (eg, one or more prophylactic or therapeutic agents). The synergistic effect of combination therapies (e.g., combination prophylactic or therapeutic agents) allows for the use of lower doses of one or more treatments (e.g., one or more prophylactic or therapeutic agents) and/or less frequent administration of all The above treatments are administered to subjects with respiratory diseases. Using a lower dose of a treatment (such as a prophylactic or therapeutic agent) and/or administering the treatment less frequently can reduce the toxicity associated with administering the treatment to a subject without reducing the effectiveness of the treatment in preventing or treating the respiratory tract. effect on disease. In addition, synergy can lead to increased efficacy of a therapy (eg, a prophylactic or therapeutic agent) in preventing or treating a respiratory disease. Finally, the synergistic effect of combination therapies (eg, prophylactic or therapeutic agents) can avoid or reduce unwanted adverse side effects associated with the use of any single therapy.

The term "T cell receptor modulator" as used herein refers to specific proteins such as cytokines that modulate the phosphorylation of T cell receptors, the activation of signal transduction pathways associated with T cell receptors, and/or the activity of T cell receptors material of expression. Such substances can directly or indirectly regulate the phosphorylation of T cell receptors, the activation of signal transduction pathways associated with T cell receptors and/or the expression of specific proteins such as cytokines associated with T cell receptor activity. Examples of T cell receptor modulators include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, and antibodies that immunospecifically bind to T cell receptors or fragments thereof. In addition, examples of T cell receptor modulators include, but are not limited to, proteins, peptides, polypeptides (such as soluble T cell receptors), fusion proteins, and antibodies that immunospecifically bind to T cell receptor ligands or fragments thereof.

The term "therapeutic agent" as used herein refers to a disease or disorder that can be used to prevent, treat and/or control, for example, a disease or disorder related to or characterized by abnormal expression and/or activity of IL-9 polypeptide, IL-9R A disease or disorder associated with or characterized by abnormal expression and/or activity of one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one of or any substance with multiple symptoms. In certain embodiments, the term "therapeutic agent" refers to an antibody that binds an IL-9 polypeptide. In certain other embodiments, the term "therapeutic agent" refers to a substance other than an antibody that immunospecifically binds an IL-9 polypeptide. Preferably, the therapeutic agent is known to be useful or has been used or is being used to prevent, treat and/or control diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity of -9R or one or more subunits thereof, or Substance with one or more symptoms. Therapeutic agents can be classified into different classes based on one or more actions that the substance produces in vivo and/or in vitro, for example, anti-inflammatory agents can also be identified as immunomodulators.

The term "therapeutically effective amount" as used herein means sufficient to reduce a disease or disorder, for example, a disease or disorder associated with or characterized by aberrant expression and/or activity of IL-9 polypeptide, IL-9R or one or more thereof A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection) associated with or characterized by aberrant expression and/or activity of the subunit, or severity of one or more symptoms thereof degree; reduce the duration of respiratory disease; alleviate the disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, abnormal expression of IL-9R or one or more subunits thereof and/or disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or one or more symptoms thereof, autoimmune disease, inflammatory disease , proliferative diseases or infections (such as respiratory tract infections), or one or more symptoms thereof; causing diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, and IL-9R or A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity of one or more subunits thereof, or one or Regression of various symptoms; or the dosage of a treatment (such as an antibody that immunospecifically binds to IL-9 polypeptide) that increases or improves the curative effect of another treatment.

The term "treatment" may refer to a disease or disorder useful for the prevention, treatment and/or control, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, IL-9R or its A disease or disorder associated with or characterized by abnormal expression and/or activity of one or more subunits, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more any regimen, method and/or substance for any symptom. In certain embodiments, the term "treatment" refers to the prevention, treatment and/or management of a disease or disorder, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of an IL-9 polypeptide, Diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative diseases or infections (such as respiratory infections), Antiviral, antibacterial, antifungal, biologic, supportive and/or other treatments known to healthcare practitioners for one or more of its symptoms.

As used herein, the term "therapeutic regimen" refers to the dosing and timing schedule for administering one or more treatments (eg, one or more therapeutic agents) that have a therapeutic effect.

The term "treatment" as used herein refers to the reduction or improvement of a disease or disorder due to the administration of one or more treatments (including but not limited to the administration of one or more preventive or therapeutic agents), such as the abnormal expression of IL-9 polypeptide and/or Diseases or disorders associated with or characterized by IL-9R or its activity, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory Progression, severity, and/or duration of disease, proliferative disease, or infection (eg, respiratory tract infection), or one or more symptoms thereof. In certain embodiments, such terms refer to reducing swelling of an organ or tissue, or reducing pain associated with respiratory disease. In other embodiments, the term refers to a reduction in inflammation or asthma-associated airway constriction. In other embodiments, this term refers to reduced replication of an infectious agent, or reduced spread of an infectious agent to other organs or tissues of the subject or to other subjects. In other embodiments, the term refers to a reduction in the release of inflammatory substances by mast cells, or a reduction in the biological effects of such inflammatory substances. In other embodiments, the term refers to a decrease in the growth, formation and/or increase in number of hyperproliferative cells (eg, cancer cells). In other embodiments, the term refers to the eradication, elimination or control (eg, minimizing or delaying the spread of cancer) of primary, localized or metastatic cancer.

The term "very little or even no decrease in biological activity" as used herein refers to antibody activity, including but not limited to: through different immunological experiments, including but not limited to ELISA and radioimmunoassay, antibody (including antibody fragments thereof) and interest Specific binding activity of antigen (such as IL-9 polypeptide). In one embodiment, as determined by an immunoassay known to those skilled in the art or as described herein, compared with a reference antibody (including antibody fragments thereof) (e.g., 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com- 2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4), the antibody (including its antibody fragment) of the preparation of the present invention keeps about 50%, preferably 55%, 60%, 65%, 70% %, 75%, 80%, 85%, 90%, 95% or 98% ability to specifically bind to an antigenic polypeptide. For example, ELISA-based assays can be used to compare antibodies (including antibody fragments thereof) to 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com - the ability of the 3D4 reference standard to immunospecifically bind IL-9 polypeptide. In this assay, called IL-9 binding ELISA, isolated IL-9 was used to coat plates and a set of concentrations of 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, The binding signal of the 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 reference standard was compared to the binding signal of the same concentration of the test antibody, including antibody fragments thereof. The term "reference standard" as used herein refers to 10 mg/ml antibody (including its antibody fragments) (such as 4D4, 4D4 H2-1D11, 4D4com-XF- 9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4), pH 6.0-6.5 composed of antibodies (including antibody fragments thereof) (such as, 4D4, 4D4 H2-1D11, 4D4com- XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4), the reference formulation generally produced a single monomer peak (≥97% area) in HPSEC. In another embodiment, the term "very little to no decrease in biological activity" as used herein refers to antibody activity, including other effector activities of the antibody.

Concentrations, amounts, cell counts, percentages and other values may be expressed herein in a range format. It should also be understood that the use of this range format is for convenience and brevity only and should be read flexibly to include, but not be limited to, the values expressly mentioned by the upper and lower limits of the range, and also to include all individual values or subranges subsumed within that range, It is as if the individual values and subranges are explicitly mentioned.

Brief description of the drawings

Figure 1A-B shows (A) the amino acid sequence of the heavy chain variable region of 4D4 (SEQ ID NO: 7), which contains the VH CDR1 (SEQ ID NO: 1) underlined starting from the leftmost VH CDR1, VHCDR2 (SEQ ID NO: 61) and VH CDR3 (SEQ ID NO: 3); (B) Amino acid sequence of the light chain variable region of 4D4 (SEQ ID NO: 8), which contains VL CDR1 starting from the leftmost VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 5) and VL CDR3 (SEQ ID NO: 6) are underlined.

Figure 2A-B shows (A) the amino acid sequence of the heavy chain variable region of 4D4H2-1D11 (SEQ ID NO: 9), which contains the VH CDR1 (SEQ ID NO: 1) underlined starting from the leftmost VH CDR1 , VH CDR2 (SEQ ID NO: 10) and VH CDR3 (SEQ ID NO: 3); (B) the amino acid sequence (SEQ ID NO: 8) of the light chain variable region of 4D4H2-1D11, which contains VL CDR1 begins with underlined VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 5) and VL CDR3 (SEQ ID NO: 6).

Figure 3A-B shows (A) the amino acid sequence of the heavy chain variable region of 4D4com-XF-9 (SEQ ID NO: 15), which contains the VH CDR1 (SEQ ID NO: 15) underlined starting from the leftmost VH CDR1 : 11), VH CDR2 (SEQ ID NO: 10) and VH CDR3 (SEQ ID NO: 12); (B) the amino acid sequence (SEQ ID NO: 16) of the light chain variable region of 4D4com-XF-9, its Contains VL CDR1 (SEQ ID NO: 13), VL CDR2 (SEQ ID NO: 14) and VL CDR3 (SEQ ID NO: 63) underlined starting from the leftmost VL CDR1.

Figure 4A-B shows (A) the amino acid sequence (SEQ ID NO: 17) of the heavy chain variable region of 4D4com-2F9, which contains the VH CDR1 (SEQ ID NO: 1) underlined starting from the leftmost VH CDR1 ), VH CDR2 (SEQ ID NO: 10) and VH CDR3 (SEQ ID NO: 12); (B) the amino acid sequence (SEQ ID NO: 18) of the light chain variable region of 4D4com-2F9, which contains VL CDR1 begins with VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 14) and VL CDR3 (SEQ ID NO: 64) underlined.

Figure 5A-B shows (A) the amino acid sequence of the heavy chain variable region of 7F3 (SEQ ID NO: 21), which contains VH CDR1 (SEQ ID NO: 19) underlined starting from the leftmost VH CDR1, VHCDR2 (SEQ ID NO: 61) and VH CDR3 (SEQ ID NO: 3); (B) Amino acid sequence of the light chain variable region of 7F3 (SEQ ID NO: 22), which contains VL CDR1 starting from the leftmost VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 5) and VL CDR3 (SEQ ID NO: 20) are underlined.

Figure 6A-B shows (A) the amino acid sequence of the heavy chain variable region of 71A10 (SEQ ID NO: 23), which contains VH CDR1 (SEQ ID NO: 19) underlined starting from the leftmost VH CDR1, VH CDR2 (SEQ ID NO: 2) and VH CDR3 (SEQ ID NO: 3); (B) Amino acid sequence of the light chain variable region of 71A10 (SEQ ID NO: 24), which contains VL CDR1 starting from the leftmost VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 5) and VL CDR3 (SEQ ID NO: 20) are underlined.

Figure 7A-B shows (A) the amino acid sequence of the heavy chain variable region of 7F3 22D3 (SEQ ID NO: 21), which contains the VH CDR1 (SEQ ID NO: 19) underlined starting from the leftmost VH CDR1 , VH CDR2 (SEQ ID NO: 61) and VH CDR3 (SEQ ID NO: 3); (B) Amino acid sequence of the light chain variable region of 7F3 22D3 (SEQ ID NO: 25), which contains VL from the leftmost CDR1 begins with VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 14) and VLCDR3 (SEQ ID NO: 20) underlined.

Figure 8A-B shows (A) the amino acid sequence of the heavy chain variable region of 7F3com-2H2 (SEQ ID NO: 27), which contains the VH CDR1 (SEQ ID NO: 26) underlined starting from the leftmost VH CDR1 ), VH CDR2 (SEQ ID NO: 2) and VH CDR3 (SEQ ID NO: 3); (B) the amino acid sequence (SEQ ID NO: 28) of the light chain variable region of 7F3com-2H2, which contains VL CDR1 begins with underlined VL CDR1 (SEQ ID NO: 62), VL CDR2 (SEQ ID NO: 65) and VL CDR3 (SEQ ID NO: 20).

Figure 9A-B shows (A) the nucleotide sequence of the heavy chain variable region of 7F3com-2H2 (SEQ ID NO: 43), which contains the VH CDR1 (SEQ ID NO: 43) underlined starting from the leftmost VH CDR1 : 44), VH CDR2 (SEQ ID NO: 45) and VH CDR3 (SEQ ID NO: 46); (B) the nucleotide sequence (SEQ ID NO: 47) of the light chain variable region of 7F3com-2H2, its Contains VL CDR1 (SEQ ID NO: 48), VL CDR2 (SEQ ID NO: 49) and VL CDR3 (SEQ ID NO: 50) underlined starting from the leftmost VL CDR1.

Figure 10A-B shows (A) the amino acid sequence of the heavy chain variable region of 7F3com-3H5 (SEQ ID NO: 29), which contains the VH CDR1 (SEQ ID NO: 19) underlined starting from the leftmost VH CDR1 ), VH CDR2 (SEQ ID NO: 2) and VH CDR3 (SEQ ID NO: 3); (B) the amino acid sequence (SEQ ID NO: 30) of the light chain variable region of 7F3com-3H5, which contains VL CDR1 begins with underlined VL CDR1 (SEQ ID NO: 4), VL CDR2 (SEQ ID NO: 14) and VL CDR3 (SEQ ID NO: 20).

Figure 11A-B shows (A) the amino acid sequence of the heavy chain variable region of 7F3com-3D4 (SEQ ID NO:31), which contains the VH CDR1 (SEQ ID NO:26) underlined starting from the leftmost VH CDR1 ), VH CDR2 (SEQ ID NO: 2) and VH CDR3 (SEQ ID NO: 3); (B) the amino acid sequence (SEQ ID NO: 32) of the light chain variable region of 7F3com-3D4, which contains VL CDR1 begins with underlined VL CDR1 (SEQ ID NO: 62), VL CDR2 (SEQ ID NO: 14) and VL CDR3 (SEQ ID NO: 20).

Figure 12 shows the nucleotide sequence of human IL-9 (SEQ ID NO:51) located in the GenBank database (accession number NM_00590).

Figure 13 shows the amino acid sequence of human IL-9 located in the GenBank database (accession numbers A60480 (SEQ ID NO:52), NP_00584 (SEQ ID NO:53) and AAC17735 (SEQ ID NO:54)).

Figures 14A-C show the nucleotides of the human IL-9R subunits found in the GenBank database (accession numbers NM_002186 (SEQ ID NO: 55), NM_176786 (SEQ ID NO: 56) and NM_000206 (SEQ ID NO: 57)) sequence. (A) Accession No. NM_002186 and (B) Accession No. NM_176786 are the nucleotide sequences of the human IL-9Rα subunit isoform precursor. (C) Accession number NM_000206 is the nucleotide sequence of human IL-9R γ chain.

Figure 15 shows the amino acid sequence of human IL-9R found in the GenBank database (accession numbers NP_002177 (SEQ ID NO: 58), NP_789743 (SEQ ID NO: 59) and NM_000197 (SEQ ID NO: 60)). Accession numbers NP_002177 and NP_789743 are the amino acid sequences of human IL-9Rα subunit isoform precursors. NP_000197 is the amino acid sequence of human IL-9Rγ chain.

Figure 16 is a schematic illustration of a protocol for preparing purified antibodies that immunospecifically bind IL-9 polypeptides.

Figures 17A-17B. Urea-induced 7F3com-2H2 unfolding in the absence of salt and in the presence of 150 mM NaCl demonstrates that antibody unfolding is a simple 2-step process in the presence of histidine alone, indicating that all structures The domains unfold cooperatively, whereas upon addition of salt, the unfolding of the 7F3com-2H2 domain is a continuous process through the formation of a population of intermediates. (A) graph, 10mM histidine, pH 6.0; (B) graph, 10mM phosphate buffer, pH 6.0.

Figures 18A-18B. (A) in 10 mM histidine without salt, 25 mM NaCl and 150 mM NaCl, pH 6.0; and (B) in 10 mM phosphate buffer without salt and 150 mM NaCl, pH 6.0 , Differential scanning calorimetry (DSC) graph of 7F3com-2H2.

Figures 19A-19B. KI (potassium iodide) quenching studies of the 7F3com-2H2 antibody. (A) SV plot (Stern-Volmerplot) of native and intermediate 7F3com-2H2 in 10 mM histidine, 150 mM NaCl, pH 6.0. (B) SV plot (Stern-Volmer plot) of native and unfolded 7F3com-2H2 in 10 mM phosphate, 150 mM NaCl, pH 6.0.

Figures 20A-20B. (A) Urea-induced 7F3com-2H2 unfolding at pH 8.1 in 10 mM histidine as measured by tryptophan fluorescence and ANS binding assays. Error bars shown were obtained from three different sets of experiments performed on different days. (B) Urea-induced 7F3com-2H2 unfolding at pH 8.1 in 10 mM phosphate as measured by tryptophan fluorescence and ANS binding assays.

Figure 21. Results of Fourier Transform Infrared Spectroscopy (FTIR) experiments used to analyze the structural non-ideality of antibody 7F3com-2H2. The results indicated that pH alone was not responsible for the binding of 7F3com-2H2 (pi=8.1) in solution; it was strongly dependent on the buffer type.

Figure 22. Urea-induced unfolding of full length, F _ab fragment and _Fc fragment of 7F3com-2H2 in the absence of salt (left panel) and presence of 150 mM NaCl (right panel), 10 mM histidine, pH 6. Unfolding of 7F3com-2H2 was determined by changing the center of spectral mass (CSM) with urea concentration. Solid lines are curves obtained with a simple two-state or non-two-state fit. The inset shows the unfolding of full-length 7F3com-2H2 in 10 mM phosphate buffer pH 6.0.

Figure 23a and b. (a) DSC studies with the full-length, _Fab and _Fc fragments of 7F3com-2H2 showed that the presence of histidine and salts resulted in reduced domain-domain interactions of the full-length mAb and The CH2 domain of the Fc region is destabilized. (b) DSC studies in phosphate buffered saline with the full-length and isolated _Fab and _Fc fragments of 7F3com-2H2 did not show any significant changes regardless of the presence or absence of salt.

Figure 24. Animation of the charge shielding effect of NaCl. At pH 6, the 7F3com-2H2 antibody is positively charged, and the imidazole side chain of histidine has a probability of more than 50% to be positively charged, thereby causing charge repulsion between histidine and the antibody.

Figure 25. Viscosity of 7F3com-2H2 antibody in two buffer systems at pH 6. The ionic strength of the solution with or without NaCl was 153 and 2.5 mM, respectively. All viscosities less than 20 cP are measured at a shear rate of 600 s ⁻¹ . The curve is a fitting curve of the third-order polynomial η=η ₀ (1+k ₁ c+k ₂ c ² +k ₃ c ³ . . . ).

Figure 26. Nephelometric turbidity (light scattering at 90°), which is a measure of the opalescence of 7F3com-2H2 under the same conditions as viscometry. Opalescence and viscosity are inversely related to the ionic strength of this antibody. Draw as a line for easy observation.

Figure 27. The osmolarity of 7F3com-2H2 solutions at pH 6 prepared with two buffer systems was determined by membrane osmometry, with ionic strength matching that of Figure 25. The second virial coefficient of the solution with higher ionic strength is negative, and its apparent molecular weight is also larger than that of the buffer system with lower ionic strength. Lines represent linear regression.

Figure 28a-c. (a) Calculation of the critical second virial coefficient from osmotic pressure data. (b) Tables showing critical virial coefficients reported in the literature are in the range of -5 to -6. (c) Table providing the linear regression of the osmolarity data of Figure 27 to the (a, left) equation and normalized to the protein volume in the (a, right) equation. The two conditions that were opalescent also had a second virial coefficient near the critical value, suggesting that opalescence may be associated with phase separation (liquid-liquid or liquid-solid).

Figure 29. Stability of anti-IL-9 antibody formulations at 40°. Different antibody preparations containing 5 g/l anti-IL-9 antibody were incubated at 40°C for up to 75 days. Antibody fragmentation levels were assessed at time intervals by size exclusion chromatography. The measured monomer concentration and antibody fragment concentration are plotted versus time.

Figure 30. Representative spectra obtained during the course of the experiment with antibody preparations containing 2.2 mM sodium phosphate. The UV absorbance curves shown correspond to the left axis. Elution data points obtained with commercially available molecular weight markers correspond to the right axis.

5. Detailed Description of the Invention

The stable liquid formulation of the present invention provides a ready-to-use formulation of a therapeutic or prophylactic antibody of interest (including antibody fragments thereof), for example, an antibody that immunospecifically binds to an IL-9 polypeptide (such as 7F3com-2H2), which Formulations are intended to be administered to a subject (eg, a human subject) without reconstituting the formulation using precise aseptic technique and waiting a period of time until the solution is clear before administering the formulation to a subject. Furthermore, such reconstitution solutions must be used within a certain period of time, making them a very expensive waste product. It simplifies the steps for a health care provider to administer a formulation to a subject.

Furthermore, due to the high stability during storage, the formulations of the invention may contain therapeutic or prophylactic antibodies of interest (including antibody fragments thereof), e.g., immunospecific Antibodies (such as 7F3com-2H2) that specifically bind to IL-9 polypeptides. This stability not only ensures the efficacy of the antibody, but also reduces the risk of possible adverse effects in the subject. Also, using fewer components in the formulation reduces the risk of contamination. Furthermore, the production process of the liquid formulation of the present invention is simple and more efficient compared to the lyophilized form, since all production stages of the liquid formulation are carried out in aqueous solution, excluding drying steps such as lyophilization and freeze-drying steps. Therefore, its cost is lower.

When an antibody is formulated in a zwitterionic buffer containing, for example, histidine, due to the zwitterionic nature of histidine, it may interact with the antibody by reducing interactions between domains, possibly breaking down salt bridges, and therefore Antibodies formulated according to the methods of the invention are characterized by certain phase properties, such as formation of unfolded intermediates, colloidal instability, soluble binding of antibody molecules, and precipitation of antibody molecules, as determined by sensitive analytical techniques including, but not Limited to: high performance size exclusion chromatography (HPSEC), tangential flow filtration (TFF) technology, static light scattering (SLS) technology, Fourier transform infrared spectroscopy (FTIR) technology, circular dichroism (CD) technology, urea- Induced protein unfolding techniques, tryptophan intrinsic fluorescence techniques, differential scanning calorimetry (DSC) and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques. This interaction can lead to reduced colloidal stability, soluble association, and precipitation, all of which can lead to protein aggregation, with undesirable consequences for antibody formulation instability. Accordingly, the present invention provides for determining whether certain buffers, such as non-zwitterionic buffers (e.g., phosphate, tris, citrate, succinate, and acetate buffers), can facilitate the binding of specific proteins (e.g., monoclonal antibodies). The reduction or reduction of certain phase characteristics such as colloidal instability, soluble binding and precipitation make them more suitable for formulation by the method of the present invention.

Antibody preparation

The stable liquid formulations of the present invention provide highly stable antibody formulations with little or no aggregation during long-term storage. In a specific embodiment, such antibody preparations are homogeneous. In one embodiment, the formulations of the invention are sterile. Formulations of the invention comprise an aqueous carrier (such as distilled water), phosphate or other non-zwitterionic buffer, optionally containing a salt such as NaCl, and a therapeutic or prophylactic antibody of interest (including antibody fragments thereof), e.g., immunospecifically binding to IL -9 polypeptide antibodies, so long as the antibody is more stable in a non-zwitterionic buffer at a pH below the pI at a concentration of about 10 mg/ml to 300 mg/ml compared to a zwitterionic buffer. In one embodiment, the formulations of the invention are free of ingredients other than water and suitable solvents. In another preferred embodiment, the water is distilled water. In a specific embodiment, the antibody that immunospecifically binds IL-9 polypeptide contained in the liquid preparation of the present invention is 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com - 2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof. In one embodiment, the antibody that immunospecifically binds IL-9 polypeptide contained in the liquid formulation of the present invention is not 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof. In one embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide contained in the liquid preparation of the present invention contains one or more of the VH CDRs listed in Table 1 above and/or one or more of the VH CDRs listed in Table 1 above. Antibodies to VL CDRs (including antibody fragments thereof). In another embodiment, the antibody (including antibody fragment thereof) immunospecifically binding to IL-9 polypeptide contained in the liquid formulation of the present invention is an antibody (including antibody fragment thereof) conjugated to another moiety, said other Some include, but are not limited to: heterologous polypeptide, another antibody (including antibody fragments thereof), marker sequences, diagnostic agents, therapeutic agents, radioactive metal ions, polymers, albumin, and solid supports. In yet another embodiment, the liquid preparation of the present invention comprises two or more antibodies or (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptide, wherein at least one antibody (including antibody fragments thereof) is 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof.

The concentration of the antibody of interest (including antibody fragments thereof) contained in the liquid preparation of the present invention, such as an antibody that immunospecifically binds to an IL-9 polypeptide, is at least 10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml , at least 30mg/ml, at least 35mg/ml, at least 40mg/ml, at least 45mg/ml, at least 50mg/ml, at least 55mg/ml, at least 60mg/ml, at least 65mg/ml, at least 70mg/ml, at least 75mg/ml , at least 80mg/ml, at least 85mg/ml, at least 90mg/ml, at least 95mg/ml, at least 100mg/ml, at least 105mg/ml, at least 110mg/ml, at least 115mg/ml, at least 120mg/ml, at least 125mg/ml , at least 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150 mg/ml, at least 175 mg/ml, at least 200 mg/ml, at least 250 mg/ml, at least 275 mg/ml, or at least 300 mg/ml. The concentration of the antibody of interest (including antibody fragments thereof), for example, an antibody that immunospecifically binds to an IL-9 polypeptide (such as 7F3com-2H2), contained in the liquid formulation of the present invention is 10 mg/ml or higher, at least 15 mg/ml or Higher, at least 20 mg/ml or higher, at least 25 mg/ml or higher, at least 30 mg/ml or higher, at least 35 mg/ml or higher, at least 40 mg/ml or higher, at least 45 mg/ml or higher , at least 50 mg/ml or higher, at least 55 mg/ml or higher, at least 60 mg/ml or higher, at least 65 mg/ml or higher, at least 70 mg/ml or higher, at least 75 mg/ml or higher, at least 80 mg/ml or higher, at least 85 mg/ml or higher, at least 90 mg/ml or higher, at least 95 mg/ml or higher, at least 100 mg/ml or higher, at least 105 mg/ml or higher, at least 110 mg/ml ml or higher, at least 115 mg/ml or higher, at least 120 mg/ml or higher, at least 125 mg/ml or higher, at least 130 mg/ml or higher, at least 135 mg/ml or higher, at least 140 mg/ml or Higher, at least 150 mg/ml or higher, at least 175 mg/ml or higher, at least 200 mg/ml or higher, at least 250 mg/ml or higher, at least 275 mg/ml or higher, or at least 300 mg/ml or higher . In a specific embodiment, the concentration of the antibody of interest (including antibody fragments thereof) contained in the liquid preparation of the present invention, such as an antibody (such as 7F3com-2H2) that immunospecifically binds to IL-9 polypeptide is about 50 mg/ml, 75 mg /ml, about 100mg/ml, about 125mg/ml, about 150mg/ml, about 175mg/ml, about 200mg/ml, about 225mg/ml, about 250mg/ml, about 275mg/ml or about 300mg/ml. In another embodiment, the concentration of the antibody of interest (including antibody fragments thereof) contained in the liquid preparation of the present invention, for example, an antibody (such as 7F3com-2H2) that immunospecifically binds to IL-9 polypeptide is 10-50 mg/ml , 15-500mg/ml, 50-300mg/ml, 50-250mg/ml, 50-200mg/ml, 50-150mg/ml, 100-200mg/ml, 50-175mg/ml, 50-150mg/ml, 50 - 125 mg/ml or 50-100 mg/ml. In a specific embodiment, a formulation of the invention comprises about 100 mg/ml of an anti-IL-9 antibody (eg, 7F3com-2H2).

The formulation may be buffered with phosphate (although other suitable buffers such as tris, citrate, succinate and acetate buffers may also be used). Phosphate is included in the liquid formulation of the present invention at a concentration ranging from 1 mM to 5 mM, 1 mM to 100 mM, 10 mM to 30 mM, 25 mM to 75 mM or 10 mM to 100 mM. In a specific embodiment, the concentration of phosphate contained in the liquid formulation of the present invention is 2mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM , 80mM, 85mM, 90mM, 95mM or 100mM. Any form of phosphate salt suitable for formulation and parenteral administration may be used. The phosphate should be at least 98%, at least 99%, or at least 99.5% pure. When referring to phosphate, the term "purity" as used herein refers to the chemical purity of phosphate as understood in the art, as in The Merck Index, 13th Ed., O'Neil et al. (Merck, Inc. ( Merck & Co.), 2001). In a specific embodiment, formulations of the invention comprise 25 mM phosphate buffer. In addition to phosphate, other buffers, especially non-zwitterionic buffers (such as tris, citrate, succinate, and acetate buffers), may also be used in the formulations of the present invention, at concentrations ranging from 1 mM to 100 mM, 25 mM to 75 mM or 10 mM to 100 mM, or at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM.

In certain embodiments, formulations of the invention comprise salts. In one embodiment, the formulation of the invention comprises a salt selected from the group consisting of NaCl, KCl, _CaCl2 and _MgCl2 . In a specific embodiment, the salt may be NaCl. NaCl may be included in the formulations of the invention at a concentration ranging from 10 mM to 300 mM, 50 mM to 200 mM, 100 mM to 200 mM or 125 mM to 175 mM. In a specific embodiment, the concentration of NaCl included in the formulations of the present invention may be about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM or about 300mM. The purity of the NaCl may be at least 98%, at least 99%, or at least 99.5%. In a specific embodiment, the concentration of NaCl included in the liquid formulation of the present invention may be about 150 mM. When referring to NaCl, the term "purity" as used herein refers to the chemical purity of NaCl as understood in the art, such as The Merck Index, 13th Ed., O'Neil et al. (Merck & Co. ), 2001).

The pH of the formulation should generally not be equal to the isoelectric point of the specific antibody (including antibody fragments thereof) used in the formulation (e.g. 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com -2H2, 7F3com-3H5 or 7F3com-3D4 has an isoelectric point in the range of 8.65 to 8.89), which may range from about 4.0 to 8.0, or may range from about 6.0 to 6.5. In a specific embodiment, a formulation of the invention comprising an anti-IL-9 antibody (eg, 7F3com-2H2) may have a pH of about 6.0.

Optionally, the preparation of the present invention can also contain other excipients, such as sugar (such as sucrose, mannose, trehalose, etc.), polyalcohols (such as mannitol, sorbitol, etc.) and surfactants (such as Tween- 20 or Tween-80). In one embodiment, the other excipient is sugar (such as sucrose). In a specific embodiment, the sugar is sucrose, which is present in the formulation at a concentration ranging from about 0% to 10%, about 1% to 20%, about 5% to 15%, or about 8% to 10%. In a specific embodiment, the sugar is trehalose, which is present in the formulation at a concentration of about 0% to 10%, about 1% to 20%, about 5% to 15%, or about 8% to 10%. In another embodiment, the excipient is a polyol. In a specific embodiment, the concentration of the polyol in the formulation is 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5% or 1%. In a specific embodiment, the polyol is sorbitol, which is present in the formulation at a concentration ranging from about 0% to 0.1%, from about 0.001% to 1%, or from about 0.01% to 0.1%. The liquid formulations of the present invention may or may not contain mannitol. In another embodiment, the excipient is a surfactant. In specific embodiments, the surfactant is Tween-20 or Tween-80, which is present in the formulation at a concentration ranging from about 0% to 0.1%, from about 0.001% to 1%, or from about 0.01% to 0.1%. In a specific embodiment, the surfactant is Tween-20 or Tween-80, and its concentration in the formulation is 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5% % or 1%.

Those skilled in the art should understand that the preparation of the present invention may be isotonic with human blood, that is, the osmotic pressure of the preparation of the present invention is substantially equal to that of human blood. The osmotic pressure of such isotonic preparations is usually about 250-350 mOSm. Isotonicity can be measured, for example, by vapor pressure or ice-type osmometers. The tonicity of the formulation is adjusted using a tonicity adjusting agent. A "tonicity modifier" is a pharmaceutically acceptable inert substance that can be added to a formulation to provide isotonicity to the formulation. Tonicity modifiers suitable for use in the present invention include, but are not limited to, sugars, salts and amino acids.

In certain embodiments, formulations of the invention may comprise about 51 mg/ml to 150 mg/ml of an anti-IL-9 antibody (e.g., 7F3com-2H2), about 10 mM to 50 mM sodium phosphate, about 100 mM to 200 mM NaCl, at a pH of About 5.5 to 7.0. In another embodiment, a formulation of the invention may comprise about 100 mg/ml anti-IL-9 antibody (eg, 7F3com-2H2), about 25 mM sodium phosphate, about 150 mM NaCl, and have a pH of about 6.0. In a specific embodiment, a formulation of the invention consists of about 100 mg/ml 7F3com-2H2 anti-IL-9 antibody, about 25 mM sodium phosphate, about 50 mM NaCl, and has a pH of about 6.0.

As evaluated by high performance size exclusion chromatography (HPSEC), the liquid formulation of the present invention has a stability of at least 15 days in the temperature range of 38°C-42°C, and in some embodiments, a stability of no more than 25 days, and a stability of no more than 25 days in the temperature range of 20°C-24°C. The temperature range of ℃ has a stability of at least 6 months, and the temperature range of 2°C-8°C (specifically 4°C) has a stability of at least 6 months, at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, A stability of at least 3 years or at least 4 years, with a stability of at least 2 years, at least 3 years, at least 4 years or at least 5 years at a temperature of -20°C. Also using tangential flow filtration (TFF) techniques, static light scattering (SLS) techniques, Fourier transform infrared spectroscopy (FTIR) techniques, circular dichroism (CD) techniques, urea-induced protein unfolding techniques, tryptophan Acid intrinsic fluorescence techniques, differential scanning calorimetry (DSC) and/or 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques were used to assess the phase properties, other physical properties and stability of the molecules. That is, the aggregation and/or fragmentation level of the liquid preparation of the present invention is so low as to be undetectable after storage for the above-mentioned defined period of time. For example, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and most preferably no more than 0.5% of antibodies (including antibody fragments thereof) as determined by HPSEC after storage for the above-mentioned defined period of time. ) to form aggregates.

Moreover, the ability of antibodies (including antibody fragments thereof) to immunospecifically bind to antigens is determined by various immunoassays, including, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, to determine the extended storage period under the above conditions, the present invention The biological activity of antibodies (including antibody fragments thereof) hardly decreases in liquid formulations. After storing the above-mentioned time, compared with the initial biological activity (such as the ability to bind IL-9 polypeptide) of the preparation before storage, the liquid preparation of the present invention retains greater than 80%, greater than 85%, greater than 90%, greater than 95%, Greater than 98%, greater than 99%, or greater than 99.5% biological activity. In some embodiments, the liquid formulation of the invention retains at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% after storage for the above defined period of time compared to the reference antibody prior to storage. % or at least 99.5% of the biological activity (such as the ability to bind IL-9 polypeptide).

Liquid formulations of the invention may be prepared in unit dosage form. For example, each vial of unit dosage form can contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml of the antibody or antibody fragment at various concentrations, which can be from about 15 mg/ml to about 300 mg/ml, about 50 mg/ml to about 300 mg/ml, about 50 mg/ml to about 150 mg/ml, about 75 mg/ml to about 300 mg/ml, about 95 mg/ml to about 300 mg/ml, about 100 mg/ml to about 300 mg/ml, about 150 mg/ml to about 300 mg/ml, about 200 mg/ml to about 300 mg/ml, about 100 mg/ml to about 200 mg/ml, about 100 mg/ml to about 150 mg/ml, or about 100 mg/ml to about 175mg/ml. These formulations can be adjusted, if necessary, to the desired concentration by adding sterile diluent to each vial.

The invention includes stable liquid formulations containing individual antibodies of interest (including antibody fragments thereof), eg, antibodies that immunospecifically bind IL-9 polypeptides. The invention also includes stable liquid formulations comprising two or more antibodies of interest (including antibody fragments thereof), eg, antibodies that immunospecifically bind IL-9 polypeptides. In a specific embodiment, the stable liquid preparation of the present invention comprises 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com- 3H5 or 7F3com-3D4 or a fragment thereof. In another embodiment, the stable liquid formulation of the present invention comprises two or more antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptide, wherein one of the antibodies (including antibody fragments thereof) is 4D4, 4D4 H2 -1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof. In another embodiment, the stable liquid formulation of the present invention comprises two or more antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptide, with the proviso that said antibodies (including antibody fragments thereof) do not include 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof.

5.1.1. Antibodies used in the formulations of the invention

Antibodies for use in the methods of the invention include, but are not limited to: detection by, for example, High Performance Size Exclusion Chromatography (HPSEC), Tangential Flow Filtration (TFF) techniques, Static Light Scattering (SLS) techniques, Fourier Transform Infrared Spectroscopy (FTIR) ) technique, circular dichroism (CD) technique, urea-induced protein unfolding technique, tryptophan intrinsic fluorescence technique, differential scanning calorimetry (DSC) and/or 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding assay, compared to antibodies formulated at the same pH and in the presence (or absence) of the same concentration of salt in the presence of a non-zwitterionic buffer (such as phosphate buffer), Shows certain phase properties when formulated at pH below the pI of the antibody in the presence of zwitterionic buffers (e.g., histidine buffer) and salts (e.g., NaCl), e.g., formation of unfolded intermediates, colloidal stability Reduced, soluble bound and precipitated antibodies. In specific embodiments, antibodies (or fragments thereof) used in the methods of the present invention include, but are not limited to: 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2 , 7F3com-3H5 or 7F3com-3D4. Preferably, the antibody (or fragment thereof) used in the methods of the invention is 7F3com-2H2. For other therapeutic or prophylactic antibodies useful in the methods of the invention see, eg, Sections 5.1.1.1 to 5.1.1.17 below.

Antibodies used in the present invention include, but are not limited to: monoclonal antibodies, synthetic antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv) (including bispecific scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv) and their epitope-binding fragments. In particular, antibodies of the invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecule of the invention may be of any type (such as IgG, IgE, IgM, IgD, IgA and IgY), class (such as IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Antibodies used in the present invention may be from any animal source, including birds and mammals (eg, humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses or chickens). Preferably, the antibody is a human or humanized monoclonal antibody. As used herein, "human" antibodies include antibodies comprising the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from mice or other animals expressing antibodies from human genes.

Antibodies used in the present invention may be monospecific, bispecific, trispecific or more specific. Multispecific antibodies can immunospecifically bind to different epitopes of a polypeptide, or can immunospecifically bind to both a polypeptide and a heterologous epitope, such as a heterologous polypeptide or a solid support material. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920 and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547-1553.

Antibodies used in the present invention include derivatives of antibodies. Mutations can be introduced in the nucleotide sequences encoding the antibodies used in the methods of the invention using standard techniques known to those skilled in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Preferably, the derivative comprises less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, relative to the original molecule. A substitution, a substitution of less than 4 amino acids, a substitution of less than 3 amino acids, or a substitution of less than 2 amino acids. In one embodiment, the derivative comprises conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. "Conservative amino acid substitutions" are substitutions of amino acid residues with amino acid residues having similarly charged side chains. Families of amino acid residues having similarly charged side chains are defined in the art. These families include, amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar Amino acids with side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (such as threonine, valine, isoleucine ) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can also be introduced randomly along all or a portion of the coding sequence (eg, by saturation mutagenesis), and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be assayed.

Antibodies used in the present invention include modified derivatives, ie covalently linked derivatives produced by covalently linking any type of molecule to the antibody. For example, but not limited to, antibody derivatives include, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other Antibodies modified by methods such as proteins. Many chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, synthesis in the presence of tunicamycin, and the like. Furthermore, the derivative may contain one or more non-classical amino acids.

Antibodies or fragments thereof used in the invention may also comprise framework regions known to those skilled in the art. In certain embodiments, one or more, preferably all, framework regions of the antibodies used in the methods of the invention are human framework regions. In certain other embodiments of the invention, fragment regions of antibodies or fragments thereof of the invention are humanized. In certain embodiments, the antibodies used in the methods of the invention are synthetic antibodies, monoclonal antibodies, intracellular antibodies, chimeric antibodies, human antibodies, humanized chimeric antibodies, humanized antibodies, glycosylated antibodies, poly Specific antibodies, human antibodies, single chain antibodies or bispecific antibodies.

In certain embodiments of the present invention, the half-life of the antibody used in the present invention in mammals, preferably humans, is greater than 12 hours, greater than 1 day, greater than 3 days, greater than 6 days, greater than 10 days, greater than 15 days, greater than 20 days , more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months or more than 5 months. Antibodies or antigen-binding fragments thereof with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies or antigen-binding fragments thereof with increased half-life in vivo can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified to be involved in the interaction of the Fc region and the FcRn receptor (see, e.g., International Publication No. WO97/34631 and U.S. Patent 7,083,784, entitled "Molecules with Extended Half-Lives, Compositions and Uses Thereof," filed December 12, 2001, by Johnson et al., which is incorporated by reference in its entirety This article). The methods known to those skilled in the art, such as the immunoassay described herein, can be used to detect the binding activity of such antibodies or antigen-binding fragments thereof to RSV antigens and the in vivo efficacy.

Additionally, antibodies or antigen-binding fragments thereof with increased in vivo half-lives can be produced by linking polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibodies or antibody fragments. PEG can be attached to the antibody or antibody fragment via site-specific coupling to the N- or C-terminus of the antibody or antibody fragment, or via the ε-amino group on a lysine residue, with or without a multifunctional linker. Said antibody or antibody fragment. Derivatization of linear or branched polymers can be utilized resulting in minimal loss of biological activity. The degree of coupling was closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecules were properly coupled to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography. Methods known to those skilled in the art, such as immunoassays described herein, can be used to detect the binding activity of PEG-derived antibodies or antigen-binding fragments thereof to RSV antigens and the in vivo efficacy.

Antibodies used in the present invention may be single chain antibodies. Methods for the design and construction of single chain antibodies are described in Marasco et al., 1993, Proc Natl Acad Sci 90: 7889-7893, which is hereby incorporated by reference in its entirety.

In certain embodiments, antibodies used in the invention are capable of binding intracellular epitopes, ie, are intrabodies. Intrabodies comprise at least a portion of an antibody capable of immunospecifically binding an antigen, preferably without sequences encoding their secretion. Such antibodies bind their antigens within the cell. In one embodiment, the intrabody comprises a single chain Fv ("sFv"). sFv are antibody fragments that contain the VH and VL regions of an antibody, where these regions are present in a single polypeptide chain. Usually, a polypeptide linker is also included between the _VH and _VL regions of the Fv polypeptide, which allows the sFv to form the desired antigen-binding structure. For a review of sFv see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, eds. Rosenburg and Moore, Springer-Verlag, New York, vol. 269 - 315 pages (1994).

In another embodiment, intrabodies preferably do not encode an operable secretory sequence and are thus retained inside the cell (see generally Marasco, WA, 1998, "Intrabodies: References in Basic Research and Clinical Gene Therapy" (Intrabodies: Basic Research and Clinical Gene Therapy Applications), Springer, New York).

5.1.1.1. IL-9 antibody

In a specific embodiment, the invention provides a preparation of an antibody that immunospecifically binds an IL-9 polypeptide, preferably a human IL-9 polypeptide. Specifically, the present invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides: 4D4 or its antigen-binding fragment, 4D4 H2-1 D11 or its antigen-binding fragment, 4D4com-XF-9 or its antigen-binding fragment, 4D4com - 2F9 or an antigen-binding fragment thereof, 7F3 or an antigen-binding fragment thereof, 71A10 or an antigen-binding fragment thereof, 7F3 22D3 or an antigen-binding fragment thereof, 7F3com-2H2 or an antigen-binding fragment thereof, 7F3com-3H5 or an antigen-binding fragment thereof and 7F3com- 3D4 or an antigen-binding fragment thereof. In one embodiment, the antibody that immunospecifically binds IL-9 polypeptide is 7F3com-2H2 or an antigen-binding fragment thereof (eg, one or more CDRs of 7F3com-2H2). The constant regions of 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 71A10, 7F322D3, 7F3com, 7F3com-2H2, 7F3com-3H5 and 7F3com-3D4 were associated with Palivizumab (Midiuni ( MedImmune, Inc.)) IgG ₁ has the same constant region (see US Patent 5,824,307, issued October 20, 1998).

The present invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising a VH region having 4D4 (FIG. 1A; SEQ ID NO: 7), 4D4H2-1D11 (FIG. 2A; SEQ ID NO: 9), 4D4com-XF-9 (Fig. 3A; SEQ ID NO: 15), 4D4com-2F9 (Fig. 4A; SEQ ID NO: 17), 7F3 (Fig. 5A; SEQ ID NO: 21), 71A10 (Fig. 6A; SEQ ID NO: 21), 71A10 (Fig. 6A; SEQ ID NO: 17) ID NO: 23), 7F3 22D3 (Fig. 7A; SEQ ID NO: 21), 7F3com-2H2 (Fig. 8A; SEQ ID NO: 27), 7F3com-3H5 (Fig. 10A; SEQ ID NO: 29) or 7F3com-3D4 (FIG. 11A; SEQ ID NO: 31) Amino acid sequence of the VH region. In one embodiment, an antibody that immunospecifically binds an IL-9 polypeptide comprises a VH region having the amino acid sequence of the VH region of 7F3com-2H2 (FIG. 8A; SEQ ID NO: 27).

The present invention provides a preparation of an antibody that immunospecifically binds to an IL-9 polypeptide, the antibody comprising VHCDR, and the VH CDR has the amino acid sequence of any one of the VH CDRs listed in Table 1 above. Specifically, the present invention provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising one, two, three, four, five or more amino acids with any one of the VH CDRs listed in Table 1 above (or consisting of) the VH CDRs of the sequence. In one embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises a VH CDR1 having the amino acids of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: 26 sequence. In another embodiment, the antibody that immunospecifically binds an IL-9 polypeptide comprises a VH CDR2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 61. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises a VH CDR3 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR1 and VH CDR2, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VH CDR2 has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 61. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR1 and VH CDR3, said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VH CDR3 has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR2 and VH CDR3, and said VH CDR2 has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 61, The VH CDR3 has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR1, VH CDR2 and VH CDR3, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or The amino acid sequence of SEQ ID NO: 26, the VH CDR2 has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 61, and the VH CDR3 has SEQ ID NO: 3 or SEQ ID NO: 12 amino acid sequence.

The present invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising a VL region having 4D4 (FIG. 1B; SEQ ID NO: 8), 4D4H2-1 D11 (FIG. 2B; SEQ ID NO : 8), 4D4com-XF-9 (Fig. 3B; SEQ ID NO: 16), 4D4com-2F9 (Fig. 4B; SEQ ID NO: 18), 7F3 (Fig. 5B; SEQ ID NO: 22), 71A10 (Fig. 6B; SEQ ID NO:24), 7F322D3 (Figure 7B; SEQ ID NO:25), 7F3com-2H2 (Figure 8B; SEQ ID NO:28), 7F3com-3H5 (Figure 10B; SEQ ID NO:30) or 7F3com-3D4 (FIG. 11B; SEQ ID NO: 32) Amino acid sequence of the VL region. In one embodiment, an antibody that immunospecifically binds an IL-9 polypeptide comprises a VL region having the amino acid sequence of the VL region of 7F3com-2H2 (FIG. 8B; SEQ ID NO: 28).

The present invention also provides a preparation of an antibody that immunospecifically binds to an IL-9 polypeptide, the antibody comprising a VLCDR, and the VLCDR has the amino acid sequence of any one of the VLCDRs listed in Table 1 above. Specifically, the present invention provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising one, two, three or more VLCDRs (or composed of it). In one embodiment, the antibody that immunospecifically binds an IL-9 polypeptide comprises VLCDR1 having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62. In another embodiment, the antibody that immunospecifically binds an IL-9 polypeptide comprises a VL CDR2 having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:14, or SEQ ID NO:65. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises a VL CDR3 having a sequence of SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 or SEQ ID NO: 64 amino acid sequence. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises VL CDR1 and VL CDR2, said VL CDR1 having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62, The VL CDR2 has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VLCDR1 and VLCDR3, and said VLCDR1 has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62, so The VL CDR3 has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 or SEQ ID NO: 64. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises VL CDR2 and VL CDR3, said VL CDR2 having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65, The VL CDR3 has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 or SEQ ID NO: 64. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VL CDR1, VL CDR2 and VL CDR3, and said VL CDR1 has SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62 Amino acid sequence, the VL CDR2 has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65, and the VL CDR3 has SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 Or the amino acid sequence of SEQ ID NO: 64, which is a part of an antibody.

The invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising the VH regions described herein and the VL regions described herein, or other VL regions (e.g., filed April 12, 2002 and published as U.S. The VL region described in U.S. Patent Application No. 10/412,703 of Patent Publication No. US 2003/0219439 A1 (herein incorporated by reference in its entirety). The present invention also provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising the VL regions described herein and the VH regions described herein, or other VH regions (e.g., filed and published as U.S. Pat. The VH region described in U.S. Patent Application No. 10/412,703, published as US 2003/0219439 A1 (herein incorporated by reference in its entirety).

The present invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising the VH CDRs listed in Table 1 above and U.S. Patent filed on April 12, 2002 and published as U.S. Patent Publication No. US2003/0219439 A1 The VL CDRs described in Application No. 10/412,703 (or consisting of them). The present invention also provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising the VL CDRs listed in Table 1 above and U.S. Patent Application filed on April 12, 2002 and published as U.S. Patent Publication No. US 2003/0219439A1 (or consisting of) the VH CDRs described in Ser. No. 10/412,703. The present invention also provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising those described herein and U.S. Patent Application No. 10/412,703 filed April 12, 2002 and published as U.S. Patent Publication No. US 2003/0219439 A1 Combinations of published VH CDRs and VL CDRs.

The present invention provides a preparation of an antibody that immunospecifically binds to an IL-9 polypeptide, the antibody comprising one or more VH CDRs and one or more VL CDRs listed in Table 1 above. Specifically, the present invention provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising VH CDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VHCDR2 and VL CDR2; VH CDR2 and VL CDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VH1 CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR2 and VL CDR2; VH CDR1, VH CDR2 and VL CDR3 ; VHCDR2, VH CDR3 and VL CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2; VH CDR1, VLCDR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1, and VL CDR3; VH CDR3, VL CDR1, and VL CDR2; VH CDR3, VL CDR1, and VL CDR3; VH CDR1, VH CDR2, VH CDR3, and VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VH CDR2, VH CDR3, and VL CDR3; VHCDR1, VH CDR2, VL CDR1, and VL CDR2; VH CDR1, VH CDR2, VL CDR1, and VL CDR3; VH CDR1, VH CDR3, VL CDR1, and VL CDR2 ; VH CDR1, VHCDR3, VL CDR1, and VL CDR3; VH CDR2, VH CDR3, VL CDR1, and VL CDR2; VH CDR2, VH CDR3, VL CDR1, and VL CDR3; VH CDR2, VH CDR3, VL CDR2, and VL CDR3; VH CDR1 , VH CDR2, VH CDR3, VL CDR1, and VL CDR2; VHCDR1, VH CDR2, VH CDR3, VL CDR1, and VL CDR3; VH CDR1, VH CDR2, VL CDR1, VL CDR2, and VL CDR3; VH CDR1, VH CDR3, VL CDR1 , VL CDR2 and VL CDR3; VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; or VH CDR and VL CDR listed in Table 1 above Any combination (or combination thereof).

In one embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises VH CDR1 and VL CDR1, said VH CDR1 having SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR1 has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR1 and VL CDR2, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR2 has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VHCDR1 and VL CDR3, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR3 has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 or SEQ ID NO: 64.

In one embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises VH CDR1 and VL CDR1, said VH CDR1 having SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR1 has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR1 and VL CDR2, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR2 has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VHCDR1 and VL CDR3, and said VH CDR1 has SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: The amino acid sequence of 26, the VL CDR3 has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 63 or SEQ ID NO: 64.

In one embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VH CDR3 and VL CDR1, said VH CDR3 has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12, and said VL CDR1 has SEQ ID The amino acid sequence of NO: 4, SEQ ID NO: 13 or SEQ ID NO: 62. In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises VHCDR3 and VL CDR2, said VH CDR3 has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12, and said VL CDR2 has SEQ ID The amino acid sequence of NO: 5, SEQ ID NO: 14 or SEQ ID NO: 65. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises VH CDR3 and VL CDR3, said VH CDR3 has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 12, and said VL CDR3 has SEQ ID NO: The amino acid sequence of ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 3 or SEQ ID NO: 64.

The present invention provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising derivatives of the VH regions, VH CDRs, VL regions or VLCDRs described herein that immunospecifically bind IL-9 polypeptides. Standard techniques known to those skilled in the art that can be used to introduce mutations (such as deletions, additions and/or substitutions) in the nucleotide sequences encoding the antibodies of the invention include, for example, site-directed mutagenesis and PCR-mediated mutagenesis to produce amino acid substitutions . Preferably, the derivative comprises less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, relative to the original molecule. A substitution, a substitution of less than 4 amino acids, a substitution of less than 3 amino acids, or a substitution of less than 2 amino acids. In one embodiment, the derivatives have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues (ie, amino acid residues that are not essential for immunospecific binding of the antibody to the IL-9 polypeptide). "Conservative amino acid substitutions" are substitutions of amino acid residues with amino acid residues having similarly charged side chains. Families of amino acid residues having similarly charged side chains are defined in the art. These families include, amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar Amino acids with side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (such as threonine, valine, isoleucine ) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can also be introduced randomly along all or a portion of the coding sequence (eg, by saturation mutagenesis), and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be assayed.

In a specific embodiment, the present invention provides a preparation of an antibody that immunospecifically binds to an IL-9 polypeptide, said antibody comprising a light chain variable region (VL) and/or a heavy chain variable region (VH) containing one or more The amino acid sequence of 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 substituted by amino acid residues. The present invention also provides antibodies immunospecifically binding to IL-9 polypeptides, said antibodies comprising 4D4, 4D4H2-1D11 containing one or more amino acid residue substitutions in one or more VL CDRs and/or one or more VH CDRs , 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 amino acid sequence. The present invention also provides antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising one or more VH frameworks and/or 4D4, 4D4 H2-containing one or more amino acid residue substitutions in one or more VL frameworks 1 Amino acid sequence of D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or its VH and/or VL regions. Can be detected in vitro and/or in vivo by detection in the VH region, VH CDR, VL region, VL CDR and/or 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com- 2H2, 7F3com-3H5 or 7F3com-3D4 framework region introduces the ability of the antibody to bind to IL-9 polypeptide, or the ability to inhibit or reduce IL-9-mediated cell proliferation, or prevent, treat and/or control Autoimmune disease, inflammatory disease, proliferative disease or respiratory tract infection or the ability of symptoms thereof.

In one embodiment, the antibody that immunospecifically binds IL-9 polypeptide is contained under stringent conditions, highly stringent conditions or other stringent hybridization conditions known to those skilled in the art (see for example, Ausubel, F.M. et al., 1989, "New Current Protocols in Molecular Biology, Volume I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, New York, pp. 6.3.1- 6.3.6 and 2.10.3 pages) with code 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or its antigen The nucleotide sequence to which the nucleotide sequence of the binding fragment hybridizes under stringent conditions comprising, for example, hybridization to filter-bound DNA at about 45° C. in 6× sodium chloride/sodium citrate (SSC), followed by 0.2 xSSC/0.1% SDS at about 50-65 ° C for one or more washes; the highly stringent conditions include, for example, in 6xSSC, hybridization with filter-bound nucleic acids at about 45 ° C, and then using 0.1xSSC/0.2% SDS in Wash one or more times at about 68°C.

In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises an amino acid sequence of a VH region or an amino acid sequence of a VL region encoded by a nucleotide sequence that is under stringent conditions or VH or VH encoding 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 under other stringent hybridization conditions known to those skilled in the art The nucleotide sequences of the VL region hybridize. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises the amino acid sequence of the VH region and the amino acid sequence of the VL region encoded by the nucleotide sequence, and the nucleotide sequence is under the stringent conditions described herein or VH and VH encoding 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 under other stringent hybridization conditions known to those skilled in the art The nucleotide sequences of the VL region hybridize. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence, the nucleotide sequence being under the stringent conditions described herein or Under other stringent hybridization conditions known to those skilled in the art, hybridize to the nucleotide sequence encoding any VH CDR or VL CDR listed in Table 1 above. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises the amino acid sequence of the VH CDR and the amino acid sequence of the VL CDR encoded by the nucleotide sequence, and the nucleotide sequence is expressed under the stringent conditions described herein or Under other stringent hybridization conditions known to those skilled in the art, hybridize to the nucleotide sequence encoding any VH CDR listed in Table 1 above and any VL CDR listed in Table 1 above.

In a specific embodiment, the antibody that immunospecifically binds to IL-9 polypeptide comprises an antibody with 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or an amino acid sequence of 7F3com-3D4 or an antigen-binding fragment thereof of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% %, at least 85%, at least 90%, at least 95%, or at least 99% identical amino acid sequences. In another embodiment, the antibody that immunospecifically binds to IL-9 polypeptide comprises an antibody with 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or The VH region of 7F3com-3D4 is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, The amino acid sequences of the VH regions are at least 90%, at least 95%, or at least 99% identical. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises an antibody associated with 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com - the VL region of 3D4 is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least The amino acid sequences of the VL regions are 90%, at least 95%, or at least 99% identical.

In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% of any of the VL CDRs listed in Table 1 above , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of one or more VL CDRs. In another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% of any VLCDR listed in Table 1 above. %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of one or more VL CDRs.

The invention includes formulations of antibodies that compete with the antibodies described herein for binding to IL-9 polypeptides. In particular, the invention includes competition for IL-binding with 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or antigen-binding fragments thereof. 9 peptide antibodies. In a specific embodiment, the present invention comprises, in the competition experiment described herein or the competition experiment well known in the art, make 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 reduce binding of IL-9 polypeptide by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% % or more, or 5-15%, 10-25%, 25%-50%, 45-75%, or 75-99% of the antibodies. In another specific embodiment, the present invention comprises 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2 in an ELISA competition assay compared to a control such as PBS , 7F3com-3H5 or 7F3com-3D4 reduce binding of IL-9 polypeptide by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% %, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, or 5-15% , 10-25%, 25%-50%, 45-75%, or 75-99% antibody formulations. In one embodiment, the ELISA competition experiment can be performed in the following manner: prepare recombinant IL-9 with a concentration of 10 μg/ml in PBS. 100 µl of this solution was added to each well of an ELISA 98-well microtiter plate and incubated overnight at 4-8°C. The ELISA plate was washed with PBS supplemented with 0.1% Tween to remove excess recombinant IL-9. Add 100 μl of bovine serum albumin (BSA) in PBS to a final concentration of 1% to block non-specific protein-protein interactions. After 1 hour of treatment at room temperature, the ELISA plates were washed. Prepare unlabeled competing antibody at concentrations ranging from 1 μg/ml to 0.01 μg/ml with blocking solution. Control wells contained blocking solution alone or control antibody at a concentration of 1 μg/ml to 0.01 μg/ml. Add horseradish peroxidase-labeled test antibody (such as 7F3com-2H2) to the competing antibody diluent, and the final concentration is fixed at 1 μg/ml. 100 [mu]l of the test and competition mixtures were added in triplicate to the ELISA wells and the plates were incubated for 1 hour at room temperature. Wash away residual unbound antibody. Add 100 [mu]l of horseradish peroxidase substrate to each well to detect bound test antibody. The plate was incubated at room temperature for 30 minutes, and the absorbance was read with an automatic microplate reader. Average values of triplicate wells were calculated. Antibodies that compete well with the test antibody reduce absorbance measurements compared to control wells. In another preferred embodiment, the invention comprises reducing the binding of 7F3com-2H2 to IL-9 polypeptide by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95% or more, or 5-15%, 10-25%, 25%-50%, 45-75%, or 75-99% of the antibodies.

In another embodiment, the present invention comprises, in a competition assay described herein or known in the art, a compound containing 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3 compared to a control such as PBS. , 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 antigen-binding fragment (such as VH region, VH CDR, VL region or VL CDR) (or consisting of) antibody binding to IL-9 polypeptide is reduced At least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, or 5-15%, 10-25%, 25%-50%, Preparations of 45-75% or 75-99% antibody. In another embodiment, the present invention includes in an ELISA competition assay, compared to a control, such as PBS, comprising 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2 , 7F3com-3H5 or 7F3com-3D4 antigen-binding fragments (such as VH region, VL region, VH CDR or VL CDR) (or consisting of) the binding of the antibody to the IL-9 polypeptide is reduced by at least 2%, at least 5%, At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, or 5-15%, 10-25%, 25%-50%, 45-75%, or 75-99% % of antibodies. In one embodiment, the invention comprises reducing the binding of an antibody comprising (or consisting of) an antigen-binding fragment of 7F3com-2H2 to an IL-9 polypeptide by at least 2% compared to a control such as PBS in an ELISA competition assay , at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, or 5-15%, 10-25%, 25%-50%, 45-75% % or 75-99% of antibodies.

The invention includes VH regions comprising polypeptides that compete with the VH regions of 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 for binding IL-9 (or consisting of) polypeptide or protein preparations. The invention also includes VL polypeptides comprising a polypeptide that competes for IL-9 binding with the VL region of 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 A preparation of a polypeptide or protein of (or consisting of) a region.

The invention includes preparations of polypeptides or proteins comprising (or consisting of) VH CDRs that compete with the VH CDRs listed in Table 1 above for binding to IL-9 polypeptides. The invention also includes polypeptides or proteins comprising (or consisting of) a VL CDR that competes with the VL CDR listed in Table 1 above for binding to an IL-9 polypeptide.

Antibodies that immunospecifically bind IL-9 polypeptides include modified derivatives, ie, covalently linked derivatives produced by covalently linking any type of molecule to the antibody. For example, but not limited to, antibody derivatives include, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other Antibodies modified by methods such as proteins. Many chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, the derivative may contain one or more non-classical amino acids.

The invention also provides preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising framework regions (eg, human or non-human framework regions) known to those of skill in the art. The framework regions may be naturally occurring or consensus framework regions. The fragment region of an antibody of the invention may be a human fragment region (see eg, a list of human framework regions in Chothia et al., 1998, J. Mol. Biol. 278:457-479, herein incorporated by reference in its entirety).

The present invention includes preparations of antibodies that immunospecifically bind IL-9 polypeptides comprising 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9 containing mutations (e.g., one or more amino acid substitutions) in the framework region , 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 amino acid sequence. In certain embodiments, the antibody that immunospecifically binds IL-9 polypeptide comprises 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-XF-9, 4D4H2-1D11, 4D4com-XF-9, Amino acid sequence of 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4. Amino acid substitutions in the framework regions can improve binding of the antibody to the IL-9 polypeptide.

In a specific embodiment, the preparation of an antibody that immunospecifically binds to an IL-9 polypeptide comprises 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or One or more CDRs of 7F3com-3D4, a VH framework region 1 having the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 33) or QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 37), a VH framework having the amino acid sequence WVRQAPGQGLEWMG (SEQ ID NO: 34) Region 2, the amino acid sequence of VH framework region 3 having the amino acid sequence RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 35) or RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 38) and VH framework region 4 having the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 36). In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide comprises 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or One or more CDRs of 7F3com-3D4, VL framework region 1 having the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 39), VL framework region 2 having the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 40), having the amino acid sequence GVPSRFSGSGSGTDFLTISSLQPEDFATYYC (SEQ ID NO: 40) ID NO: 41) VL framework region 3 and the amino acid sequence of VL framework region 4 having the amino acid sequence FGGGTKVEIK (SEQ ID NO: 42). In yet another embodiment, the antibody that immunospecifically binds to an IL-9 polypeptide comprises 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 Or one or more CDRs of 7F3com-3D4, VH framework region 1 having amino acid sequence SEQ ID NO: 33 or SEQ ID NO: 37, VH framework region 2 having amino acid sequence SEQ ID NO: 34, having amino acid sequence SEQ ID NO : 35 or a VH framework region of SEQ ID NO: 38 3, a VH framework region 4 having an amino acid sequence of SEQ ID NO: 36, a VL framework region 1 having an amino acid sequence of SEQ ID NO: 39, having an amino acid sequence of SEQ ID NO: 40 The amino acid sequences of the VL framework region 2, the VL framework region 3 having the amino acid sequence of SEQ ID NO: 41 and the VL framework region 4 having the amino acid sequence of SEQ ID NO: 42.

The invention also includes preparations of antibodies that immunospecifically bind IL-9 polypeptides, said antibodies comprising 4D4, 4D4H2-1D11, 4D4com- Amino acid sequence of XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4. Amino acid substitutions in the variable and framework regions can improve binding of the antibody to the IL-9 polypeptide.

The invention also provides formulations of antibodies of the invention comprising constant regions known to those skilled in the art. The constant regions of the antibodies of the present invention or fragments thereof may be human.

The present invention includes preparations of antibodies that immunospecifically bind IL-9 polypeptides expressed by immune cells such as activated T cells or mast cells. The invention also includes antibodies that immunospecifically bind IL-9 polypeptides and modulate the activity or function of T cells, B cells, mast cells, neutrophils and/or eosinophils. The present invention also includes antibodies that immunospecifically bind IL-9 polypeptides and inhibit or reduce the infiltration of inflammatory cells into tissues, joints or organs of a subject and/or inhibit or reduce proliferation of epithelial cells.

The present invention includes preparations of antibodies that immunospecifically bind IL-9 polypeptides found in the environment, ie, that do not bind IL-9R or a subunit thereof. Antibodies that immunospecifically bind IL-9 polypeptides that bind to a soluble IL-9Ra subunit are also encompassed by the invention. The present invention also includes antibodies that immunospecifically bind IL-9 polypeptides that bind to cell membrane-bound IL-9R or a subunit thereof.

In one embodiment, in an in vivo and/or in vitro assay (e.g., an immunoassay such as an ELISA) described herein or known to those skilled in the art, the immunospecific binding to IL- The antibody of 9 polypeptide can inhibit and/or reduce the interaction between IL-9 polypeptide and IL-9R or its subunit by about 25%, about 30%, about 35%, about 45%, about 50%, about 55% %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98%. In another embodiment, immunospecifically binding IL- Antibodies to the 9 polypeptide do not inhibit the interaction between the IL-9 polypeptide and IL-9R or its subunits. In another embodiment, in (for example) immunization experiment such as ELISA, compared with control such as PBS or IgG control antibody, the antibody that immunospecifically binds to IL-9 polypeptide has the difference between IL-9 polypeptide and IL-9R. The inhibition of the interaction is less than 20%, less than 15%, less than 10% or less than 5%.

In one embodiment, in vivo and/or in vitro experiments described herein or well known to those skilled in the art (for example, using IL-9-dependent cell lines such as IL-9-dependent mouse T cells expressing human IL-9R In a cell proliferation assay of the same line), an antibody that immunospecifically binds to an IL-9 polypeptide can bind the IL-9 polypeptide to IL-9R or one or The interaction of multiple subunits is inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In another embodiment, in vivo and/or in vitro experiments described herein or well known to those skilled in the art (for example, using IL-9-dependent cell lines such as IL-9-dependent mouse T cells expressing human IL-9R) In cell proliferation experiments of cell lines), compared with controls such as PBS or IgG control antibodies, antibodies that immunospecifically bind IL-9 polypeptides do not inhibit the interaction between IL-9 polypeptides and IL-9R or one or more subunits thereof Interaction. In another embodiment, in vivo and/or in vitro experiments described herein or well known to those skilled in the art (for example, using IL-9-dependent cell lines such as IL-9-dependent mouse T cells expressing human IL-9R) In cell proliferation experiments of cell lines), compared with controls such as PBS or IgG control antibodies, the antibody that immunospecifically binds to IL-9 polypeptides has an effect on the interaction between IL-9 polypeptides and IL-9R or one or more subunits thereof The inhibition of action is less than 20%, less than 15%, less than 10% or less than 5%.

The invention encompasses immunospecifically binding IL-9 polypeptides and does not induce or reduce cytokine expression and/or release relative to controls such as PBS or IgG control antibodies in in vivo and/or in vitro assays described herein or known in the art Antibody preparations. In one embodiment, the immunospecifically binds IL-9 polypeptide and does not induce a cytokine, such as IFN, in the serum of a subject administered a control, such as PBS or IgG control antibody, relative to the concentration of such cytokine in the serum of a subject administered a control antibody such as PBS or IgG. - Antibodies with increasing concentrations of gamma, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15 and IL-23. In another embodiment, in an in vitro and/or in vivo assay described herein or well known in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces cytokine expression and/or relative to a control such as PBS or an IgG control antibody. or release. In a specific embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide induces a cytokine, such as IFN, in the serum of a subject administered the antibody relative to the concentration of these cytokines in the serum of a subject administered a control such as PBS or an IgG control antibody. -γ, IL-2, IL-12 and IL-15 concentrations increased. In another specific embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide induces the production of Th1 cells in a subject administered the antibody relative to the concentration of these cytokines in the serum of the subject administered a control such as PBS or an IgG control antibody. Increased concentrations of cytokines such as IFN-γ and IL-12. In a specific embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide induces a cytokine, such as IL, in the serum of a subject administered the antibody relative to the concentration of these cytokines in the serum of a subject administered a control such as PBS or an IgG control antibody. -4, IL-5, IL-10, IL-13 and IL-23 concentrations decreased. In another specific embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide induces mast cell production in the serum of a subject administered the antibody relative to the concentration of these cytokines in the serum of a subject administered a control such as PBS or an IgG control antibody. Cytokines, such as TNF-α, IL-4 and IL-13 concentrations were decreased. In another specific embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide induces the production of Th2 cells in the serum of a subject administered the antibody relative to the concentration of these cytokines in the serum of a subject administered a control such as PBS or an IgG control antibody. Cytokines, such as IL-4, IL-5, IL-13, and IL-10 concentrations were decreased. Serum concentrations of cytokines can be determined by any technique known to those skilled in the art, such as ELISA or Western blot experiments.

In one embodiment, in an in vivo and/or in vitro assay described herein or known to those skilled in the art (such as a trypan blue assay or a 3H-thymidine assay), the immune specific Antibodies that specifically bind IL-9 polypeptides can reduce the proliferation of inflammatory cells (such as mast cells, T cells, B cells, macrophages, neutrophils, basophils and/or eosinophils) and/or or inhibit at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, or at least 98%. In another embodiment, in an in vivo and/or in vitro experiment described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces inflammatory cells relative to a control such as PBS or IgG control antibody. Reduction or inhibition of infiltration of the upper airway and/or lower airway by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In another embodiment, in an in vivo and/or in vitro experiment described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces inflammatory cells relative to a control such as PBS or IgG control antibody. Reduction and/or inhibition of infiltration of the upper airway and/or lower airway by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or ^at least 98%; In the H-thymidine test), the antibody that immunospecifically binds to the IL-9 polypeptide can reduce and/or inhibit the proliferation of inflammatory cells by at least 25%, at least 30%, at least 35%, relative to a control such as PBS or an IgG control antibody , at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

In certain embodiments, mast cell degranulation assays are performed in vivo and/or in vitro assays described herein or known to those skilled in the art (see, e.g., Windmiller and Backer, 2003, J. Biol. Chem. 278:11874-78) example), relative to a control such as PBS or an IgG control antibody, an antibody that immunospecifically binds to an IL-9 polypeptide can reduce mast cell degranulation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide inhibits mast cell activation relative to a control, such as a PBS or IgG control antibody, in an in vivo and/or in vitro assay described herein or known to those skilled in the art. or at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay described herein or known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces mast cell activation and / or the expression and / or release of the degranulation product is inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

In a specific embodiment, in an in vivo and/or in vitro assay described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide inhibits mast cell protease relative to a control such as PBS or an IgG control antibody. , such as expression, activity, serum concentration and/or release of chymotrypsin and tryptase inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In one embodiment, mast cell activity can be determined by culturing primary mast cells or mast cell lines in vitro in the presence of 10 ng/ml IL-9. Baseline levels of proteases (such as chymotrypsin and tryptase) and leukotrienes in the supernatant were measured by commercially available ELISA kits. IL-9-reactive antibodies or control antibodies were added directly to cell cultures at a concentration of 1 μg/ml to assess the ability of the antibodies to modulate protease or leukotriene levels. Protease and leukotriene levels were assessed at 24 and 36 hour time points. In another specific embodiment, in an in vivo and/or in vitro experiment described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces mast cell Expression, activity, serum concentration and/or release of leukotrienes, such as C4, D4 and E4, are inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60% %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In another specific embodiment, the immunospecific binding to IL relative to a control such as PBS or an IgG control antibody in an in vivo and/or in vitro assay described herein or known to those skilled in the art (such as an ELISA or Western blot assay) -9 polypeptide antibodies to mast cell cytokines, such as TNF-α, IL-4 and IL-13 expression, activity, serum concentration and / or release inhibition or reduction of at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

In other embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide inhibits mast cell infiltration relative to a control, such as PBS or a control IgG antibody, in an in vivo and/or in vitro assay described herein or known to those skilled in the art. or at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, Antibodies that immunospecifically bind IL-9 polypeptides inhibit or reduce mast cell proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In yet another embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide infiltrates mast cells relative to a control such as PBS or a control IgG antibody in an in vivo and/or in vitro assay described herein or known to those skilled in the art Inhibit or reduce by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, or at least 98%; and in in vivo and/or in vitro experiments described herein or well known to those skilled in the art (such as trypan blue assay, FACS or ³ H thymidine assay), relative to Control such as PBS or control IgG antibody, the antibody that immunospecifically binds to IL-9 polypeptide inhibits or reduces mast cell proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In one embodiment, animals are sensitized with ovalbumin to measure reduction in mast cell infiltration in vivo. Briefly, on days 1 and 21, 100 μg ovalbumin complexed with aluminum adjuvant was administered subcutaneously. During the three-week sensitization process, animals were dosed with IL-9 reactive antibody or control antibody at 10 mg/kg every 5-7 days. On days 29, 30 and 31, animals were exposed to ovalbumin without adjuvant by aerosol delivery, or by intranasal instillation of 100 μl of a 1 μg/ml solution in PBS. On day 31, 6 hours after the last challenge with ovalbumin, animals were sacrificed and lung tissue fixed by formalin perfusion. Mast cell infiltration was assessed histologically by counting mast cells per field of view of lung epithelial tissue sections. Using this experimental design, mast cell precursors can be distinguished from mast cells in the lung epithelium by assessing, for example, the presence of heterochromatic granules and/or immunohistochemistry using differentiation-dependent cell surface markers such as FcεRI .

In other embodiments, in an in vivo and/or in vitro assay described herein or known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces mast cell precursors relative to a control such as PBS or a control IgG antibody. Inhibition or reduction of infiltration of the upper airway and/or lower airway by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, Antibodies that immunospecifically bind IL-9 polypeptides inhibit or reduce proliferation of mast cell precursors by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65% %, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In another embodiment, in an in vivo and/or in vitro experiment described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide causes mast cells to pro- Inhibition and/or reduction of infiltration of the upper airway and/or lower airway of the body by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%; and in vivo and/or in vitro experiments (such as trypan blue test, In FACS or ³ H-thymidine experiment), relative to control such as PBS or control IgG antibody, the antibody that immunospecifically binds to IL-9 polypeptide can make the proliferation of mast cell precursor and/or reduce by at least 25%, at least 30%. %, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or At least 98%. In one embodiment, mast cell precursor infiltration is determined in vivo by a mast cell infiltration assay, as described above.

In certain embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide mediates depletion of peripheral blood T cells by inducing increased apoptosis of T cells, especially Th2 cells. In one embodiment, animals can be sensitized with ovalbumin to measure Th2 T lymphocyte depletion in vivo. Briefly, on days 1 and 21, 100 μg ovalbumin complexed with aluminum adjuvant was administered subcutaneously. During the three-week sensitization process, animals were dosed with IL-9 reactive antibody or control antibody at 10 mg/kg every 5-7 days. On day 28, animals were given a 100 [mu]g booster dose of ovalbumin without adjuvant intravenously. Animals were sacrificed 2 days after intravenous booster doses. Splenocytes were recovered and analyzed by flow cytometry. Splenic Th2 T lymphocytes, as identified by cytoplasmic IL-4 staining, should be reduced in animals receiving IL-9 neutralizing antibodies compared to control antibody recipients. In another embodiment, in an in vivo and/or in vitro assay (such as FACS) described herein or well known to those skilled in the art, the antibody that immunospecifically binds to the IL-9 polypeptide relative to a control such as PBS or a control IgG antibody Inhibit or reduce Th1 and Th2 differentiation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% %, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, in vivo and/or in vitro experiments well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, an antibody that immunospecifically binds to an IL-9 polypeptide induces an upper respiratory tract and/or lower respiratory tract T-cell infiltration, particularly Th2 cell infiltration, is inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, Antibodies that immunospecifically bind IL-9 polypeptides inhibit or reduce T cell proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in the in vivo and/or in vitro experiments described herein or well-known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, an antibody that immunospecifically binds to an IL-9 polypeptide causes the upper respiratory tract and/or or T cell infiltration in the lower respiratory tract, especially Th2 cell infiltration inhibition and / or reduction of at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65% , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%, T cell proliferation, especially Th2 cell proliferation inhibition and/or reduction of at least 25%, at least 30% %, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or At least 98%, and/or increased T cell apoptosis.

In certain embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide reduces macrophage infiltration by at least 25% relative to a control, such as PBS or a control IgG antibody, in in vivo and/or in vitro assays well known to those skilled in the art. %, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 95% or at least 98%. In one embodiment, animals are sensitized with ovalbumin to measure the reduction of macrophage infiltration in vivo. Briefly, on days 1 and 21, 100 μg ovalbumin complexed with aluminum adjuvant was administered subcutaneously. During the three-week sensitization process, animals were dosed with IL-9 reactive antibody or control antibody at 10 mg/kg every 5-7 days. On days 29, 30 and 31, animals were exposed to ovalbumin without adjuvant by aerosol delivery, or by intranasal instillation of 100 μl of a 1 μg/ml solution in PBS. On day 31, 6 hours after the last challenge with ovalbumin, animals were sacrificed and lung tissue fixed by formalin perfusion. Macrophage infiltration was assessed by immunohistochemistry by counting CD14-positive cells per field of view of lung tissue sections. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, Antibodies that immunospecifically bind IL-9 polypeptides inhibit or reduce macrophage proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in the in vivo and/or in vitro experiments described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, an antibody that immunospecifically binds to an IL-9 polypeptide can make macrophages Infiltration inhibition and/or reduction by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 98%, and in in vivo and/or in vitro experiments described herein or known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, the macrophage Inhibition or reduction of proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, or at least 98%.

In certain embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide infiltrates B cells relative to a control, such as PBS or a control IgG antibody, in in vivo and/or in vitro assays described herein or known to those skilled in the art Reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In one embodiment, animals are systemically sensitized with ovalbumin to measure the reduction in B lymphocyte infiltration in vivo. Briefly, on days 1 and 21, 100 μg ovalbumin complexed with aluminum adjuvant was administered subcutaneously. During the three-week sensitization process, animals were dosed with IL-9 reactive antibody or control antibody at 10 mg/kg every 5-7 days. On days 29, 30 and 31, animals were exposed to ovalbumin without adjuvant by aerosol delivery, or by intranasal instillation of 100 μl of a 1 μg/ml solution in PBS. On day 31, 6 hours after the last challenge with ovalbumin, animals were sacrificed and lung tissue fixed by formalin perfusion. B lymphocyte infiltration was assessed by immunohistochemistry by counting CD19-positive cells per field of view of lung tissue sections. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, Antibodies that immunospecifically bind IL-9 polypeptides inhibit or reduce B cell proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, an antibody that immunospecifically binds to an IL-9 polypeptide is capable of infiltrating B cells relative to a control such as PBS or a control IgG antibody in an in vivo and/or in vitro assay described herein or known to those skilled in the art Inhibit and/or reduce by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, and inhibits B cell proliferation relative to a control such as PBS or a control IgG antibody in an in vivo and/or in vitro assay described herein or known to those skilled in the art or at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, or at least 98%.

In certain embodiments, in vivo and/or in vitro assays described herein or known to those skilled in the art (see, e.g., Li et al., 2000, Am. J. Respir. Cell Mol. Biol. 25:644-51) wherein, relative to a control such as PBS or a control IgG antibody, the antibody that immunospecifically binds to the IL-9 polypeptide reduces the infiltration of eosinophils in the upper airway and/or lower airway by at least 25%, at least 30%, at least 35%, At least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay described herein (see Section 5.6) or well known to those skilled in the art (eg trypan blue assay, FACS or ³ H thymidine assay), relative to a control such as PBS Or a control IgG antibody, an antibody that immunospecifically binds to an IL-9 polypeptide inhibits or reduces eosinophil proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60% %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay described herein or known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide is capable of rendering eosinophils relative to a control such as PBS or a control IgG antibody. Inhibition and/or reduction of cellular infiltration by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, or at least 98%, and in in vivo and/or in vitro experiments described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, eosinophilic Inhibition or reduction of granulocyte proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 98%.

In other embodiments, in an in vivo and/or in vitro assay described herein or well known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide induces neutrophils relative to a control such as PBS or a control IgG antibody. Infiltration is reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay (such as a trypan blue assay, FACS or ³ H thymidine assay) described herein or well known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, An antibody that immunospecifically binds to an IL-9 polypeptide inhibits or reduces neutrophil proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65% , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In other embodiments, in an in vivo and/or in vitro assay described herein or known to those skilled in the art, an antibody that immunospecifically binds to an IL-9 polypeptide is capable of rendering neutrophils relative to a control such as PBS or a control IgG antibody. Inhibition and/or reduction of cellular infiltration by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, or at least 98%, and in in vivo and/or in vitro experiments described herein or well-known to those skilled in the art, neutralize relative to a control such as PBS or a control IgG antibody Inhibition or reduction of granulocyte proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 98%.

In one embodiment, an antibody that immunospecifically binds to an IL-9 polypeptide can neutralize or inhibit IL-9-mediated biological effects, including but not limited to inflammatory cell recruitment, epithelial hyperplasia, epithelial cell production of mucin, and hypertrophy Cell activation, degranulation, proliferation and/or infiltration.

In a specific embodiment, in the in vivo and/or in vitro experiments described herein or well-known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, the antibody that immunospecifically binds to IL-9 polypeptide and the antibody that antagonizes IgE Expression, function, and/or activity of proteinaceous substances (such as peptides, polypeptides, or proteins (including antibodies)) and/or nonproteinaceous substances that act synergistically to reduce or inhibit mast cell activation, degranulation, proliferation, and/or infiltration At least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 95%, or at least 98%.

In another embodiment, in the in vivo and/or in vitro experiments described herein or well-known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, an antibody that immunospecifically binds to an IL-9 polypeptide interacts with an antagonistic mast cell Expression, function, and/or activity of proteases Proteinaceous (such as peptides, polypeptides, or proteins (including antibodies)) and/or nonproteinaceous agents act synergistically to reduce mast cell activation, degranulation, proliferation, and/or infiltration or inhibit at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 98%.

In another embodiment, in the in vivo and/or in vitro experiments described herein or well-known to those skilled in the art, relative to a control such as PBS or a control IgG antibody, the antibody that immunospecifically binds to IL-9 polypeptide and the antagonistic stem cell factor The expression, function and/or activity of proteinaceous substances (such as peptides, polypeptides and proteins (including antibodies)) and/or non-proteinaceous substances act synergistically to reduce the activation, degranulation, proliferation and/or infiltration of mast cells or Inhibit at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In one embodiment, primary mast cells or mast cell lines are cultured in vitro in the presence of 1 ng/ml IL-9 and 1 ng/ml stem cell factor. Baseline levels of proteases (such as chymotrypsin and tryptase) and leukotrienes in the supernatant were measured by commercially available ELISA kits. IL-9-reactive antibodies or control antibodies were added directly to cell cultures at a concentration of 1 μg/ml to assess the ability of the antibodies to modulate protease or leukotriene levels. Protease and leukotriene levels were assessed at 24 and 36 hour time points.

Preparations of antibodies of the invention that immunospecifically bind IL-9 polypeptides may be monospecific, bispecific, trispecific or more specific. Multispecific antibodies can be specific for different epitopes of IL-9 polypeptide, or can be specific for heterologous epitopes of IL-9 polypeptide, such as heterologous polypeptides or solid support materials. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920 and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992).

为至少10⁵M^-1s^-1、至少1.5X10⁵M^-1s^-1、至少2X10⁵M^-1s^-1、至少2.5X10⁵M^-1s^-1、至少5X10⁵M^-1s^-1、至少10⁶M^-1s^-1、至少5X10⁶M^-1s^-1、至少10⁷M^-1s^-1、至少5X 10⁷M^-1s^-1或至少10⁸M^-1s^-1，或者10⁵-10⁸M^-1s^-1、1.5X10⁵M^-1s^-1-1X10⁷M^-1s^-1、2X 10⁵-1X10⁶M^-1s^-1或4.5X10⁵X10⁷M^-1s^-1。在一个实施方式中，经BIAcore实验测定，免疫特异性结合IL-9多肽的抗体的k_结合为至少2X10⁵M^-1s^-1、至少2.5X10⁵M^-1s^-1、至少5X10⁵M^-1s^-1、至少10⁶M^-1s^-1、至少5X10⁶M^-1s^-1、至少10⁷M^-1s^-1、至少5X10⁷M^-1s^-1或至少10⁸M^-1s^-1，该抗体在本文所述的微量中和实验中中和人IL-9。在一个实施方式中，经BIAcore实验测定，免疫特异性结合IL-9多肽的抗体的k_结合为至多10⁸M^-1s^-1、至多10⁹M^-1s^-1、至多10¹⁰M^-1s^-1、至多10¹¹M^-1s^-1或至多10¹²M^-1s^-1，该抗体在本文所述的微量中和实验中中和人IL-9。按照这些实施方式，这类抗体可包含4D4、4D4H2-1D11、4D4com-XF-9、4D4com-2F9、7F3、71A10、7F3 22D3、7F3com-2H2、7F3com-3H5或7F3com-3D4的VH区和/或VL区，或者4D4、4D4H2-1 D11、4D4com-XF-9、4D4com-2F9、7F3、71A10、7F3 22D3、7F3com-2H2、7F3com-3H5或7F3com-3D4的VH CDR和/或VL CDR。The present invention provides antibodies with high binding affinity to IL-9 polypeptides. In a specific embodiment, the on-rate constant or k- _on- rate of the antibody that immunospecifically binds to the IL-9 polypeptide

at least 10 ⁵ M ^-1 s ^-1 , at least 1.5X10 ⁵ M ^{-1 s} -1 , at least 2X10 ⁵ M ^-1 s ^-1 , at least 2.5X10 ⁵ M ^-1 s ^-1 , at least 5X10 ⁵ M ^-1 s ^-1 , at least 10 ⁶ M ^-1 s ^-1 , at least 5X10 ⁶ M ^-1 s ^-1 , at least 10 ⁷ M ^-1 s ^-1 , at least 5X 10 ⁷ M ^-1 s ^-1 or at least 10 ⁸ M ^-1 s ^-1 , or 10 ⁵ -10 ⁸ M ^-1 s ^-1 , 1.5X10 ⁵ M ^-1 s ^-1 -1X10 ⁷ M ^-1 s ^-1 , 2X 10 ⁵ -1X10 ⁶ M ^-1 s ^-1 or 4.5 X10 ⁵ X10 ⁷ M ^-1 s ^-1 . In one embodiment, as determined by BIAcore assay, the k _binding of the antibody that immunospecifically binds to IL-9 polypeptide is at least 2X10 ⁵ M ^-1 s ^-1 , at least 2.5X10 ⁵ M ^-1 s ^-1 , at least 5X10 ⁵ M ^-1 s ^-1 , at least 10 ⁶ M ^-1 s ^-1 , at least 5X10 ⁶ M ^-1 s ^-1 , at least 10 ⁷ M ^-1 s ^-1 , at least 5X10 ⁷ M ^-1 s ^-1 or at least 10 ⁸ M ^-1 s ^-1 , the antibody neutralizes human IL-9 in the microneutralization assay described herein. In one embodiment, as determined by BIAcore assay, the k _binding of the antibody that immunospecifically binds to IL-9 polypeptide is at most 10 ⁸ M ⁻¹ s ⁻¹ , at most 10 ⁹ M ⁻¹ s ⁻¹ , at most 10 ¹⁰ M ^{−1 1} s ⁻¹ , up to 10 ¹¹ M ⁻¹ s ⁻¹ , or up to 10 ¹² M ⁻¹ s ⁻¹ , the antibody neutralizes human IL-9 in the microneutralization assay described herein. According to these embodiments, such antibodies may comprise the VH region and/or VL region, or VH CDR and/or VL CDR of 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4.

为小于10^-3s^-1、小于5X10^-3s^-1、小于10^-4s^-1、小于2x10^-4s^-1、小于5X10^-4s^-1、小于10^-5s^-1、小于5X10^-5s^-1、小于10^-6s^-1、小于5X10^-6s^-1、小于10^-7s^-1、小于5X10^-7s^-1、小于10^-8s^-1、小于5X10^-8s^-1、小于10^-9s^-1、小于5X10^-9s^-1或小于10^-10s^-1，或者10^-3-10^-10s^-1、10^-4-10^-8s^-1或10^-5-10^-8s^-1。在一个实施方式中，经BIAcore实验测定，免疫特异性结合IL-9多肽的抗体的k_解离为10^-5s^-1、小于5X10^-5s^-1、小于10^-6s^-1、小于5X10^-6s^-1、小于10^-7s^-1、小于5X10^-7s^-1、小于10^-8s^-1、小于5X10^-8s^-1、小于10^-9s^-1、小于5X10^-9s^-1或小于10^-10s^-1，该抗体在本文所述的微量中和实验中中和人IL-9。在另一优选实施方式中，免疫特异性结合IL-9多肽的抗体的k_解离大于10^-13s^-1、大于10^-12s^-1、大于10^-11s^-1、大于10^-10s^-1、大于10^-9s^-1或大于10^-8s^-1。按照这些实施方式，这类抗体可包含4D4、4D4 H2-1 D11、4D4com-XF-9、4D4com-2F9、7F3、71A10、7F3 22D3、7F3com-2H2、7F3com-3H5或7F3com-3D4的VH区和/或VL区，或者4D4、4D4 H2-1 D11、4D4com-XF-9、4D4com-2F9、7F3、71A10、7F3 22D3、7F3com-2H2、7F3com-3H5或7F3com-3D4的VH CDR和/或VLCDR。In another embodiment, the antibody that immunospecifically binds IL-9 polypeptide

less than 10 ^-3 s ^-1 , less than 5X10 ^-3 s ^-1 , less than 10 ^-4 s ^-1 , less than 2x10 ^-4 s -1 , less than 5X10 ^-4 s ^-1 , less than 10 ^-5 s ^-1 , ^less than 5X10 ^-5 s ^-1 , less than 10 ^-6 s ^-1 , less than 5X10 ^-6 s ^-1 , less than 10 ^-7 s ^-1 , less than 5X10 ^-7 s ^-1 , less than 10 ^-8 s ^-1 , less than 5X10 ^{- 8} s ^-1 , less than 10 ^-9 s ^-1 , less than 5X10 ^-9 s ^-1 or less than 10 ^-10 s ^-1 , or 10 ^-3 -10 ^-10 s ^-1 , 10 ^-4 -10 ^-8 s ^{- 1} or 10 ^-5 -10 ^-8 s ^-1 . In one embodiment, as determined by BIAcore assay, the k _dissociation of the antibody that immunospecifically binds to IL-9 polypeptide is 10 ^-5 s ^-1 , less than 5X10 ^-5 s -1 , less than 10 -6 s ^-1 , less than 10 ^-6 s ^-1 , less than 5X10 ^-6 s ^-1 , less than 10 ^-7 s ^-1 , less than 5X10 ^-7 s ^-1 , less than 10 ^-8 s ^-1 , less than 5X10 ^-8 s ^-1 , less than 10 ^-9 s ^-1 , less than 5X10 ^{- 9} s ^-1 or less than 10 ^-10 s ^-1 , the antibody neutralizes human IL-9 in the microneutralization assay described herein. In another preferred embodiment, the k _dissociation of the antibody that immunospecifically binds IL-9 polypeptide is greater than 10 ^-13 s ^-1 , greater than 10 ^-12 s ^-1 , greater than 10 ^-11 s ^-1 , greater than 10 ^-10 s ^-1 , greater than 10 ^-9 s ^-1 or greater than 10 ^-8 s ^-1 . According to these embodiments, such antibodies may comprise the VH region and and/or the VL region, or the VH CDR and/or VLCDR of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4.

In another embodiment, the affinity constant or _Ka (k _on /k _dissociation ) of an antibody that immunospecifically binds to an IL-9 polypeptide is at least 10 ² M ⁻¹ , at least 5×10 ² M ⁻¹ , at least 10 ³ M ^-1 , at least 5X10 ³ M ^-1 , at least 10 ⁴ M ^-1 , at least 5X10 ⁴ M ^-1 , at least 10 ⁵ M ^-1 , at least 5X10 ⁵ M ^-1 , at least 10 ⁶ M ^-1 , at least 5X10 ⁶ M ^-1 , at least 10 ⁷ M ^-1 , at least 5X10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 5X10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 5X10 ⁹ M ^-1 , at least 10 ¹⁰ M ^{-1 1.} At least 5X10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 5X10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 5X10 ¹² M ^-1 , at least 10 ¹³ M ^-1 , at least 5X10 ¹³ M ^-1 , at least 10 ¹⁴ M ^-1 , at least 5X10 ¹⁴ M ^-1 , at least 10 ¹⁵ M ^-1 or at least 5X10 ¹⁵ M ^-1 , or 10 ² -5X10 ⁵ M ^-1 , 10 ⁴ -1X10 ¹⁰ M ^-1 or 10 ⁵ -1X10 ⁸ M ^-1 . In another embodiment, the _Ka of the antibody that immunospecifically binds IL-9 polypeptide is at most 10 ¹¹ M ^-1 , at most 5X10 ¹¹ M ^-1 , at most 10 ¹² M ^-1 , at most 5X10 ¹² M ^-1 , at most 10 ¹³ M ⁻¹ , up to 5×10 ¹³ M ⁻¹ , up to 10 ¹⁴ M ⁻¹ , or up to 5×10 ¹⁴ M ⁻¹ . In another embodiment, the dissociation constant or K _d (k _dissociation /k _binding ) of the antibody that immunospecifically binds IL-9 polypeptide is less than 10 ⁻⁵ M, less than 5×10 ⁻⁵ M, less than 10 ⁻⁶ M, Less than 5X10 ^-6 M, less than 10 ^-7 M, less than 5X10 ^-7 M, less than 10 ^-8 M, less than 5X10 ^-8 M, less than 10 ^-9 M, less than 5X10 ^-9 M, less than 10 ^-10 M, less than 5X10 ^-10 M, less than 10 ^-11 M, less than 5X10 ^-11 M, less than 10 ^-12 M, less than 5X10 ^-12 M, less than 10 ^-13 M, less than 5X10 ^-13 M, less than 10 ^-14 M, less than 5X 10 ^{- 14} M, less than 10 ^-15 M or less than 5X 10 ^-15 M, or 10 ^-2 M-5X 10 ^-5 M, 10 ^-6 -10 ^-15 M or 10 ^-8 -10 ^-14 M. In one embodiment, as determined by BIAcore assay, the K _d of the antibody that immunospecifically binds to IL-9 polypeptide is less than 10 ⁻⁹ M, less than 5×10 ⁻⁹ M, less than 10 ⁻¹⁰ M, less than 5×10 ⁻¹⁰ M, less than 1×10 ^-11 M, less than 5X10 ^-11 M, less than 1X10 ^-12 M, less than 5X10 ^-12 M, less than 10 ^-13 M, less than 5X10 ^-13 M or less than 1X10 ^-14 M, or 10 ^-9 M-10 ^-14 M, the antibody neutralizes human IL-9 in the microneutralization assay described herein. In another preferred embodiment, the _Kd of the antibody that immunospecifically binds IL-9 polypeptide is greater than 10 ^-9 M, greater than 5X10 ^-9 M, greater than 10 ^-10 M, greater than 5X10 ^-10 M, greater than 10 ^-11 M , greater than 5X10 ^-11 M, greater than 10 ^-12 M, greater than 5X10 -12 M, greater than 6X10 ^-12 M, greater than 10 ^-13 M, greater than 5X10 ^-13 M ^, greater than 10 ^-14 M, greater than 5X10 ^-14 M or greater ^10-9M - ^10-14M . According to these embodiments, such antibodies may comprise the VH region and /or VL region, or VH CDR and/or VL CDR of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 .

In certain embodiments, preparations of antibodies of the invention do not comprise antibodies known in the art that immunospecifically bind IL-9 polypeptides. Non-limiting examples of known antibodies that immunospecifically bind IL-9 polypeptides include 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4.

In a specific embodiment, the antibody preparations of the invention bind to peptides and polypeptides of IL-9 bearing antigenic epitopes comprising at least 4 of IL-9 found in any species, At least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25 , at least 30, at least 40, at least 50 contiguous amino acid residues, or an amino acid sequence of about 15-30 contiguous amino acids, or consist thereof. A polypeptide containing an immunogenic or antigenic epitope can be at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 amino acid residues in length.

Peptides, polypeptides and fragments thereof bearing IL-9 epitopes can be produced by any conventional means. See, e.g., Houghten, R.A. (1985) "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interactions on single amino acid epithelium" ( 1986).

The invention provides formulations of peptides, polypeptides and/or proteins comprising one or more variable or hypervariable regions of the antibodies described herein. Peptides, polypeptides and/or proteins comprising one or more variable or hypervariable regions of an antibody of the invention also comprise heterologous amino acid sequences. In certain embodiments, such heterologous amino acid sequences may comprise at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 Contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 75 contiguous amino acid residues, at least 100 contiguous amino acid residues, or more contiguous amino acid residues Residues. Such peptides, polypeptides and/or proteins may be referred to as fusion proteins.

In a specific embodiment, the length of the peptide, polypeptide and/or protein preparation containing one or more variable regions or hypervariable regions of the antibody of the present invention is 10 amino acid residues, 15 amino acid residues, 20 amino acid residues residues, 25 amino acid residues, 30 amino acid residues, 35 amino acid residues, 40 amino acid residues, 45 amino acid residues, 50 amino acid residues, 75 amino acid residues, 100 amino acid residues , 125 amino acid residues, 150 amino acid residues, or more amino acid residues. In certain embodiments, peptides, polypeptides and/or proteins comprising one or more variable or hypervariable regions of an antibody of the invention immunospecifically bind to an IL-9 polypeptide. In other embodiments, peptides, polypeptides and/or proteins comprising one or more variable or hypervariable regions of an antibody of the invention do not immunospecifically bind IL-9 polypeptide.

In a specific embodiment, the invention provides formulations of peptides, polypeptides and/or proteins comprising the VH region and/or VL region (see Table 1 above) of one of the antibodies described herein. In one embodiment, the invention provides a peptide, polypeptide and/or protein comprising one or more CDRs having the amino acid sequence of any of the CDRs listed in Table 1 above. According to these embodiments, the peptide, polypeptide or protein may also comprise a heterologous amino acid sequence.

Peptides, polypeptides or proteins containing one or more variable regions or hypervariable regions can be used, for example, to produce anti-idiotypic antibodies, which in turn can be used for the prevention, treatment and/or control of diseases or disorders (such as autoimmune diseases, One or more symptoms associated with an inflammatory disease, a proliferative disease, or an infection (such as a respiratory infection). The anti-idiotypic antibodies produced can also be used in immunoassays, such as ELISA, to detect antibodies containing variable or hypervariable regions contained in the peptide, polypeptide or protein used to produce the anti-idiotypic antibodies.

5.1.1.2. Antibodies that immunospecifically bind RSV antigens

Formulations of the invention comprise isolated antibodies that immunospecifically bind to RSV antigens. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In certain embodiments, the anti-RSV antigen antibody is Palivizumab. Palivizumab is a humanized monoclonal antibody currently used to prevent RSV infection in pediatric patients. The invention provides preparations of antibodies that immunospecifically bind one or more RSV antigens. Antibodies used in the present invention immunospecifically bind one or more RSV antigens, regardless of the RSV strain. The invention also provides antibodies that bind differentially or preferentially to RSV antigens of one RSV strain compared to another RSV strain. In one embodiment, the formulation comprises an antibody that immunospecifically binds to RSV F glycoprotein, G glycoprotein or SH protein. In another embodiment, the formulation comprises an antibody that immunospecifically binds RSV F glycoprotein. In another embodiment, the formulation comprises an antibody that binds to the A, B or C antigenic site of RSV F glycoprotein. See also U.S. Application No. 11/473,537, filed June 23, 2006, entitled "Antibody Formulations Having Optimized Aggregation and Fragmentation Profiles" for other RSV antibodies that can be formulated using the methods of the invention , which is incorporated herein by reference in its entirety. 5.1.1.3 Antibodies that immunospecifically bind to human postpneumonia virus (hMPV)

Formulations of the invention may comprise isolated antibodies and compositions comprising such antibodies that specifically bind human postpneumonia virus (hMPV) antigens. The term "anti-hMPV-antigen antibody" refers to an antibody or antibody fragment thereof that immunospecifically binds to an hMPV antigen. hMPV antigen refers to hMPV polypeptides or fragments thereof, such as hMPV nucleoprotein, hMPV phosphoprotein, hMPV matrix protein, hMPV small hydrophobic protein, hMPV RNA-dependent hMPV polymerase, hMPV F protein and hMPV G protein. hMPV antigens also refer to hMPV polypeptides or fragments thereof, such as hMPV nucleoprotein, hMPV phosphoprotein, hMPV matrix protein, hMPV small hydrophobic protein, hMPV RNA-dependent hMPV polymerase, hMPV F protein and hMPV G protein. of polypeptides.

The anti-hMPV-antigen antibody used in the present invention may be a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In some preferred embodiments, the anti-hMPV antibodies of the invention are those described in U.S. Patent Application No. 10/628,088, filed July 25, 2003, published May 20, 2004, U.S. Patent Publication No. US 2004/0096451 A1 Antibody.

The anti-hMPV-antigen antibodies of this section are available in accordance with U.S. Patent Application No. 10/628,088, filed July 25, 2003, published May 20, 2004, U.S. Patent Publication No. US 2004/0096451 A1 (incorporated by reference which is incorporated herein in its entirety) for preparation, formulation, administration, therapeutic use or prophylactic use.

5.1.1.4. Immunospecific binding to integrin alpha _v beta ₃ antibody

和

的组合物或制剂参见例如国际公开号WO98/33919、WO 00/78815和WO 02/070007；2002年3月4日提交、2002年11月12日公开为美国专利公开号US 2002/0168360的美国申请序列号10/091,236，通过引用将其全文纳入本文。Formulations of the invention may also comprise isolated antibodies and compositions comprising such antibodies that specifically bind integrin [alpha] _v [beta] ₃ . The antibody can be a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In some preferred embodiments, the anti-integrin α _v β ₃ antibody of the invention is MEDI-522 (vitacin

and

See, for example, International Publication Nos. WO 98/33919, WO 00/78815 and WO 02/070007; U.S. Patent Publication No. US 2002/0168360, filed March 4, 2002, published Nov. 12, 2002 as US Patent Publication No. US 2002/0168360 Application Serial No. 10/091,236, which is hereby incorporated by reference in its entirety.

或

的抗原结合片段。免疫特异性结合整联蛋白α_vβ₃的已知抗体的例子包括但不限于：11D2(Searle)、鼠单克隆LM609(Scripps，国际公开号WO 89/05155和美国专利号5,753,230(通过引用将其全文纳入本文)，国际公开号WO98/33919和WO 00/78815(通过引用将其全文纳入本文))，17661-37E和17661-37E 1-5(USBiological)，MON 2032和2033(CalTag)，ab7166(BV3)和ab7167(BV4)(Abcam)和WOW-1(Kiosses等，Nature Cell Biology，3：316-320)。In other _embodiments , the antibody that immunospecifically binds integrin _αvβ3 is not

or

antigen-binding fragments. _Examples of known antibodies that immunospecifically bind integrin _αvβ3 include, but are not limited to: 11D2 (Searle), murine monoclonal LM609 (Scripps, International Publication No. WO 89/05155, and U.S. Patent No. 5,753,230 (incorporated by reference which are incorporated herein in their entirety), International Publication Nos. WO 98/33919 and WO 00/78815 (which are incorporated herein by reference in their entirety)), 17661-37E and 17661-37E 1-5 (US Biological), MON 2032 and 2033 (CalTag), ab7166(BV3) and ab7167(BV4) (Abeam) and WOW-1 (Kiosses et al., Nature Cell Biology, 3:316-320).

_αvβ3 integrins have been found on new blood vessels, as well as on the surface of many solid tumors, activated macrophages _, monocytes and osteoclasts. Likewise, the anti-integrin _αvβ3 antibodies of this section are useful, for example, as research antibodies, or in the prevention or treatment _of several devastating diseases.

The anti-integrin α _v β ₃ antibodies of this section may be prepared, formulated, administered, used therapeutically, or used prophylactically as described in: filed March 4, 2002 and published in November 2002 U.S. Patent Application No. 10/091,236 published as U.S. Patent Publication No. US 2002/0168360 A1 on the 12th; U.S. Patent Application No. 10/769,712 filed on January 30, 2004 and published as U.S. Patent Publication No. US 2004/0208870 A1; U.S. Patent Application No. 10/769,720 filed January 30, 2004 and published September 9, 2004 as U.S. Patent Publication No. 2004/0176272; filed March 4, 2003 and published as U.S. Patent Publication No. US 2005/ 0084489 A1, U.S. Patent Application No. 10/379,145; U.S. Patent Application No. 10/379,189, filed March 4, 2003 and published as U.S. Patent Publication No. US 2004/0001835; PCT Application No. PCT, filed January 30, 2004 /US04/02701; International Application Publication No. WO 00/78815 A1, _entitled "Anti-α _{v β 3} recombinant human antibodies, nucleic acid encoding same and methods" ), Huse et al.; and International Application Publication No.: WO 98/33919 A1, entitled "Anti-alpha-V-beta-3 recombinant humanized antibody, its encoding nucleic acid and method of use" (Anti-alpha-V-veta -3 recombinant humanized antibodies, nucleic acids encoding same and methods of use), Huse et al; International Publication No. WO 89/05155, which is incorporated herein by reference in its entirety.

5.1.1.5. Antibodies that immunospecifically bind to CD2

弗吉尼亚州玛纳萨斯大学大街10801号(10801 University Boulevard，Manassas，Virginia20110-2209)，保藏号为HB 11423的细胞系产生的单克隆抗体的氨基酸序列。Formulations of the invention may also comprise isolated antibodies that immunospecifically bind CD2 and compositions comprising such antibodies. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In some preferred embodiments, the anti-CD2 antibody of the invention is cilizumab (MEDI-507). Silizumab can selectively bind to cells expressing CD2 antigen (especially T cells, natural killer cells and thymocytes), and can be used, for example, to prevent and treat T cell lymphoma or other related diseases. MEDI-507 is described in, for example, International Publication No. WO 99/03502, International Application Nos. PCT/US02/22273 and PCT/US02/06761 and U.S. Patent No. 6,849,258, U.S. Application Serial No. 10/258 published as US 2003/0068320 A1 091,268 and 10/091,313 published as US 2003/0044406A1, which are hereby incorporated by reference in their entirety. MEDI-507 is a monoclonal antibody of the humanized IgG1κ type, which immunospecifically binds to human CD2 polypeptide. The construction method of MEDI-507 is to insert the CDR of rat monoclonal antibody LO-CD2a/BTI-322 into the framework region of human IgG1 by molecular technology. For the amino acid sequence in LO-CD2a/BTI-322 see, e.g., U.S. Patents 5,730,979, 5,817,311, and 5,951,983; and U.S. Application Serial No. 09/056,072 and U.S. Patent No. 6,849,258 (incorporated herein by reference in their entirety), or published in July 1993. Deposited at the American Type Culture Collection (

10801 University Boulevard, Manassas, Virginia (10801 University Boulevard, Manassas, Virginia 20110-2209), the amino acid sequence of the monoclonal antibody produced by the cell line with deposit number HB 11423.

The anti-CD2 antibodies of this section may be prepared, formulated, administered, used therapeutically, or used prophylactically as described in: U.S. Patent filed on March 4, 2002 and published on April 15, 2003 U.S. Patent Application No. 10/091,268, Publication No. US 2003/0068320; U.S. Patent Application No. 10/091,313, filed March 4, 2002 and published as U.S. Patent Publication No. US 2003/0044406 on March 6, 2003; and U.S. Patent Application No. 10/657,006, filed September 5, 2003 and published December 30, 2004 as U.S. Patent Publication No. US 2004/0265315, is hereby incorporated by reference in its entirety.

5.1.1.6 Antibodies that immunospecifically bind to CD19

Formulations of the invention may also comprise isolated antibodies that immunospecifically bind CD19 and compositions comprising such antibodies. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody. In some preferred embodiments, the anti-CD19 antibody of the invention is MT-103. MT-103 is the latest clinical representative of a new class of antibody derivatives called Bi-Specific T Cell Engagers (BiTE ^™ ). The BiTE compound MT-103 directs and activates the patient's own immune system to fight cancer cells, stimulating T cells (a very important type of white blood cell) to destroy B tumor cells (cancerous white blood cells). MT-103 specifically targets a specific protein (the CD19 antigen) that is present on cancerous B cells but not other blood cell types or healthy tissue, thus avoiding the side effects of traditional chemotherapy.

The anti-CD19 antibodies of this section can be prepared, formulated, administered, used therapeutically or used prophylactically as described in US Patent 6,723,538 and US Patent Publication No. 2004/0162411.

The human CD19 molecule is a structurally distinct cell surface receptor expressed on the surface of human B cells. The present invention relates to immunotherapeutic compositions and methods for the prevention and treatment of GVHD, humoral rejection, and post-transplantation lymphoproliferative disorders; autoimmune diseases and disorders; and cancer in human subjects using therapeutic antibodies that bind the human CD19 antigen.

Hybridomas producing HB12a and HB12b anti-CD19 antibodies have been deposited with the ATCC under accession numbers PTA-6580 and PTA-6581. See also copending US Application No. (Attorney Docket No. 11605-006-999) and US Application No. 11/355,905 filed 2/15/2006, which are hereby incorporated by reference in their entirety.

5.1.1.7 Antibodies that immunospecifically bind to EphA2

Formulations of the invention may also comprise isolated antibodies that immunospecifically bind EphA2 and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies. In some embodiments, an anti-EphA2 antibody of the invention is EA2. In some preferred embodiments, the EA2 antibody is a human or humanized antibody. In other embodiments, the antibody is EA5. In some preferred embodiments, the EA5 antibody is a human or humanized antibody. Hybridomas producing the anti-EphA2 antibodies of the present invention have been deposited with the American Type Culture Collection (ATCC, PO Box 1549, Manassas, VA, pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, 20108) and assigned a deposit number (incorporated herein by reference), as shown in Table 2.

Table 2: Anti-EphA2 antibodies.

EphA2 antibody deposit number Date of preservation EA2.31 PTA-4380 May 22, 2002 EA5.12 PTA-4381 May 22, 2002 Eph099B-102.147 PTA-4572 August 7, 2002 Eph099B-208.261 PTA-4573 August 7, 2002 Eph099B-210.248 PTA-4574 August 7, 2002 Eph099B-233.152 PTA-5194 May 12, 2003 Eph101.530.241 PTA-4724 September 26, 2002

EphA2 is a 130kDa receptor tyrosine kinase expressed in human epithelium. It is expressed at low levels in human epithelium and is enriched in the site of cell adhesion (Zantek et al., Cell Growth & Differentiation 10:629, 1999; Lindberg, et al., Molecular & Cellular Biology 10:6316, 1990). EphA2 is upregulated in many aggressive cancer cells. Anti-EphA2 antibodies of the invention are useful, for example, in the treatment of various tumors, including breast, colon, prostate, lung, and skin cancers, and in the prevention of metastasis.

The anti-EphA2 antibodies of this section may be prepared, formulated, administered, used therapeutically, or used prophylactically as described in: U.S. Patent filed on April 12, 2004 and published on March 3, 2005 U.S. Patent Application No. 10/823,259, Publication No. US 2005/0049176 A1; U.S. Patent Application No. 10/823,254, filed April 12, 2004 and published as U.S. Patent Publication No. US 2005/0059592 A1 on March 17, 2005; U.S. Patent Application No. 10/436,782 filed May 12, 2003 and published February 12, 2004 as U.S. Patent Publication No. 2004/0028685; filed May 12, 2003 and published May 13, 2004 as U.S. Patent Application No. 10/436,783 of U.S. Patent Publication No. 2004/0091486; U.S. Patent Application No. 11/004,794 filed on December 3, 2004 and published as U.S. Patent Publication No. US 2005/0153923A1 on July 14, 2005; U.S. Patent Application No. 10/994,129, filed November 19, 2004, and published July 14, 2005 as U.S. Patent Publication No. US2005/0152899A1; filed December 3, 2004, and published July 7, 2005 as U.S. Patent Application No. 11/004,795 of U.S. Patent Publication No. US 2005/0147593A1; and U.S. Provisional Application Nos. 60/662,517, 60/622,711, 60/622,489, filed October 27, 2004, which are incorporated herein by reference in their entirety.

5.1.1.8 Antibodies that immunospecifically bind to EphA4

Formulations of the invention may comprise isolated antibodies and compositions comprising such antibodies that immunospecifically bind to the EphA4 antigen. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies. The hybridoma producing the anti-EphA4 antibody used in the method of the present invention has been deposited with the American Type Culture Collection (ATCC, Post Box 1549, Manassas, VA, 20108) and assigned accession number PTA-6044 (incorporated herein by reference).

EphA4 is a receptor expressed in the brain, heart, lung, muscle, kidney, placenta, pancreas (Fox et al., Oncogene 10:897, 1995) and melanocytes (Easty et al., Int. J. Cancer 71:1061, 1997) Tyrosine kinase. EphA4 is overexpressed in many cancers. The anti-EphA4 antibody of this chapter can be used to treat pancreatic cancer and the like to reduce the expression of EphA4.

The anti-EphA4 antibodies of this section may be prepared, formulated, administered, used therapeutically, or used prophylactically as described in: U.S. Patent filed on June 7, 2004 and published on January 20, 2005 U.S. Patent Application No. 10/863,729, Publication No. US 2005/0013819 A1; U.S. Patent Application No. 11/004,794, filed December 3, 2004 and published as U.S. Patent Publication No. US 2005/0153923 A1 on July 14, 2005; U.S. Patent Application Nos. 11/004,794 and 11/004,795, filed December 3, 2004 and published as U.S. Patent Publication No. US 2005/0147593 Al on July 7, 2005, are hereby incorporated by reference in their entirety.

5.1.1.9. Antibodies that immunospecifically bind to HMG1

Formulations of the invention may comprise antibodies that immunospecifically bind to HMG1 and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

Early proinflammatory cytokines (such as TNF, IL-1, etc.) mediate inflammation and induce the late release of high mobility group box 1 (HMG1) (also known as HMG-1, HMG1, and HMGB1), which is present in serum Proteins that accumulate and mediate delayed lethality and further induction of early proinflammatory cytokines.

It has also been demonstrated that HMG1 can be actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of this molecule, where HMG1 is translocated from the nucleus into secretory lysosomes, resulting in the secretion of the acetylated form of HMG1. See PCT/IB2003/005718 for passive release by necrotic cells. Thus, the molecules of HMG1 passively released by necrotic cells and HMGB1 actively secreted by inflammatory cells are different.

In addition, HMG1 may be a cytokine mediator of delayed death in endotoxemia. See, eg, US Patents 6,468,533 and 6,448,223. More specifically, bacterial endotoxin (lipopolysaccharide (LPS)) has been shown to activate monocytes/macrophages to release HMG1 as a late response to activation, leading to toxic elevated serum HMG1 levels. Antibodies to HMG1 have been shown to prevent endotoxin lethality even though administration of the antibodies is delayed until after an early cytokine response has developed. Like other proinflammatory cytokines, HMG1 is a potent activator of monocytes. Intratracheal administration of HMG1 caused acute lung injury, and anti-HMG1 antibodies prevented endotoxin-induced pulmonary edema. Furthermore, serum HMG1 levels were elevated in critically ill patients with sepsis or hemorrhagic shock, with levels in nonsurvivors being significantly higher than those in survivors.

The anti-HMG1 antibodies of this section can be prepared, formulated, administered, used therapeutically, or used prophylactically as described in U.S. Patent Publication No. 2006-0099207A1, filed October 21, 2005, which is incorporated by reference in its entirety incorporated into this article. Three clones, S6, S16 and G4, have been deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209), respectively assigned ATCC deposit number PTA -6143 (Date of Deposit August 4, 2004), PTA-6259 (Date of Deposit October 19, 2004) and PTA-6258 (Date of Deposit October 19, 2004) (respectively referred to herein as "S6" , "S16" and "G4"), as described in US Patent Publication No. 2006-0099207A1, filed October 21, 2005, which is incorporated herein by reference in its entirety.

5.1.1.10 Antibodies that immunospecifically bind to ALK

Formulations of the invention may comprise antibodies that immunospecifically bind to ALK and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

Monoclonal Antibody to ALK and Hybridoma Cell Lines Producing ALK Monoclonal Antibodies 8B10, 16G2-3, and 9C10-5 (Deposited with the American Type Culture Collection (10801 University Avenue, Manassas, VA, 20110-2209) , ATCC deposit number to be assigned), such as U.S. Patent Application No. 09/880,097 filed June 14, 2001 and published as U.S. Patent Publication No. 2002/0034768 on March 21, 2002 (herein incorporated by reference in its entirety) mentioned.

Pleiotropin (PTN) is a 136 amino acid secreted heparin-binding cytokine that has multiple functions, including a role in angiogenesis. PTN can specifically bind receptor tyrosine kinase, anaplastic lymphoma enzyme (ALK), this binding leads to autophosphorylation of receptor, which subsequently leads to many signal transduction molecules such as IRS-1, PLC-γ, PI3 kinase Phosphorylation of and Shc and activation of cell survival pathways. See PCT Patent Application Publication No. WO 01/96364. Thus substances and therapeutic methods that modulate ALK-mediated signal transduction pathways may affect one or more ALK-regulated functions, including, for example, angiogenesis. ALK is involved in various disease states, including cancer and diseases associated with poor or excessive angiogenesis. In addition, ALK is involved in certain processes, such as certain pathways in wound healing. ALK and/or PTN are frequently expressed at high levels in many tumors. Thus, substances that downregulate ALK and/or PTN function may affect tumors by directly affecting tumor cells, indirectly affecting tumor-recruited angiogenesis processes, or a combination of direct and indirect effects.

5.1.1.11. Antibodies that immunospecifically bind to CD20

Formulations of the invention may comprise antibodies that immunospecifically bind to CD20 and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

CD20 is only expressed by B lymphocytes (Stashenko et al. (1980) J Immunol 125: 1678-1685; Tedder et al., 1988a). CD20 forms homo- or heterotetrameric complexes and has important functions in the regulation of cell cycle progression and signal transduction in B lymphocytes (Tedder and Engel, 1994). CD20 also regulates transmembrane Ca++ conductance, which may be a functional component of Ca++-permeable cation channels (Bubien et al., J Cell Biol 121:1121-1132; Kanzaki et al. (1997a) J Biol Chem 272:14733-14739; Kanzaki et al. (1997b) J Biol Chem 272:4964-4969; Kanzaki et al. (1995) J Biol Chem 270:13099-13104). CD20 antibodies can effectively treat non-Hodgkin's lymphoma (McLaughlin et al. (1998) Oncology 12: 1763-1769; Onrust et al. (1989) J Biol Chem 264: 15323-15327; Weiner (1999) Semin Oncol 26: 43-51 ).

See also US Patent Application No. 10/433,287, filed September 30, 2003, published July 15, 2004 as US 20040137566, which is hereby incorporated by reference in its entirety.

5.1.1.12. Antibodies that immunospecifically bind to CD22

Formulations of the invention may comprise antibodies that immunospecifically bind to CD22 and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

6,183,744; 6,187,287; 6,254,868; 6,306,393; and Tuscano et al., Blood 94(4):1382-92 (1999) (herein incorporated by reference in its entirety). For a review of the use of monoclonal antibodies, including anti-CD22 antibodies, in the treatment of non-Hodgkin's lymphoma see, eg, Renner et al., Leukemia 11 (suppl 2): S5509 (1997).

Humanized CD22 antibodies have been documented in the treatment of autoimmune diseases (see Tedder, U.S. Patent Application Publication No. US2003/0202975) and B cell malignancies such as lymphomas and leukemias (see Tuscano U.S. Patent Application Publication No. U.S. 2004/0001828 ) in the application. The use of humanized CD22 antibodies targeting specific epitopes on CD22 in immunoconjugates for the treatment of cancer has been described (see US Patents 5,789,554 and 6,187,287 to Leung).

Exemplary VH and VK antibody regions of the invention are deposited with the American Type Culture Collection (ATCC). Specifically, a plasmid encoding the humanized anti-CD22 VH sequence of the present invention, called RHOv2, was deposited with the ATCC under the accession number PTA-7372 on February 9, 2006. A plasmid encoding the humanized anti-CD22 VH sequence of the present invention, called RHOv2ACD, was deposited with the ATCC under the accession number PTA-7373 on February 9, 2006. The plasmid RKA encoding the humanized anti-CD22VK sequence of the present invention was deposited in ATCC with the deposit number PTA-7370 and the deposit date was February 9, 2006. The plasmid RKC encoding the humanized anti-CD22VK sequence of the present invention was deposited in ATCC with the deposit number PTA-7371 and the deposit date was February 9, 2006.

See also U.S. Provisional Application No. TBA, Attorney Docket No. BC320P1, filed March 6, 2006, which is incorporated herein by reference in its entirety.

5.1.1.13. Antibodies that immunospecifically bind chitinase

The formulations of the invention may comprise antibodies that immunospecifically bind chitinase and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

Blocking chitinase/chitinase-like proteins in vivo has been documented to protect bone and cartilage and attenuate weight loss in mouse models of RA. These results support a role for chitinases/chitinase-like proteins in chronic inflammatory diseases, and more specifically chitinases/chitinase-like proteins in OCL-related disorders, including bone metabolism and connective tissue disorders and diseases . Moreover, these result in inflammatory, human chitinase/chitinase-like proteins as potential therapeutic targets for the prevention and treatment of OCL-related diseases.

See also US Patent Application No. 10/202,436, filed July 23, 2002, published March 13, 2003 as US 20030049261, which is incorporated herein by reference in its entirety.

5.1.1.14. Antibodies that immunospecifically bind interferon alpha

Formulations of the invention may comprise antibodies that immunospecifically bind to interferon alpha and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

The invention provides methods of treating an interferon-alpha-mediated disease or disorder in a subject, the methods comprising administering to the subject an anti-IFN-alpha antibody of the invention, so as to treat the interferon-alpha-mediated disease in the subject. Examples of treatable diseases include autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, insulin-dependent diabetes mellitus, inflammatory bowel disease, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, and glomerulonephritis ), transplant rejection and graft-versus-host disease.

Anti-interferon alpha monoclonal antibodies are also described in US Application Serial No. 11/009,410, filed December 10, 2004, which is incorporated herein by reference in its entirety.

5.1.1.15. Antibodies that immunospecifically bind to interferon alpha receptors

The formulations of the present invention may comprise antibodies that immunospecifically bind interferon alpha receptors and compositions comprising such antibodies. Antibodies of the invention may be monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies.

The present invention also provides a method for inhibiting the biological activity of type I interferon on cells expressing interferon alpha receptor 1, said method comprising contacting said cell with an antibody of the invention to inhibit the biological activity of type I interferon active. The invention also provides a method of treating a type I interferon-mediated disease or disorder in a subject in need thereof, said method comprising administering to the subject an antibody of the invention, or an antigen-binding portion thereof, to treat the subject's type I interferon-mediated disease or disorder. leading diseases. A type I interferon-mediated disease can be, for example, an interferon alpha-mediated disease.

Examples of diseases or disorders treatable by the methods of the invention include systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and graft versus host disease.

Anti-interferon receptor monoclonal antibodies are described in U.S. Patent Publication No. 2006-0029601 Al, filed June 20, 2005, and published February 9, 2006, which is incorporated herein by reference in its entirety.

5.1.1.16. Antibodies with therapeutic use

The preparation of the present invention contains antibodies with therapeutic use, including but not limited to the antibodies listed in Table 3.

Table 3: Therapeutic antibodies useful in the invention

5.1.1.17 Antibodies that can be used for inflammatory diseases or autoimmune diseases

The formulations of the invention also comprise any antibody known in the art to be useful in the treatment and/or prophylaxis of autoimmune or inflammatory diseases. Non-limiting examples of antibodies that can be engineered according to the present invention for treating or preventing inflammatory diseases are shown in Table 4A, and non-limiting examples of antibodies for treating or preventing autoimmune diseases are shown in Table 4B.

Table 4A. Antibodies useful in the treatment of inflammatory and autoimmune diseases according to the invention

Antibody name target antigen product type isotype sponsor Indications 5G1.1 Complement (C5) Humanization IgG AlexionPharm Inc Rheumatoid Arthritis 5G1.1 Complement (C5) Humanization IgG Allison Pharmaceuticals SLE 5G1.1 Complement (C5) Humanization IgG Allison Pharmaceuticals Nephritis 5G1.1-SC Complement (C5) Humanization ScFv Allison Pharmaceuticals cardiopulmonary bypass 5G1.1-SC Complement (C5) Humanization ScFv Allison Pharmaceuticals myocardial infarction 5G1.1-SC Complement (C5) Humanization ScFv Allison Pharmaceuticals Angioplasty ABX-CBL CBL people Abugini GvHD ABX-CBL CD147 mouse IgG Abugini Allograft rejection ABX-IL8 IL-8 people IgG2 Abugini Psoriasis Antegren VLA-4 Humanization IgG Athena Company (Athena) / Allen Company (Elan) multiple sclerosis Anti-CD11a CD11a Humanization IgG1 Genentech/Soma Psoriasis Anti-CD18 CD18 Humanization Fab'2 Genentech myocardial infarction Anti-LFA1 CD18 mouse Fab'2 PM Company (Pasteur-Merieux) / Immunotech Company (Immunotech) Allograft rejection Antova CD40L Humanization IgG Biogen Allograft rejection Antova CD40L Humanization IgG Baiji Company SLE BTI-322 CD2 rat IgG Midie Muni GvHD, psoriasis CDP571 TNF-α Humanization IgG4 Celtek Corporation (Celltech) Crohn's disease CDP571 TNF-α Humanization IgG4 Celtec Corporation Rheumatoid joints

Antibody name target antigen product type isotype sponsor Indications Inflammation CDP850 E-selectin Humanization Celtec Corporation Psoriasis Corsevin M Factor VII Chimeric Centoc anticoagulant D2E7 TNF-α people CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 Humanization ICOS Pharmaceuticals multiple sclerosis Hu23F2G CD11/18 Humanization IgG ICOS Pharmaceuticals stroke IC14 CD14 ICOS Pharmaceuticals Toxic shock ICM3 ICAM-3 Humanization ICOS Pharmaceuticals Psoriasis IDEC-114 CD80 primatization IDEC Pharmaceuticals / Mitsubishi Psoriasis IDEC-131 CD40L Humanization IDEC Pharmaceutical Company/Wesai Company (Eisai) SLE IDEC-131 CD40L Humanization IDEC Pharmaceutical Company/Wesai Company (Eisai) multiple sclerosis IDEC-151 CD4 primatization IgG1 IDEC Pharmaceuticals/GlaxoSmithKline Rheumatoid Arthritis IDEC-152 CD23 primatization IDEC Pharmaceuticals Asthma/Allergies Infliximab TNF-α Chimeric IgG1 Centoc Rheumatoid Arthritis Infliximab TNF-α Chimeric IgG1 Centoc Crohn's disease LDP-01 β2-integrin Humanization IgG Millennium Corporation (LS Corporation (LeukoSite Inc.)) stroke LDP-01 β2-integrin Humanization IgG Millennium Company (LS Company) Allograft rejection LDP-02 α4β7 Humanization Millennium Company (LS Company) Ulcerative colitis MAK-195F TNF-α mouse Fab'2 Knoll Pharmaceuticals (KnollPharm, BASF) Toxic shock MDX-33 CD64(FcR) people Midex Corporation/Centeon Corporation (Centeon) autoimmune blood disease MDX-CD4 CD4 people IgG Medelex Company/Weicai Company/Genmab Company (Genmab) Rheumatoid Arthritis MEDI-507 CD2 Humanization Midie Muni Psoriasis MEDI-507 CD2 Humanization Midie Muni GvHD OKT4A CD4 Humanization IgG OrthoBiotech Allograft rejection OrthoClone OKT4A CD4 Humanization IgG Ossur Biotech autoimmune disease Orthoclone/anti-CD3OKT3 CD3 mouse mIgG2a Ossur Biotech Allograft rejection RepPro/Abciximab gpIIbIIIa Chimeric Fab Centoc/Lilly Complications of coronary angioplasty rhuMab-E25 IgE Humanization IgG1 Genentech/Novartis/Tanox Biosystems Asthma/Allergies SB-240563 IL5 Humanization GlaxoSmithKline Asthma/Allergies SB-240683 IL-4 Humanization GlaxoSmithKline Asthma/Allergies

Antibody name target antigen product type isotype sponsor Indications SCH55700 IL-5 Humanization Celltech/Schering Asthma/Allergies Simulect CD25 Chimeric IgG1 Novartis Pharmaceuticals Allograft rejection SMARTa-CD3 CD3 Humanization Protein Design Lab autoimmune disease SMARTa-CD3 CD3 Humanization Protein Design Lab Allograft rejection SMARTa-CD3 CD3 Humanization IgG Protein Design Lab Psoriasis Zenapax CD25 Humanization IgG1 Protein Design Laboratories/Roche (Hoffman-La Roche) Allograft rejection

Table 4B. Antibodies useful in the treatment of autoimmune diseases according to the invention

Antibody Indications target antigen ABX-RB2 Antibodies to CBL antigens on T cells, B cells and NK cells Fully human antibodies from Xeno mice 5c8 (anti-CD-40 ligand antibody) In October 1999, the phase II clinical trial was terminated because "adverse events" were detected CD-40 IDEC 131 Systemic lupus erythematosus (SLE) Anti-CD40 humanization IDEC 151 Rheumatoid Arthritis Primatization; anti-CD4 IDEC 152 Asthma Primatization; anti-CD23 IDEC 114 Psoriasis Primatized anti-CD80 MEDI-507 rheumatoid arthritis; multiple sclerosis Crohn's disease psoriasis Anti-CD2 LDP-02 (anti-b7 monoclonal antibody) Inflammatory Bowel Disease Crohn's Disease Ulcerative Colitis a4b7 integrin receptor on white blood cells (leukocytes) SMART anti-gamma interferon antibody autoimmune disease anti-gamma interferon Verteportin Rheumatoid Arthritis MDX-33 Autoimmune Hemolysis Idiopathic Thrombocytopenic Purpura (ITP) Monoclonal Antibody to FcRI Receptor MDX-CD4 Treat rheumatoid arthritis and other autoimmune diseases Monoclonal antibody to CD4 receptor molecule VX-497 autoimmune disease multiple sclerosis Inosine monophosphate dehydrogenase inhibitors (enzymes needed to make new RNA and DNA

Antibody Indications target antigen Rheumatoid Arthritis Inflammatory Bowel Disease Lupus Psoriasis For the production of nucleotides required for lymphocyte proliferation) VX-740 Rheumatoid Arthritis ICE inhibitor interleukin-1β (converting enzyme controls pathway leading to aggressive immune response) VX-745 Inflammation-specific chemical signal transduction involved in the initiation and progression of inflammatory immune responses P38MAP Kinase Inhibitor Mitogen-Activated Protein Kinase Etanercept (Enbrel) (etanercept) Target TNF (Tumor Necrosis Factor) IL-8 Fully human monoclonal antibody to IL-8 (Interleukin 8) Apogen MP4 Recombinant antigens selectively destroy disease-associated T cells and induce apoptosis Eliminate T cells through programmed cell death and no longer attack their own cells Specific Aperkin targets specific T cells

5.1.1.18. Antibodies with extended half-life

The present invention provides formulations of antibodies and antibody fragments that immunospecifically bind an antigen of interest, such as an IL-9 polypeptide, with extended half-lives in vivo. Specifically, the present invention provides an in vivo half-life in animals, preferably mammals (such as humans), greater than 3 days, greater than 7 days, greater than 10 days, preferably greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days , more than 45 days, more than 2 months, more than 3 months, more than 4 months or more than 5 months of preparations of antibodies and antibody fragments that immunospecifically bind to an antigen of interest (such as an IL-9 polypeptide).

To prolong the serum circulation time of antibodies (such as monoclonal antibodies and single-chain antibodies) or antibody fragments (such as Fab fragments) in vivo, for example, inert polymer molecules such as high molecular weight polyethylene glycol ( PEG) is linked to the antibody (including antibody fragments thereof), specifically by site-directed coupling of PEG to the N- or C-terminus of the antibody or via the ε-amino group on a lysine residue. Derivatization of linear or branched polymers can be utilized resulting in minimal loss of biological activity. The degree of coupling was closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecules were properly coupled to the antibody. Unreacted PEG can be separated from antibody-PEG conjugates by size exclusion or ion exchange chromatography. The binding activity and in vivo efficacy of PEG-derived antibodies (including antibody fragments thereof) can be tested using methods known to those skilled in the art, such as the immunoassay described herein.

Antibodies with increased half-life in vivo can also be produced by introducing one or more amino acid modifications (ie substitutions, insertions or deletions) in the IgG constant region or its FcRn binding fragment (preferably an Fc or hinge-Fc region fragment). See, eg, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375, which are incorporated herein by reference in their entirety.

In addition, antibodies (including antibody fragments thereof) can be conjugated to albumin to produce antibodies (including antibody fragments thereof) that are more stable in vivo or have a longer half-life in vivo. These techniques are well known in the art, see for example International Publication Nos. WO 93/15199, WO 93/15200 and WO 01/77137; and European Patent No. EP 413,622, all of which are incorporated herein by reference.

5.1.1.19. Antibody conjugates

The present invention provides recombinant fusion or chemical coupling (including covalent and non-covalent coupling) to heterologous proteins or polypeptides (or at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 polypeptide fragments of at least 70, at least 80, at least 90 or at least 100 amino acids) to generate antibodies (including antibody fragments thereof) that immunospecifically bind to an antigen of interest (such as an IL-9 polypeptide) of a fusion protein preparation. In particular, the invention provides antigen-binding fragments (such as Fab fragments, Fd fragments, Fv fragments, F(ab) ₂ fragments, VH regions, VH CDRs, VL regions or VL CDRs) and heterologous proteins comprising the antibodies described herein, Preparation of polypeptides or fusion proteins of peptides. Heterologous proteins, polypeptides, or peptides that can be fused to antibodies (including antibody fragments thereof) can be used to target antibodies to airway epithelial cells, mast cells, neutrophils, eosinophils, B cells, macrophages, or activated T cell. For example, antibodies that immunospecifically bind to cell surface receptors expressed by specific cell types (e.g., airway epithelial cells, mast cells, neutrophils, eosinophils, B cells, macrophages, or activated T cells) can be fused to or conjugated to antibodies of the invention (including antibody fragments thereof). In a specific embodiment, an antibody that immunospecifically binds IL-9 polypeptide is fused or conjugated to an anti-stem cell factor or an anti-kit ligand. Methods are known in the art for fusing or conjugating proteins, polypeptides, peptides to antibodies, including antibody fragments thereof. See, eg, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO91/06570; Ashkenazi et al. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154: 5590-5600; and Vil et al., 1992, Proc. Natl. Acad. cited in its entirety).

Other fusion proteins can be produced by the techniques of gene shuffling, motif shuffling, exon shuffling and/or codon shuffling (collectively "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the invention (eg, an antibody or fragment thereof with higher affinity and lower off-rate). See generally U.S. Patents 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al, 1997, Curr. 1999, J. Mol. Biol., 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications is incorporated herein by reference). Antibodies (including antibody fragments thereof) may be altered or encoded by random mutagenesis followed by recombination by error-prone PCR, random nucleotide insertion or other methods. A polynucleotide encoding an antibody (including antibody fragments thereof) may be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

Furthermore, antibodies (including antibody fragments thereof) can be fused to marker sequences, such as peptides, to facilitate purification. The marker amino acid sequence can be a hexahistidine peptide, such as the tag provided by the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), etc. , many of which are commercially available. As described by Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, hexahistidine provides a convenient method for purification of fusion proteins. Other peptide tags used for purification include, but are not limited to: the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the "flag" Label.

In other embodiments, antibodies of the invention or fragments thereof are conjugated to diagnostic or detection reagents. Such antibodies are useful as part of a clinical testing program for monitoring or diagnosing the emergence, occurrence, progression and/or severity of a disease or disorder such as an autoimmune disease, an inflammatory disease, a proliferative disease or an infection such as a respiratory infection extent, for example to measure the effect of a particular treatment. Such diagnosis and detection can be performed by coupling the antibody to a detectable substance including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-semi Lactosylase or acetylcholinesterase; prosthetic groups such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent substances such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate esters, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances, such as but not limited to luciferase, luciferin and Photoproteins; radioactive substances such as but not limited to iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In , ¹¹² In and ¹¹¹ In) and technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum (99Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F ), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, and ¹¹⁷ Sn; positron-emitting metals with various positron emission tomography and nonradioactive (noradioactive) Paramagnetic metal ions.

)，以及它们的药学上可接受的盐、溶剂合物、包合物和前药。The invention also includes the use of antibodies or fragments thereof conjugated to therapeutic moieties. Antibodies or fragments thereof may be conjugated to therapeutic moieties, such as cytotoxins, such as cytostatic and cytocidal agents, therapeutic agents, or radioactive metal ions, such as alpha-emitters. A cytotoxin or cytotoxic agent includes any substance that is harmful to cells. Therapeutic moieties include, but are not limited to, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, carbamide), alkylating agents (such as dichloroethylene methylamine, thioepa, chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, chain Ureamycin, mitomycin C, and cis-dichlorodiamidoplatinum(II) (DDP) and cisplatin), anthracyclines (such as daunomycin and doxorubicin), antibiotics (such as dactinomyces bleomycin, mithramycin, and anthranimycin (AMC)); auristatin molecules (eg, auristatin PHE, bryostatin 1, and solastatin10; see Woyke et al., Antimicrob. Agents Chemother. 46: 3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother. 45: 3580-4 (2001), Mohammad et al., Anti Cancer Drugs 12: 735-40 (2001), Wall et al., Biochem .Biophys.Res.Commun.266: 76-80 (1999), Mohammad et al., Int.J.Oncol.15: 367-72 (1999), the above-mentioned documents are incorporated herein by reference); hormones (such as glucocorticoids, progestins, androgens, and estrogens), DNA repair enzyme inhibitors (such as etoposide or topotecan), kinase inhibitors (such as compound T1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res.8 (7): 2167-76 (2002)); cytotoxic agents (such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide , vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydro Testosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologues, and U.S. Patents 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196、6,218,410、6,218,372、6,057,300、6,034,053、5,985,877、5,958,769、5,925,376、5,922,844、5,911,995、5,872,223、5,863,904、5,840,745、5,728,868、5,648,239、5,587,459所述的化合物)；法尼基转移酶抑制剂(例如，R115777，BMS-214662和美国专利6,458,935、6,451,812、6,440,974、6,436,960、6,432,959、6,420,387、6,414,145、6,410,541、6,410,539、6,403,581、6,399,615、6,387,905、6,372,747、6,369,034、6,362,188、6,342,765、6,342,487、6,300,501、6,268,363、6,265,422、 6,248,756、6,239,140、6,232,338、6,228,865、6,228,856、6,225,322、6,218,406、6,211,193、6,187,786、6,169,096、6,159,984、6,143,766、6,133,303、6,127,366、6,124,465、6,124,295、6,103,723、6,093,737、6,090,948、6,080,870、6,077,853、6,071,935、6,066,738、6,063,930、6,054,466、 6,051,582, 6,051,574, and 6,040,305); topoisomerase inhibitors (eg, camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX -8951f; IST-622; Rubitecan; Pyrazolinacridine; XR-5000; 506; ED-110; NB-506; and rebeccamycin); Bulgarein; DNA minor groove binders such as Hoescht dye 33342 and Hoechst dye 33258; berberine; metoxyberine; beta-lapachone; BC-4-1; bisphosphonates (eg, alendronate, cimadronte, clodronate, tiglu Phosphonates, etidronate, ibandronate, neridronate, olpandronate, risedronate, pyridronate, pamidronate, zoledronate Phosphonate (zolendronate)), HMG-CoA reductase inhibitors (such as lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lycox, livastatin Proto, rosuvastatin, and atorvastatin); antisense oligonucleotides (such as described in U.S. Patents 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminase inhibitors (such as fludarabine phosphate and 2-chlorodeoxyadenosine); ilimomab Tositumomab

), and their pharmaceutically acceptable salts, solvates, clathrates and prodrugs.

Additionally, antibodies or fragments thereof may be conjugated to therapeutic or drug moieties that alter a given biological response. It should not be considered that the therapeutic portion or the drug portion is limited to classical chemotherapeutic agents. For example, the drug moiety can be a biologically active protein, peptide or polypeptide. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, cholera toxin or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, Nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis agent, such as TNF-α, TNF-β, AIM I (see International Publication No. WO97/33899), AIM II (see International Publication No. WO 97/34911), Fas ligand (Takahashi et al., 1994, J.Immunol., 6:1567-1574) and VEGF (see International Publication No. WO 99/23105), anti-angiogenic agents, such as angiostatin, endothelial Statins or components of the coagulation pathway (such as tissue factor); or biological response modifiers, such as lymphokines (such as interferon gamma ("IFN-gamma"), interleukin-1 ("IL-1"), interleukin- 2 ("IL-2"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-7 ("IL-7"), interleukin-10 ("IL-10 ”), interleukin-12 (“IL-12”), interleukin-15 (“IL-15”), interleukin-23 (“IL-23”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”) ”) and granulocyte colony-stimulating factor ("G-CSF")) or growth factors (such as growth hormone ("GH")) or coagulation agents (such as calcium, vitamin K, tissue factors such as, but not limited to, Hagman factor (Factor XII), High Molecular Weight Kininogen (HMWK), Prekallikrein (PK), Coagulation Protein Factor II (Prothrombin), Factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X. Phospholipids, fibrinopeptides A and B) from the alpha and beta chains of fibrinogen as fibrin monomers. In a specific embodiment, the antibody that immunospecifically binds IL-9 polypeptide is conjugated to a leukotriene antagonist (such as montelukast, zafirlukast, pranlukast, and zyleuton).

Furthermore, the antibody can be conjugated to a therapeutic moiety, such as a radioactive metal ion such as an alpha emitter such as ²¹³ Bi, or to a radioactive metal ion, including but not limited to: ¹³¹ In, ¹³¹ L, ¹³¹ Y, ¹³¹ Ho , ¹³¹ Sm macrocyclic chelating agent coupled with polypeptide or any of the above-mentioned substances. In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be The molecule is linked to the antibody. These linker molecules are well known in the art, see Denardo et al., 1998, ClinCancer Res.4(10):2483-90; Peterson et al., 1999, Bioconjua.Chem.10(4):553-7; and Zimmerman et al., 1999 , Nucl. Med. Biol. 26(8):943-50, which is hereby incorporated by reference in its entirety.

Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy. and Cancer Therapy" (Monoclonal Antibodies And Cancer Therapy), Reisfeld et al. (Ed.), pp. 243-56 (ARL Company (Alan R. Liss, Inc.) 1985); Hellstrom et al., "Antibodies for Drug Delivery" ( Antibodies For Drug Delivery), in "Controlled Drug Delivery" (Controlled Drug Delivery) (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (MD Company (Marcel Dekker, Inc). 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al (Ed.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", In "Monoclonal Antibodies For Cancer Detection And Therapy" (Monoclonal Antibodies For Cancer Detection And Therapy), Baldwin et al (eds), pp. 303-16 (Academic Press (Academic Press) 1985) and Thorpe et al, 1982 , Immunol. Rev. 62:119-58.

Alternatively, the antibody can be conjugated to a second antibody to form a heterologous antibody conjugate as described by Segal in US Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated to an antigen of interest (such as an IL-9 polypeptide) or a fragment thereof should be selected so as to achieve the desired prophylactic or therapeutic effect in a subject of a particular disease or disorder, including, for example, , a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof A disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof. When deciding on a therapeutic moiety or drug to be conjugated to an antibody of interest, e.g., an antibody or fragment thereof that immunospecifically binds IL-9 polypeptide, the clinician or medical practitioner should consider the following factors: the nature of the disease, the severity of the disease, and the subject overall situation.

Antibodies can also be attached to solid supports, which is particularly useful in immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

A therapeutic moiety or drug conjugated to an antibody of interest (including antibody fragments thereof), such as an antibody that immunospecifically binds IL-9 polypeptide, should be selected to achieve the desired prophylactic or therapeutic effect on a particular disease in a subject. When deciding on a therapeutic moiety or drug to be conjugated to an antibody of interest, e.g., an antibody (including antibody fragments thereof) that immunospecifically binds IL-9 polypeptides, the clinician or medical staff should consider the following factors: the nature of the disease, the severity of the disease The extent and general condition of the subject.

5.2. Methods for preparing antibody preparations

The present invention provides methods for preparing liquid formulations of antibodies or derivatives, analogs or fragments thereof that immunospecifically bind an antigen of interest, such as an IL-9 polypeptide. Figure 16 is a schematic diagram showing a protocol for preparing purified anti-IL-9 antibodies. The method for preparing the liquid preparation of the present invention may comprise: purifying antibodies (including antibody fragments thereof) from conditioned medium (single batch or mixed batches of medium) and concentrating the purified antibody (including antibody fragments thereof) components to a final concentration About 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml , about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 250 mg/ml, or about 300 mg/ml. Conditioned medium containing antibodies (including antibody fragments thereof), such as antibodies that immunospecifically bind IL-9 polypeptide, can be subjected to CUNO filtration and HS50 cation exchange chromatography to the filtered antibodies. Fractions from HS50 cation exchange chromatography were then subjected to r-Protein A affinity chromatography followed by low pH treatment. Following low pH treatment, antibody (including antibody fragments thereof) fractions are subjected to super Q 650 anion exchange chromatography followed by nanofiltration. Then, the antibody (including antibody fragments thereof) fraction obtained after nanofiltration was diafiltered and ultrafiltered into the exchange buffer, and the same membrane was used to concentrate the antibody (including its antibody fragments) fraction into the formulation buffer. See Section 6 below for details on preparation of antibody formulations.

The formulation buffers of the present invention comprise a phosphate ion (or other non-zwitterion, such as tris, citrate, succinate, and acetate). In a specific embodiment, the formulation buffer of the present invention comprises a concentration of about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM , about 90 mM, about 95 mM or about 100 mM phosphate ion (or other non-zwitterions such as tris, citrate, succinate and acetate). The formulation may have a pH in the range of about 4.0-8.0, such as about 6.0-6.5.

Liquid formulations of the invention can be prepared in unit dosage form by preparing vials containing aliquots of the liquid formulation for single administration. For example, the unit dose per vial may contain 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 15ml or 20ml of antibodies (including antibodies thereof) that immunospecifically bind to IL-9 polypeptides in different concentrations. Fragment) at a concentration ranging from about 10 mg/ml to 300 mg/ml. These formulations can be adjusted, if necessary, to the desired concentration by adding sterile diluent to each vial. In a specific embodiment, the liquid formulation of the present invention is formulated as a sterile liquid containing 50 mM phosphate buffer, pH 6.2, and 150 mM sodium chloride, in single-dose vials. Each 1.0 mL solution contains 100 mg of antibody (including antibody fragments thereof), 50 mg, and 1 mg of sodium chloride in water. In one embodiment, antibodies of the invention (including antibody fragments thereof) are provided at 100 mg/ml in 3cc USP Type I borosilicate amber vials (West Pharmaceutical Services - Part No. 6800-0675). The target canning volume is 1.2 mL.

The liquid preparation of the present invention can be sterilized by various sterilization methods, including filter sterilization, radiation and the like. In the most preferred embodiment, the difiltrated antibody preparation is filter sterilized using a pre-sterilized 0.2 micron filter. The sterilized liquid formulation of the present invention can be administered to a subject to prevent, treat and/or manage a disease or disorder (e.g., a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, associated with A disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection)) or one or more of its symptoms.

Although the present invention relates to non-lyophilized formulations, it should be noted that the formulations of the present invention can also be lyophilized if desired. Accordingly, the invention includes lyophilized forms of the formulations of the invention.

5.3. Methods of preparing antibodies

Antibodies (including antibody fragments thereof) that immunospecifically bind to an antigen can be produced by any method for synthesizing antibodies known in the art, particularly by chemical synthesis, or preferably by recombinant expression techniques.

血蓝蛋白、二硝基酚和可能有用的人佐剂，如BCG(卡介苗)和短小棒状杆菌(corynebacterium parvum)。这些佐剂也是本领域熟知的。Polyclonal antibodies immunospecific for an antigen can be produced by various methods well known in the art. For example, a human antigen can be administered to various host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the human antigen. Various adjuvants can be used to enhance the immune response according to the type of host, these adjuvants include but not limited to: Freund's adjuvant (complete or incomplete), mineral gel, such as aluminum hydroxide, surfactant, such as hemolyzed egg Phospholipids, Pluronic polyols, polyanions, peptides, oil emulsions, keyholes

Hemocyanin, dinitrophenol and possibly useful human adjuvants such as BCG (BCG) and corynebacterium parvum. These adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using various techniques known in the art, including the use of hybridomas, recombinant techniques, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, which is known in the art, see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (Cold SpringHarbor Laboratory Press), 2nd ed., 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier) , New York, 1981 and Harlow et al., "Using Antibodies: A laboratory Manual", Cold Spring Harbor Laboratory Press (1999), the entirety of which is incorporated herein by reference. The term "monoclonal antibody" is used herein Not limited to antibodies produced by hybridoma technology.The term "monoclonal antibody" refers to antibodies derived from a single clone, including eukaryotic, prokaryotic, or phage clones, and does not limit the method of production thereof.

Methods of producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with a non-mouse antigen, and once an immune response is detected, for example, antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The spleen cells are then fused with any suitable hybridoma cells, such as cells of the cell line SP20 available from ATCC, by well-known techniques. Hybridomas were selected and cloned by limiting dilution. Additionally, animals can be immunized using the RIMMS (Repeated Immunization at Multiple Sites) technique (Kilpatrack et al., 1997, Hybridoma 16:381-9, herein incorporated by reference in its entirety). The hybridoma clones are then assayed for cells that secrete antibodies capable of binding a polypeptide of the invention using methods known in the art. Ascites fluid, which usually contains large amounts of antibodies, can be produced by immunizing mice with positive hybridoma clones.

The present invention provides a method for producing a monoclonal antibody and the antibody produced by the method, said method comprising culturing hybridoma cells secreting the antibody of the present invention, preferably by fusing splenocytes isolated from mice immunized with non-mouse antigens with myeloma cells Hybridomas are generated, and the resulting hybridomas are screened for hybridoma clones that secrete antibodies capable of binding the antigen.

Antibody fragments that specifically recognize a particular epitope can be generated by any technique known to those of skill in the art. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The F(ab')2 fragment contains the variable region, the constant region of the light chain and the CH1 domain of the heavy chain. In addition, antibodies of the present invention can also be produced by various phage display methods known in the art.

In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Specifically, DNA sequences encoding the _VH and _VL regions are amplified from an animal cDNA library (eg, a cDNA library of human or murine diseased tissue). The DNA encoding the _VH and _VL regions was recombined with the scFv linker by PCR and cloned into a phagemid vector. The vector was electroporated into E. coli, which was infected with helper phage. Half of the phages used in these methods are filamentous phages, including fd and M13, usually with recombinant fusions of the VH and VL regions to either phage gene III or gene VIII. Antigens, such as labeled antigens or antigens bound or captured on solid surfaces or beads, can be used to select or identify phage expressing an antigen binding domain capable of binding a particular antigen. Examples of phage display methods that can be used to prepare antibodies of the present invention include Brinkman et al., 1995, J.Immunol.Methods 182:41-50; Ames et al., 1995, J.Immunol.Methods 184:177-186; .J.Immunol.24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; International Application No. PCT/GB91/01134; International Publication No. WO90 /02809、WO91/10737、WO92/01047、WO92/18619、WO93/11236、WO95/15982、WO95/20401和WO97/13844；以及美国专利号5,698,426、5,223,409、5,403,484、5,580,717、5,427,908、5,750,753、5,821,047、5,571,698 , 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, 5,969,108, 6,33,187, 5,824,520 and 5,702,892, which are incorporated herein by reference in their entirety.

Following phage selection, as described in the above references, the antibody coding regions of the phage can be isolated and used to generate intact antibodies, including human antibodies or any other desired antigen-binding fragment, and grown in any desired host, including mammalian cells, Expression in insect cells, plant cells, yeast and bacteria, as described below. Methods known in the art, such as PCT Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., The method disclosed in Science 240: 1041-1043, 1988, using techniques for the recombinant production of Fab, Fab' and F(ab') ₂ fragments (which is hereby incorporated by reference in its entirety).

To generate intact antibodies, PCR primers, including the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites, can be used to amplify the VH or VL sequences in scFv clones. The PCR-amplified VH region can be cloned into a vector expressing a VH constant region, such as a human γ4 constant region, using cloning techniques known to those skilled in the art, and the PCR-amplified VL region can be cloned into a vector expressing a VL constant region, such as In the vector of human kappa or lambda constant region. Vectors for expression of VH or VL regions may contain the EF-la promoter, secretion signals, variable regions, cloning sites for constant regions and a selectable marker such as neomycin. The VH and VL regions can also be cloned into a vector expressing the desired constant regions. The heavy chain transforming vector and the light chain transforming vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies such as IgG.

In some applications, including in vivo use of antibodies in humans and in vitro assays, it may be preferable to use humanized or chimeric antibodies. Fully human and humanized antibodies are particularly preferred for human therapeutic applications. Human antibodies can be prepared by various methods known in the art, including phage display methods described above using antibody libraries generated from human immunoglobulin sequences. See also US Patents 4,444,887 and 4,716,111; and International Publication Nos. WO98/46645, WO98/50433, WO98/24893, WO98/16654, WO96/34096, WO96/33735, and WO91/10741; each incorporated herein by reference in its entirety.

Transgenic mice that do not express functional endogenous immunoglobulins, but that express human immunoglobulin genes, can also be used to produce human antibodies. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells by random methods or homologous recombination. Alternatively, human variable, constant and multivariable regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Human immunoglobulin loci can be introduced by homologous recombination, rendering mouse heavy and light chain immunoglobulin genes separately or simultaneously nonfunctional. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then mated to produce homozygous offspring that express the human antibodies. Transgenic mice are immunized in the normal manner with an antigen of choice, eg, all or a portion of a polypeptide of the invention. Monoclonal antibodies to this antigen can be obtained from immunized transgenic mice by conventional hybridoma technology. Transgenic mice harbor human immunoglobulin transgenes that rearrange during B-cell differentiation, followed by class switching and somatic mutation. Thus, it is possible to use this technology to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technique for producing human antibodies see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies and for protocols for producing such antibodies see, e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, incorporated herein by reference in their entirety. In addition, companies such as Abgeni (Freemont, CA), Genpharm (San Jose, CA), and others are able to provide a sample of the antigen of choice using techniques similar to those described above. human antibody.

Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. Methods of making chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Citations are incorporated herein in their entirety.

A humanized antibody is an antibody or variant or fragment thereof capable of binding a predetermined antigen and comprising framework regions substantially having the amino acid sequence of a human immunoglobulin and CDRs substantially having the amino acid sequence of a non-human immunoglobulin. A humanized antibody comprises substantially the entire sequence of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , Fabc, Fv), wherein all or substantially all of the CDR regions correspond to non-human immune domains. The globulin (ie, donor antibody), all or substantially all of the framework regions are those of the human immunoglobulin consensus sequence. Preferably, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Typically, antibodies contain a light chain and at least the variable region of a heavy chain. An antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of a heavy chain. Humanized antibodies may be selected from any immunoglobulin class including IgM, IgG, IgD, IgA and IgE and any isotype including IgGl, IgG2, IgG3 and IgG4. Usually, when the humanized antibody is required to have cytotoxic activity, the constant region is a complement fixation constant region, and the type is generally IgG ₁ . When such cytotoxic activity is not required, the constant region may be of the IgG ₂ type. A humanized antibody may comprise sequences of more than one type or isotype, and it is within the ability of one of ordinary skill in the art to select particular constant regions to optimize desired effector functions. The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequence, for example, a donor CDR or consensus framework region can be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR at that site Or framework region residues do not correspond to consensus or imported antibodies. However, such variation should not occur widely. Usually, at least 75% of the humanized antibody residues will correspond to the parental framework region and CDR sequences, more often 90% and more. Humanized antibodies can be produced using various techniques known in the art, including but not limited to: CDR grafting (see, eg, European Patent EP 239,400; International Publication WO 91/09967; and US Patents 5,225,539, 5,530,101, and 5,585,089) , veneering or resurfacing (resurfacing) (European patents EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka et al., 1994, Protein Engineering, 7 (6): 805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (US Pat. J. Immunol., 169: 1119--25 (2002), Caldas et al., Protein Eng., 13(5): 353--60 (2000), Morea et al., Methods, 20(3): 267--79( 2000), Baca et al., J.Biol.Chem., 272(16):10678--84(1997), Roguska et al., Protein Eng., 9(10):895--904(1996), Couto et al., Cancer Res., 55 (23 Supplement): 5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717--22 (1995), Sandhu JS, Gene, 150(2): 409--10 (1994) and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Framework residues in the framework regions are often substituted by corresponding residues of the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues that are important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions groups (see, eg, Queen et al., US Patent No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, herein incorporated by reference in their entirety).

Single domain antibodies, eg, antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann et al., 1999, J. Immuno. 231: 25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3): 253-263; Muylderman, 2001, J. Biotechnol. 74(4): 277302 ; U.S. Patent No. 6,005,079; and International Publication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, which are incorporated herein by reference in their entirety.

In addition, anti-idiotypic antibodies that "mimic" antigens can be generated using antibodies that immunospecifically bind to antigens (such as IL-9 polypeptides) by techniques well known to those skilled in the art (see, for example, Greenspan and Bona, 1989, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).

5.3.1. Recombinant expression of antibodies

Recombinant expression of an antibody contained in a formulation of the invention (eg, a heavy or light chain of an antibody of the invention or a fragment thereof, or a single chain antibody of the invention) may require the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, a heavy or light chain of an antibody, or a fragment thereof (preferably but not necessarily containing a heavy or light chain variable region) is obtained, antibody production can be obtained by recombinant DNA techniques well known in the art. molecular carrier. Thus, described herein are methods of making proteins by expressing a polynucleotide comprising a nucleotide sequence encoding an antibody. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Accordingly, the invention provides a molecule encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable region of an antibody (including antibody fragments thereof), or a heavy or light chain comprising a molecule operably linked to a promoter. A replicable vector of the nucleotide sequences of the CDRs. Such vectors can comprise nucleotide sequences encoding constant regions of antibody molecules (see, e.g., International Publication Nos. WO 86/05807 and International Publication Nos. WO 89/01036; and U.S. Patent No. 5,122,464), into which antibody variable regions can be cloned. vectors to express the entire heavy chain, the entire light chain, or both the heavy and light chains.

The expression vector is transferred into host cells by conventional techniques, and then the transfected cells are cultured by conventional techniques to produce the antibodies of the present invention. Accordingly, the present invention includes host cells comprising a polynucleotide encoding an antibody of the present invention or a fragment thereof, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody of the present invention, said polynucleotide being operably linked to an iso source promoter. In a preferred embodiment, for the expression of diabodies, vectors encoding the heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule, as detailed below.

A variety of host expression vector systems can be utilized to express the antibody molecules of the invention (see eg, US Pat. No. 5,807,715). Such host expression systems represent vectors from which the coding sequence of interest can be produced and subsequently purified, but also represent cells capable of expressing the antibody molecule of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. They include but are not limited to microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences (such as Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis)); Yeast (such as Pichia) transformed with a recombinant yeast expression vector containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (such as baculovirus) containing antibody coding sequences; recombinant viral expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) infected plant cell systems or transformed with recombinant plasmid expression vectors (e.g. Ti plasmids) containing antibody coding sequences; or mammalian cell systems (e.g. COS, CHO , BHK, 293, NSO and 3T3 cells) carrying promoters derived from mammalian cell genomes (e.g. metallothionein promoter) or mammalian viruses (e.g. adenovirus late promoter; vaccinia virus 7.5K promoter) recombinant expression constructs. Recombinant antibody molecules are expressed using bacterial cells such as Escherichia coli and eukaryotic cells (especially when expressing intact recombinant antibody molecules). For example, mammalian cells such as Chinese hamster ovary cells (CHO) combined with a vector such as the major intermediate early gene promoter element from human cytomegalovirus are efficient expression systems for antibodies (Foecking et al., 1986, Gene 45:101 and Cockett et al., 1990, Bio/Technology 8:2). In a specific embodiment, the expression of the nucleotide sequence encoding the antibody, derivative, analog or fragment thereof of the invention is regulated by a constitutive promoter, an inducible promoter or a tissue-specific promoter.

In bacterial systems, a number of expression vectors can be advantageously selected according to the intended application of the antibody molecule being expressed. For example, when large quantities of such antibodies are to be produced, vectors capable of mediating high-level expression of fusion protein products that are readily purified may be required in order to produce pharmaceutical compositions of the antibody molecules. Such vectors include, but are not limited to: Escherichia coli expression vector pUR278 (Ruther et al., 1983, EMBO, 12:1791)), wherein the antibody coding sequence can be ligated into the vector alone, in the same reading frame as the lacZ coding region, to generate Fusion proteins; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem., 24: 5503-5509); and the like. The pGEX vector can also be used to express the fusion protein of foreign polypeptide and glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione sepharose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or Factor Xa protease cleavage site to release the cloned target gene product from the GST moiety.

In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned separately into non-essential regions of the virus (such as the polyhedrin gene) and placed under the control of an AcNPV promoter (such as the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems are available. Where adenovirus is used as the expression vector, the antibody coding sequence of interest can be linked to an adenoviral transcriptional/translational control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (such as the El or E3 regions) will produce recombinant viruses that are live and capable of expressing antibody molecules in infected hosts (see, for example, Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). Specific initiation signals may also be required to enable efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Additionally, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. These exogenous translational control signals and initiation codons can be of various natural and synthetic origin. Expression efficiency can be increased by inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell line can be selected that modulates the expression of the inserted sequence, or modifies and processes the gene product in the specific manner desired. Such modifications (such as glycosylation) and processing (such as cleavage) of protein products may be critical for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells containing the cellular machinery capable of appropriately processing primary transcripts, glycosylation, and phosphorylation of gene products can be employed. Such mammalian host cells include, but are not limited to: CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, BT483, Hs578T, HTB2, BT2O, and T47D, NSO (do not endogenously produce any immunoglobulins protein chain mouse myeloma cell line), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered to stably express antibody molecules. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA and selectable markers controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introducing the foreign DNA, the engineered cells are grown in nutrient-rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selective pressure, allowing cells to stably integrate the plasmid into their chromosomes, grow to form foci, and then be cloned and expanded into cell lines. This approach can be advantageously used to engineer cell lines that express the antibody molecule. Such engineered cell lines are particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1992, Proc. Natl. Acad.Sci.USA 48:202) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes, which can be used in tk-, hgprt- or aprt-cells, respectively. Additionally, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, conferring resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA, 77:357; O'Hare et al., 1981, Proc.Natl.Acad.Sci.USA, 78:1527); gpt, produces mycophenolic acid resistance (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA, 78:2072); neo, produces aminoglycosides G-418 resistance (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol.32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev.Biochem.62: 191-217; May, 1993, TIB TECH 11 (5): 155-215); and hygro, produce hygromycin resistance (Santerre et al., 1984, Gene, 30 :147). Methods well known in the art of recombinant DNA technology are generally used to select desired recombinant clones, see for example: Ausubel et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons ), NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al. (eds.) , Chapters 12 and 13 of Current Protocols in Human Genetics, John Wellison Co., NY (1994); Colberre-Garapin et al., 1981, J.Mol.Biol., 150:1, via It is incorporated herein by reference in its entirety.

The expression level of antibody molecules can be increased by vector amplification (reviewed in Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning (Using vectors based on gene amplification in DNA cloning to express mammalian Cloned Genes in Animal Cells), Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is amplifiable, increased levels of the inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is connected to the antibody gene, it will also increase the yield of the antibody (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

Host cells can be co-transfected with two expression vectors of the present invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker enabling equivalent expression of heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of expressing both heavy and light chain polypeptides may be used. In this case, the light chain should precede the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, 1980, Proc. Natk Acad. Sci. USA, 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, including, for example, chromatography (e.g., ion exchange chromatography, affinity chromatography, especially on protein A This is followed by affinity chromatography for the specific antigen, as well as size column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. In addition, antibodies of the invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

5.4. Monitoring methods for the stability and aggregation of antibody preparations

Various methods are available to assess the stability of protein formulations, including antibody formulations, based on the protein's physical and chemical structure and its biological activity. For example, to study protein denaturation, methods such as charge transfer absorption, thermal analysis, fluorescence spectroscopy, circular dichroism (CD), NMR and HPSEC, tangential flow filtration (TFF) techniques, static light scattering (SLS) are available technology, Fourier transform infrared spectroscopy (FTIR), urea-induced protein unfolding, tryptophan intrinsic fluorescence, differential scanning calorimetry, and/or 1-anilino-8-naphthalenesulfonic acid (ANS ) protein binding technology. See, eg, Wang et al., 1988, J. of Science & Technology 42 (suppl): S4-S26.

rCGE and HPSEC are the most common and easiest methods to evaluate protein aggregate formation, protein degradation, and protein fragmentation. Therefore, the stability of the liquid formulations of the present invention can be assessed by these methods.

For example, the stability of liquid formulations of the invention can be assessed by HPSEC or rCGE, where the percent frontal area represents undegraded antibody or undegraded antibody fragments. Specifically, about 250 μg of antibody (including antibody fragments thereof) (about 25 μl of a liquid preparation containing 10 mg/ml of said antibody or antibody fragment) was injected into a TosoH Biosep TSK G3000SW equipped with a TSK SW x1 guard column (6.0mm CX 4.0cm) _XL column (7.8mmx30cm). Antibodies (including antibody fragments thereof) were eluted isocratically with 0.1 M disodium phosphate containing 0.1 M sodium sulfate and 0.05% sodium azide at a flow rate of 0.8 to 1.0 mL/min. Eluted proteins were detected by UV absorbance at 280 am. A reference standard was used as a control in this experiment and the results are reported as the area percent of the product monomer peak to all other peaks except the internal volume peak observed at about 12 to 14 minutes. Peaks eluting earlier than the monomer peak were reported as percent aggregate.

As determined by any of the above methods, the liquid preparation of the present invention has a low to undetectable aggregation level, that is, the aggregate weight of the protein does not exceed 5%, does not exceed 4%, does not exceed 3%, does not exceed 2%, does not exceed 1 % and not more than 0.5%, and have a low to undetectable level of fragmentation, that is, the proportion of the peak area representing the intact antibody (including antibody fragments thereof) to the total peak area is 80% or more, 85% or more High, 90% or higher, 95% or higher, 98% or higher, or 99% or higher, or 99.5% or higher. In the case of SDS-PAGE, the density or radioactivity of each band stained or labeled with a radioisotope can be determined, and the % density or % radioactivity of the bands representing undegraded antibodies (including antibody fragments thereof) can be obtained.

The stability of liquid formulations of the invention can also be assessed by any assay that measures the biological activity of the antibodies in the formulation. Biological activities of antibodies include, but are not limited to: antigen binding activity, complement activation activity, Fc-receptor binding activity, and the like. Antigen-binding activity of antibodies (including antibody fragments thereof) can be determined by any method known to those skilled in the art, including but not limited to ELISA, radioimmunoassay, Western blot, and the like. Complement activation activity is measured in the system by the C3a/C4a assay, in which antibodies react in the presence of complement components with cells expressing the antigen to which the antibody immunospecifically binds. See also Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) (herein incorporated by reference in its entirety). For example, ELISA-based assays can be used to compare antibodies (including antibody fragments thereof) to 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com- The ability of the 3D4 (or any other antibody in the formulation of the invention) reference standard to immunospecifically bind IL-9 polypeptide. In this assay, called VnR binding ELISA, plates are coated with isolated IL-9 polypeptide and a set of concentrations of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10 , 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 reference standards were compared to binding signals of the test antibody (including antibody fragments thereof) at the same concentration.

The purity of liquid antibody preparations of the invention can be determined by methods well known to those skilled in the art, such as HPSEC. Assess the sterility of the liquid antibody preparation as follows: filter the liquid antibody preparation through a sterile filter with a nominal porosity of 0.45 µm, inoculate sterile soybean-casein digest medium with liquid thioglycolic acid with the test liquid antibody preparation salt medium. When using the Sterisure ^™ or Steritest ^™ method, aseptically fill each filter unit with approximately 100 ml of sterile soybean-casein digest medium or liquid thioglycollate medium. Using conventional methods, the stimulated filter is aseptically transferred to 100 ml of sterile soybean-casein digest medium or liquid thioglycollate medium. Inoculate the medium at an appropriate temperature and observe three times during 14 days to confirm bacterial or fungal growth.

5.5. Prophylactic and therapeutic application of antibody preparations

The invention also relates to antibody therapy, comprising administering to a subject, preferably a human, a liquid antibody formulation (or "antibody formulation" or "liquid formulation") of the invention to prevent, treat and/or manage a disease or disorder, e.g., in combination with IL-9 A disease or disorder associated with or characterized by abnormal expression and/or activity of a polypeptide, a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, itself Immune disease, inflammatory disease, proliferative disease or infection (such as respiratory tract infection), or one or more symptoms thereof (see U.S. Provisional Application No. 60/477,801, entitled "Methods of Preventing or Treating Respiratory Conditions," filed April 12, 2004 and published as U.S. Application No. 10/ of U.S. Patent Publication No. US 2005/0147607A1 823810, entitled "Methods of Preventing or Treating Respiratory Conditions," and U.S. Provisional Application (Attorney Docket No. 10271-113-999), filed concurrently therewith on April 12, 2004, entitled " Methods of Preventing or Treating Respiratory Conditions", which is incorporated herein by reference in its entirety). In a specific embodiment, the liquid formulation of the invention comprises an antibody (including antibody fragments thereof) that immunospecifically binds to IL-9 polypeptide. The liquid formulation of the present invention may comprise a single antibody (including antibody fragments thereof) that immunospecifically binds IL-9 polypeptide (such as 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2 , 7F3com-3H5 or 7F3com-3D4). The liquid formulations of the invention may also comprise two or more antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides. In a specific embodiment, the antibody (including antibody fragments thereof) contained in such liquid formulations is 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com - 3H5 or 7F3com-3D4 or a fragment thereof. In another embodiment, the antibody (including antibody fragments thereof) contained in such liquid formulations is not 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2 , 7F3com-3H5 or 7F3com-3D4 or a fragment thereof. In yet another embodiment, the liquid preparation of the present invention comprises an antibody (including antibody fragment thereof) that immunospecifically binds IL-9 polypeptide, and the antibody (including antibody fragment thereof) is also coupled to another moiety, including but not limited to: iso Source protein, peptide or polypeptide, another antibody (including antibody fragments thereof), marker sequences, diagnostic agents, therapeutic agents, radioactive metal ions and solid supports.

As a therapeutic agent, the liquid preparation of the present invention can be applied locally or systemically to the body. Specifically, the liquid formulation of the present invention can be used to prevent, treat and/or control diseases or disorders, for example, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity of one or more subunits thereof, or one or Various symptoms. Formulations of the invention can be used to modulate the activity of cells expressing IL-9R. In a specific embodiment, the preparation of the present invention can be used to regulate various activities of the body, including but not limited to: immune function. The formulations of the invention are also suitably administered in combination with one or more other treatments (e.g., one or more other prophylactic or therapeutic agents), preferably for the prevention, treatment and/or management of a disease or disorder, e.g. , a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof A disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof. Where one or more other treatments (eg, prophylactic or therapeutic agents) are used, they may be administered alone, in any suitable form and by any suitable route. Therapeutic or prophylactic agents include, but are not limited to: small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (such as RNA and DNA nucleotides, including, but not limited to: antisense nucleotide sequences, triple helices, RNAi, and Nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetics and synthetic or natural organic molecules.

As described herein, any treatment (such as a prophylactic or therapeutic agent) known to be useful, has been used or is currently being used to prevent, treat and/or manage one or more symptoms may be combined with the liquid antibody formulations of the invention. In combination, the one or more symptoms are diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, abnormal expression of IL-9R or one or more subunits thereof Diseases or disorders, autoimmune diseases, inflammatory diseases, proliferative diseases, or associated symptoms of infections (such as respiratory tract infections) associated with or characterized by and/or activity. See, e.g., Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy), Berkow, M.D. et al (eds.), 17th ed., Merck Sharp and Dohme Research Laboratories, Rahway, NJ, 1999; and Cecil Textbook of Medicine (Textbook of Medicine), 20th edition, Bennett and Plum (eds.), W.B.Saunders, Philadelphia, 1996, related to the abnormal expression and/or activity of IL-9 polypeptides that have been used or are currently being used to prevent, treat and/or control or characterized by diseases or disorders, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative Information on the treatment of a disease or infection (such as a respiratory infection), or one or more symptoms thereof, especially prophylactic or therapeutic agents. Examples of prophylactic and therapeutic agents include, but are not limited to, immunomodulators, anti-inflammatory agents (eg, adrenocorticoids, corticosteroids (eg, beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone) Nylon, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, NSAIDs (eg, aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), and leukotriene antagonists (such as montelukast, methylxanthines, zafirlukast, and zileuton), beta2-agonists (such as albuterol, biterol, fenoterol, isoetharyl ), ocinaline, pirbuterol, albuterol, terbutaline formoterol, salmeterol, and salbutamol terbutaline), anticholinergics (such as ipratropium bromide and oxitropium bromide ), sulfasalazine, penicillamine, dapsone, antihistamines, antimalarials (such as hydroxychloroquine), antivirals, and antibiotics (such as actinomycin D (formerly known as actinomycin) , bleomycin, erythromycin, penicillin, mithramycin and antramycin (AMC)).

The liquid formulation of the present invention can be administered to a mammal, preferably a human, simultaneously with one or more other treatments (such as one or more other prophylactic or therapeutic agents) that can be used to prevent, treat and/or control IL- Diseases or disorders related to or characterized by abnormal expression and/or activity of 9 polypeptides, diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, Autoimmune disease, inflammatory disease, proliferative disease or infection (such as respiratory tract infection), or one or more symptoms thereof. The term "simultaneously" is not limited to the precise simultaneous administration of prophylactic or therapeutic agents/treatments, it means that the liquid formulation of the present invention and other agents/treatments are sequentially administered to mammals at certain time intervals, so that the immunospecifically binding substances contained in the liquid formulation Antibodies to IL-9 polypeptides (including antibody fragments thereof) may act in combination with other agents/therapies to provide a therapeutic effect superior to that administered alone. For example, the liquid formulations of the present invention and are used for the prevention, treatment and/or control of diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or one or more subunits thereof A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection) associated with or characterized by aberrant expression and/or activity of a unit, or one or more of its symptoms or multiple other prophylactic or therapeutic agents may be administered at the same time or sequentially in any order at different time points; however, if not at the same time, they should be administered sufficiently close enough to provide the desired desired therapeutic and preventive effects.

In various embodiments, the liquid formulation of the present invention and one or more other treatments (such as one or more other prophylactic or therapeutic agents), preferably for the prevention, treatment and/or management of abnormalities related to IL-9 polypeptide Diseases or disorders associated with or characterized by expression and/or activity, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases , an inflammatory disease, a proliferative disease, or an infection (such as a respiratory infection), or one or more symptoms thereof, administered at intervals of less than 1 hour, about 1 hour, about 1 hour to 2 hours, about 2 hours to 3 hours, about 3 hours to 4 hours, about 4 hours to 5 hours, about 5 hours to 6 hours, about 6 hours to 7 hours, about 7 hours to 8 hours, about 8 hours to 9 hours, about 9 hours to 10 hours hours, from about 10 hours to 11 hours, from about 11 hours to 12 hours, up to 24 hours, or up to 48 hours. In a preferred embodiment, the liquid preparation of the present invention and one or more other treatments (such as one or more other preventive or therapeutic agents), are preferably used to prevent, treat and/or control abnormal expression of IL-9 polypeptide and/or activity associated with or characterized by diseases or disorders, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, The treatment of an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof, is administered during the same patient visit. In other embodiments, the liquid preparation of the present invention and one or more other treatments (such as one or more other preventive or therapeutic agents) are preferably used to prevent, treat and/or control abnormal expression of IL-9 polypeptide and/or activity associated with or characterized by diseases or disorders, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, Inflammatory diseases, proliferative diseases or infections (such as respiratory tract infections), or the treatment of one or more symptoms thereof are administered at intervals of about 2-4 days, about 4-6 days, about 1 week, about 1-2 days week or more than 2 weeks. In a preferred embodiment, the liquid preparation of the present invention and one or more other treatments (such as prophylactic or therapeutic agents) are preferably used to prevent, treat and/or control the abnormal expression and/or activity of IL-9 polypeptide or characterized by diseases or disorders, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative Treatment of a disease or infection (such as a respiratory infection), or one or more symptoms thereof, is administered within the time frame in which both drugs are active. One skilled in the art can determine this time frame by determining the half-life of the administered drug.

In certain embodiments, the liquid formulation of the present invention and one or more other treatments (such as one or more other prophylactic or therapeutic agents), preferably used for preventing, treating and/or controlling the interaction with IL-9 polypeptide Diseases or disorders associated with or characterized by aberrant expression and/or activity, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmunity Treatment of a disease, inflammatory disease, proliferative disease or infection (eg, a respiratory infection), or one or more symptoms thereof, is administered periodically to the subject. Cycling therapy involves administering a first agent for a period of time, followed by a second agent and/or a third agent for a period of time, and repeating this sequence of administrations. Cycling therapy can reduce the development of resistance to one or more treatments, avoid or reduce side effects of one of the treatments, and/or improve efficacy.

In certain embodiments, the liquid formulation of the present invention and one or more other treatments (such as one or more other prophylactic or therapeutic agents), preferably used for preventing, treating and/or controlling the interaction with IL-9 polypeptide Diseases or disorders associated with or characterized by aberrant expression and/or activity, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmunity Treatment of disease, inflammatory disease, proliferative disease or infection (such as respiratory tract infection), or one or more symptoms thereof, with a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days, or about once a week give object. A cycle may include administration of the therapeutic or prophylactic agent by infusion, for about 90 minutes, about 1 hour, or about 45 minutes per cycle. Each cycle may include a drug rest period of at least 1 week, a drug rest period of at least 2 weeks, or a drug rest period of at least 3 weeks. The number of cycles given is about 1-12 cycles, more typically 2-10 cycles, more typically 2-8 cycles.

In other embodiments, the liquid preparation of the present invention and one or more other treatments (such as prophylactic or therapeutic agents) are preferably used to prevent, treat and/or control the abnormal expression and/or activity of the IL-9 polypeptide. or characterized by diseases or disorders, diseases or disorders associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative The treatment of a disease or infection (such as a respiratory infection), or one or more symptoms thereof, is administered by a metronomic dosing regimen, such as continuous infusion or frequent dosing without prolonged withdrawal periods. Such metronomic dosing regimens may include dosing at constant intervals with no rest periods. Generally lower doses of prophylactic or therapeutic agents, particularly cytotoxic agents, are used. Such dosing regimens involve long-term administration of relatively low daily doses over an extended period of time. In preferred embodiments, lower doses are used to minimize toxic side effects and eliminate drug withdrawal periods. In certain embodiments, the prophylactic and therapeutic agents are delivered by chronic low-dose infusion or continuous infusion over a period of about 24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3 weeks , to about 1 month, to about 2 months, to about 3 months, to about 4 months, to about 5 months, to about 6 months.

In one embodiment, the liquid formulation of the present invention is administered with a dosage regimen that maintains the plasma concentration of the immune-specific antibody (including antibody fragments thereof) of the IL-9 polypeptide at a desired level (such as about 0.1 to 100 μg/ml), To continuously block IL-9R activity. In a specific embodiment, the plasma concentration of the antibody (including antibody fragments thereof) is maintained at 0.2 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6μg/ml, 7μg/ml, 8μg/ml, 9μg/ml, 10μg/ml, 15μg/ml, 20μg/ml, 25μg/ml, 30μg/ml, 35μg/ml, 40μg/ml, 45μg/ml or 50μg/ml ml. Desired plasma concentrations in a subject will depend on several factors including, but not limited to, the nature of the disease or disorder, the severity of the disease or disorder, and the general condition of the subject. Such dosing regimens are particularly suitable for the prevention, treatment and/or management of chronic diseases or disorders.

In one embodiment, the plasma concentration of antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides is maintained at a level that blocks at least 40%, at least 50%, at least 55%, at least 60%, at least 65% , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the dosing regimen of the level of IL-9R binding to the IL-9 polypeptide, the liquid formulation of the present invention is administered to appear associated with IL-9 Diseases or disorders associated with or characterized by abnormal expression and/or activity of the -9 polypeptide, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof , an autoimmune disease, an inflammatory disease, a proliferative disease, or an infection (such as a respiratory infection), or one or more symptoms thereof. In a specific embodiment, in the occurrence of a disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, abnormal expression and/or IL-9R or one or more subunits thereof In subjects associated with or characterized by a disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or one or more symptoms thereof, immunospecific binding to IL- Plasma concentrations of antibodies to the 9 polypeptide (including antibody fragments thereof) are maintained at about 0.1 μg/ml to about 100 μg/ml.

In some embodiments, a liquid formulation of the invention comprising an antibody (including antibody fragments thereof) conjugated to a moiety (eg, a therapeutic agent or toxin) is administered intermittently to a subject.

For preventing, treating and/or controlling diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptide, abnormal expression of IL-9R or one or more subunits thereof and/or disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as respiratory tract infection), or other treatment of one or more symptoms thereof (such as prophylactic and/or or therapeutic agents) in combination, the liquid preparation of the present invention and other treatments can produce additive or synergistic effects. The present invention contemplates administration of the liquid formulation of the present invention in combination with other treatments, such as prophylactic or therapeutic agents, preferably for prevention, treatment and/or management, by the same or different routes of administration, such as oral and parenteral routes Diseases or disorders associated with or characterized by abnormal expression and/or activity of IL-9 polypeptides, diseases associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as respiratory tract infection), or the treatment of one or more symptoms thereof. In some embodiments, when the liquid preparation of the present invention is administered simultaneously with one or more treatments (such as prophylactic or therapeutic agents) that may cause toxic side effects (including but not limited to: toxicity), it is preferable to use a dose lower than the dose that causes toxicity. The treatment (eg, prophylactic or therapeutic agent) is administered at a dosage threshold for side effects.

5.5.1. Cancer treatment

The liquid formulation of the present invention can be administered to a subject in need thereof for the prevention, treatment and/or management of cancer or one or more symptoms thereof. The liquid formulation of the present invention may also be administered to a subject in need thereof in combination with one or more other treatments, preferably for the prevention, control or treatment of cancer, to prevent, treat and/or manage cancer or one or more symptoms thereof Treatment (including but not limited to preventive or therapeutic agents described in Section 5.5.1.1 below). In a specific embodiment, the present invention provides a method of preventing, treating and/or controlling cancer or one or more symptoms thereof, said method comprising administering a preventive or therapeutically effective amount of the liquid of the present invention to a subject in need preparation. In another embodiment, the present invention provides a method of preventing, treating and/or controlling cancer or one or more symptoms thereof, said method comprising administering to a subject in need a dose of a preventively or therapeutically effective amount of the liquid of the present invention formulation and a prophylactically or therapeutically effective amount of one or more treatments (eg, a prophylactic or therapeutic agent other than an antibody (including antibody fragments thereof) that immunospecifically binds IL-9 polypeptide).

The liquid formulations of the present invention can be used as first-line, second-line, third-line or fourth-line cancer therapy. The present invention provides a method of preventing, treating and/or managing one or more symptoms of cancer in a subject refractory to conventional treatments, said method comprising administering to said subject a prophylactically or therapeutically effective amount of a liquid formulation of the present invention . A cancer is determined to be refractory to treatment when at least a substantial portion of the cancer cells have not been killed or their cell division has been blocked by treatment. "Refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of a treatment on cancer cells, using the art-accepted meaning of "refractory". In a specific embodiment, the cancer is refractory when the number of cancer cells is not significantly reduced, or is increased.

The present invention provides a method of preventing, treating and/or managing cancer or one or more symptoms thereof in a subject refractory to existing single drug therapy of the cancer, said method comprising administering to said subject a dose of a prophylactic or therapeutic An effective amount of the liquid formulation of the present invention and a dose of a prophylactically or therapeutically effective amount of treatment (such as a prophylactic agent or a therapeutic agent), wherein the one or more treatments are antibodies (including antibodies) other than immunospecifically binding IL-9 polypeptides Fragment) other than treatment. The present invention also provides a method of preventing, treating and/or controlling cancer, that is, administering the liquid preparation of the present invention in combination with any other treatment (such as radiotherapy, chemotherapy or surgery) to patients who have proved refractory to other treatments and are no longer treated. The invention also provides methods of managing or treating patients with cancer who are immunosuppressed due to other prior cancer treatments. The invention also provides other methods for the prevention, treatment and/or management of cancer or one or more symptoms thereof, wherein the toxicity of chemotherapy, radiotherapy, hormone therapy and/or biological therapy/immunotherapy for the subject being treated Too large or possibly too toxic to cause unacceptable or unaffordable side effects. The invention also provides a method of preventing recurrence of cancer in a patient who has been treated and has no active disease by administering the liquid formulation of the invention.

Cancers treatable by the methods of the present invention include, but are not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth. Cancer can be primary or metastatic cancer. Cancers may or may not express IL-9R. Specific examples of cancers that may be treated with the methods of the present invention include, but are not limited to: head, neck, eye, oral cavity, throat, esophagus, breast, bone, lung, colon, rectum, stomach, prostate, breast, ovary, kidney, Cancers of the liver, pancreas, and brain. Other cancers include, but are not limited to: leukemias, such as, but not limited to: acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, such as myeloid leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, Nuclear cell leukemia, erythroleukemia and myelodysplastic syndrome, chronic leukemia such as but not limited to: chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphoma, For example, but not limited to: Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, such as but not limited to: smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, isolated malignant and extramedullary plasmacytoma; Waldenstrom macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective Histological sarcomas such as but not limited to: osteosarcoma, myelomatous bone disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, Paget's disease of bone, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma , angiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to: glioma, astrocytoma , brainstem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal tumor, pine Fruiting cell tumor, primary brain lymphoma; breast cancer, including but not limited to: adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary carcinoma breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal gland cancers such as but not limited to: pheochromocytoma and adrenocortical carcinoma;

Thyroid cancer, such as but not limited to: papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, such as but not limited to: insulinoma, gastrinoma, glucagonoma, Angiogutoma, somatostatin-secreting and carcinoid tumors or islet cell tumors; pituitary carcinomas such as, but not limited to: Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer , such as but not limited to: ocular melanoma, such as iris, choroid, and ciliary body melanoma, and retinoblastoma; vaginal cancer, such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, such as squamous squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, such as but not limited to: squamous cell carcinoma and adenocarcinoma; uterine cancer, such as but not limited to: endometrial cancer and Uterine sarcomas; ovarian cancers such as, but not limited to: epithelial ovarian carcinoma, borderline tumors, germ cell tumors, and stromal tumors;

Esophageal cancers such as, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell ) carcinoma; gastric cancer such as but not limited to: adenocarcinoma, mycosis fungoides (polypoid) carcinoma, ulcer carcinoma, superficial spreading carcinoma, extensive spreading carcinoma, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon carcinoma; rectal carcinoma Carcinoma; liver cancer, such as but not limited to: hepatocellular carcinoma and hepatotoxic tumor, gallbladder cancer, such as adenocarcinoma; cholangiocarcinoma, such as but not limited to: papillary, nodular and diffuse carcinoma; lung cancer, such as non-small cell carcinoma Lung cancer, squamous cell carcinoma (epidermoid), adenocarcinoma, large cell carcinoma, and small cell lung carcinoma; testicular cancer such as, but not limited to: germ cell tumor, seminoma, undifferentiated carcinoma, classical (typical) carcinoma, Spermatoma, nonseminoma, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor), prostate cancer such as but not limited to: adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cavity cancer, Such as but not limited to: squamous cell carcinoma; basal carcinoma; salivary gland carcinoma such as but not limited to: adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; laryngeal carcinoma such as but not limited to: squamous cell carcinoma and verrucous cancer;

Skin cancers such as but not limited to: basal cell carcinoma, squamous cell carcinoma and melanoma, melanoma with superficial spread, nodular melanoma, lentigo maligna melanoma, acral melanoma; kidney cancer, such as But not limited to: renal cell carcinoma, adenocarcinoma, adrenal adenoid tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and/or ureter); Wilms tumor; bladder cancer such as but not limited to: transitional cell carcinoma, squamous Cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myoma, osteogenic sarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovial tumor, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary Carcinoma and papillary adenocarcinoma (for a review of these diseases see Fishman et al., 1985, Medicine, 2nd ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: Cancer Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America ). It is also contemplated that cancers arising from aberrant apoptosis may also be treated using the methods and compositions of the present invention. Such cancers may include, but are not limited to: follicular lymphoma, p53-mutated cancers, hormone-dependent breast, prostate, and ovarian tumors, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes .

5.5.1.1. Anticancer therapy

The present invention provides a method for preventing, treating and/or controlling cancer or one or more symptoms thereof, the method comprising administering the liquid preparation of the present invention and one or more immunospecifically binding IL-9 polypeptides to a subject in need thereof. Treatment other than antibodies (including antibody fragments thereof) (eg, prophylactic or therapeutic agents). Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules. Any agent or treatment known to be useful, has been or is being used in the prevention, treatment and/or management of cancer or one or more of its symptoms (such as chemotherapy, radiotherapy, hormonal therapy and/or biological therapy/immunotherapy) Combinations with the liquid formulations of the invention described herein may be used.

In certain embodiments, the anticancer agent is an immunomodulator, such as a chemotherapeutic agent. In certain other embodiments, the anticancer agent is an immunomodulator other than a chemotherapeutic agent. In other embodiments, the anticancer agent is not an immunomodulator. In a specific embodiment, the anticancer agent is an antiangiogenic drug. In other embodiments, the anticancer agent is not an antiangiogenic drug. In a specific embodiment, the anticancer agent is an anti-inflammatory agent. In other embodiments, the anticancer agent is not an anti-inflammatory agent.

In a specific embodiment, anticancer agents include, but are not limited to: acivicin; aclarubicin; alcodazole hydrochloride; acronine; Amanthaquinone; Amantranone Acetate; Aminoglutethimide; Amsacridine; Anastrozole; Marestat; Benzotepa; Bicalutamide; Bisantrene hydrochloride; ), zoledronic acid (Zometa), alendronate (Fosamax), etidronate, ibandronate, incadronate, risedronate, and tiludronate); Bizelisin; Bleomycin Sulfate; Buquina Sodium; Bropirilamine; Busulfan; Actinomycin C; Carrubicin hydrochloride; Carrubicin; Cedifenoxine; Chlorambucil; Siromycin; Cisplatin; Cladribine; Clinator mesylate; Cyclophosphamide; Glycoside; Dacarbazine; Actinomycin D; Daunorubicin hydrochloride; Decitabine; Dexomaplatin; Dezaguanine; Dezaguanine mesylate; Ruubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifene citrate; For example, anti-EphA2 antibodies that cause EphA2 phosphorylation and EphA2 degradation (see U.S. Patent Application No. 60/418,213, which is hereby incorporated by reference in its entirety); elsamitrucin; enloplatin; enprabamate; Pyridine; Epirubicin Hydrochloride; Ebrozole; Esorubicin Hydrochloride; Estramustine; Estramustine Sodium Phosphate; Etanidazole; Etoposide; Etoposide Phosphate; ; Fadrozole hydrochloride; Fazarabine; Fenretinide; Azuridine; Fludarabine phosphate; Fluorouracil; Flucitabine; Idarubicin hydrochloride; ifosfamide; imofosine; interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3 ; Interferon β-Ia; Interferon γ-Ib; Isoproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Stine; loxoanthraquinone hydrochloride; masoprofen; maytansine; nitrogen mustard hydrochloride; anti-CD2 antibody (eg, cilizumab (MedImmune Inc.); /098370, which is hereby incorporated by reference in its entirety)); Progesterone Acetate; Methutepa; Mituodamide; Mitokacin; Mitoerythrin; Mitojieline; Mitomectin; Mitomycin; Mitospex; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Noramycin; Omaplatin; Oxysulam; Paclitaxel; Pegaspargase; Pelithromycin; Nemustine; Pelomycin Sulfate; Promycin; Plomestane; Porfier Sodium; Porfimycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; ; Roglutimide; Safingo; Safingo hydrochloride; Semustine; Octtrazine; Sodium phosphoacetylaspartate; Spamycin; Germanospiramine hydrochloride; Spiromustine; Spiroplatinum; Streptomycin; streptozocin; sulfoclofenamide; talithromycin; ticogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; tiroxiron; Testosterone; thiometapine; thioguanine; thiotepa; thiazofurin; tirapazamine; toremifene citrate; Trimethrexate; Triptorelin; Tobrazole Hydrochloride; Ulamustine; Uretepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vinblastine; Vinblastine Sulfate Dixin; Vinblastine Sulfate; Vinblastin Sulfate; Vinblastine Sulfate; Vinorelbine Tartrate; Vinblastine Sulfate; star.

Other anticancer agents include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; arubicin; acylfulvenes; adecypenol; Dolaisin; aldesleukin; ALL-TK antagonist; hexamethylmelamine; amustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacridine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis Inhibitor; Antagonist D; Antagonist G; Anrelix; Anti-Dorsalization-Forming Protein-1; Antiandrogen, Prostate Cancer; Antiestrogens ; antineoplaston; antisense oligonucleotide; aphidicolin glycine; apoptosis gene regulator; apoptosis regulator; apurinic nucleic acid; ara-CDP-DL-PTBA; arginine deamination Enzyme; asulacrine; atamestane; amomustine; axinastatin 1; axistatin 2; acistatin 3; azasetron; Tyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonist; benzochlorin; benzoyl staurosporine; beta-lactam Derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; diaziridine spermine; dinaphard; Bistrane A; Breflate; Breflate; Bropirimin; Budotitanium; Butylthionine; Calcipotriol; Calphostin C; Hi Dentin derivative; Canarypox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxamide triazole; CaRestM3; CARN 700; Inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Dihydrophyllin (chlorlns); Chloroquinoxaline sulfonamide; Cicaprost; Enclomid Analogues; Clotrimazole; Collismycin A; Clindamycin B; Combretastatin A4; Combretastatin Analogues; Conagenin; Crambescidin 816; Clinato; Cryptophycin 8 from Nostoc; Cryptophycin A Derivative from Nostoc; Curacin A; Cyclopentanthraquinone; Cycloplatam ; cypemycin (cypemycin); cytarabine sodium octadecyl phosphate; cell lysis factor; hexestrol phosphate (cytostatin); dalcimab; decitabine; Delorelin; Dexamethasone; Dexifosf Amide; Dexrazoxane; Dexverapamil; Deacroquinone; Daidaining B; Dihydroxybenzohydroxamic acid (didox); Diethyl-norspermine; Dihydro-5-azacytidine; Hydropaclitaxel, 9-; Dioxamycin; Diphenylspiromustine; Docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Cannabidiol; Duocamycin SA; Ebselen; Ecomustine; Edefosine; Edrolomab; Eflornithine; Eristeride; Estramustine analogues; Estrogen agonists; Estrogen antagonists; Itanidazole; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; finasteride; flavopiridol; fludlastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; ; formestane; fostrixine; formestine; gadolinium texaphyrin; gallium nitrate; gallocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; HMG CoA reductase inhibitors (such as atorvastatin, cerivastatin, fluvastatin, Lysoco, Lipitor, lovastatin, rosuvastatin, and simvastatin); hepsulfam; heregulin; cyclohexylbisacetamide; hypericin; ibandronic acid; flavobilin; edoxifene; ; imiquimod; immunostimulatory peptide; insulin-like growth factor-1 receptor inhibitor; interferon agonist; interferon; interleukin; La; Isoladine; Isobengazole (isobengazole); Isohomohalicondrin B; Itasetron; lamellarin)-N triacetic acid; lanreotide; leinamycin (leinamycin); legrastim; mushroom polysaccharide sulfate; leptolstatin (leptolstatin); letrozole; leukemia inhibitory factor; leukocyte interferon alpha; Proterelin+Estrogen+Progesterone; Leuprolide; Levamisole; LFA-3TIP (Biogen, Cambridge, MA; International Publication No. WO93/0686 and U.S. Patent No. 6,162,432); Riazol; linear polyamine analogs; lipophilic diglycopeptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; Suoxanthraquinone; lovastatin; loxoribine; letotecan; lutetium texaphyrin; lysofyll ine); cytolytic peptide; maytansine; mannostatin A; marimastat; masorol; maspin; matrix lytic factor inhibitors; matrix metalloproteinase inhibitors; menoril ; Thiobatidine; Metirelin; Methionase; Metoclopramide; MIF inhibitors; Mifepristone; Miltefosine; Miristim; Mismatched double-stranded RNA; Toguanidine hydrazone; Dibromodulbutol; Mitomycin analogs; Rastim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A + mycobacterial cell wall skeleton; mopedadol; multidrug resistance gene inhibitor; multi-tumor suppressor 1-based therapy; mustard Anticancer agent; Indian Ocean sponge (mycaperoxide) B; mycobacterial cell wall extract; myriaporone; N-acetylgenaline; N-substituted benzamides; nafarelin; (nagrestip); naloxone + analgesic; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptide Enzyme; Nilutamide; Nisamycin; Nitrox Modulator; Nitroxide Antioxidant; Nitrullyn; O6-Benzylguanine; Octreotide; Okicenone Oligonucleotide; Onapristone; Ondansetron; Ondansetron; Oracin; Oral Cytokine Inducer; Omaplatin; Oxateron; Oxaliplatin; Oxaonor oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; ;Paziptin; Pegaspargase; Peidesin; Pentosulfide Sodium; Pentostatin; Pentozole (pentrozole); Perfluorobromane; (phenazinomycin); phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; ; Plasminogen Activator Inhibitor; Platinum Complex; Platinum Compound; Platinum-Triamine Complex; J2; Proteasome Inhibitor; Protein A-Based Immunomodulator; Protein Kinase C Inhibitor; Protein Kinase C Inhibitor, Microalgae (microalgal); Protein Tyrosine Phosphatase Inhibitor; Purine Nucleoside Phosphatase Inhibitor ; purpurin; pyrazolinacridine; pyridoxalylated hemoglobin polyoxyethylene conjugate; raf antagonist; raltitrexed; ramosetron; ras method Nitinyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated reteptine; etidronate rhenium Re186; Roglutimide; Rohitukine; Romotide; Roquimex; Rubiginone B1; Ruboxyl; Safingo; Saintopin; SarCNU; Sarcophytol A; Sargragrastim; Sdi 1 Mimetic; Semustine; Senescence-Derived Inhibitor 1; Sense Oligonucleotide; Signal Transduction Inhibitor; Signal Transduction Regulator; Single-chain antigen-binding protein; sizoran; sorbudzoxan; borcassodium; sodium phenylacetate; solverol; somatomodulin-binding protein; sonamine; spicamycin) D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitors; stem cell division inhibitors; stipiamide; stromalysin inhibitors; Sulfinosine; potent vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic mucopolysaccharides; tamustine; 5-fluorouracil; folinic acid; other Moxifen mediodide; tazamustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; Niposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; tiacolaline; thrombopoietin; thrombopoietin mimic; thymofasin; thymopoietin receptor Body agonist; Thymustrinam; Thyroid-stimulating hormone; Benpurin ethyl tin; Tirapazamine; Dichlorocyclopentadienyl titanium; Topsentin (topsentin); Toremifene; Totipotent stem cell factor; Translational inhibition Retinoic acid; Triacetyl uridine; Tricilibine; Trimetrexate; Triptorelin; Tropisetron; Torosteride; Tyrosine kinase inhibitors; Tyrosine phosphorylation inhibitors ( tyrphostin); UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitors; urea kinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; Verteporphine; Viraresol; Veratramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin ^™ (see US Patent Publication No. US2002/ 0168360 A1, November 14, 2002, entitled "Administration of Integrin α _v β ₃ Antagonists and Other Preventive or therapeutic agent "(Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylacticor Therapeutic Agents)); Volozole; Zanol Tron; geniplatin; benzyretin C; and net statins. In another preferred embodiment, antibody derivatives, such as MT103, part of a class of antibody derivatives known as bispecific T-cell conjugates (BiTE ^™ ; Midiuni Corporation), may also be combined with one or more A combination of liquid formulations of the present invention.

(IDEC和SKB)、mAB 4162W94、奥索克隆(Orthoclone)和OKTcdr4a(JC公司(Janssen-Cilag)))，抗-CD3抗体(如努为昂(Nuvion)(产品设计实验室(Product Design Labs))，OKT3(强生公司(Johnson&Johnson))或利妥昔(IDEC))，抗-CD5抗体(如抗-CD5蓖麻毒蛋白-连接的免疫偶联物)，抗-CD7抗体(如CHH-380(诺华公司))，抗-CD8抗体，抗CD40配体单克隆抗体(如IDEC-131(IDEC))，抗-CD52抗体(如坎帕斯1H(Ilex))，抗-CD2抗体(如MEDI-507(米迪缪尼公司，国际公开号WO 02/098370和WO 02/069904)，抗-CD11a抗体(如仙耐林(Xanelim)(基因泰克公司))和抗-B7抗体(如IDEC-114)(IDEC))；抗-细胞因子受体抗体(如抗-IFN受体抗体、抗-IL-2受体抗体(如Zenapax(蛋白质设计实验室))，抗-IL-4受体抗体，抗-IL-6受体抗体，抗-IL-10受体抗体和抗-IL-12受体抗体)，抗-细胞因子抗体(如抗-IFN抗体、抗-TNF-α抗体、抗-IL-1β抗体、抗-IL-6抗体、抗-IL-8抗体(如ABX-IL-8(阿布基尼克斯公司(Abgenix)))和抗-IL-12抗体))；CTLA4-免疫球蛋白；LFA-3TIP(百集公司，国际公开号WO 93/08656和美国专利号6,162,432)；可溶性细胞因子受体(如TNF-α受体的胞外结构域或其片段、IL-1β受体的胞外结构域或其片段和IL-6受体的胞外结构域或其片段)；细胞因子或其片段(如白介素(IL)-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-15、TNF-α、TNF-β、干扰素(IFN)-α、IFN-β、IFN-γ和GM-CSF)；和抗-细胞因子抗体(如抗-IL-2抗体、抗-IL-4抗体、抗-IL-6抗体、抗-IL-10抗体、抗-IL-12抗体、抗-IL-15抗体、抗-TNF-α抗体和抗-IFN-γ抗体)和免疫特异性结合肿瘤相关抗原的抗体(如

)。Other examples of anticancer drugs include, but are not limited to, angiogenesis inhibitors, topoisomerase inhibitors, and immunomodulators, (e.g., chemotherapeutics and non-therapeutic immunomodulators, including, but not limited to, anti-T cell receptor antibodies (such as cM-T412 (Boeringer), IDEC-

(IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (JC Company (Janssen-Cilag)), anti-CD3 antibodies (such as Nuvion (Product Design Labs) ), OKT3 (Johnson & Johnson) or rituximab (IDEC)), anti-CD5 antibody (such as anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibody (such as CHH-380 (Novartis)), anti-CD8 antibody, anti-CD40 ligand monoclonal antibody (such as IDEC-131 (IDEC)), anti-CD52 antibody (such as Campas 1H (Ilex)), anti-CD2 antibody (such as MEDI -507 (Midiuni, International Publication Nos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies (such as Xanelim (Genentech)) and anti-B7 antibodies (such as IDEC- 114) (IDEC)); anti-cytokine receptor antibody (eg, anti-IFN receptor antibody, anti-IL-2 receptor antibody (eg, Zenapax (Protein Design Laboratories)), anti-IL-4 receptor antibody , anti-IL-6 receptor antibody, anti-IL-10 receptor antibody and anti-IL-12 receptor antibody), anti-cytokine antibodies (such as anti-IFN antibody, anti-TNF-α antibody, anti- IL-1β antibody, anti-IL-6 antibody, anti-IL-8 antibody (such as ABX-IL-8 (Abgenix) and anti-IL-12 antibody)); CTLA4-immunosphere protein; LFA-3TIP (Baiji Company, International Publication No. WO 93/08656 and U.S. Patent No. 6,162,432); soluble cytokine receptors (such as the extracellular domain of TNF-α receptor or its fragments, IL-1β receptor extracellular domain or fragments thereof and IL-6 receptor extracellular domain or fragments thereof); cytokines or fragments thereof (such as interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, Interferon (IFN)-α, IFN- β, IFN-γ, and GM-CSF); and anti-cytokine antibodies (eg, anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-10, anti-IL -12 antibody, anti-IL-15 antibody, anti-TNF-α antibody and anti-IFN-γ antibody) and antibodies that immunospecifically bind tumor-associated antigens (such as

).

The invention also encompasses the administration of the liquid formulations of the invention in combination with radiation therapy, which includes the use of X-rays, gamma rays, and other sources of radiation to destroy cancer cells. In a preferred embodiment, radiation therapy is administered in the form of external beam radiation therapy or teletherapy, in which radiation is delivered from a remote source. In other preferred embodiments, radiation therapy is administered in the form of internal therapy or brachytherapy, wherein the radiation source is placed in the body close to the cancer cells or tumors.

他莫昔芬，他莫昔芬和细胞毒性化疗联用。在某些实施方式中，将预防或治疗有效量的一种或多种本发明液体制剂与有效量的紫杉烷如多西他赛和紫杉醇联合给予转移性乳腺癌患者。在其它实施方式中，将预防或治疗有效量的本发明液体制剂与预防或治疗有效量的紫杉烷以及标准多柔比星和环磷酰胺联用，以便辅佐治疗结节阳性的局限性乳腺癌。In a specific embodiment, a preventive or therapeutic effective amount of the liquid preparation of the present invention is combined with a preventive or therapeutic effective amount of one or more other agents for treating breast cancer, and the other agents include but not Limited to: doxorubicin, epirubicin, combination of doxorubicin and cyclophosphamide (AC), combination of cyclophosphamide, doxorubicin and 5-fluorouracil (CAF), cyclophosphamide, epirubicin Combination of Bixing and 5-fluorouracil (CEF),

Tamoxifen, the combination of tamoxifen and cytotoxic chemotherapy. In certain embodiments, a prophylactically or therapeutically effective amount of one or more liquid formulations of the present invention is administered to a patient with metastatic breast cancer in combination with an effective amount of taxanes such as docetaxel and paclitaxel. In other embodiments, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is combined with a prophylactically or therapeutically effective amount of a taxane and standard doxorubicin and cyclophosphamide to adjunctively treat nodular positive localized breast cancer.

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is combined with a prophylactically or therapeutically effective amount of one or more other agents for treating prostate cancer, including but not Limited to: external-beam radiotherapy, interstitial implants of radioisotopes (ie, I ¹²⁵ , palladium, iridium), leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide, bismuth karutamide), steroidal antiandrogens (cyproterone acetate), leuprolide and flutamide in combination, estrogens such as DES, chlorestradiol, ethinyl estradiol, conjugated estradiol Hormone USP, DES diphosphate, radioisotopes such as strontium-89, combination of external beam radiotherapy and strontium-89, second-line hormone therapy such as amglutethimide, hydrocortisone, flutamide withdrawal, progesterone and Ketoconazole, low-dose prednisone, or other chemotherapy regimens reported to produce subjective symptom improvement and lower PSA levels, including docetaxel, paclitaxel, estramustine/docetaxel, estramustine/retinol Poside, estramustine/vinblastine, and estramustine/paclitaxel. In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is combined with a prophylactically or therapeutically effective amount of one or more other agents for treating ovarian cancer, including but not Limited to: intraperitoneal radiotherapy, such as P ³² therapy, whole abdomen radiotherapy and pelvic radiotherapy, cisplatin, paclitaxel (taxol) or docetaxel (taxotere) in combination with cisplatin or carboplatin, cyclophosphamide and cisplatin Platinum, cyclophosphamide and carboplatin, 5-FU and folinic acid, etoposide, liposomal doxorubicin, gemcitabine, or topotecan. It is contemplated that a prophylactically or therapeutically effective amount of the liquid formulation of the invention is administered in combination with Taxol to a patient suffering from platinum refractory disease. Treatment of patients with refractory ovarian cancer includes the administration of ifosfamide to patients with platinum-refractory disease, hexamethylmelamine (HMM) as salvage therapy after failure of cisplatin-based combination regimens, and tamoxifen Xifen was administered to patients whose tumors had detectable levels of cytoplasmic estrogen receptors. In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to an osteosarcoma patient in combination with a prophylactically or therapeutically effective amount of one or more other agents for the treatment of osteosarcoma, including but not Limited to: doxorubicin, ifosfamide, cisplatin, high-dose methotrexate, cyclophosphamide, etoposide, vincristine, actinomycin D, and surgery.

In a specific embodiment, a preventive or therapeutic effective amount of the liquid formulation of the present invention is combined with a preventive or therapeutic effective amount of one or more other agents for treating bone metastatic tumors to a patient whose tumor has metastasized to bone, said Other agents include, but are not limited to: agents or treatments for mechanistically treating malignancies (non-limiting examples are hormone inhibitors and surgery when prostate or breast cancer metastasizes to the bone), radiation therapy (non-limiting examples are strontium 89 and samarium 153, which are osteophilic radionuclides, have antitumor effects and can relieve symptoms) and bisphosphonates.

Cancer treatments and their dosages, routes of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (56th Edition, 2006).

5.5.2. Proliferative diseases

The liquid antibody formulations of the present invention can be used to prevent, treat and/or manage proliferative diseases or one or more symptoms thereof. In a specific embodiment, the proliferative disease is characterized by abnormal proliferation (eg, uncontrolled proliferation or lack of proliferation) of cells from which IL-9 mediates growth, including but not limited to: T cells, erythroid Progenitor cells, B cells, mast cells, eosinophils, neutrophils, and fetal thymocytes.

The present invention provides methods of preventing, treating and/or managing one or more symptoms of non-cancerous diseases (i.e., diseases without metastatic potential) associated with IL-9-mediated hyperproliferation of cells, particularly are epithelial (eg, asthma, COPD, pulmonary fibrosis, bronchial hyperresponsiveness, psoriasis, lymphoproliferative disorders, and seborrheic dermatitis) and endothelial (eg, restenosis, hyperproliferative vascular disease, Behcet syndrome, atherosclerosis and macular degeneration), the method comprising administering to a subject in need thereof an effective amount of one or more antibodies of the invention. The present invention also provides a method for preventing, treating and/or controlling non-cancer diseases related to IL-9-mediated cell hyperproliferation, said method comprising administering to a subject in need thereof an effective amount of one or more antibodies of the present invention and an effective An amount of one or more other treatments (for example, one or more prophylactic or therapeutic agents) that are not the antibodies of the invention for the prevention, treatment and/or management of the disease.

The present invention provides methods of preventing, treating and/or managing one or more symptoms of non-cancerous diseases associated with IL-9-mediated cellular hyperproliferation in a subject refractory to conventional therapy, said method comprising administering the An effective amount of one or more antibodies, compositions or combination therapies of the invention is administered to a subject. In certain embodiments, a patient with a non-cancerous disease associated with IL-9-mediated cellular hyperproliferation is refractory to treatment when the hyperproliferation is not eliminated and/or symptoms are not alleviated. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of a treatment for a non-cancerous hyperproliferative disease, using the art-accepted meaning of "refractory". In various embodiments, a patient with a non-cancerous disease associated with IL-9-mediated cellular hyperproliferation is said to be refractory when the patient's IL-9 levels remain abnormal and/or if cellular proliferation is not seen to decrease. The present invention also provides a method of preventing, treating and/or controlling non-cancer diseases associated with IL-9-mediated cell hyperproliferation in a subject refractory to conventional treatments, the method comprising administering to a subject in need thereof an effective amount of a One or more antibody preparations of the present invention and an effective amount of one or more other treatments (for example, one or more prophylactic or therapeutic agents) that are not used to prevent, treat and/or control the disease Antibody preparations of the present invention.

In a specific embodiment, an effective amount of one or more antibodies of the present invention is combined with an effective amount of an antibody (including antibody fragments thereof) (such as 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9 , 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4) liquid formulations of the invention are administered in combination to a subject at risk of or suffering from a proliferative disease. The liquid antibody formulation of the invention or the combination therapy of the invention can be used as the first, second, third, fourth or fifth treatment to prevent, treat and/or control a proliferative disease or one or Various symptoms. The invention also includes methods of preventing, treating and/or managing proliferative diseases or one or more symptoms thereof in patients who are being treated for other diseases or disorders. The invention includes methods of preventing, treating and/or managing a proliferative disease or one or more symptoms thereof in a patient prior to any adverse reaction or intolerance to therapy other than the antibodies of the invention. The invention also includes methods of preventing, treating and/or managing proliferative diseases or their symptoms in patients prone to adverse reactions to conventional treatments.

The invention includes methods of preventing, treating and/or managing proliferative diseases or symptoms thereof in patients who have proven refractory to treatments other than the antibodies, compositions or combinations of the invention. In certain embodiments, when the proliferative disease is not resolved and/or symptoms are not alleviated, the patient with the proliferative disease is refractory to treatment. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of a treatment for a proliferative disease, using the art-accepted meaning of "refractory". In various embodiments, a patient with a proliferative disease is said to be refractory when the patient's IL-9 levels remain abnormal and/or cell proliferation does not decrease.

The present invention provides methods of preventing, treating and/or managing proliferative diseases or one or more symptoms thereof as an alternative to other conventional treatments. In a specific embodiment, the patients managed or treated according to the methods of the present invention are patients who are refractory to other treatments or prone to adverse effects from other treatments. The patient may be a person with a suppressed immune system (such as postoperative patients, patients undergoing chemotherapy, and patients with immunodeficiency diseases), a person with impaired renal or hepatic function, the elderly, children, infants, people with neuropsychiatric disorders, or those taking psychoactive People who are on medication, have a history of epilepsy, or are taking medications that conflict with conventional medications used to control or treat proliferative disorders.

Treatments for the prevention, treatment and/or management of proliferative diseases or one or more symptoms thereof, as well as dosages, routes of administration and recommended usage for treatment are known in the art, see, e.g., Physicians' Desk Reference ) (60th edition, 2006).

5.5.3. Treatment of inflammatory diseases

The liquid formulations of the present invention can be administered to a subject in need thereof for the prevention, treatment and/or management of inflammatory diseases such as asthma or one or more symptoms thereof. The liquid formulation of the present invention can also be administered in combination with one or more other treatments, preferably treatments for the prevention, treatment and/or control of inflammatory diseases (including but not limited to the preventive or therapeutic agents listed in Section 5.5.3.1 below) A subject in need thereof, to prevent, treat and/or manage an inflammatory disease or one or more symptoms thereof. In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling an inflammatory disease or one or more symptoms thereof, the method comprising administering a preventive or therapeutically effective dose of the present invention to a subject in need thereof. Invention of liquid formulations. In another embodiment, the present invention provides a method of preventing, treating and/or controlling an inflammatory disease or one or more symptoms thereof, the method comprising administering a preventive or therapeutically effective amount of the present invention to a subject in need thereof. Invention of a liquid formulation and a dose of a prophylactically or therapeutically effective amount of one or more treatments (eg, a prophylactic or therapeutic agent other than an antibody (including antibody fragments thereof) that immunospecifically binds IL-9 polypeptide).

The present invention provides prevention, treatment and/or management of one or more symptoms of inflammatory diseases in subjects refractory to conventional treatments such as methotrexate and TNF-alpha antagonists such as REMICADE ^™ or ENBREL ^™ A method comprising administering to the subject a prophylactically or therapeutically effective amount of the liquid formulation of the present invention. The present invention also provides a method of preventing, treating and/or managing one or more symptoms of an inflammatory disease in a subject refractory to existing single drug therapy of the inflammatory disease, said method comprising administering to said subject a A preventive or therapeutic effective amount of the liquid preparation of the present invention and a preventive or therapeutic effective amount of treatment (such as prophylactic or therapeutic agent), said one or more treatments are antibodies in addition to immunospecifically binding IL-9 polypeptide (including antibody fragments thereof). The present invention also provides a method of controlling or treating an inflammatory disease by administering the liquid formulation of the present invention in combination with any other therapy to a patient who has proven refractory to other therapy and is no longer on these treatments. The present invention also provides alternative methods of treating inflammatory diseases when other treatments are or may be too toxic for the subject being treated, ie, result in unacceptable or unaffordable side effects. For example, a liquid formulation of the invention can be administered to a subject, wherein the subject is refractory to TNF antagonists or methotrexate. The invention also provides a method of preventing recurrence of an inflammatory disease in a patient who has been treated and has no active disease by administering the liquid formulation of the invention.

Inflammatory diseases treatable by the methods of the present invention include, but are not limited to: asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disease, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy , undifferentiated arthropathy, arthritis, osteoarthritis, spondyloarthropathies (eg, psoriatic arthritis, ankylosing spondylitis, Reiter syndrome (active arthritis), inflammatory osteolysis, Wilson disease, and chronic Chronic inflammation caused by viral or bacterial infection. As described in Section 5.5.4.1 herein, some autoimmune diseases are associated with inflammatory conditions.

Anti-inflammatory treatments and their dosages, routes of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (60th Edition, 2006).

5.5.3.1. Anti-inflammatory treatment

The present invention provides a method for preventing, treating and/or controlling inflammatory diseases or one or more symptoms thereof, the method comprising administering the liquid preparation of the present invention and one or more immune-specific binding IL-9 to a subject in need thereof. Therapeutic (eg, prophylactic or therapeutic) other than antibodies to polypeptides (including antibody fragments thereof). Any agent or treatment known to be useful, used or being used in the prevention, treatment and/or management of an inflammatory disease or one or more symptoms thereof may be used in combination with the liquid formulations of the invention described herein.

Any anti-inflammatory agent, including agents known to those skilled in the art for the treatment of inflammatory diseases, can be used in the compositions and methods of the present invention. Non-limiting examples of anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (e.g., atropine sulfate, atropine methylnitrate, and ipratropium bromide (ATROVENT ^™ )), beta2 - Agonists (e.g., abuterol (VENTOLIN ^TM and PROVENTIL ^TM ), bitoterol (TORNALATE ^TM ), levalbuterol (XOPONEX ^TM ), orcinalin (ALUPENT ^TM ), pirbuterol (MAXAIR TM ) ^TM ), terbutaline (BRETHAIRE ^TM and BRETHINE ^TM ), albuterol (PROVENTIL ^TM , REPETABS ^TM and VOLMAX ^TM ), formoterol (FORADILAEROLIZER ^TM ) and salmeterol (SEREVENT ^TM and SEREVENTDISKUS ^TM )) and methyl Xanthines (eg, theophylline (UNIPHYL ^™ , THEO-DUR ^™ , SLO-BID ^™ and TEHO-42 ^™ )). Examples of NSAIDs include, but are not limited to: aspirin, ibuprofen, celecoxib (CELEBREX ^™ ), diclofenac (VOLTAREN ^™ ), etodolac (LODINE ^™ ), fenoprofen (NALFON ^™ ), indomethacin (INDOCIN ^TM ), ketoralac (TORADOL ^TM ), oxaprozin (DAYPRO ^TM ), nabumetone (RELAFEN ^TM ), sulindac (CLINORIL ^TM ), tolmentin (TOLECTIN ^™ ), rofecoxib (VIOXX ^™ ), naproxen (ALEVE ^™ , NAPROSYN ^™ ), ketoprofen (ACTRON ^™ ), and nabumetone (RELAFEN ^™ ). Such NSAIDs work by inhibiting cyclooxygenases such as COX-1 and/or COX-2. Examples of steroidal anti-inflammatory drugs include, but are not limited to: glucocorticoids, dexamethasone (DECADRON ^™ ), corticosteroids (eg, methylprednisolone (MEDROL ^™ )), cortisone, hydrocortisone, prednisone ( PREDNISONE ^TM and DELTASONE ^TM ), prednisolone (PRELONE ^TM and PEDIAPRED ^TM ), triamcinolone, sulfasalazine, and eicosanoid inhibitors (such as prostaglandins, thromboxanes, and leukotrienes (leukotriene See Table 2)) below for non-limiting examples and typical dosages of such agents.

In a specific embodiment, an effective amount of one or more antibodies of the present invention and an effective amount of Vitacin ^™ (Midiuni Corporation, International Publication No. WO 00/78815, International Publication No. WO 02/070007A1, September 12, 2002, entitled "Methods of Preventing or _Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV _Beta3 Antagonists" ), International Publication No. WO 03/ _075957A1 , September ₁₈ , 2003, entitled "The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3Antagonists in Combination with Other Agents" (The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3Antagonists in Combination With Other Agents), U.S. Patent Publication No. US 2002/0168360A1, November 14, 2002, entitled "Prevention or Treatment of Inflammatory Diseases by Combination Administration of Integrin α _v β ₃ Antagonists and Other Prophylactic or Therapeutic Agents" or autoimmune diseases" (Methods of Preventing or Treating Inflammatory or AutoimmuneDisorders by Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylactic or Therapeutic Agents) and International Publication No. WO 03/075741A2, September 18, 2003, titled "Methods of Preventing or Treating Disorders by Administering an Integrin α _{v β 3 Antagonist in} Combination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate), which is incorporated herein by reference in its entirety), is administered to a subject in combination to prevent, treat and/or control an inflammatory disease or one or more symptoms thereof. In another embodiment, an effective amount of one or more antibodies of the present invention and an effective amount of cilizumab (Midiuni, International Publication No. WO 02/069904) are administered to the subject in combination to prevent, treat and/or control an inflammatory disease or one or more symptoms thereof. In another embodiment, an effective amount of one or more antibodies of the invention is combined with an effective amount of one or more EphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (Midiuni; International Publication No. WO 02/102974A4, December 27, 2002, entitled Mutant Proteins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure, International Publication No. 03/094859 A2 , November 20, 2003, entitled "EphA2 Monoclonal Antibodies and Methods of Use Thereof," U.S. Application No. 10/436,783 and U.S. Patent Publication No. US 2004/0091486A1; and U.S. Application No. 10/994,129 and US Patent Publication No. US 2005/0152899A1 , which are incorporated herein by reference in their entirety)) in combination for the prevention, treatment and/or management of an inflammatory disease or one or more symptoms thereof. In another preferred embodiment, an effective amount of one or more antibodies of the present invention is administered to an object in combination with an effective amount of Vitasine ^™ , Silizumab and/or EphA2 inhibitors to prevent, treat and/or To control an inflammatory disease or one or more symptoms thereof.

In one embodiment, an effective amount of one or more antibody preparations of the invention is administered to a subject at risk of or suffering from an inflammatory disease in combination with a mast cell protease inhibitor. In another embodiment, the mast cell protease inhibitor is a tryptase kinase inhibitor, such as but not limited to: GW-45, GW-58, and genistein. In a specific embodiment, the mast cell protease inhibitor is a phosphatidylinositol-3'(PI3)-kinase inhibitor, such as but not limited to: carverstatin C. In another embodiment, the mast cell protease inhibitor is a protein kinase inhibitor, such as but not limited to staurosporine. According to this embodiment, the mast cell protease inhibitor is preferably administered locally to the affected area.

(IDEC和SKB)、mAB4162W94、奥索克隆和OKTcdr4a(JC公司))、抗-CD3抗体(如努为昂(产品设计实验室)、OKT3(强生公司)或利妥昔(IDEC))、抗-CD5抗体(如抗-CD5蓖麻毒蛋白-连接的免疫偶联物)、抗-CD7抗体(如CHH-380(诺华公司))、抗-CD8抗体、抗CD40配体单克隆抗体(如IDEC-131(IDEC))、抗-CD52抗体(如坎帕斯1H(Ilex))、抗-CD2抗体(如MEDI-507(米迪缪尼公司，国际公开号WO02/098370和WO 02/069904)、抗-CD11a抗体(如仙耐林(基因泰克公司))和抗-B7抗体(如IDEC-114)(IDEC))；抗-细胞因子受体抗体(如抗-IFN受体抗体、抗-IL-2受体抗体(如Zenapax(蛋白质设计实验室))、抗-IL-4受体抗体、抗-IL-6受体抗体、抗-IL-10受体抗体和抗-IL-12受体抗体)、抗-细胞因子抗体(如抗-IFN抗体、抗-TNF-α抗体、抗-IL-1β抗体、抗-IL-6抗体、抗-IL-8抗体(如ABX-IL-8(阿布基尼克斯公司))和抗-IL-12抗体))；CTLA4-免疫球蛋白；LFA-3TIP(百集公司，国际公开号WO93/08656和美国专利号6,162,432)；可溶性细胞因子受体(如TNF-α受体的胞外结构域或其片段、IL-1β受体的胞外结构域或其片段和IL-6受体的胞外结构域或其片段)；细胞因子或其片段(如白介素(IL)-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-15、TNF-α、TNF-β、干扰素(IFN)-α、IFN-β、IFN-γ和GM-CSF)；和抗-细胞因子抗体(如抗-IL-2抗体、抗-IL-4抗体、抗-IL-6抗体、抗-IL-10抗体、抗-IL-12抗体、抗-IL-15抗体、抗-TNF-α抗体和抗-IFN-γ抗体)。Specific examples of immunomodulators that can be administered to patients with inflammatory diseases in combination with the liquid formulations of the present invention include, but are not limited to: methotrexate, leflunomide, cyclophosphamide, cyclophosphamide, Imuran, cyclosporine A , minocycline, azathioprine, antibiotics (such as FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus) Division), mizoribine, deoxyspergualin, buquinal, malononitrile amides (such as leflunamide), anti-T cell receptor antibodies (for example, anti-CD4 antibodies (such as cM-T412 (Bollinger Corporation), IDEC

(IDEC and SKB), mAB4162W94, Osoclon and OKTcdr4a (JC Company)), anti-CD3 antibodies (such as Nuweiang (Product Design Laboratories), OKT3 (Johnson & Johnson) or Rituximab (IDEC)), anti- - CD5 antibody (eg anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibody (eg CHH-380 (Novartis)), anti-CD8 antibody, anti-CD40 ligand monoclonal antibody (eg IDEC-131 (IDEC)), anti-CD52 antibodies (such as Campas 1H (Ilex)), anti-CD2 antibodies (such as MEDI-507 (Midiuni Corporation, International Publication Nos. WO02/098370 and WO 02/069904 ), anti-CD11a antibodies (such as Xanalin (Genentech)) and anti-B7 antibodies (such as IDEC-114) (IDEC)); anti-cytokine receptor antibodies (such as anti-IFN receptor antibodies, anti - IL-2 receptor antibodies (such as Zenapax (Protein Design Laboratories)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibody), anti-cytokine antibody (such as anti-IFN antibody, anti-TNF-α antibody, anti-IL-1β antibody, anti-IL-6 antibody, anti-IL-8 antibody (such as ABX-IL- 8 (Abukinix Company)) and anti-IL-12 antibody)); CTLA4-immunoglobulin; LFA-3TIP (Baiji Company, International Publication No. WO93/08656 and U.S. Patent No. 6,162,432); soluble cytokine receptor body (such as the extracellular domain of TNF-α receptor or its fragment, the extracellular domain of IL-1β receptor or its fragment and the extracellular domain of IL-6 receptor or its fragment); cytokine or its Fragments (such as interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF); and anti-cytokine antibodies (such as anti-IL-2 antibody, anti- IL-4 antibody, anti-IL-6 antibody, anti-IL-10 antibody, anti-IL-12 antibody, anti-IL-15 antibody, anti-TNF-α antibody and anti-IFN-γ antibody).

Any TNF-[alpha] antagonist known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples of TNF-alpha antagonists that can be administered to patients with inflammatory diseases in combination with the liquid formulations of the present invention include proteins, polypeptides, peptides that block, reduce, inhibit or neutralize the function, activity and/or expression of TNF-alpha , fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal antibodies, Fv, ScFv, Fab fragments, F(ab) ₂ fragments, and antigen-binding fragments thereof) such as immunospecifically binding TNF- Alpha antibodies, nucleic acid molecules (such as antisense molecules or triple helices), organic molecules, inorganic molecules and small molecules. In various embodiments, the TNF-α antagonist reduces the function, activity and/or expression of TNF-α by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, or at least 99%. Examples of antibodies that immunospecifically bind TNF-alpha include, but are not limited to: Infliximab (REMICADE ^™ ; Centacor), D2E7 (Albert Laboratories, Olive Hill, NJ/Noll Pharmaceuticals ( Abbott Laboratories/Knoll Pharmaceuticals Co., Mt. Olive, NJ)), CDP571 (also known as HUMICADE ^™ ) and CDP-870 (all from Celltech/Pharmacia, Slough, UK )) and TN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91: 2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA 89: 7375-7379). The present invention also includes the use of antibodies immunospecifically binding to TNT-α disclosed in the following U.S. patents for use in the compositions and methods of the present invention: 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412; 5,959,087; 5,968,741; 5,994,510; Examples of soluble TNF-alpha receptors include, but are not limited to: sTNF-R1 (Amgen), etanercept (ENBREL ^™ ; Immunex) and its rat homologue RENBREL ^™ , a soluble inhibitor of TNF-α derived from TNFrI, TNFrII (Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335) and TNF-αInh (Seckinger et al., 1990, Proc. . Sci. USA 87:5188-5192).

Other TNF-alpha antagonists encompassed by the present invention include, but are not limited to: IL-10 known to block TNF-alpha production by interferon gamma-activated macrophages (Oswald et al., 1992, Proc. Natl. Acad. Sci. .USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc.Natl.Acad.Sci.USA88:10535-10539), mouse product TBP-1 (SY company (Serono/Yeda)), vaccine CytoTAb (Protherics), antisense molecule 104838 (ISIS), peptide RDP-58 (SS (SangStat)), thalidomide (Celgene), CDC-801 (Celgene) Base Company), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco )), NPI-13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax), Ridaura (SmithKline Beecham Pharmaceuticals), quinol Clin (mepacrindichlorohydrate), Tenidap (Enablex), Melanin (Large Scale Biological), and Uriazi's Anti-p38 MAPK agents.

Non-limiting examples of anti-inflammatory agents that can be administered to patients with inflammatory diseases in combination with the liquid formulations of the invention include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-agonists, anticholinergics, and methylxanthines . Examples of NSAIDs include, but are not limited to: aspirin, ibuprofen, celecoxib (CELEBREX ^™ ), diclofenac (VOLTAREN ^™ ), etodolac (LODINE ^™ ), fenoprofen (NALFON ^™ ), indomethacin (INDOCIN ^TM ), ketoralac (TORADOL ^TM ), oxaprozin (DAYPRO ^TM ), nabumetone (RELAFEN ^TM ), sulindac (CLINORIL ^TM ), tolmentin (TOLECTIN ^™ ), rofecoxib (VIOXX ^™ ), naproxen (ALEVE ^™ , NAPROSYN ^™ ), ketoprofen (ACTRON ^™ ), and nabumetone (RELAFEN ^™ ). Such NSAIDs work by inhibiting cyclooxygenases such as COX-1 and/or COX-2. Examples of steroidal anti-inflammatory drugs include, but are not limited to: corticosteroids, dexamethasone (DECADRON ^™ ), cortisone, hydrocortisone, prednisone (DELTASONE ^™ ), prednisolone, triamcinolone Long, sulfasalazine and eicosanoids such as prostaglandins, thromboxanes and leukotrienes.

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to a patient with osteoarthritis in combination with other agents or treatments for the prevention, treatment and/or control of osteoarthritis, including but Without limitation: analgesics (non-limiting examples are acetaminophen at doses up to 4000 mg/d; phenacetin; tramadol at doses ranging from 200 to 300 mg per day); NSAIDs (non-limiting examples include but not Limited to: aspirin, diflunisal, diclofenac, etodolac, fenamate, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methyl salicylate, Nabumetone, naproxen, piperazine, phenylbutazone, piroxicam, sulindac, and tolmetin. Low-dose NSAIDs are preferred, eg, 1200 mg/d ibuprofen, 500 mg/d naproxen. Stomach Protective agents such as misoprostol, famotidine, or omeprazole are preferably used concurrently with NSAIDs; non-acetylated salicylates including but not limited to salicylates; cyclooxygenase (Cox)-2 - Specific inhibitors (CSI) including but not limited to celecoxib and rofecoxib; intra-articular or periarticular injection of long-acting corticosteroid preparations; intra-articular injection of hyaluronic acid; capsaicin cream; Inflamed knee is carried out sufficient lavage (copious irrigation) to flush out fibrin, cartilage fragments and other debris; And arthroplasty.The liquid formulation of the present invention can also be combined with other non-pharmacological means of preventing, treating and/or controlling osteoarthritis Use, these means include, but are not limited to: reduce joint loading (non-limiting examples are correcting poor posture, supporting lumbar hyperlordosis, avoiding excessive loading of damaged joints, avoiding prolonged standing, kneeling and squatting); Joint heating; aerobic exercise and other physical therapy.

In a specific embodiment, a preventive or therapeutically effective amount of the liquid formulation of the present invention is administered to patients with rheumatoid arthritis in combination with other agents or treatments for the prevention, treatment and/or control of rheumatoid arthritis, said other agents Or treatments include, but are not limited to: NSAIDs (non-limiting examples include, but are not limited to: aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methyl salicylate, nabumetone, naproxen, piperazine, phenylbutazone, piroxicam, sulindac, and tolmetin); analgesics (non- Limiting examples are acetaminophen, phenacetin, and tramadol); CSIs, including but not limited to: celecoxib and rofecoxib; corticosteroids (preferably low-dose oral corticosteroids such as <7.5mg/d prednisone, or monthly pulse high-dose corticosteroids, or intra-articular glucocorticoids); disease-modifying antirheumatic drugs (DMARDs), including but not limited to: methotrexate (preferably given intermittent low doses, such as 7.5-30mg once a week), gold compounds (such as gold salts), D-penicillamine, antimalarials (such as chloroquine) and sulfasalazine; TNF-α neutralizers, including but not Limited to: etanercept and infliximab; immunosuppressants and cytotoxic agents (examples include but are not limited to: azathioprine, leflunomide, cyclosporine and cyclophosphamide) and surgery (examples include but not limited to Limited to: arthroplasty, total joint replacement, hand reconstruction, open or arthroscopic synovectomy, and early tenosynovectomy of the wrist). The liquid formulations of the present invention may also be used in combination with other means to prevent, treat and/or manage rheumatoid arthritis, including but not limited to: rest, splinting to reduce unnecessary movement of inflamed joints, exercise , with various orthotic and assistive devices, and other physical therapy. The liquid formulations of the present invention may also be used in conjunction with non-traditional approaches to the prevention, treatment and/or management of rheumatoid arthritis, including but not limited to: dietary (e.g., supplementation with omega- 3 fatty acids such as eicosapentaenoic acid to replace omega-6 essential fatty acids in meat), vaccines, hormones and topical preparations.

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to a COPD patient in combination with other agents or treatments for the prevention, treatment and/or management of chronic obstructive pulmonary disease (COPD), said other agents or treatments Including, but not limited to: bronchodilators, including, but not limited to: short-acting and long-acting _beta2 -adrenergic agonists (examples of short-acting _beta2- agonists include, but are not limited to: salbutamol, pirbuterol, terbuterol Taline and Oxinaline; examples of long-acting _beta2 agonists include, but are not limited to, oral extended-release albuterol and inhaled salmeterol), anticholinergics (examples include, but are not limited to, ipratropium bromide), and Theophylline and its derivatives (therapeutic range of theophylline is preferably 10-20 μg/mL); glucocorticoids; exogenous α ₁ AT (eg, α ₁ AT derived from pooled human plasma given intravenously, weekly 60 mg/kg); oxygen; lung transplantation; lung volume reduction surgery; endotracheal intubation, assisted breathing; annual influenza and pneumococcal vaccines (23-valent polysaccharide); exercise; and smoking cessation.

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to a patient with pulmonary fibrosis in combination with an effective amount of one or more other agents for the treatment of pulmonary fibrosis, including but not limited to : Oxygen; corticosteroids (non-limiting example is prednisone given daily, starting at 1-1.5 mg/kg/d (up to 100 mg/d) for 6 weeks, gradually tapering to minimum maintenance over 3-6 months dose 0.25 mg/kg/d); cytotoxic drugs (non-limiting examples are cyclophosphamide, 100-120 mg orally once daily; azathioprine, 3 mg/kg orally once daily, up to 200 mg); bronchodilators (non-limiting non-limiting examples are _{diphenhydramine} and doxylamine ).

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to an asthma patient in combination with an effective amount of one or more other agents for the treatment of asthma. Non-limiting examples of such agents include adrenergic stimulants (e.g., catecholamines (e.g., epinephrine, isoproterenol, and ethyl isoproterenol), resorcinols (e.g., butaline and fenoterol) and salicyl alcohol (such as albuterol), adrenocorticoids, glucocorticoids, corticosteroids (such as beclomethadonse, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, and prednisone), other steroids, beta2-agonists (eg, albuterol (albtuerol), bitoterol, fenoterol, ethylisoproterenol, Naline, pirbuterol, albuterol, terbutaline, formoterol, salmeterol, and albutamol (terbutaline), anti-cholinergics (such as ipratropium bromide and oxytropium) bromide), IL-4 antagonists (including antibodies), IL-5 antagonists (including antibodies), IL-13 antagonists (including antibodies), PDE4-inhibitors, NF-κ-β inhibitors, VLA-4 Inhibitors, CpG, anti-CD23, selectin antagonists (TBC1269), mast cell protease inhibitors (such as tryptase kinase inhibitors (such as GW-45, GW-58 and genistein), phosphatidylinositol- 3'(PI3)-kinase inhibitors (such as carvestatin C) and other kinase inhibitors (such as staurosporine) (see Temkin et al., 2002J Immunol 169(5):2662-2669; Vosseller et al., 1997 Mol. Biol. Cell 8(5):909-922; and Nagai et al., 1995 Biochem Biophys Res Commun 208(2):576-581)), C3 receptor antagonists (including antibodies), immunosuppressants (such as methotrexate and gold salts), mast cell modulators (such as cromolyn sodium (INTAL ^TM ) and nedocromil sodium (TILADE ^TM )), and mucolytic agents (such as acetylcysteine)). In a specific embodiment, the anti-inflammatory agent is a leukotriene inhibitor (such as montelukast (SINGULAIR ^™ ), zafirlukast (ACCOLATE ^™ ), pranlukast (ONON ^™ ) or zileuton (ZYFLO ^™ ) (see Table 5)).

Table 5: Leukotriene inhibitors for the treatment of asthma.

Leukotriene modifier Usual daily dose Montelukast (SINGULAIR ^TM ) 2-5 years old 4mg6-15 years old 5mg15 years old and above 10mg Zafirlukast (ACCOLATE ^TM ) 5-12 years old 10mg twice a day 12 years old and above 20mg twice a day Pranlukast (ONON ^TM ) Only available in Asia Zileuton (ZYFLO ^TM ) Over 12 years old 600mg four times a day

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to an allergy patient in combination with an effective amount of one or more other agents for treating allergy. Non-limiting examples of such agents include anti-mediator drugs (e.g., antihistamines, see Table 6 below for non-limiting examples of antihistamines and typical doses of such agents), corticosteroids, decongestants, Sympathomimetic drugs (such as α-adrenergic and β-adrenergic drugs), TNX901 (Leung et al., 2003, N Engl J Med348(11):986-993), IgE antagonists (such as antibody rhuMAb-E25 Omar Zizumab (see Finn et al., 2003 J Allergy Clin Immuno 111(2): 278-284; Corren et al., 2003 J Allergy Clin Immuno 111(1): 87-90; Busse and Neaville, 2001 Curr Opin Allergy Clin Immuno 1(1): 105-108; and Tang and Powell, 2001, Eur J Pediatr 160(12):696-704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int Arch Allergy Immuno 128(1):24-32) and mABHu -901 (see van Neerven et al., 2001 Int Arch Allergy Immuno 124(1-3):400), theophylline and its derivatives, glucocorticoids, and immunotherapy (eg, repeated chronic allergen injections, short-term desensitization and venom immunotherapy).

Table 6: H ₁ Antihistamines

Chemical types and representative drugs Usual daily dose ethanolamine diphenhydramine clemastine 25-50mg every 4-6 hours 0.34-2.68mg every 12 hours ethylenediamine tripyramine 25-50mg every 4-6 hours Alkylamine Brompheniramine Chlorpheniramine Triprolidine (1.25mg/5ml) 4 mg every 4-6 hours; or 8-12 mg SR form every 8-12 hours 4 mg every 4-6 hours; or 8-12 mg SR form 2.5 mg every 4-6 hours phenothiazine promethazine 25mg before going to bed Piperazine Hydroxyzine 25mg every 6-8 hours Piperidine Astemizole (no sedative effect) Azatadine Cetirizine Cyproheptadine Fexofenadine (no sedative effect) Loratadine (no sedative effect) 10mg/d 1-2mg every 12 hours 10mg/d every 6-8 hours 4mg every 12 hours 60mg every 24 hours 10mg

5.5.4. Treatment of autoimmune diseases

The liquid formulations of the present invention can be administered to a subject in need thereof to prevent, treat and/or manage autoimmune diseases or one or more symptoms thereof. The liquid preparation of the present invention can also be administered to a subject in need in combination with one or more other treatments to prevent, treat and/or control autoimmune diseases or one or more symptoms thereof, and said treatment is preferably used for preventing, controlling Or treatment for autoimmune diseases (including but not limited to the preventive or therapeutic agents described in Section 5.5.4.1 below). In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling an autoimmune disease or one or more symptoms thereof, the method comprising administering to a subject in need a dose of a prophylactically or therapeutically effective amount of Liquid formulations of the invention. In another embodiment, the present invention provides a method of preventing, treating and/or controlling an autoimmune disease or one or more symptoms thereof, said method comprising administering to a subject in need a dose of a prophylactically or therapeutically effective amount of The liquid formulation of the present invention and a prophylactically or therapeutically effective amount of one or more treatments (for example, prophylactic or therapeutic agents other than antibodies (including antibody fragments) that immunospecifically bind IL-9 polypeptide).

The present invention provides a method of preventing, treating and/or controlling an autoimmune disease or one or more symptoms thereof in a subject refractory to conventional treatment, said method comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of Liquid formulations of the invention. The present invention also provides a method of preventing, treating and/or managing an autoimmune disease or one or more symptoms thereof in a subject refractory to existing single drug therapy of the autoimmune disease, said method comprising administering the Said subject has a dose of the liquid preparation of the present invention in a preventive or therapeutic effective dose and a dose of preventive or therapeutic effective dose of treatment (such as a preventive agent or a therapeutic agent), and the one or more treatments are immune-specific binding IL-9 Therapy other than antibodies to polypeptides (including antibody fragments thereof). The invention also provides a method of preventing, treating and/or managing an autoimmune disease or one or more symptoms thereof, i.e. administering a liquid formulation of the invention in combination with any other treatment which has proven refractory to the other treatment and which is no longer administered of patients. The present invention also provides alternative methods of managing or treating autoimmune diseases when other treatments are or may be too toxic for the subject being treated, ie, cause unacceptable or unaffordable side effects. In particular, the present invention provides alternative methods of managing or treating autoimmune diseases in patients refractory to other treatments. The invention also provides a method of preventing relapse of an autoimmune disease in a patient who has been treated and does not have active disease by administering the liquid formulation of the invention.

In an autoimmune disease, the immune system mounts an immune response when there is no foreign substance to fight against, and the body's normal protective immune system damages its own tissues by mistakenly attacking itself. There are many different autoimmune diseases, which affect the body in different ways. For example, the midbrain is affected in individuals with multiple sclerosis, the digestive tract is affected in individuals with Crohn's disease, and the synovium, bone, and cartilage of various joints are affected in individuals with rheumatoid arthritis. As autoimmune disease progresses, it may result in the destruction of one or more body tissue types, abnormal growth of organs, or changes in organ function. Autoimmune diseases may affect only one organ or tissue type, or multiple organs or tissues. Organs and tissues commonly affected by autoimmune diseases include red blood cells, blood vessels, connective tissue, endocrine glands (such as the thyroid or pancreas), muscles, joints, and skin. Examples of autoimmune diseases that may be treated using the methods of the present invention include, but are not limited to: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic Anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet syndrome, bullous pemphigoid, cardiomyopathy, sprue-dermatitis, chronic fatigue immune dysfunction Syndrome (CFIDS), Chronic Inflammatory Demyelinating Polyneuropathy, Chu-Schter syndrome, Pemphigoid cicatrix, CREST syndrome, Cold agglutinin disease, Crohn's disease, Discoid lupus, Idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic Thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's syndrome, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, Pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agamma globulin Hyperemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Systemic myotonic syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, transient arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Weger Sodium granulomatous disease.

Autoimmune therapy and its dosage, route of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (60th Edition, 2006).

5.5.4.1. Autoimmune disease therapy

The present invention provides methods for preventing, treating and/or controlling autoimmune diseases or one or more symptoms thereof, the method comprising administering the liquid formulation of the present invention and one or more immune-specific binding IL- 9 Treatment (such as prophylactic or therapeutic agents) other than antibodies to polypeptides (including antibody fragments thereof). Any agent or treatment known to be useful, used or being used in the prevention, treatment and/or management of an autoimmune disease or one or more symptoms thereof may be used in combination with the liquid formulations of the invention described herein. Examples of such agents include, but are not limited to, immunomodulators, anti-inflammatory agents, and TNF-alpha antagonists. Specific examples of immunomodulators, anti-inflammatory agents and TNF-α antagonists that can be used in combination with the liquid preparation of the present invention to prevent, treat and/or control autoimmune diseases are as described above.

In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the invention is administered to a MS patient in combination with other agents or treatments for the prevention, treatment and/or management of multiple sclerosis (MS), including But not limited to: IFN-β1b (Betaseron) (eg, eight million international units (8.0 MIU) given subcutaneously every other day); IFN-β1a (Avonex) (eg, 6.0 MIU given weekly IM); acetic acid Glatiramer (Copaxone) (eg, 20 mg daily by subcutaneous injection); mitoxantrone (eg, 12 mg/ ^m2 by IV infusion every three months); azathioprine (eg, daily by oral 2 to 3 mg/kg body weight); methotrexate (eg, 7.5 mg orally once weekly); cyclophosphamide; IV immune globulin (eg, 0.15 to 0.2 g/kg body weight monthly for up to 2 years); Corticosteroids; methylprednisolone (eg, in high doses every 2 months); 2-chlorodeoxyadenosine (cladribine); baclofen (eg, 15 to 80 mg/day in divided doses) , or higher doses up to 240 mg/day orally, or intrathecally via an indwelling catheter); cyclobenzaprine hydrochloride (eg, 5-10 mg bid or thrice daily); clonazepam (eg, 0.5-1.0 mg three times daily, including bedtime dose); clonidine hydrochloride (eg, 0.1-0.2 mg tid, including bedtime dose); carbamazepine (eg, 100-1200 mg/d in divided doses, dose increasing); gabapentin (eg, 300-3600 mg/d); phenytoin (eg, 300-400 mg/d); amitriptyline (eg, 25-150 mg/d); baclofen (eg, 10-80 mg/d) ; primidone (eg, 125-250 mg bid or thrice daily); ondansetron (eg, 4-8 mg bid or thrice daily); isoniazid (eg, up to 1200 mg in divided doses given); oxybutynin (eg, 5 mg bid or thrice daily); tolterodine (eg, 1-2 mg bid); propantheline (eg, 7.5-15 mg bid ); bethanecol (eg, 10-50 mg three times daily or four times daily); terazosin hydrochloride (eg, 1-5 mg at bedtime); sildenafil citrate (eg, 50- 100 mg po prn); amantadine (eg, 100 mg bid); pemoline (eg, 37.5 mg bid); high-dose vitamins; calcium orotate; ganciclovir; antibiotics; and plasma exchange.

In a specific embodiment, the preventive or therapeutic effective amount of the liquid formulation of the present invention is administered to psoriasis patients in combination with other agents or treatments for the prevention, treatment and/or control of psoriasis, including but not limited to: topical Steroid creams or ointments; tars (examples include but are not limited to: Estar, Psorigel, Fototar creams, and 10% LCD in Nutraderm lotion or mixed directly with triamcinolone 0.1% cream); occlusion; Topical vitamin D analogs (non-limiting example is calcipotriene ointment); ultraviolet light; PUVA (psoralen and ultraviolet A irradiation); methotrexate (eg, up to 25 mg three times at intervals to allow separate administration); synthetic retinoids (non-limiting example is acitretin, such as a dose of 0.5-1 mg/kg/d); immunomodulatory therapy (non-limiting example is cyclosporine) ; sulfasalazine (eg, at a dose of 1 g three times daily).

和其缓释形式

特别是高剂量给药，如

1g每天四次和

0.8-1.2g每天四次)、皮质类固醇、免疫调节药(如硫唑嘌呤(1-2mg/kg)、巯嘌呤(50-00mg)、环孢霉素和氨甲喋呤)、抗生素、TNF抑制剂(如英利昔单抗

)、免疫抑制剂(如他克莫司、麦考酚酸吗乙酯和沙利度胺)、抗炎细胞因子(如IL-10和IL-11)、营养治疗、以要素饮食进行肠道治疗(如维沃，服用4周)和胃肠道外全面营养。In a specific embodiment, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered to a patient with Crohn's disease in combination with other agents or treatments for the prevention, treatment and/or management of Crohn's disease, said other agents or treatments Including but not limited to: antidiarrheal drugs (for example, loperamide 2-4 mg, up to 4 times a day; diphenoxylate and atropine 1 tablet, up to 4 times a day; opium tincture 8-15 drops, up to 4 times a day; Letyramine 2-4g or colestipol 5g, once or twice a day), antispasmodics (for example, propantheline 15mg, dicyclvirine 10-20mg or hyoscyamine 0.125mg, taken before meals), 5- Aminosalicylic acid agents (eg, sulfasalazine 1.5 to 2 g twice daily; mesalamine

and its extended release form

Especially at high doses, such as

1g four times a day and

0.8-1.2g four times a day), corticosteroids, immunomodulators (such as azathioprine (1-2mg/kg), mercaptopurine (50-00mg), cyclosporine, and methotrexate), antibiotics, TNF inhibitors ( infliximab

), immunosuppressants (eg, tacrolimus, mycophenolate mofetil, and thalidomide), anti-inflammatory cytokines (eg, IL-10 and IL-11), nutritional therapy, elemental diet for bowel therapy (such as Vivo, taking 4 weeks) and parenteral comprehensive nutrition.

In a specific embodiment, a preventive or therapeutically effective amount of the liquid preparation of the present invention is administered to a lupus patient in combination with other agents or treatments for the prevention, treatment and/or control of lupus erythematosus, including but not limited to : antimalarials (including but not limited to hydroxychloroquine); glucocorticoids (for example, low-dose, high-dose or high-dose intravenous pulse therapy can be used); immunosuppressants (including but not limited to: cyclophosphamide, benzene butyrate and azathioprine); cytotoxic agents (including but not limited to methotrexate and mycophenolate mofetil); androgenic sex steroids (including but not limited to danazol); and anticoagulants (including but not limited to not limited to warfarin).

In a specific embodiment, an effective amount of one or more liquid antibody preparations of the present invention and an effective amount of Vitacin ^™ (Midiuni Corporation, International Publication No. WO 00/78815, International Publication No. WO 02/070007A1, September 12, 2002, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administration of Integrin α _v β ₃ Antagonists" Administering IntegrinAlphaV Beta3 Antagonists), International Publication No. WO 03/075957 A1, September 18, 2003, entitled "Integrin α _v β ₃ antagonists combined with other agents to prevent or treat cancer" (The Prevention or Treatment of Cancer Using Integrin _AlphaVBeta3 Antagonists in Combination With Other Agents), U.S. Patent _Publication No. US 2002/0168360A1, November 14, 2002, entitled "Prevention of Or the method for treating inflammatory diseases or autoimmune diseases" (Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylactic or Therapeutic Agents) and International Publication No. WO 03/075741A2, 2003 September ₁₈ , entitled "Methods of Preventing or Treating Disorders by _{Administering} an Integrin α _v β ₃ Antagonist in Combination With an HMG-CoA Reductase Inhibitor or aBisphosphonate), which is incorporated by reference in its entirety herein) in combination with an object for the prevention, treatment and/or control of autoimmune diseases or one or more of its symptoms. In another preferred embodiment, an effective amount of one or more antibodies of the present invention and an effective amount of silizumab (Midiuni, International Publication No. WO 02/069904) are administered to the subject in combination to prevent, Treating and/or managing an autoimmune disease or one or more symptoms thereof. In another embodiment, an effective amount of one or more antibodies of the invention is combined with an effective amount of one or more EphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (Midiuni; International Publication No. WO 02/102974 A4, December 27, 2002, entitled Mutant Proteins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure, International Publication No. 03/094859 A2, November 20, 2003, entitled "EphA2 Monoclonal Antibodies and Methods of Use Thereof," U.S. Application No. 10/436,783 and U.S. Patent Publication No. US 2004/0091486 A1; and U.S. Application No. 10/994,129 and US Patent Publication No. US2005/0152899 A1 , which are incorporated herein by reference in their entirety)) are administered in combination to a subject to prevent, treat and/or manage an autoimmune disease or one or more symptoms thereof. In another embodiment, an effective amount of one or more antibodies of the present invention is administered to a subject in combination with an effective amount of Vitacin ^™ , Silizumab and/or an EphA2 inhibitor to prevent, treat and/or control Autoimmune disease or one or more symptoms thereof.

The antibody preparation of the present invention or the combination therapy of the present invention can be used as the first, second, third, fourth or fifth treatment to prevent, treat and/or control autoimmune diseases or one or Various symptoms. The invention also includes methods of preventing, treating and/or managing an autoimmune disease or one or more symptoms thereof in a patient who is being treated for another disease or disorder. The invention includes methods of preventing, treating and/or managing an autoimmune disease or one or more symptoms thereof in a patient prior to any adverse reaction or intolerance to a treatment other than the antibodies of the invention. The invention also includes methods of preventing, treating and/or managing autoimmune diseases or symptoms thereof in refractory patients. The invention includes methods of preventing, treating and/or managing proliferative diseases or symptoms thereof in patients who have proven refractory to treatments other than the antibodies, compositions or combinations of the invention. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of a treatment for an autoimmune disease, using the art-accepted meaning of "refractory". In certain embodiments, a patient with an autoimmune disease is said to be refractory to treatment when one or more symptoms of the autoimmune disease cannot be prevented, controlled and/or alleviated. The invention also includes methods of preventing, treating and/or managing autoimmune diseases or their symptoms in patients prone to adverse reactions to conventional treatments.

The present invention includes methods of preventing, treating and/or managing autoimmune diseases or one or more symptoms thereof as an alternative to other conventional treatments. In a specific embodiment, the patients managed or treated according to the methods of the present invention are patients who are refractory to other treatments or prone to adverse effects from other treatments. Patients may be immunosuppressed (eg, postoperative patients, chemotherapy patients, patients with immunodeficiency disorders, patients with broncho-pulmonary dysplasia, congenital heart disease, cystic fibrosis, acquired or congenital heart disease patients, and patients with infections), persons with impaired renal or hepatic function, the elderly, children, infants, premature infants, neuropsychiatric patients or persons taking psychotropic drugs, persons with a history of epilepsy, or persons receiving and preventing , people on conflicting medications with conventional medications for the treatment and/or control of viral respiratory infections or one or more symptoms thereof.

Autoimmune therapy and its dosage, route of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (60th Edition, 2006).

5.5.5. Virus infection

One or more antibody formulations of the invention can be administered to a subject to prevent, treat and/or manage viral infection or one or more symptoms thereof. One or more antibody preparations of the present invention can be combined with one or more other treatments (such as one or more prophylactic or therapeutic agents) are administered in combination to subjects susceptible to or suffering from viral infections, preferably respiratory viral infections. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acids that inhibit and/or reduce adhesion of the virus to its receptors, internalization of the virus into the cell, replication of the virus, or release of the virus from the cell molecules, organic molecules, inorganic molecules and small molecules. Specifically, antiviral agents include, but are not limited to: nucleoside analogs (e.g., zidovudine, acyclovir, ganciclovir, vidarabine, iodine, trifluridine, and ribavirin ), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferon and other interferons, and AZT.

In a specific embodiment, the antiviral agent is an immunomodulator with immunospecificity for a viral antigen. The term "viral antigen" as used herein includes, but is not limited to: any viral peptide, polypeptide and protein capable of eliciting an immune response (such as HIV gp120, HIVnef, RSV F glycoprotein, RSV G glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin , HTLVtax, herpes simplex virus glycoproteins (such as gB, gC, gD and gE) and hepatitis B surface antigen). Antibodies used for the treatment of viral infectious diseases in the present invention include but are not limited to: antibodies to antigens of the following pathogenic viruses, including but not limited to: Adenoviridae (such as mammalian adenovirus and avian adenovirus), herpes Viridae (eg, HSV 1, HSV 2, HSV 5, and HSV 6), Smoothoviridae (eg, Glaciovirus, Enterobacteriophage (phase) MS2, Heterobacteriophage), Pox Viridae (such as Vertebpopoxvirinae, Parapoxvirus, Fowlpoxvirus, Cappopoxvirus, Lapopoxvirus, Hyoppoxvirus, Molluspovirus, and Entomopoxvirinae), milkpoxvirus Povaviridae (eg, polyomavirus and papillomavirus), Paramyxoviridae (eg, paramyxovirus, parainfluenzavirus 1), morbillivirus (eg, measles virus), mumpsvirus (eg, parotid Pneumoviruses), Pneumoviridae (such as Pneumovirus, Human Respiratory Syncytial Virus) and Postpneumovirus (such as Avian Pneumovirus and Human Postpneumovirus)), Picornaviridae (such as Enterovirus, Rhinoviruses, Hepaviruses (e.g. Human Hepatitis A Virus), Cardioviruses and Foot-and-Mouth Viruses), Reoviridae (e.g. Orthoreoviruses, Orbiviruses, Rotaviruses, Polygonal Virus, Fijivirus, Phytoreovirus, and Oryzavirus), Retroviridae (such as mammalian B-type retrovirus, mammalian C-type retrovirus, avian C-type retrovirus , group D retroviruses, BLV-HTLV retroviruses, lentiviruses (such as HIV-1 and HIV-2), foamyviruses), Flaviviridae (such as hepatitis C virus), Hepadnaviridae (eg, Hepatitis B), Togaviridae (eg, Alphaviruses (eg, Sindbisvirus) and Rubellaviruses (eg, Rubivirus)), Rhabdoviridae (eg, Vesivirus, lyssaviruses, ephemeral rhabdoviruses, rhabdoviruses, and rhabdoviruses), arenaviridae (eg, arenavirus, lymphocytic choriomeningitis virus, Ipai virus, and Lassa virus ) and Coronaviridae (such as Coronaviridae and Orbiviridae).

米迪缪尼公司；国际公开号WO 02/43660)，它是用于治疗RSV的人源化抗体。Specific examples of antibodies that can be used to treat viral infectious diseases include, but are not limited to: PRO542 (Progenics), which is a CD4 fusion antibody used to treat HIV infection; Ostavir (Protein Design Laboratories, Inc., California). Labs, Inc., CA)), which is a human antibody for the treatment of hepatitis B virus; and Protovir (California Protein Design Laboratories, Inc.), which is a humanized IgG1 antibody for the treatment of cytomegalovirus (CMV) ; and palivizumab (

Midiuni Corporation; International Publication No. WO 02/43660), which is a humanized antibody for the treatment of RSV.

In a specific embodiment, the antiviral agent used in the compositions and methods of the present invention can inhibit or reduce lung or respiratory virus infection, inhibit or reduce the replication of virus that causes lung or respiratory tract infection, or inhibit or reduce lung or respiratory tract infection. The infected virus spreads to other cells or objects. In another preferred embodiment, the antiviral agent used in the compositions and methods of the present invention can inhibit or reduce the infection of RSV, hMPV or PIV, inhibit or reduce the replication of RSV, hMPV or PIV, or inhibit or reduce the Or PIV spreads to other cells or objects. Examples of such agents and methods of treating RSV, hMPV and/or PIV infections include, but are not limited to: nucleoside analogs such as zidovudine, acyclovir, ganciclovir, vidarabine, iodine , trifluridine and ribavirin, and foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir and interferon-alpha. See U.S. Patent Application No. 10/628,088, filed July 25, 2003 and published as U.S. Patent Publication No. US 2004/0096451 A1, entitled "Treatment and Prevention of RSV, The method of HMPV and PIV (Methods of Treating and Preventing RSV, HMPV, and PIV Using Anti-RSV, Anti-HMPV, and Anti-PIV Antibodies)" and submitted and published as US Patent Publication No. US 2004/ on February 21, 2003 US Patent Application No. 10/371,122 for 0005544A1, incorporated herein by reference in its entirety.

In a preferred embodiment, the viral infection is RSV and the anti-viral antigen antibody is an antibody that immunospecifically binds an RSV antigen. In certain embodiments, the anti-RSV-antigen antibody immunospecifically binds an RSV antigen of a group A RSV. In other embodiments, the anti-RSV-antigen antibody immunospecifically binds an RSV antigen of group B RSV. In other embodiments, the antibodies bind to an antigen of a certain group of RSV and cross-react with similar antigens of other groups. In a specific embodiment, the anti-RSV-antigen antibody immunospecifically binds to RSV nucleoprotein, RSV phosphoprotein, RSV matrix protein, RSV small hydrophobin, RSV RNA-dependent RNA polymerase, RSV F protein and/or RSV G protein. In other specific embodiments, the anti-RSV-antigen antibody binds to an RSV nucleoprotein, an RSV nucleocapsid protein, an RSV phosphoprotein, an RSV matrix protein, an RSV adhesion glycoprotein, a RSV fusion glycoprotein, an RSV nucleocapsid protein, Allelic variants of RSV matrix protein, RSV small hydrophobin, RSV RNA-dependent RNA polymerase, RSV F protein, RSV L protein, RSV P protein, and/or RSV G protein.

(帕丽珠单抗)是人源化单克隆抗体，目前用于预防儿科患者的RSV感染。在一个具体实施方式中，本发明方法所用抗体是帕丽珠单抗或其抗体结合片段(如，含有帕丽珠单抗的一个或多个互补决定区(CDR)，优选可变区的片段)。帕丽珠单抗的氨基酸序列参见例如，Johnson等，1997，J.Infectious Disease176：1215-1224和美国专利号5,824,307和国际申请公开号WO 02/43660，题为“给予抗-RSV抗体进行预防和治疗的方法(Methods of Administering/DosingAnti-RSV Antibodies for Prophylaxis and Treatment)”，Young等，通过引用全文纳入本文。It will be appreciated that antibodies that immunospecifically bind RSV antigens are known in the art. For example,

(palivizumab) is a humanized monoclonal antibody currently used to prevent RSV infection in pediatric patients. In a specific embodiment, the antibody used in the method of the invention is palivizumab or an antibody-binding fragment thereof (e.g., a fragment comprising one or more complementarity determining regions (CDRs), preferably variable regions, of palivizumab ). For the amino acid sequence of Palivizumab, see, e.g., Johnson et al., 1997, J. Infectious Disease 176:1215-1224 and U.S. Patent No. 5,824,307 and International Application Publication No. WO 02/43660, entitled "Administration of Anti-RSV Antibodies for Prophylactic and Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment", Young et al., incorporated herein by reference in its entirety.

The invention can also use one or more antibodies or antigen-binding fragments thereof that immunospecifically bind RSV antigens, comprising an Fc region that has a higher affinity for the FcRn receptor than the Fc region of Palivizumab. Such antibodies are described in US Patent Application No. 10/020,354, filed December 12, 2001, which is incorporated herein by reference in its entirety. In addition, anti-RSV-antigen antibodies A4B4; P12f2P12f4; P11d4; A1e9; A12a6; A13c4; A17d4; A4B4; 1X-493L1; 6H8; L1-7E5; L2-15B10; A13a11; A1h5; A4B4(1); A4B4-F52S; or A4B4L1FR-S28R. These antibodies are described in International Application Publication No. WO02/43660, entitled "Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment", Young et al.; and Jul. 25, 2003 U.S. Patent Application No. 10/628,088, filed and published as U.S. Patent Publication No. US 2004/0096451A1, entitled "Methods of Treating and Preventing RSV, HMPV, and PIV Using Anti-RSV, Anti-HMPV, and Anti-PIV Antibodies" Treating and Preventing RSV, HMPV, and PIV Using Anti-RSV, Anti-HMPV, and Anti-PIV Antibodies)", which is incorporated herein by reference in its entirety.

In certain embodiments, the anti-RSV-antigen antibody is U.S. Application No. 09/724,531 filed November 28, 2000; 09/996,288 filed November 28, 2001 by Young et al., incorporated herein by reference in their entirety; and 09/996,265 submitted and published as U.S. Patent Publication No. US2003/0091584A1 on November 28, 2001, both titled "Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment by Giving Anti-RSV Antibodies for Prophylaxis and Treatment )" described anti-RSV-antigen antibody or the anti-RSV-antigen antibody prepared by the method described therein. For methods of making and compositions of stable antibody formulations useful in the methods of the invention see U.S. Provisional Application No. 60/388,921, filed June 14, 2002, and U.S. Patent Publication No. US 2004/0018200A1, filed May 7, 2003 and published as US Patent Application No. 10/461,863, which is incorporated herein by reference in its entirety.

Antiviral therapy and its dosage, route of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (60th Edition, 2006). Additional information on respiratory viral infections can be found in the Cecil Textbook of Medicine (18th ed., 1988).

5.5.5.1. Treatment of viral infections

In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling viral respiratory tract infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present invention to a subject in need thereof. Antibody preparations. In another embodiment, the present invention provides a method of preventing, treating and/or controlling viral respiratory tract infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more An antibody formulation of the invention and an effective amount of one or more treatments (eg, one or more prophylactic or therapeutic agents) in addition to the antibody formulation of the invention.

In certain embodiments, an effective amount of one or more antibody formulations of the invention is administered to a subject in need thereof in combination with an effective amount of one or more treatments (e.g., one or more prophylactic or therapeutic agents), The treatment is, has been or is known to be useful in preventing, treating and/or managing a viral infection, such as a viral respiratory infection, or one or more symptoms thereof. Treatment of viral infections, such as viral respiratory infections, includes, but is not limited to, antiviral agents such as amantadine, oseltamivir, ribavirin, palivizumab (SYNAGIS ^™ ) and anamivir. In certain embodiments, an effective amount of one or more antibody preparations of the present invention is administered to a subject in need in combination with one or more supportive measures to prevent, treat and/or control viral infection or one or more Various symptoms. Non-limiting examples of supportive measures include humidification of the air by ultrasonic nebulizer, administration of aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, catheterization, antipyretics (eg, ibuprofen, acetyl aminophenols) and antibacterial and/or antifungal therapy (ie, to prevent or treat secondary bacterial infections).

Any type of viral infection or a condition caused by or associated with a viral infection (such as a respiratory condition) can be prevented, treated and/or controlled according to the methods of the present invention, said method comprising alone or in combination with an effective amount of another treatment (such as in addition to this prophylactic or therapeutic agent other than the antibody preparation of the invention) in combination with an effective amount of one or more antibody preparations of the invention. Examples of viruses that cause viral infections include, but are not limited to: retroviruses (such as types I and II human T-cell lymphotrophic virus (HTLV) and human immunodeficiency virus (HIV)), herpes viruses (such as types I and II herpes simplex virus (HSV), Epstein-Barr virus, HHV6-HHV8 and cytomegalovirus), arenaviruses (such as Lassa fever virus), paramyxoviruses (such as measles virus, human respiratory syncytial virus, mumps virus, hMPV pneumoviruses), adenoviruses, bunyaviruses (such as hantavirus), coronaviruses, filoviruses (such as Ebola virus), flaviviruses (such as hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (eg, hepatitis B virus (HBV)), orthomyxoviruses (eg, influenza A, B, and C, and PIV), papovaviruses (eg, papillomavirus), picoribose Nucleic acid viruses (eg, rhinoviruses, enteroviruses, and hepatitis A), poxviruses, reoviruses (eg, rotaviruses), togaviruses (eg, rubella virus), and rhabdoviruses (eg, rabies virus). Biological responses to viral infection include, but are not limited to, increased levels of IgE antibodies, increased proliferation and/or infiltration of T cells, increased proliferation and/or infiltration of B cells, epithelial hyperplasia, and mucin production. In a specific embodiment, the present invention also provides methods for preventing, treating and/or controlling viral respiratory tract infections, which are related to the common cold, viral pharyngitis, viral laryngitis, viral croup, viral bronchitis , influenza, parainfluenza virus ("PIV") disease (eg, croup, bronchiolitis, bronchitis, pneumonia), respiratory syncytial virus ("RSV") disease, postpneumonic viral disease, and adenovirus disease (eg, febrile Respiratory diseases, croup, bronchitis, pneumonia) related to or causing these diseases, the method comprises administering an effective amount of one or more antibody preparations of the invention alone or in combination with an effective amount of another therapy.

奥塞米韦

金刚乙胺和金刚烷胺(

)。In a specific embodiment, influenza virus infection, PIV infection, hMPV infection, adenovirus infection and/or RSV infection, or one or more symptoms thereof, can be prevented, treated and/or controlled according to the method of the present invention. In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling RSV respiratory infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more antibody preparations of the present invention alone or Administer to a subject in need in combination with one or more antiviral agents such as, but not limited to: amantadine, rimantadine, oseltamivir, zanamivir, ribavirin, RSV- IVIG (ie Intravenous Immunoglobulin Molecular Infusion) (RESPIGAM ^™ ) and Palivizumab (SYNAGIS ^™ ). In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling PIV infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibody preparations of the present invention alone or with An effective amount of one or more antiviral agents is jointly administered to a subject in need, such as but not limited to: amantadine, rimantadine, oseltamivir, zanamivir, ribavirin, RSV-IVIG (ie Intravenous Immunoglobulin Molecular Infusion) (RESPIGAM ^™ ) and Palivizumab (SYNAGIS ^™ ). In another specific embodiment, the present invention provides a method for preventing, treating and/or controlling hMPV infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more antibody preparations of the present invention alone or Administer to a subject in need in combination with an effective amount of one or more antiviral agents such as but not limited to: amantadine, rimantadine, oseltamivir, zanamivir, ribavirin , RSV-IVIG (ie Intravenous Immunoglobulin Molecular Infusion) (RESPIGAM ^™ ) and Palivizumab (SYNAGIS ^™ ). In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling influenza, the method comprising administering an effective amount of one or more antibody preparations of the present invention alone or in combination with an effective amount of an antiviral agent. subjects, the antiviral agents such as but not limited to: Zanamivir

Oseltamivir

Riantadine and amantadine (

).

The invention provides methods of preventing asthma in a subject having or having had a viral respiratory infection comprising administering an effective amount of one or more antibody formulations of the invention, alone or in combination with an effective amount of another therapy. In a specific embodiment, the subject is a premature infant, infant or child. In another specific embodiment, the subject has had or has had an RSV infection.

In a specific embodiment, the present invention provides a method of preventing, treating and/or managing one or more secondary reactions of a primary viral infection, said method comprising alone or in combination with an effective amount of other treatments (such as other prophylactic or therapeutic agent) in combination with an effective amount of one or more antibody preparations of the present invention. Examples of secondary responses to primary viral infections, particularly primary viral respiratory infections, include, but are not limited to: asthma-like responses to mucosal irritation, increased total respiratory resistance, secondary viral, bacterial, and fungal There is increased susceptibility to infection, and the development of conditions such as, but not limited to, pneumonia, croup, and febrile bronchitis.

In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling viral infection or one or more symptoms thereof, the method comprising combining an effective amount of one or more antibody preparations of the present invention and an effective amount Vitacin ^™ (Midiuni Corporation, International Publication No. WO _00/78815 , International Publication No. WO 02/070007A1, September ₁₂ , 2002, entitled "Prevention of Or the method for treating inflammatory diseases or autoimmune diseases" (Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3Antagonists), International Publication No. WO 03/075957A1, September 18, 2003, entitled "Integrin α _v β ₃ antagonists combined with other agents to prevent or treat cancer" (The Prevention or Treatment of Cancer Using Integrin AlphaVBeta3 Antagonists in CombinationWith Other Agents), US Patent Publication No. US 2002/0168360 A1, November 14, 2002, Titled "Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin α _v β ₃ Antagonists and Other Preventive or Therapeutic Agents _" β ₃ Antagonists in Combination With Other Prophylactic or Therapeutic Agents) and International Publication No. WO 03/075741A2, September 18, 2003, entitled "Integrin α _v β ₃ Antagonists in Combination With Other Prophylactic or Therapeutic Agents and HMG-CoA Reductase Inhibition Methods of Preventing or Treating Disorders by Administering an Integrin α _{v β 3} Antagonist in Combination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate”, which is hereby incorporated by reference in its entirety Incorporated into this article) jointly given to the objects in need. In another specific embodiment, the present invention provides a method for preventing, treating and/or controlling viral infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibodies of the present invention with an effective amount Silizumab (Midiuni, International Publication No. WO 02/069904) was administered to subjects in need. In another embodiment, the present invention provides methods for preventing, treating and/or controlling viral infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibodies of the present invention with an effective amount of One or more EphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (Midiuni Corporation; International Publication No. WO02/102974A4, December 27, 2002, entitled "Mutins, highly potent inhibitory Antibody and FIMCH Crystal Structure" (Mutant Proteins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure), International Publication No. 03/094859A2, November 20, 2003, entitled "EphA2 Monoclonal Antibodies and Methods of Use thereof" (EphA2 Monoclonal Antibodies and Methods of UseThereof), U.S. Application No. 10/436,783 and U.S. Patent Publication No. US 2004/0091486A1; and U.S. Application No. 10/994,129 and U.S. Patent Publication No. US 2005/0152899A1, which are hereby incorporated by reference in their entirety)) to those in need object. In yet another embodiment, the present invention provides a method for preventing, treating and/or controlling viral infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibodies of the present invention with an effective amount of Vitasine ^™ , Silizumab and/or EphA2 are administered in combination to subjects in need.

In one embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of one or more anti-IgE antibodies to prevent, treat and/or control viral infections or one or more of them. Various symptoms. In a specific embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of anti-IgE antibody TNX901 to prevent, treat and/or control viral infection or one or more symptoms thereof . In a specific embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of anti-IgE antibody rhuMAb-E25 omalizumab to prevent, treat and/or control viral infection or its one or more symptoms. In another embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of an anti-IgE antibody HMK-12 to prevent, treat and/or control viral infections or one or more of them. symptoms. In a specific embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of anti-IgE antibody 6HD5 to prevent, treat and/or control viral infection or one or more symptoms thereof . In another embodiment, an effective amount of one or more antibody preparations of the present invention is administered to a subject in combination with an effective amount of anti-IgE antibody MAbHu-901 to prevent, treat and/or control viral infections or one or more of them. symptoms.

The invention includes patients who are expected to develop viral infections or are at increased risk of developing such infections, such as patients with suppressed immune systems (such as organ transplant recipients, AIDS patients, patients undergoing chemotherapy, the elderly, premature infants, infants , children, patients with obstructive esophageal cancer, patients with tracheobronchial fistula, patients with neurological diseases (such as stroke, amyotrophic lateral sclerosis, multiple sclerosis and diseases caused by myopathy) and patients who have developed respiratory tract infections) to prevent the occurrence of Viral infections, such as viral respiratory infections. Patients may or may not have been treated for respiratory infections.

The antibody preparation of the present invention or the combination therapy of the present invention can be used as the first, second, third, fourth or fifth treatment to prevent, treat and/or control viral infections, such as viral respiratory tract infections or one or more of its symptoms. The present invention also includes other diseases or disorders related to or characterized by aberrant expression and/or activity of IL-9 polypeptide, aberrant expression and/or activity of IL-9R or one or more subunits thereof being treated. Prevention, treatment and/or management in patients associated with or characterized by a disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or one or more symptoms thereof A viral infection, such as a viral respiratory infection, or one or more of its symptoms. The present invention includes methods of preventing, treating and/or managing viral infections, such as viral respiratory infections or one or more symptoms thereof, in a patient prior to any adverse reaction or intolerance to treatments other than the antibody formulations of the present invention. The present invention also includes methods of preventing, treating and/or managing viral infections, such as viral respiratory tract infections or symptoms thereof, in refractory patients. In certain embodiments, a patient with a viral infection, such as a viral respiratory infection, is said to be refractory to treatment when the infection has not been significantly resolved and/or the symptoms have not been significantly relieved. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of treatment against an infection, using the art-accepted meaning of "refractory". In various embodiments, a patient with a viral respiratory infection is considered refractory when viral replication is not decreased or increased. The invention also includes methods of preventing the occurrence or recurrence of a viral respiratory infection in a patient at risk of developing a viral respiratory infection. The invention also includes methods of preventing, treating and/or managing viral infections, such as viral respiratory tract infections or symptoms thereof, in patients prone to adverse reactions to conventional treatments. The present invention also includes methods of preventing, treating and/or managing viral infections for which no antiviral therapy is available, such as viral respiratory infections.

The invention includes methods of preventing, treating and/or managing viral infections, such as viral respiratory tract infections, or symptoms thereof, in patients who have proven refractory to therapies other than the antibody formulations of the invention and are no longer on such therapies. In certain embodiments, the patients managed or treated in accordance with the methods of the present invention are patients who have been treated with antibiotics, antivirals, antifungals, or other biological/immunotherapy. Among these patients, those who are too young to be candidates for conventional therapy and those whose viral infection recurs despite control or treatment with existing therapies are refractory patients.

The present invention includes methods of preventing, treating and/or managing viral infections, such as viral respiratory infections or one or more symptoms thereof, as an alternative to other conventional treatments. In a specific embodiment, the patients managed or treated according to the methods of the present invention are patients who are refractory to other treatments or prone to adverse effects from other treatments. The patient may be a person with a suppressed immune system (such as postoperative patients, chemotherapy patients, and patients with immunodeficiency diseases), a person with impaired renal or hepatic function, the elderly, children, infants, premature infants, persons with neuropsychiatric disorders or persons taking psychotropic medications, persons with a history of epilepsy, or persons who are receiving medications that conflict with conventional medications for the prevention, treatment and/or control of viral infections or one or more of their symptoms.

Viral infection treatment and its dosage, route of administration and recommended usage are known in the art, such literature includes, for example, Physician's Desk Reference (60th Edition, 2006).

5.5.6. Bacterial infection

The present invention provides methods of preventing, treating and/or managing bacterial infections or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an effective amount of one or more antibody preparations of the present invention. In another embodiment, the present invention provides a method of preventing, treating and/or controlling bacterial infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more of the present An inventive antibody formulation and an effective amount of one or more treatments (eg, one or more prophylactic or therapeutic agents) other than the inventive antibody formulation. Antibacterial agents and antimicrobial treatments well known to those skilled in the art for the prevention, treatment and/or management of bacterial infections can be used in the compositions and methods of the present invention. Non-limiting examples of antimicrobial agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules and Small molecule. Specifically, examples of antibacterial agents include, but are not limited to: penicillins, cephalosporins, imipenem, axtreonam, norvancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin , clindamycin, tetracycline, streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin , rifampin, polymyxin, amphotericin B, nystatin, ketoconazole, isoniazid, metronidazole, and pentamidine.

In one embodiment, an antimicrobial agent is a substance that inhibits or reduces bacterial infection of the lungs or respiratory tract, inhibits or reduces the replication of bacteria that cause lung or respiratory tract infections, or inhibits or reduces the spread of bacteria that cause lung or respiratory tract infections to other subjects. In cases where the bacterial infection of the lungs or respiratory tract is a mycoplasma infection (such as pharyngitis, organ bronchitis and pneumonia), the antibacterial agent is, for example, tetracycline, erythromycin or spectinomycin. When the lung or respiratory bacterial infection is pneumonia caused by aerobic gram-negative bacteria (GNB), the antimicrobial is preferably penicillin, first- or third-generation cephalosporins (such as cefaclor, cefadroxil, cephalexin or cefazolin), erythromycin, clindamycin, aminoglycosides (such as gentamicin, tobramycin, or amikacin), or monolactams (such as aztreonam). In case the bacterial infection of the lungs or respiratory tract is tuberculosis, the antibacterial agents are preferably rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin. In cases where the respiratory infection is recurrent inhaled, the antimicrobial is preferably a penicillin, an aminoglycoside, or a second- or third-generation cephalosporin.

5.5.6.1. Treatment of bacterial infections

Any type of bacterial infection or conditions caused by or associated with a bacterial infection (eg, a respiratory infection) can be prevented, treated and/or managed according to the methods of the present invention. Examples of bacteria that cause bacterial infections include, but are not limited to: Aquaspirillum, Azospirillum, Azotobacteraceae, Bacteroidaceae, Bartonella, Bacteroides Bdellovibrio, Campylobacter, Chlamydia (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae (e.g., Citrobacter, Edwardsiella, Enterobacter aerogenes, Erwinia, Escherichia coli, Hafnia, Klebsiella, Morganella, Proteus vulgaris, Providencia, Salmonella, Serratia marcescens and Shigella flexneri), Gardnerellaceae ( Gardinella, Haemophilus influenzae, Halobacteriaceae, Helicobacter, Legionellaceae, Listeria, Methylococcaceae, Clade mycobacteria (eg, Mycobacterium tuberculosis), Neisseriaceae, Oceanospirillum, Pasteurellaceae, Pneumococcus, Pseudomonas (Pseudomonas), Rhizobiaceae, Spirillum, Spirosomaceae, Staphylococcus (such as methicillin-resistant Staphylococcus aureus and pyrogenic Staphylococcus pyrogenes), Streptococcus (eg Streptococcus enteritidis (Streptococcus enteritidis), Streptococcus fasciae and Streptococcus pneumoniae), Vampirovibr Helicobacter and Vampirovibrio.

In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present invention to a subject in need thereof. Antibody preparations. In another embodiment, the present invention provides a method of preventing, treating and/or controlling bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more The antibody preparation of the present invention and an effective amount of one or more treatments (such as prophylactic or therapeutic agents) other than the antibody preparation of the present invention.

In certain embodiments, the invention provides methods for preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections, or one or more symptoms, comprising administering one or more antibody preparations of the invention and an effective amount of one or more treatments (such as one or more preventive agents or therapeutic agents) are administered to the object in need, and the one or more treatments are used for prevention, treatment other than the antibody preparation of the present invention and/or treatment to control bacterial infections. Treatment of bacterial infections, particularly bacterial respiratory infections, includes, but is not limited to: antibacterial agents (eg, aminoglycosides (eg, gentamicin, tobramycin, amikacin, netilmicin), aztreonam, cephalosporins Mycocins (eg, cefaclor, cefadroxil, cephalexin, cefazolin), clindamycin, erythromycin, penicillins (eg, penicillin V, crystalline penicillin G, procaine penicillin G), spectinomycin and tetracyclines (eg, chlortetracycline, doxycycline, oxytetracycline) and supportive airway therapy such as supplemental and mechanical ventilation. In certain embodiments, one or more antibody preparations of the invention are administered to a subject in need thereof in combination with one or more supportive measures to prevent, treat and/or manage bacterial infection or one or more symptoms thereof . Non-limiting examples of supportive measures include humidification of the air by ultrasonic nebulizer, administration of aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, catheterization, antipyretics (eg, ibuprofen, acetyl aminophenol), more preferably antibacterial or antiviral therapy (ie, prevention or treatment of secondary infection).

The present invention provides methods of preventing, treating and/or managing biological responses to bacterial infections, such as bacterial respiratory infections, including, but not limited to, elevated levels of IgE antibodies, mast cell proliferation, degranulation and/or infiltration, B Increased cell proliferation and/or infiltration and increased T cell proliferation and/or infiltration, the method comprising using an effective amount of one or more antibody preparations of the present invention alone or with an effective amount of one or more antibody preparations other than the antibody preparations of the present invention Multiple treatments (eg, prophylactic or therapeutic agents) are administered in combination to a subject in need thereof. The invention also provides methods of preventing, treating and/or managing respiratory disorders caused by or associated with bacterial infections, including, for example, bacterial respiratory infections such as, but not limited to, pneumococcal pneumonia, aerobic gram-negative bacteria Pneumonia, recurrent aspiration pneumonia, Legionnaires' disease, streptococcal disease, infection caused by Hemophilus, whooping cough, meningitis or tuberculosis, said method comprising administering an effective amount of one or more antibody preparations of the present invention Administered to a subject in need thereof, alone or in combination with an effective amount of another treatment.

In a specific embodiment, the method of the present invention is used to prevent, treat and/or control Pneumococcus (Pneumonococcus), Mycobacteria (Mycobacteria), aerobic Gram-negative bacteria, Streptococcus (Streptococcus) or Haemophilus (Hemophilus) caused bacterial respiratory infection or one or more symptoms thereof, the method comprises using an effective amount of one or more antibody preparations of the present invention alone or with an effective amount of one or more antibody preparations other than the antibody preparations of the present invention Additional treatments, such as one or more prophylactic or therapeutic agents, are administered in combination to a subject in need thereof.

In a specific embodiment, the present invention provides a method for preventing, treating and/or managing a primary bacterial infection, such as one or more secondary conditions of a primary bacterial respiratory infection or a reaction thereto, The methods include administering to a subject in need thereof an effective amount of one or more antibody formulations of the invention alone or in combination with an effective amount of other treatments (eg, other prophylactic or therapeutic agents). Examples of conditions or reactions secondary to primary bacterial infection, particularly bacterial respiratory infection, include, but are not limited to: asthma-like response to mucosal irritation, increased total respiratory resistance, secondary viral, bacterial, and fungal infections increased susceptibility to the development of conditions such as, but not limited to, pneumonia, croup, and febrile bronchitis.

In a specific embodiment, the method of the present invention is used to prevent, treat and/or control bacterial infection, such as bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present invention Antibody and effective amount of Vitacin ^™ (Midiuni Corporation, International Publication No. WO 00/78815, International Publication No. WO 02/070007 A1, September 12, 2002, entitled "By Administering Integrin _αv Beta ₃ antagonists prevent or treat inflammatory diseases or autoimmune diseases" (Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3 Antagonists), International Publication No. WO 03/075957 A1, September 18, 2003, Titled "The Prevention or Treatment of Cancer _Using Integrin _AlphaVBeta3 Antagonists in Combination With Other Agents", U.S. Patent Publication No. US2002/0168360A1, 2002 November 14, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Diseases by Combining Administration of Integrin α _v β ₃ Antagonists and Other Preventive or Therapeutic Agents" Disorders by Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylacticor Therapeutic Agents) and International Publication No. WO 03/075741A2, September 18, 2003, entitled "Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylacticor Therapeutic Agents and HMG- CoA reductase inhibitors or bisphosphonates prevent or treat diseases" (Methods of Preventing or Treating Disorders by Administering an Integrin α _v β ₃ Antagonist in Combination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate), by citing with the entirety of which is incorporated herein) in combination with the subject in need. In another specific embodiment, the method of the present invention is used to prevent, treat and/or control bacterial infection, such as bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present The inventive antibody was administered to a subject in need in combination with an effective amount of cilizumab (Midiuni, International Publication No. WO02/069904). In another embodiment, the method of the present invention is used to prevent, treat and/or control bacterial infection, such as bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present invention An antibody and an effective amount of one or more EphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (Midiuni Corporation; International Publication No. WO 02/102974 A4, December 27, 2002, entitled "Mutant Proteins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure," International Publication No. 03/094859A2, November 20, 2003, entitled "EphA2 Monoclonal Antibodies and Their Uses Methods" (EphA2Monoclonal Antibodies and Methods of UseThereof), U.S. Application No. 10/436,783 and U.S. Patent Publication No. US 2004/0091486A1; and U.S. Application No. 10/994,129 and U.S. Patent Publication No. US 2005/0152899A1, which are incorporated by reference in their entirety This article)) is jointly given to the object in need. In yet another embodiment, the present invention provides a method for preventing, treating and/or controlling bacterial infection, such as bacterial respiratory tract infection or one or more symptoms thereof, said method comprising administering in combination an effective amount of one or more Antibody of the present invention and effective amount of Vitasine ^™ , Silizumab and/or EphA2.

The invention includes patients who are expected to develop bacterial respiratory infections or are at increased risk of developing such infections, such as patients with suppressed immune systems (eg, organ transplant recipients, AIDS patients, patients undergoing chemotherapy, the elderly, premature infants) , infants, children, patients with obstructed esophageal cancer, patients with tracheobronchial fistula, patients with neurological diseases (such as stroke, amyotrophic lateral sclerosis, multiple sclerosis and diseases caused by myopathy) and those who have developed infections, especially respiratory tract infections patients) to prevent the development of bacterial infections, such as bacterial respiratory infections. Patients may or may not have been treated for infection.

The antibody preparation of the present invention or the combination therapy of the present invention can be used as the first, second, third, fourth or fifth treatment to prevent, treat and/or control bacterial infections, such as bacterial respiratory infections or one or more of its symptoms. The invention also includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections or one or more symptoms thereof, in a patient being treated for other diseases or disorders. The present invention includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections or one or more symptoms thereof, in a patient prior to any adverse reaction or intolerance to treatments other than the antibody formulations of the present invention. The invention also includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections or symptoms thereof, in refractory patients. In certain embodiments, a patient with a bacterial respiratory infection is said to be refractory to treatment when the infection is not substantially resolved and/or symptoms are not significantly relieved. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of treatment against an infection, using the art-accepted meaning of "refractory". In various embodiments, a patient with a bacterial respiratory infection is considered refractory when bacterial replication is not decreased or increased. The invention also includes methods of preventing the occurrence or recurrence of a bacterial infection, such as a bacterial respiratory infection, in a patient at risk of developing a bacterial infection. The invention also includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections or symptoms thereof, in patients prone to adverse reactions to conventional treatments. The invention also includes methods of preventing, treating and/or managing bacterial infections for which no antimicrobial therapy is available, such as bacterial respiratory infections.

The invention includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections, or symptoms thereof, in patients who have proven refractory to therapies other than the antibody formulations of the invention and are no longer on such therapies. In certain embodiments, the patients managed or treated according to the methods of the present invention are patients who have been treated with anti-inflammatory drugs, antibiotics, antiviral agents, antifungal agents or other biological therapy/immunotherapy. Among these patients, those who are too young to be candidates for conventional therapy and those whose bacterial infection recurs despite control or treatment with existing therapies are refractory patients.

The present invention includes methods of preventing, treating and/or managing bacterial infections, such as bacterial respiratory infections, or one or more symptoms thereof, as an alternative to other conventional treatments. In a specific embodiment, the patients managed or treated according to the methods of the present invention are patients who are refractory to other treatments or prone to adverse effects from other treatments. The patient may be a person with a suppressed immune system (such as postoperative patients, chemotherapy patients, and patients with immunodeficiency diseases), a person with impaired renal or hepatic function, the elderly, children, infants, premature infants, persons with neuropsychiatric disorders or persons taking psychotropic medications, persons with a history of epilepsy, or persons who are receiving medications that conflict with conventional medications for the prevention, treatment, and/or control of bacterial infections, such as bacterial respiratory infections, or one or more of their symptoms.

Bacterial infection treatment and its dosage, route of administration and recommended usage are known in the art, such literature includes, for example, Physician's Desk Reference (60th Edition, 2006).

5.5.7. Fungal infection

乙酸卡泊芬净

咪唑、三唑(如氟康唑

)和伊曲康唑

)、多烯(如制霉菌素、两性霉素B两性霉素B脂质复合物(“ABLC”)

、两性霉素B胶体分散体(“ABCD”)

、脂质体两性霉素B

)、碘化钾(KI)、嘧啶(如、氟胞嘧啶

和伏立康唑

)。具体抗真菌剂和其推荐剂量的列表参见例如，下表7。Antifungal agents and antifungal treatments well known to those skilled in the art for the prevention, treatment and/or management of fungal infections or one or more symptoms thereof (eg, fungal respiratory infections) can be used in the compositions and methods of the present invention. Non-limiting examples of antifungal agents include proteins, polypeptides, peptides, fusion proteins, antibodies, Nucleic acid molecules, organic molecules, inorganic molecules and small molecules. Specific examples of antifungal agents include, but are not limited to: azoles (e.g. miconazole, ketoconazole

caspofungin acetate

imidazole, triazole (such as fluconazole

) and itraconazole

), polyenes (such as nystatin, amphotericin B Amphotericin B Lipid Complex ("ABLC")

, Amphotericin B Colloidal Dispersion ("ABCD")

, liposomal amphotericin B

), potassium iodide (KI), pyrimidines (eg, flucytosine

and voriconazole

). See, eg, Table 7 below, for a list of specific antifungal agents and their recommended dosages.

Table 7: Antifungal agents.

In certain embodiments, an antifungal agent is a substance that inhibits or reduces a fungal infection of the respiratory tract, inhibits or reduces the replication of a fungus that causes a lung or respiratory infection, or inhibits or reduces the spread of a fungus that causes a lung or respiratory infection to other subjects. In case the fungal infection of the lung or respiratory tract is Blastomyces dermatitidis, the antifungal agent is preferably itraconazole, amphotericin B, fluconazole or ketoconazole. In case the pulmonary or respiratory fungal infection is pulmonary aspergillosis, the antifungal agent is preferably amphotericin B, liposomal amphotericin B, itraconazole or fluconazole. In case the fungal infection of the lung or respiratory tract is histoplasmosis, the antifungal agent is preferably amphotericin B, itraconazole, fluconazole or ketoconazole. In cases where the pulmonary or respiratory fungal infection is coccidioidomycosis, the antifungal agent is preferably fluconazole or amphotericin B. In cases where the pulmonary or respiratory fungal infection is cryptococcosis, the antifungal agent is preferably amphotericin B, fluconazole or a combination of both. In case the fungal infection of the lungs or respiratory tract is chromomycosis, the antifungal agent is preferably itraconazole, fluconazole or flucytosine. In case the pulmonary or respiratory fungal infection is mucormycosis, the antifungal agent is preferably amphotericin B or liposomal amphotericin B. In case the pulmonary or respiratory fungal infection is pseudomycosis, the antifungal agent is preferably itraconazole or miconazole.

Antifungal treatments and their dosages, routes of administration and recommended usage are known in the art, see for example Dodds et al., 2000 Pharmacotherapy 20(11) 1335-1355, Physicians' Desk Reference (60th Edition, 2006) and The Merck Manual of Diagnosis and Therapy (17th ed., 1999).

5.5.7.1. Antifungal therapy

One or more antibody formulations of the invention may be administered to a subject according to the methods of the invention to prevent, treat and/or manage a fungal infection or one or more symptoms thereof. One or more antibody preparations of the present invention may also be combined with one or more other treatments other than the antibody preparations of the present invention (such as a or multiple preventive or therapeutic agents) are administered to the subject in combination to prevent, treat and/or control fungal infection and/or one or more symptoms thereof.

In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling fungal infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more antibody preparations of the present invention . In another embodiment, the present invention provides a method of preventing, treating and/or controlling fungal infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more of the present invention An antibody formulation and an effective amount of one or more treatments (eg, prophylactic or therapeutic agents) other than the antibody formulation of the invention.

Any type of fungal infection or conditions caused by or associated with fungal infections (eg, respiratory tract infections) can be prevented, treated and/or managed according to the methods of the present invention. Examples of fungi that cause fungal infections include, but are not limited to: Absidia (e.g., Absidia corymbifera and Absidia ramosa), Aspergillus (e.g., Aspergillus flavus ( Aspergillus flavus), Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida ) (eg, Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida parapsilosis, Candida pseudotropis Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Candida stellatoidea and Candida tropicalis), Coccidioides immitis , Conidiobolus, Cryptococcus neoforms, Cunninghamella, dermatophytes, Histoplasma capsulatum, Microsporum gypseum, tiny Mucor pusillus, Paracoccidioides brasiliensis, Pseudallescheriaboydii, Rhinosporidium seeberi, Pneumocystiscarinii, Rhizopus ( Rhizopus) (such as Rhizopus arrhizus, Rhizopusoryzae, and Rhizopus microsporus), Saccharomyces, Sporothrix schenckii, Zygomycetes, and Such as Zygomycetes (Zygomycetes Mycetes), Ascomycetes, Basidiomycetes, Deuteromycetes and Oomycetes.

In a specific embodiment, the present invention provides a method for preventing, treating and/or controlling fungal respiratory infection or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present invention to a subject in need thereof. Antibody preparations. In another embodiment, the present invention provides a method of preventing, treating and/or controlling fungal respiratory infection or one or more symptoms thereof, said method comprising administering to a subject in need thereof an effective amount of one or more An antibody formulation of the invention and an effective amount of one or more treatments (eg, one or more prophylactic or therapeutic agents) in addition to the antibody formulation of the invention.

、乙酸卡泊芬净、咪唑、三唑(如氟康唑

)和伊曲康唑

多烯(如制霉菌素、两性霉素B胶体分散体、两性霉素B脂质复合物(“ABLC”)两性霉素B胶体分散体(“ABCD”)

、脂质体两性霉素B)、碘化钾(KI)、嘧啶(如氟胞嘧啶

)和伏立康唑

在些实施方式中，将有效量的一种或多种本发明抗体制剂与一种或多种支持性手段联合给予需要的对象，以预防、治疗和/或控制真菌感染或其一种或多种症状。支持性手段的非限制性例子包括通过超声喷雾器对空气增湿、给予气雾化外消旋肾上腺素、口服地塞米松、静脉内液体、插管、解热剂(如布洛芬和对乙酰氨基酚)以及抗病毒或抗菌治疗(即，预防或治疗继发性病毒或细菌感染)。In certain embodiments, an effective amount of one or more antibody formulations is combined with an effective amount of one or more treatments (e.g., one or more prophylactic or therapeutic agents) other than an antibody formulation of the invention Administered to a subject in need thereof, the treatment is, has been or is known to be useful in the prevention, treatment and/or management of fungal infections, such as fungal respiratory infections. Treatment of fungal infections includes, but is not limited to: antifungal agents such as azoles such as miconazole, ketoconazole

, caspofungin acetate , imidazole, triazole (such as fluconazole

) and itraconazole

Polyenes (eg, nystatin, amphotericin B colloidal dispersion, amphotericin B lipoplex (“ABLC”) Amphotericin B Colloidal Dispersion ("ABCD")

, liposomal amphotericin B ), potassium iodide (KI), pyrimidines (such as flucytosine

) and voriconazole

In some embodiments, an effective amount of one or more antibody preparations of the present invention is administered to a subject in need in combination with one or more supportive measures to prevent, treat and/or control fungal infections or one or more of them. symptoms. Non-limiting examples of supportive measures include humidification of the air by ultrasonic nebulizer, administration of aerosolized racemic epinephrine, oral dexamethasone, intravenous fluids, catheterization, antipyretics (eg, ibuprofen and acetaminophen, aminophenols) and antiviral or antibacterial therapy (ie, to prevent or treat secondary viral or bacterial infections).

The present invention also provides methods of preventing, treating and/or managing biological responses to fungal respiratory infections including, but not limited to: elevated levels of IgE antibodies, elevated levels of nerve growth factor (NGF), mast cell proliferation , degranulation and/or infiltration, increased proliferation and/or infiltration of B cells, and increased proliferation and/or infiltration of T cells, the method comprising administering an effective amount of one or more of An antibody preparation that immunospecifically binds IL-9 polypeptide.

In a specific embodiment, the present invention provides a method of preventing, treating and/or controlling a primary fungal infection, such as one or more secondary conditions of a primary fungal respiratory infection or a reaction thereto, The method comprises administering to a subject in need thereof an effective amount of one or more antibody preparations of the invention alone or in combination with an effective amount of other treatments other than the antibody preparations of the invention (eg, other prophylactic or therapeutic agents). Examples of secondary conditions or reactions to primary fungal infections, particularly primary fungal respiratory infections, include, but are not limited to: asthma-like responses to mucosal irritation, increased total respiratory resistance, secondary viral, fungal increased susceptibility to and fungal infections, and the development of conditions such as, but not limited to, pneumonia, croup, and febrile bronchitis.

In a specific embodiment, the present invention provides methods for preventing, treating and/or controlling fungal infections, such as fungal respiratory infections or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present Invention of Antibodies and Effective Amounts of Vitacin ^™ (Midiuni Corporation, International Publication No. WO 00/78815, International Publication No. WO 02/070007A1, September 12, 2002, entitled "By Administering Integrin α _v β ₃ antagonists to prevent or treat inflammatory diseases or autoimmune diseases" (Methods of Preventing or Treating Inflammatory or Autoimmune Disorders by Administering Integrin AlphaV Beta3Antagonists), International Publication No. WO 03/075957A1, September 18, 2003, entitled " Integrin α _v β ₃ antagonists combined with other agents to prevent or treat cancer" (The Prevention or Treatment of Cancer Using IntegrinAlpha VBeta3 Antagonists in Combination With Other Agents), US Patent Publication No. US 2002/0168360 A1, 2002 November 14, entitled "Methods of Preventing or Treating Inflammatory or Autoimmune Diseases by Combining Administration of Integrin α _v β ₃ Antagonists and Other Preventive or Therapeutic Agents" Disorders by Administering Integrin _{α v} β ₃ Antagonists in Combination With Other Prophylacticor Therapeutic Agents) and International Publication No. WO 03/075741A2, September 18, 2003, entitled "Administering Integrin α _v β ₃ Antagonists in Combination With Other Prophylacticor Therapeutic Agents and HMG-CoA Reductase inhibitors or bisphosphonates prevent or treat diseases" (Methods of Preventing or Treating Disorders by Administering an Integrin α _v β ₃ Antagonist in Combination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate), cited (the entirety of which is incorporated herein) is jointly administered to the object in need. In another specific embodiment, the present invention provides a method for preventing, treating and/or controlling fungal respiratory infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibodies of the present invention with An effective amount of cilizumab (Midiuni, International Publication No. WO 02/069904) was co-administered to subjects in need. In another embodiment, the present invention provides a method for preventing, treating and/or controlling fungal respiratory infection or one or more symptoms thereof, said method comprising combining an effective amount of one or more antibodies of the present invention with an effective amount of one or more EphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (Midiuni Corporation; International Publication No. WO 02/102974A4, December 27, 2002, entitled "Mutins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure" (Mutant Proteins, High Potency Inhibitory Antibodies and FIMCH Crystal Structure), International Publication No. 03/094859A2, November 20, 2003, entitled "EphA2 monoclonal antibody and method of use thereof" (EphA2 Monoclonal Antibodies and Methods of Use Thereof), U.S. Application No. 10/436,783; and U.S. Application No. 10/994,129 and U.S. Patent Publication No. US 2005/0152899A1, which are incorporated herein by reference in their entirety)) to a subject in need. In yet another embodiment, the present invention provides methods for preventing, treating and/or controlling fungal infections, such as fungal respiratory tract infections or one or more symptoms thereof, said method comprising administering an effective amount of one or more of the present The inventive antibody is administered to a subject in need in combination with an effective amount of Vitasine ^™ , Silizumab and/or EphA2.

The present invention includes methods of preventing the development of fungal respiratory infections in patients predicted to develop fungal infections, such as fungal respiratory infections, or at increased risk of developing such infections. Such subjects include, but are not limited to: patients with suppressed immune systems (such as organ transplant recipients, AIDS patients, patients undergoing chemotherapy, patients with obstructed esophageal cancer, patients with tracheobronchial fistula), neurological diseases (such as stroke, muscular Atrophic lateral sclerosis, multiple sclerosis, and myopathy) and patients who have developed respiratory disorders, especially respiratory infections). In a specific embodiment, the patient suffers from bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis and/or acquired or congenital immunodeficiency. In another specific embodiment, the patient is a premature infant, an infant, a child, an elderly person, or a person living in a correctional home, nursing home, or some other type of institution. The present invention also includes methods of preventing, treating and/or managing a respiratory disorder or one or more symptoms thereof in patients prone to adverse reactions to conventional treatments for the respiratory disorder and thus unavailable for treatment.

The antibody preparation of the present invention or the combination therapy of the present invention can be used as the first, second, third, fourth or fifth treatment to prevent, treat and/or control fungal infections, such as fungal respiratory infections or one or more of its symptoms. The invention also includes methods of preventing, treating and/or managing fungal infections, such as fungal respiratory infections or one or more symptoms thereof, in a patient who is being treated for other diseases or disorders. The invention includes methods of preventing, treating and/or controlling fungal infections, such as fungal respiratory tract infections or one or more symptoms thereof, in a patient prior to any adverse reaction or intolerance to treatments other than the antibody formulations of the invention. The invention also includes methods of preventing, treating and/or managing fungal infections, such as fungal respiratory infections or symptoms thereof, in refractory patients. In certain embodiments, a patient with a fungal infection, such as a fungal respiratory tract infection, is said to be refractory to treatment when the infection has not been significantly resolved and/or the symptoms have not been significantly relieved. Whether a patient is "refractory" can be determined in vivo or in vitro by any method known in the art for determining the effectiveness of treatment against an infection, using the art-accepted meaning of "refractory". In various embodiments, a patient with a fungal infection, such as a fungal respiratory infection, is considered refractory when fungal replication is not decreased or increased. The invention also includes methods of preventing the occurrence or recurrence of a fungal infection, such as a fungal respiratory infection, in a patient at risk of developing a fungal infection. The invention also includes methods of preventing, treating and/or managing fungal infections, such as fungal respiratory infections or symptoms thereof, in patients prone to adverse reactions to conventional treatments. The invention also includes methods of preventing, treating and/or managing fungal infections for which no antifungal therapy is available, such as fungal respiratory infections.

The invention includes methods of preventing, treating and/or controlling fungal infections, such as fungal respiratory tract infections, or symptoms thereof, in patients who have proven refractory to therapies other than the antibody formulations of the invention and are no longer on such therapies. In certain embodiments, the patients managed or treated in accordance with the methods of the present invention are patients who have been treated with antibiotics, antivirals, antifungals, or other biological/immunotherapy. Among these patients, those who are too young to be candidates for conventional therapy and those whose fungal infection recurs despite control or treatment with existing therapies are refractory patients.

The present invention provides methods of preventing, treating and/or managing fungal infections, such as fungal respiratory infections, or one or more symptoms thereof, as an alternative to other conventional treatments. In a specific embodiment, the patients managed or treated according to the methods of the present invention are patients who are refractory to other treatments or prone to adverse effects from other treatments. The patient may be a person with a suppressed immune system (such as postoperative patients, chemotherapy patients, and patients with immunodeficiency diseases), a person with impaired renal or hepatic function, the elderly, children, infants, premature infants, persons with neuropsychiatric disorders or persons taking psychotropic medications, persons with a history of epilepsy, or persons who are receiving medications that conflict with conventional medications for the prevention, treatment, and/or control of fungal infections, such as fungal respiratory infections, or one or more of their symptoms.

Treatment of fungal infections and their dosages, routes of administration and recommended usage are known in the art, such literature includes, for example, the Physician's Desk Reference (60th Edition, 2006).

5.6. Methods of administering antibody preparations

The present invention provides prevention, treatment and/or control of diseases, for example, diseases associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof, by administering to a subject an effective amount of the liquid formulation of the present invention. Disease, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or one or more symptoms thereof. A variety of delivery systems are known and can be used to administer the liquid formulations or prophylactic or therapeutic agents of the invention. Methods of administering antibody liquid formulations or treatments (such as prophylactic or therapeutic agents) of the invention include, but are not limited to: parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, preferably subcutaneous), epidural Administration, topical administration, and mucosal administration (eg, intranasal and oral routes). In a specific embodiment, the liquid formulation of the invention is administered intramuscularly, intravenously or subcutaneously. In one embodiment, the liquid formulations of the invention are administered subcutaneously. The formulation can be administered by any conventional route, such as infusion or bolus injection, absorbed through the epithelium or mucocutaneous lining (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In a specific embodiment, the liquid formulation of the invention is administered intratumorally or at a site of inflammation.

In a specific embodiment, the antibody formulation of the invention comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is water for injection, USP, 5% dextrose in water (D5W) or saline.

Typically, the antibodies (including antibody fragments thereof) that immunospecifically bind to IL-9 polypeptides contained in the liquid formulations of the present invention are derived from subjects of the same species origin or species reactivity as the recipients of the liquid formulations of the present invention. Accordingly, in one embodiment, a liquid formulation of the invention comprising a human or humanized antibody that immunospecifically binds IL-9 polypeptide is administered to a human patient for treatment or prophylaxis.

The present invention also provides that the liquid preparation of the present invention is packaged in a sealed container, such as an ampoule or a sachet, on which the content of the antibody (including antibody fragments thereof) is indicated. Preferably, the liquid preparation of the present invention is contained in a sealed container indicating the content and concentration of the antibody (including antibody fragments thereof). Preferably, the liquid preparation of the present invention is loaded into a sealed container, containing at least 10mg/ml, 15mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 150mg/ml, 175mg/ml, 200mg/ml, 250mg/ml or 300mg/ml of antibodies (including antibody fragments) that immunospecifically bind to IL-9 polypeptides, the content is 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 15ml or 20ml, most preferably 1.2ml. In a specific embodiment of the present invention, the liquid formulation of the present invention is contained in a sealed container and contains at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml ml, at least 175 mg/ml, at least 200 mg/ml, at least 250 mg/ml, or at least 300 mg/ml of antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides for intravenous injection (for example, 4D4, 4D4 H2 - 1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof) and at least 15mg/ml, 20mg/ml, 50mg/ml, 80mg/ml, 100mg/ml, 150mg/ml, 175mg/ml, 200mg/ml, 250mg/ml or 300mg/ml for repeated subcutaneous administration of antibodies (including antibody fragments thereof) that immunospecifically bind IL-9 polypeptides (such as 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or fragments thereof). In a specific embodiment, antibody formulations of the invention can be produced by lyophilization of aqueous antibody formulations. In a specific embodiment, the aqueous antibody solution of the lyophilized antibody can be reconstituted with a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutically acceptable carrier is water for injection, USP, 5% dextrose in water (D5W) or saline.

The effective prevention, treatment and/or control of diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity of one or more subunits thereof, or one or The dosage of the liquid preparation of the present invention for various symptoms. The exact dosage of the formulation to be used will also depend on the route of administration and the severity of the inflammatory disease, autoimmune disease or cancer, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves obtained in vitro or in animal model test systems.

For antibody, protein, polypeptide, peptide, and fusion protein formulations encompassed by the invention, the dose administered to a patient can be calculated by multiplying the administered dose expressed in mg/kg by the patient's body weight in kilograms (kg). The volume (mL) required for administration is then determined by dividing the desired dose in milligrams by the concentration of the antibody formulation. The antibody formulations of the invention are administered by collecting the contents of the desired number of vials into syringes to achieve the final calculated desired volume. The drug is administered by combining the contents of the required number of vials into a syringe to obtain the final calculated required volume. The maximum volume of antibody preparation that can be injected per site is 2.0 mL. The dose (mL) can be calculated by the following formula: dose (mL) = [volunteer body weight] (kg) x [dose] mg/kg ÷ 100 mg/mL antibody preparation. Generally, human antibodies have a longer half-life in the human body than antibodies from other sources because of the immune response to foreign polypeptides. Accordingly, human antibodies may be administered at lower doses and less frequently. In addition, the liquid formulations of the present invention can be reduced by increasing the concentration of antibodies (including antibody fragments thereof) in the formulation, increasing the affinity and/or avidity of antibodies (including antibody fragments thereof) and/or prolonging the half-life of antibodies (including antibody fragments thereof). Dosage, volume and frequency of administration.

In a specific embodiment, the patient's body weight in kilograms (kg) is multiplied by the administered dose expressed in mg/kg to calculate the dose administered to the patient. The volume (mL) required for administration is then determined by dividing the desired dose in milligrams by the concentration of antibody (including antibody fragments thereof) in the formulation (100 mg/mL). The drug is administered by combining the contents of the required number of vials into a syringe to obtain the final calculated required volume. The maximum volume of antibody (including antibody fragments) preparations that can be injected at each site is 2.0 mL.

Exemplary dosages of small molecules include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram/kg to about 500 mg/kg, about 100 microgram/kg to about 5 mg/kg, or about 1 µg/kg to about 50 µg/kg).

In a specific embodiment, 0.1-20 mg/kg/week, 1-15 mg/kg/week, 2-8 mg/kg/week, 3-7 mg/kg/week or 4-6 mg/kg Antibodies (including antibody fragments thereof) (such as 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or fragments thereof) administered to patients with inflammatory diseases, autoimmune diseases or cancer. In another embodiment, a subject is administered one or more doses of a prophylactically or therapeutically effective amount of a liquid formulation of the invention, wherein the prophylactically or therapeutically effective amount is different in each dose.

In one embodiment, the liquid formulations of the invention are administered with a regimen that maintains plasma concentrations of immunospecific antibodies to _αvβ3 at desired levels (e.g., _about 0.1 to 100 μg/ml) to continuously block IL- 9 polypeptide activity. In a specific embodiment, the plasma concentration of the antibody is maintained at 0.2 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml or 50 μg/ml. Desired plasma concentrations in a subject will depend on several factors including, but not limited to, the nature of the disease or disorder, the severity of the disease or disorder, and the general condition of the subject. Such dosing regimens are particularly suitable for the prevention, treatment and/or management of chronic diseases or disorders.

In a specific embodiment, the liquid formulation of the invention comprising an immunospecifically conjugated antibody (including antibody fragments thereof) to an IL-9 polypeptide is administered intermittently. As used herein, the term "conjugated antibody or antibody fragment" refers to an antibody (including antibody fragments thereof) that is conjugated or fused to another moiety, including but not limited to: a heterologous peptide or polypeptide, another antibody (including antibody fragments thereof), fragments), marker sequences, diagnostic agents, therapeutic moieties, therapeutic drugs, radioactive metal ions, polymers, albumin, and solid supports.

In another embodiment, one or more doses of antibodies (including antibody fragments thereof) (such as 4D4, 4D4H2-1D11) that immunospecifically bind to IL-9 polypeptides in the liquid formulation of the present invention in a prophylactically or therapeutically effective amount , 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or a fragment thereof) to a subject, preferably a human, wherein as the treatment proceeds, the subject will be administered Prophylactically or therapeutically effective doses of antibodies (including antibody fragments thereof) in the liquid formulation of the present invention are increased, for example, 0.01 μg/kg, 0.02 μg/kg, 0.04 μg/kg, 0.05 μg/kg, 0.06 μg/kg, 0.08 μg/kg μg/kg, 0.1μg/kg, 0.2μg/kg, 0.25μg/kg, 0.5μg/kg, 0.75μg/kg, 1μg/kg, 1.5μg/kg, 2μg/kg, 4μg/kg, 5μg/kg, 10μg/kg, 15μg/kg, 20μg/kg, 25μg/kg, 30μg/kg, 35μg/kg, 40μg/kg, 45μg/kg, 50μg/kg, 55μg/kg, 60μg/kg, 65μg/kg, 70μg/kg kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg or 125 μg/kg.

In another embodiment, one or more doses of antibodies (including antibody fragments thereof) (such as 4D4, 4D4H2-1D11) that immunospecifically bind to IL-9 polypeptides in the liquid formulation of the present invention in a prophylactically or therapeutically effective amount , 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or a fragment thereof) to a subject, preferably a human, wherein as the treatment proceeds, the subject will be administered Prophylactically or therapeutically effective doses of antibodies (including antibody fragments thereof) in the liquid formulations of the present invention are reduced, for example, 0.01 μg/kg, 0.02 μg/kg, 0.04 μg/kg, 0.05 μg/kg, 0.06 μg/kg, 0.08 μg/kg, 0.1μg/kg, 0.2μg/kg, 0.25μg/kg, 0.5μg/kg, 0.75μg/kg, 1μg/kg, 1.5μg/kg, 2μg/kg, 4μg/kg, 5μg/kg, 10μg/kg, 15μg/kg, 20μg/kg, 25μg/kg, 30μg/kg, 35μg/kg, 40μg/kg, 45μg/kg, 50μg/kg, 55μg/kg, 60μg/kg, 65μg/kg, 70μg/kg kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg or 125 μg/kg.

Dosing of prophylactic or therapeutic agents is described in Physicians' Desk Reference (60th Edition, 2006).

5.7. Biological experiments

Antibodies (including antibody fragments thereof) of the liquid formulations of the invention can be identified in a variety of ways well known to those skilled in the art. For example, antibodies (including antibody fragments thereof) of liquid formulations of the invention can be assayed for their ability to immunospecifically bind an antigen. Such assays can be performed in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991, Nature 354:82-84), on a chip (Fodor, 1993, Nature 364:555-556 ), on bacteria (US Patent No. 5,223,409), on spores (US Patents 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al., 1992, Proc. Scott and Smith, 1990, Science 249:386-390; Cwirla et al., 1990, Proc.Natl.Acad.Sci.USA 87:6378-6382; and Felici, 1991, J.Mol.Biol.222:301-310) (Each of these references is incorporated herein by reference in its entirety). For example, the specificity and affinity of antibodies (including antibody fragments thereof) identified to immunospecifically bind IL-9 polypeptides to IL-9 polypeptides can be determined.

Antibodies (including antibody fragments thereof) of liquid formulations of the invention can be assayed for immunospecific binding to antigens and cross-reactivity with other antigens by any method known in the art. Immunological assays that can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to: using methods such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin Competitive and non-competitive assay systems for reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such experiments are routine experiments well known in the art (see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). ., New York), incorporated by reference in its entirety). Exemplary immunoassays are briefly described below (but not limited to).

Immunoprecipitation protocols usually involve cells being treated with a lysis buffer such as RIPA buffer (1% NP-40 Or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate pH 7.2, 1% Trasylol) to lyse the cell population, and add the antibody of interest to the cell lysis Incubate for a period of time (such as 1-4 hours) at 40°C, add protein A and/or protein G agarose beads to the cell lysate, incubate at 40°C for about one hour or longer, wash the beads with lysis buffer, And resuspend beads in SDS/sample buffer. The ability of an antibody of interest to immunoprecipitate a particular antigen can be assessed by, for example, Western blot analysis. Those skilled in the art will recognize that parameters can be varied to improve antibody antigen binding and reduce background (eg, pre-cleaning of cell lysates with sepharose beads). For further discussion of immunoprecipitation protocols see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York , 10.16.1.

Western blot analysis typically involves: preparing protein samples, electrophoresis of protein samples on a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein samples from the polyacrylamide gel to a membrane such as On nitrocellulose, PVDF or nylon membranes, incubate the membrane in blocking buffer (e.g., PBS containing 3% BSA or nonfat dry milk), wash the membrane with washing buffer (e.g., PBS-Tween 20), wash with blocking buffer Incubate the membrane with the primary antibody (antibody of interest) diluted in buffer solution, wash the membrane with washing buffer, and conjugate to an enzyme substrate (such as horseradish peroxidase or alkaline phosphatase) or The membrane is incubated with a secondary antibody (eg, anti-human antibody) to a radioactive molecule (eg, ^{32p or125I} ), which recognizes the primary antibody, washed with wash buffer, and the presence of the antigen detected. Those skilled in the art recognize that parameters can be varied to increase the detected signal and reduce background noise. For further discussion of Western blotting protocols see, eg, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York , 10.8.1.

ELISA involves preparing antigen, coating the wells of a 96-well microtiter plate with the antigen, and adding an antibody of interest conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells , incubated for a period of time and tested for the presence of antigen. In an ELISA, the antibody of interest need not be conjugated to a detectable compound; a secondary antibody (recognizing the antibody of interest) linked to the detectable compound can also be added to the wells. Alternatively, instead of coating the wells with antigen, wells can be coated with antibodies. In such cases, a secondary antibody conjugated to a detectable compound can be added, followed by addition of the antigen of interest to the coated wells. Those skilled in the art will recognize that parameters can be changed to increase the signal detected and to achieve other ELISA changes known in the art. In one embodiment, high binding 96-well microtiter plates (Costar) can be coated overnight with 2 μg/ml rhu-IL-9 in PBS for ELISA. After three washes with PBS, the plates were incubated with three-fold serially diluted Fabs for 1 hour at 25°C. After washing three times with PBS, 1 μg/ml anti-human κ-alkaline phosphatase conjugate was added, and the plate was incubated at 25°C for 1 hour. After three washes with PBST, alkaline phosphatase activity was measured with 50 μl/AMP/PPMP substrate. The reaction was terminated, and the absorbance at 560 nm was measured with a VMAX microplate reader. For further discussion of ELISA see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, 11.2 .1.

The binding affinity of the antibody to the antigen and the dissociation rate of the antibody-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (eg, ³ H or ¹²⁵ I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting antibody bound to the labeled antigen. Scatchert plot analysis can be used to determine the affinity and binding and dissociation rate of the antibody or fragment thereof contained in the antibody preparation of the present invention to the specific antigen from the data. Competition with secondary antibodies can also be determined using radioimmunoassays. In one example, the IL-9 polypeptide is incubated with an antibody conjugated to a labeled compound (eg, ^{3H or125I} ) in the presence of increasing amounts of an unlabeled secondary antibody.

For example, in one embodiment, BIAcore kinetic analysis is used to determine the association and dissociation rates of antibodies from liquid formulations of the invention to IL-9 polypeptides. BIAcore kinetic analysis includes analyzing the binding and dissociation of IL-9 polypeptide to the antibody of the present invention immobilized on the chip surface. BIAcore kinetic studies generally include injecting 250 μL of different concentrations of antibody reagents (mAb, Fab) prepared in HBS buffer containing 0.005% Tween-20 on the surface of the antigen-immobilized sensor chip. The flow rate was maintained at 75 μL/min. Collect dissociation data for 15 minutes or more, as needed. After each injection/dissociation cycle, a 1 minute pulse brief treatment with dilute acid, typically 10-100 mM HCl, removes mAb bound on the antigen surface, but other regenerating agents are used as circumstances warrant. More specifically, to measure the on-rate kon and _off-rate koff _, standard amine coupling chemistry, the EDC/NHS method (EDC=N-diethylaminopropyl)-carbodiimide) was used to combine Antigens are immobilized directly on the surface of the sensor chip. Briefly, 5-100 nM antigen solutions were prepared in 10 mM NaOAc, pH 4 or pH 5, and passed over the EDC/NHS-activated surface until approximately 30-50 RU values of antigen were immobilized. Then, unreacted active ester was "killed" by injection of 1M Et-NH2. A blank surface without antigen was prepared under the same fixation conditions for reference purposes. After preparation of suitable surfaces, appropriate serial dilutions of each antibody were prepared in HBS/Tween-20 and passed over serially coupled antigen and reference cell surfaces. Antibody concentrations were prepared in varying ranges, depending on the estimated equilibrium binding constant, KD. After each injection/dissociation cycle, remove bound antibody with a suitable regenerating agent, as described above.

If the antibody (including antibody fragments thereof) of the liquid formulation of the present invention is immunospecific for a receptor ligand, the ability of the antibody to inhibit the binding of the ligand to its receptor can also be tested using techniques known to those skilled in the art. For example, cells expressing the IL-9 receptor are contacted with IL-9 in the presence or absence of a liquid formulation of the antibody (including antibody fragments thereof), and the antibody can be determined, for example, by flow cytometry or scintillation assays ( including antibody fragments thereof) inhibit the binding of IL-9. Antibodies (including antibody fragments thereof) contained in IL-9 or liquid preparations can be detected with compounds such as radioactive labels (such as ³² P, ³⁵ S and ¹²⁵ I) or fluorescent labels (such as fluorescein isothiocyanate, rhodamine, Phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine) were labeled to detect the interaction between IL-9 and its host cell receptor. Alternatively, the ability of antibodies (including antibody fragments thereof) of the liquid formulations of the invention to inhibit the binding of ligands to their receptors can be assayed by cell-free assays. For example, an IL-9 polypeptide can be contacted with an antibody (including antibody fragments thereof) in a liquid formulation of the invention, and the ability of the antibody (including antibody fragments thereof) to inhibit binding of the IL-9 polypeptide to its host cell receptor can be determined. Preferably, the antibody (including antibody fragments thereof) of the liquid formulation of the present invention is immobilized on a solid support, and the IL-9 polypeptide is labeled with a detectable compound. Alternatively, the IL-9 polypeptide is immobilized on a solid support, and the antibody (including antibody fragments thereof) contained in the liquid formulation of the present invention is labeled with a detectable compound. IL-9 can be partially or fully purified (eg, partially or completely free of other polypeptides), or part of a cell lysate. Alternatively, the IL-9 polypeptide may be a fusion protein comprising IL-9, derivatives, analogs or fragments thereof and domains such as glutathione-S-transferase. Alternatively, IL-9 polypeptides can be biotinylated using techniques well known to those skilled in the art (eg, Biotinylation Kit, Pierce Chemicals (Rockford, IL)).

再培育细胞4-5小时。用荧光计，以555nm激发和590nm发射定量测定续表代谢。In a specific embodiment, the ability of the antibody (including antibody fragments thereof) of the liquid formulation of the present invention to inhibit the binding of a ligand to its host cell receptor can be determined by a cell proliferation assay. For example, the murine TS1-RA3T cell line expressing both human and murine IL-9Ra can be cultured continuously on growth medium (DMEM) containing rhuIL-9 (25 ng/ml, R&D Systems). After removal of rhuIL-9, TS1-RA3 cell death occurred within 18-24 hours. TS1-RA3 cells were cultured in RPMI 1640 (ATCC) medium supplemented with 10% FBS and 25 ng/ml rHu-IL9. Before the experiment, cells were washed with IL-9-free medium and resuspended in medium containing 2ng/ml rhuIL-9 at 5×10 ⁵ cells/ml. Cells were aliquoted into black clear bottom non-binding 96-well microtiter plates (100 μl cells/well) and 100 ml serially diluted variant Fabs were added to the plates. Incubate the plate for 72 hours at 37°C, 5% _CO2 . Add 20 μl/well alamar blue

Cells were incubated for an additional 4-5 hours. Continuing table metabolism was quantitatively determined with a fluorometer with excitation at 555nm and emission at 590nm.

5.7.1. In vitro studies

The ability of an antibody, composition or combination therapy of the invention to modulate the biological activity of immune cells (such as T cells, neutrophils and mast cells), endothelial cells and epithelial cells can be tested in vitro and/or in vivo. Modulation of immune cells (such as T cells, B cells, mast cells, macrophages, neutrophils, and eosinophils), endothelial cells, and epithelial cells can be assessed by the following methods: Capacity for biological activity: Detection of antigens (eg, IL-9 activating genes such as but not limited to: mucin genes (eg, MUC2, MUC5AC, MUC5B, and MUC6) and genes involved in lymphocyte activation (eg, Lγ-6A/E) ) expression; detecting the proliferation of immune cells, endothelial cells and/or epithelial cells; detecting the activation of signal transduction molecules (such as phosphorylation of Stat2, phosphorylation of JAK3 or phosphorylation of IL-9R); detecting immune cells (such as , effector functions of T cells, B cells, mast cells, macrophages, neutrophils, and eosinophils), endothelial cells, and/or epithelial cells; or to detect differentiation of immune cells, endothelial cells, and/or epithelial cells . These activities can be measured using techniques known to those skilled in the art. For example, cell proliferation can be measured by 3H-thymidine incorporation assay and trypan blue cell count. Antigen expression can be analyzed, for example, by immunological assays including, but not limited to, using methods such as Western blot, immunohistochemistry, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation Competitive and non-competitive assay systems for experiments, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, and FACS analysis . Activation of signaling molecules can be measured by, for example, kinase assays and electrophoretic mobility shift assays (EMSA). For example, serotonin (5-HT) release or histamine release can be measured using high performance liquid chromatography to analyze mast cell degranulation (see, e.g., Taylor et al., 1995 Immunology 86(3):427-433 and Kurosawa et al., 1998 Clin ExpAllergy 28(8):1007-1012).

Antibodies, compositions or combination therapies of the invention are preferably studied in vitro and then in vivo for desired therapeutic or prophylactic activity prior to use in humans. For example, assays that can be used to determine which particular pharmaceutical composition should be administered include cell culture assays, in which patient tissue samples are grown, exposed or contacted to the pharmaceutical composition, and the effect of such composition on the tissue sample is observed. A patient's tissue sample may be obtained by biopsy. Such testing enables the identification of the most therapeutic treatment (eg, prophylactic or therapeutic agent) for each individual patient. In various embodiments, diseases or disorders related to or characterized by aberrant expression and/or activity of IL-9 polypeptides, aberrant expression of IL-9R or one or more subunits thereof may be utilized. and/or activity related to or characterized by diseases or disorders, inflammatory diseases, autoimmune diseases, proliferative diseases or infection (such as respiratory tract infection) of representative cells of cell types for in vitro testing to determine the drug of the present invention Whether the composition has the desired effect on these cell types. -{}-

The effect of an antibody, composition or combination therapy of the invention on peripheral blood lymphocyte counts can be monitored/assessed using standard techniques known to those skilled in the art. Lymphocytes can be separated from other components of peripheral blood, such as plasma, by, for example, obtaining a sample of peripheral blood from a subject; using, for example, Ficoll-Hypaque (Pharmacia) gradient centrifugation; Lymphocytes were counted with trypan blue to determine the subject's peripheral blood lymphocyte count. Lymphocytes can be separated from other components of peripheral blood, such as plasma, by, for example, using Ficoll-Hypaque (Pharmacia) gradient centrifugation; Antibodies to T cell antigens label T cells, and the number of T cells is determined by FACS to determine the peripheral blood T cell count of the subject.

The ability of the methods of the invention for preventing, treating and/or managing viral respiratory infections or one or more symptoms thereof to inhibit viral replication or reduce viral load can be tested in vitro. For example, viral replication can be determined by plaque assay, see eg Johnson et al., 1997, Journal of Infectious Diseases 176: 1215-1224. The ability of an antibody, composition or combination therapy administered according to the methods of the invention to inhibit or downregulate expression of a viral polypeptide can also be determined. Expression of viral polypeptides can be determined using techniques known to those skilled in the art including, but not limited to, Western blot analysis, northern blot analysis, and RT-PCR.

The activity of the methods of the invention for preventing, treating and/or controlling respiratory tract infection or one or more symptoms thereof against bacteria causing respiratory tract infection can be tested in in vitro assays well known in the art. In vitro assays known in the art can also be used to detect the presence or development of bacterial resistance to a treatment of the invention, such as an antibody of the invention, other prophylactic or therapeutic agents, combination therapies or combinations thereof. For such in vitro assays see Gales et al., 2002, Diag.Nicrobiol.Infect.Dis.44(3):301-311; Hicks et al., 2002, Clin.Microbiol.Infect.8(11):753-757; and Nicholson et al. , 2002, Diagn. Microbiol. Infect. Dis. 44(1): 101-107.

Treatment of the present invention for the prevention, treatment and/or control of respiratory tract infection or one or more symptoms thereof (e.g., the antibody of the liquid formulation of the present invention is used alone or in combination with a prophylactic or therapeutic agent other than the antibody of the present invention) Antifungal activity against different fungi. Any standard antifungal assay well known in the art can be used to assess the antifungal activity of a treatment. The antifungal effect against different fungi can be tested. Tests recommended by the National Committee for Clinical Laboratories (NCCLS) are available (see National Committee for Clinical Laboratories Standards, 1995, proposed standard M27T, Villanova, PA , Pa.), which is incorporated herein by reference in its entirety) and other methods known to those skilled in the art (Pfaller et al., 1993, Infectious Dis. Clin. N. Am. 7: 435-444) to assess the antifungal effect of the treatment . The antifungal properties of treatments can be determined by fungal lysis assays, among other methods, including growth inhibition assays, fluorescence-based fungal viability assays, flow cytometry analysis and other standard assays known to those skilled in the art, and the like.

For example, methods well known to those skilled in the art can be used by means of major dilution and/or microdilution (see, e.g., Clancy et al., 1997 Journal of Clinical Microbiology, 35(11): 2878-82; Ryder et al., 1998, Antimicrobial Agents and Chemotherapy, 42(5):1057-61; U.S. 5,521,153; U.S. 5,883,120, U.S. 5,521,169, all incorporated herein by reference in their entirety) to test the antifungal activity of the treatments. Briefly, the fungal strain is grown in a suitable liquid medium at a suitable temperature for a period of time depending on the particular fungal strain used. The inoculum is then prepared photometrically so that the turbidity of the suspension is equal to that of a standard, such as the McFarland standard. The effect of treatment on inoculum turbidity was determined by visual observation or spectrophotometry. The therapeutic minimum inhibitory concentration ("MIC"), which is defined as the lowest concentration of lead compound that prevents observable growth of the inoculum as measured by culture turbidity, is determined.

The antifungal activity of treatments can also be determined using colorimetric based assays well known to those skilled in the art. An exemplary colorimetric assay that can be used to assess the antifungal activity of a treatment is found in Pfaller et al. (1994, Journal of Clinical Microbiology, 32(8): 1993-6, which is incorporated herein by reference in its entirety; see also Tiballi et al., 1995, Journal of Clinical Microbiology, 33(4):915-7). The assay uses a redox indicator (Alamar Biosciences, Inc., Sacramento, CA) to determine the colorimetric assay endpoint.

Treatments can also be assayed for antifungal activity using photometric assays well known to those skilled in the art (see, e.g., Clancy et al., 1997 Journal of Clinical Microbiology, 35(11): 2878-82; Jahn et al., 1995, Journal of Clinical Microbiology, 33( 3): 661-667, which is hereby incorporated by reference in its entirety). The basis of this spectrophotometric analysis is the quantification of Analysis of mitochondrial respiration in live fungi. The MIC determined by this assay is defined as the highest concentration of test therapeutic associated with the first sudden drop in optical density. In some embodiments, the antifungal activity of the treatments is determined in parallel using the macrodilution, microdilution, and MTT assays.

Additionally, any in vitro assay known to those of skill in the art may be used to evaluate the prophylactic and/or therapeutic use of the antibodies, compositions and combination treatments described herein in respiratory infection or one or more symptoms thereof.

5.7.2. In vivo experiments

Antibodies, compositions or combination therapies of the invention can be tested in appropriate animal models prior to use in humans. Such animal models include, but are not limited to: rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system known in the art can be used. Several aspects of the procedural steps may vary; such aspects include, but are not limited to, the timing of administration of treatments (eg, prophylactic and/or therapeutic agents), whether the treatments are administered separately or as a mixture, and the frequency with which treatments are administered.

Animal models of autoimmune disease can also be used to assess the efficacy of the antibodies, compositions or combination treatments of the invention. Animal models of autoimmune diseases such as type 1 diabetes, thyroid autoimmune disease, systemic lupus erythematosus and glomerulonephritis have been developed (Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81: 511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

Reduction of one or more symptoms of an autoimmune disease, reduction in mean absolute value of lymphocyte count, reduction in T cell activation, reduction in T cell proliferation, reduction in cell production of cytokines or the ability to modulate one or more specific cytokine properties to demonstrate efficacy in the prevention, treatment and/or management of autoimmune diseases. For example, by detecting the antibody or fragment thereof of the present invention or the composition of the present invention, one or more symptoms of psoriasis can be relieved, the average value of the absolute value of lymphocyte count can be reduced, the production of cytokines can be reduced, and one or more specific psoriasis can be modulated. Cytokine properties, reduce desquamation, reduce erythema, reduce plaque height, reduce T cell activation in the dermis or epidermis of the diseased area, reduce T cell infiltration into the dermis or epidermis of the diseased area, reduce PASI, improve doctor's Overall score or ability to improve quality of life to demonstrate efficacy in preventing or treating psoriasis.

Animal models of cancer can be used to assess the efficacy of antibodies, compositions, or combination therapies of the invention. Examples of animal models of lung cancer include, but are not limited to: the animal model of lung cancer described by Zhang and Roth (1994, In vivo 8(5):755-69) and a transgenic mouse model with disrupted p53 function (see, e.g., Morris et al., 1998 , J La State Med Soc 150(4):179-85). Examples of animal models of breast cancer include, but are not limited to, transgenic mice overexpressing cyclin D1 (see eg, Hosokawa et al., 2001, Transgenic Res 10(5):471-8). Examples of colon cancer animal models include, but are not limited to, TCR b and p53 double knockout mice (see eg, Kado et al., 2001, Cancer Res 61(6):2395-8). Examples of animal models of pancreatic cancer include, but are not limited to: Panc02 murine pancreatic cancer metastasis model (see e.g., Wang et al., 2001, IntJ Pancreatol 29(1):37-46) and nu-nu mice that subcutaneously generate pancreatic tumors (see For example, Ghaneh et al., 2001, Gene Ther 8(3):199-208). Examples of animal models of non-Hodgkin's lymphoma include, but are not limited to: severe combined immunodeficiency ("SCID") mice (see, e.g., Bryant et al., 2000, Lab Invest 80(4):553-73) and IgHmu-HOX11 transgenic Mice (see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA 95(23):13853-8). Examples of animal models of esophageal cancer include, but are not limited to, mice transgenic for the human papillomavirus type 16 E7 oncogene (see, eg, Herber et al., 1996, J Virol 70(3): 1873-81). Examples of animal models of colorectal cancer include, but are not limited to: Apc mouse model (see, e.g., Fodde and Smits, 2001, Trends Mol Med 7(8): 369-73 and Kuraguchi et al., 2000, Oncogene 19(50): 5755 -63).

Arthritis and Allied Conditions: A Textbook of Rheumatism, Arthritis and Allied Conditions: A Textbook of Rheumatology), McCarty (ed.), Chapter 30 (Lee and Febiger, 1993) described various experimental animal models of inflammatory arthritis to determine the anti-inflammatory activity of the antibodies, compositions or combination therapy of the present invention. Experimental and spontaneous animal models of inflammatory arthritis and autoimmune rheumatic diseases can also be used to evaluate the anti-inflammatory activity of the antibodies, compositions or combination therapies of the invention.

Also available is Winter C.A. et al., "Carrageenan-Induced Edema in Hind Paw of the Rat as an Assay for Anti-inflammatory Drugs" (Carrageenan-Induced Edema in Hind Paw of the Rat as an Assay for Anti-inflammatory Drugs) Proc.Soc.Exp A variation of the method described in Biol Med. 111, 544-547 (1962) to assess the anti-inflammatory activity of the antibodies, compositions or combination treatments of the invention by measuring the inhibition of carrageenan-induced paw edema in rats. This assay has been used as a preliminary in vivo screening method for the anti-inflammatory activity of most NSAIDs and is believed to be predictive of efficacy in humans. Anti-inflammatory activity of a test treatment (eg, prophylactic or therapeutic) is expressed as percent inhibition of hind paw weight gain in the test group compared to the vehicle control group.

In a specific embodiment of the present invention, the experimental animal model used is an adjuvant-induced arthritis rat model, and the body weight can be measured relative to the control group to determine the anti-inflammatory activity of the antibody, composition or combination therapy of the present invention.

Animal models of allergy and asthma are known in the art, such as the constant-fow inflation with end-inspiratory occlusion described by Ewart et al., 1995 J Appl Physiol 79(2):560-566 And such as Komai et al, 2003Br J Pharmacol 138(5):912-920; Kenyon et al, 2003Toxicol Appl Pharmacol 186(2):90-100; Path et al, 2002Am J Respand Critical Care Med 166(6):818-826; Martins et al., 1990 Crit Care Med 19:515-519; Nicolaides et al., 1997 Proc Natl Acad Sci USA 94:13175-13180; McLane et al., 1998 19:713-720; and Temann et al., 1998J Exp Med 188(7)::1307 -Other experiments described in 1320. For example, a murine adoptive transfer model is an animal model used to assess the efficacy of an antibody, composition or combination therapy of the invention in preventing, treating and/or controlling asthma. In a murine adoptive transfer model, aeroallergen provocation of TH1 or TH2 recipient mice results in migration of TH effector cells into the airways and in response to strong neutrophil (TH1) and eosinophilic (TH2) lung mucosal inflammation Correlation (Cohn et al., 1997, J. Exp. Med. 1861737-1747). Mice can be induced with ovalbumin (Tomkinson et al., 2001, J. Immunol. 166:5792-5800) or Schistosoma mansoni egg antigen (Tesciuba et al., 2001, J. Immunol. 167:1996-2003). airway hypersensitivity reactions.

Alleviation of one or more symptoms of an inflammatory disease, reduction of T cell activation, reduction of T cell proliferation, modulation of one or more cytokine profiles, reduction of cytokine Proof of prophylaxis or treatment of inflammatory diseases by reducing joint, organ or tissue inflammation or improving quality of life.

Changes in inflammatory disease activity were also assessed by tender and swollen joint counts, patient and physician global scores for pain and disease activity, and ESR/CRP. Progression of structural joint damage can be assessed by quantitative scoring (Sharp method) of radiographs of the hands, wrists, and feet. The Health Assessment Questionnaire (HAQ) can be used to evaluate the changes in the functional status of patients with inflammatory diseases, and the SF can be used to evaluate the changes in the quality of life.

The effectiveness of the antibodies, compositions or combination treatments of the invention in the prevention, treatment and/or management of IgE can be assessed by determining their ability to induce in vitro anti-IgE antibodies that inhibit the binding of IgE to receptors on mast cells or basophils. Efficacy in type allergy. IgE levels can be determined by immunoassay, visual observation after gel electrophoresis, radioimmunosorbent assay (RIST), radioallergensorbent assay (RAST), or any other method known to those skilled in the art.

Animal models of viral infection can also be used to assess the efficacy of the antibodies, compositions or combination treatments of the invention. Viral infections have developed, such as EBV-related disease, gamma herpes virus, infectious mononucleosis, simian immunodeficiency virus ("SIV"), Borna disease virus infection, hepatitis, varicella virus infection, viral pneumonia , Epstein-Barr virus disease, feline immunodeficiency virus (“FIV”), HTLV type 1 infection, human rotavirus, and animal models of genital herpes (see, for example, Hayashi et al., 2002, Histol Histopathol 17 (4): 1293-310; Arico et al., 2002, J Interferon Cytokine Res 22(11): 1081-8; Flano et al., 2002, Immunol Res 25(3): 201-17; Sauermann, 2001, Curr Mol Med1(4): 515-22; Pletnikov et al. , 2002, Front Biosci 7: d593-607; Engler et al., 2001, Mol Immunol 38(6): 457-65; White et al., 2001, Brain Pathol 11(4): 475-9; Davis and Matalon, 2001, News Physiol Sci 16:185-90; Wang, 2001, Curr Top Microbiol Immunol.258:201-19; Phillips et al., 2000, J Psychopharmacol.14(3):244-50; Kazanji, 2000, AIDS Res Hum Retroviruses.16( 16):1741-6; Saif et al., 1996, ArchVirol Suppl. 12:153-61; and Hsiung et al., 1984, Rev Infect Dis. 6(1):33-50).

Viral respiratory infections can also be employed, such as, but not limited to: PIV (see eg, Shephard et al., 2003 Res Vet Sci 74(2):187-190; Ottolini et al., 2002 J Infect Dis 186(12):1713-1717), RSV (See eg, Culley et al., 2002 J Exp Med 196(10):1381-1386; and Curtis et al., 2002 Exp Biol Med 227(9):799-802) for animal models. In a specific embodiment, according to the method of the present invention, the antibody, the composition or the combination therapy of the present invention is administered to cotton rats, stimulated with 10 ⁵ pfu of RSV, and the rats are sacrificed four or more days later, and the RSV titer and anti-RSV Antibody serum titers. Thus, a dose that results in a 2 log reduction or a 99% reduction in RSV titers in cotton rats challenged with 10 ⁵ pfu RSV compared to cotton rats challenged with 10 ⁵ pfu RSV but not administered the formulation is acceptable for administration to humans. Dosages of formulations for the prevention, treatment and/or management of one or more symptoms associated with RSV infection. Additionally, according to this embodiment, histological changes in tissues (eg, lung tissue) of sacrificed rats can be detected.

Antibodies, compositions or combination treatments of the invention can be tested for their ability to reduce the time course of viral infection. Antibodies, compositions or combination treatments of the invention can also be tested for their ability to prolong the survival of virally infected patients by at least 25%, at least 50%, at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% . Additionally, the antibodies, compositions or combination treatments of the invention can be tested for their ability to reduce hospital stay in virally infected patients by at least 60%, at least 75%, at least 85%, at least 95%, or at least 99%. The in vivo function of an antibody, composition or combination therapy of the invention can be assayed using techniques known to those skilled in the art.

Animal models of bacterial infection can also be used to assess the efficacy of the antibodies, compositions or combination treatments of the invention. Has developed bacterial infections such as Helicobacter pylori (H. pylori) infection, genital mycoplasmosis, primary sclerosing cholangitis, cholera, chronic lung infection with Pseudomonas aeruginosa, Legionnaires' disease, gastric - Duodenal ulcer, bacterial meningitis, gastric Helicobacter infection, pneumococcal otitis media, experimental allergic neuritis, leprosy neuropathy, mycobacterial infection, endocarditis, aeromonas Bacteria-associated enteritis, Bacteroides fragilis infection, syphilis, streptococcal endocarditis, acute hematogenous osteomyelitis, human scrub typhus, toxic shock syndrome, anaerobic infection, Escherichia coli (Escherichia coli) infection and animal models of Mycoplasma pneumoniae (Mycoplasmapneumoniae) infection (see, e.g., Sugiyama et al., 2002, J Gastroenterol.37 Suppl. 13:6-9; Brown et al., 2001, Am J Reprod Immunol.46(3): 232-41; Vierling, 2001, Best Pract Res Clin Gastroenterol.15(4):591-610; Klose, 2000, Trends Microbiol.8(4):189-91; Stotland et al., 2000, Pediatr Pulmonol.30(5) : 413-24; Brieland et al., 2000, Immunopharmacology 48(3): 249-52; Lee, 2000, Baillieres Best Pract Res Clin Gastroenterol.14(1): 75-96; Koedel and Pfister, 1999, Infect Dis Clin North Am. 13(3):549-77; Nedrud, 1999, FEMS Immunol Med Microbiol.24(2):243-50; Prellner et al., 1999, Microb Drug Resist.5(1):73-82; Vriesendorp, 1997, J Infect Dis.176 Supplement 2:S164-8; Shetty and Antia, 1996, Indian J Lepr.68(1):95-104; Balasubramanian et al., 1994, Immunobiology 191(4-5):395-401; Carbon et al., 1994, Int J Biomed Comput. 36(1-2): 59- 67; Haberberger et al., 1991, Experientia.47(5):426-9; Onderdonk et al., 1990, Rev Infect Dis.12 Supplement 2:S169-77; Wicher and Wicher, 1989, Crit Rev Microbiol.16(3): 181-234; Scheldt, 1987, JAntimicrob Chemother.20 Supplement A:71-85; Emslie and Nade, 1986, Rev Infect Dis.8(6):841-9; Ridgway et al., 1986, Lab Anim Sci.36(5 ):481-5; Quimby and Nguyen, 1985, Crit Rev Microbiol.12(1):1-44; Onderdonk et al., 1979, Rev Infect Dis.1(2):291-301; Smith, 1976, Ciba Found Symp .(42): 45-72 and Taylor-Robinson, 1976, Infection. 4 (Suppl. 1): 4-8).

The antibody, composition or combination therapy of the invention can be tested to reduce the time course of a bacterial infection, such as a bacterial respiratory infection, by at least 25%, at least 50%, at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% %Ability. Antibodies, compositions or combination treatments of the invention can also be tested for their ability to prolong the survival time of patients with bacterial infections by at least 25%, at least 50%, at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% . In addition, it can be detected that the antibody, composition or combination therapy administered according to the method of the present invention reduces the length of hospitalization of patients with bacterial infections, such as bacterial respiratory tract infections, by at least 60%, at least 75%, at least 85%, at least 95%, or at least 99%. Ability. The in vivo function of an antibody, composition or combination therapy of the invention can be assayed using techniques known to those skilled in the art.

Animal models of infection can be used to assess the efficacy of the antibodies, compositions or combination treatments of the invention in preventing, treating and/or controlling fungal infections. Fungal infections have developed, such as Candida infection, zygomycosis, candidal mastitis, progressive disseminated trichosporosis and latent trichosporon bacteremia, disseminated candidiasis, pulmonary paralysis Coccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioides meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis , paranasal sinus mycosis, Aspergillus fumigatus endocarditis, tibial chondrodysplasia, candidal vaginitis glabrata, oropharyngeal candidiasis, X-linked chronic granulomatous disease, athlete's foot, cutaneous candidiasis, mycotic placenta Inflammation, disseminated trichosporosis, allergic bronchopulmonary aspergillosis, fungal keratitis, Cryptococcus neoformans infection, fungal peritonitis, Curvularia geniculata infection, staphylococcal ocular Animal models of enditis, sporotrichosis, and dermatophytosis (see, e.g., Arendrup et al., 2002, Infection 30(5):286-91; Kamei, 2001, Mycopathologia 152(1):5-13; Guhad et al. , 2000, FEMS Microbiol Lett.192(1):27-31; Yamagata et al., 2000, J Clin Microbiol.38(9):32606; Andrutis et al., 2000, J Clin Microbiol.38(6):2317-23; Cock et al., 2000, Rev Inst Med Trop Sao Paulo42(2):59-66; Shibuya et al., 1999, Microb Pathog.27(3):123-31; Beers et al., 1999, J Lab Clin Med.133(5) Najvar et al., 1999, Antimicrob Agents Chemother.43(2):413-4; Williams et al., 1988, J Infect Dis.178(4):1217-21; Yoshida, 1988, Kansenshokaku Zasshi.1998 Jun;72(6):621-30; Alexandrakis et al., 1998, BrJ Ophthalmol.82(3):306-11; Chakrabarti et al., 1997, J Med Vet Mycol.35(4):295-7; Martin et al. , 1997, Antimicrob Agents Chemother.41 (1): 13-6; Chu et al., 1996 , Avian Dis.40(3):715-9; Fidel et al., 1996, J Infect Dis.173(2):425-31; Cole et al., 1995, FEMS Microbiol Lett.15; 126(2):177-80; Pollock et al., 1995, Nat Genet.9(2):202-9; Uchida et al., 1994, Jpn J Antibiot.47(10):1407-12; Maebashi et al., 1994, J Med VetMycol.32(5):349 -59; Jensen and Schonheyder, 1993, J Exp Anim Sci.35(4): 155-60; Gokaslan and Anaissie, 1992, Infect Immun.60(8): 3339-44; Kurup et al., 1992, JImmunol.148( 12): 3783-8; Singh et al., 1990, Mycopathologia.112(3): 127-37; Salkowski and Balish, 1990, Infect Immun.58(10): 3300-6; Ahmad et al., 1986, Am J Kidney Dis .7(2):153-6; Alture-Werber E, Edberg SC, 1985, Mycopathologia.89(2):69-73; Kane et al., 1981, Antimicrob Agents Chemother.20(5):595-9; Barbee et al., 1977, Am J Pathol. 86(1):281-4; and Maestrone et al., 1973, Am J Vet Res. 34(6):833-6). Fungal respiratory infections have also been developed, such as Candida albicans, Aspergillus fumigatus, invasive pulmonary aspergillosis, Pneumocystis carinii, pulmonary cryptococcosis, Pseudomonas aeruginosa, Cunninghamella bertholletia) (see, e.g., Aratani et al., 2002 Med Mycol 40(6):557-563; Bozza et al., 2002 Microbes Infect 4(13):1281-1290; Kurup et al., 2002 Int Arch Allergy Immunol 129(2):129 -137; Hori et al., 2002Eur J Immuno 32(5):1282-1291; Rivera et al., 2002J Immuno 168(7):3419-3427; Vassallo et al., 2001, Am J Respir Cell Mol Biol 25(2):203- 211; Wilder et al., 2002Am J Respir Cell Mol Biol 26(3):304-314; Yonezawa et al., 2000J Infect Chemother 6(3):155-161; et al., 1998 Mycopathologia 144(3):141-146).

An antibody, composition or combination therapy of the invention can be tested for its ability to reduce the time course of a fungal respiratory infection by at least 25%, at least 50%, at least 60%, at least 75%, at least 85%, at least 95%, or at least 99%. It can also be tested that the antibody, composition or combination therapy of the invention prolongs the survival time of patients with fungal respiratory infection by at least 25%, at least 50%, at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% Ability. Additionally, an antibody, composition or combination therapy administered according to the methods of the invention can be tested for its ability to reduce the length of hospital stay in patients with fungal respiratory infection by at least 60%, at least 75%, at least 85%, at least 95%, or at least 99%. The in vivo function of an antibody, composition or combination therapy of the invention can be assayed using techniques known to those skilled in the art.

In addition, any test known to those skilled in the art can be used to evaluate the effectiveness of the antibodies, compositions or combination therapy described herein in preventing, treating, controlling and/or alleviating the abnormal expression and/or activity of IL-9 polypeptides or related to the abnormal expression and/or activity of IL-9 polypeptides. Disease or disorder characterized by, disease or disorder associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection), or its prophylactic and/or therapeutic use in one or more of its symptoms.

5.7.3. Toxicity test

Can measure LD50 (the dose that makes 50% population dead) and ED50 (the dose that effectively treats in 50% population) in cell culture or experimental animal by standard pharmaceutical method, to determine the prevention and/or treatment scheme of the present invention Toxicity and/or Efficacy. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD50/ED50. Therapies with large therapeutic indices are preferred. While treatments with toxic side effects are available, delivery systems that target such drugs to diseased tissue sites should be carefully designed to minimize potential damage to uninfected cells and thereby reduce side effects.

Dosage ranges for prophylactic and/or therapeutic agents in humans can be formulated using data obtained from cell culture experiments and animal studies. The dosage of such drugs lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration employed. For any treatment employed in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dosage in animal models can be adjusted to achieve a circulating plasma concentration range (as determined in cell culture) that includes the IC50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. For example, plasma levels can be determined by high performance liquid chromatography.

In addition, any assay known to those skilled in the art can be used to assess the efficacy of the antibodies, compositions or combination therapy described herein in diseases or disorders associated with or characterized by abnormal expression and/or activity of IL9 polypeptides, IL- A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity of 9R or one or more subunits thereof, or one of prophylactic and/or therapeutic use in one or more symptoms.

5.8. Diagnostic application of antibody preparations

Antibodies (including molecules comprising, or consisting of, antibody fragments or variants thereof) of the invention that immunospecifically bind to an antigen of interest (such as an IL-9 polypeptide) can be used for diagnostic purposes to detect, diagnose, predict or Monitoring a disease or disorder, for example, a disease or disorder associated with or characterized by abnormal expression and/or activity of an IL9 polypeptide, associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof It is a characterized disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof. The present invention provides a method for detecting abnormal expression of IL-9, the method comprising: (a) measuring IL-9 in a biological sample of an individual with one or more antibodies that immunospecifically bind to the liquid preparation of the IL-9 polypeptide of the present invention; 9 expression; and (b) comparing the IL-9 level with a standard level of IL-9, such as a standard level in a normal biological sample, an increase or decrease in the measured level of IL-9 compared to the standard level of IL-9 indicates a relationship with A disease or disorder associated with or characterized by abnormal expression and/or activity of IL-9 polypeptide, associated with or characterized by abnormal expression and/or activity of IL-9R or one or more subunits thereof A disease or disorder, an autoimmune disease, an inflammatory disease, a proliferative disease or an infection (such as a respiratory infection), or one or more symptoms thereof. In a specific embodiment, an abnormal expression level of IL-9 indicates suffering from an autoimmune disease, or a disease or condition related thereto. In another specific embodiment, an abnormal expression level of IL-9 indicates suffering from an inflammatory disease, or a disease or condition related thereto, such as asthma. In a preferred embodiment, an abnormal expression level of IL-9 indicates a respiratory tract infection, such as but not limited to RSV, PVI or hMPV.

In certain embodiments, labeled antibodies of the liquid formulations of the invention that immunospecifically bind IL-9 are used for diagnostic purposes to detect, diagnose, predict or monitor respiratory tract infections, such as RSV infection, PIV infection or hMPV. The invention provides a method for detecting a respiratory infection, the method comprising: (a) detecting the expression of IL-9 in a cell or tissue sample of a subject using one or more antibodies that immunospecifically bind IL-9; and (b) The level of IL-9 is compared to a control level, eg, in an uninfected normal tissue sample, and an increase in the measured level of IL-9 compared to the IL-9 control level indicates the presence of a respiratory tract infection.

IL-9 levels in biological samples can be determined using antibodies in liquid formulations of the invention by classical immunohistological methods as described herein or known to those skilled in the art (see e.g. Jalkanen et al., 1985, J. Cell. Biol. 101 : 976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105: 3087-3096). Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable labels for antibody assays are known in the art and include enzyme labels such as glucose oxidase; radioactive isotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the present invention is the detection and diagnosis of diseases or disorders associated with abnormal expression of IL-9 in animals, preferably mammals, most preferably humans. In one embodiment, the diagnosis comprises: a) administering (e.g., parenterally, subcutaneously or intraperitoneally) an effective amount of a labeled antibody (including an antibody fragment comprising an antibody fragment) of the liquid formulation of the invention that immunospecifically binds to an IL-9 polypeptide or a variant thereof, or a molecule composed thereof); b) waiting for a period of time after administration to allow for preferential concentration of the labeled antibody at sites expressing IL-9 in the subject (and clearance of unbound labeled molecule to background levels ); c) determining the background level; and d) detecting the labeled antibody in the subject, detection of the labeled antibody (including antibody fragments thereof) above the background level and above or below the level in a person not suffering from the disease or disorder indicates that the The subject suffers from a particular disease or disorder associated with abnormal expression of IL-9. Background levels can be determined by a variety of methods, including comparison of the detected molecular weight of the marker to previously determined standards for the particular system. Aberrant expression of IL-9 can occur especially in lymphoid and myeloid cell types. Diagnosing respiratory infections with greater certainty could allow health care workers to initiate preventive or aggressive treatment earlier, preventing the onset or progression of infection.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. Where radioisotopic moieties are used, the amount of radiation injected typically ranges from about 5-20 millicuries ^of99Tc for human subjects. The labeled antibody then preferentially accumulates at the site of the cell that contains the specific protein. For in vivo tumor imaging see SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Tumor Imaging: The Radiochemical Detection of Cancer), p. 13 Chapter, eds. SW Burchiel and BARhodes, Masson Publishing Inc. (1982)). Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to preferentially concentrate the labeled molecule at a site in the subject and clear the unbound labeled molecule to background levels is 6 to 48 hours, 6 to 24 hours or 6 to 12 hours. In another embodiment, the interval between events following administration is 5 to 20 days or 5 to 10 days.

In one embodiment, the disease or disorder is monitored by repeating the method of diagnosing the disease or disorder, eg, one month after the initial diagnosis, 6 months after the initial diagnosis, or 1 year after the initial diagnosis, etc.

The presence or absence of labeled IL-9 antibody in a patient can be detected using methods known in the art for in vivo scanning. These methods depend on the type of marker used. Those skilled in the art will be able to determine methods suitable for detecting a particular marker. Methods and devices that may be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound imaging.

In a specific embodiment, the IL-9 antibody is labeled with a radioactive isotope and detected in a patient using a radiation-responsive surgical device (Thurston et al., US Patent No. 5,441,050). In another embodiment, the IL-9 antibody is labeled with a fluorescent compound and detected in a patient using a fluorescence-responsive scanning device. In another embodiment, the IL-9 antibody is labeled with a positron emitting metal and detected in a patient using positron emission tomography. In yet another embodiment, the IL-9 antibody is labeled with a paramagnetic marker and detected in a patient using magnetic resonance imaging (MRI).

Antibodies of the invention (including molecules comprising or consisting of antibody fragments or variants thereof) can be used to perform immunophenotyping of cell lines and biological samples based on IL-9 expression or IL-9 receptor expression. Cell populations (cell populations expressing IL-9 and/or IL-9 receptors, particularly immune cells, i.e. T and B lymphocytes, Mast cells, eosinophils, macrophages, neutrophils, and epithelial cells or IL-9 receptors, including magnetic separation using antibody-coated magnetic beads, using antibodies attached to solid substrates (i.e., plates) and Flow cytometry "panning" (see eg, US Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques enable the screening of specific cell populations, such as those found in hematological malignancies (i.e., minimal residual disease (MRD) in acute leukemia patients) and "non-self" cells in transplants, for the prevention of graft-versus-host disease ( GVHD). Alternatively, these techniques can screen for hematopoietic stem and progenitor cells capable of proliferation and/or differentiation, such as those found in human umbilical cord blood.

5.9. Medicine box

The invention provides a pharmaceutical pack or kit comprising one or more containers containing a liquid formulation of the invention. In a specific embodiment, the liquid preparation of the present invention comprises an antibody (including antibody fragments thereof) recombinantly fused or chemically coupled to another part, including but not limited to: heterologous protein, heterologous polypeptide, heterologous peptide , macromolecules, small molecules, labeling sequences, diagnostic or detection reagents, therapeutic moieties, drug moieties, radioactive metal ions, secondary antibodies, and solid supports. The present invention also provides a pharmaceutical pack or kit comprising one or more first containers containing the liquid formulation of the present invention and one or more second containers containing one or more of the second containers. Various other prophylactic or therapeutic agents for the prevention, control or treatment of diseases or disorders, for example, diseases or disorders associated with or characterized by abnormal expression and/or activity of IL9 polypeptides, IL-9R or one of its A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory infection) associated with or characterized by abnormal expression and/or activity of multiple subunits, or one or more of symptom. In one embodiment, liquid formulations of the invention are formulated in single dose vials to form sterile liquids containing 50 mM phosphate buffer, pH 6.2, and 150 mM sodium chloride. The formulations of the invention can be packaged in 3cc USP Type I borosilicate glass amber vials (West Pharmaceutical Serices - No. 6800-0675) with a target volume of 1.2 mL. The container is optionally affixed with a notice issued by a governmental agency for the manufacture, use or sale of the pharmaceutical or biological product, which notice reflects the approval by the regulatory agency of manufacture, use or sale for use in humans.

The present invention provides kits useful in the above methods. In one embodiment, a kit comprises one or more containers containing a liquid formulation of the invention. In another embodiment, the kit comprises the liquid formulation of the present invention in one or more containers, and one or more of the drugs for preventing, controlling or treating a disease or disorder in another one or more containers. Other prophylactic or therapeutic agents, the disease or disorder may be related to or characterized by abnormal expression and/or activity of IL-9 polypeptide, and IL-9R or one or more subunits thereof A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (such as a respiratory tract infection) associated with or characterized by abnormal expression and/or activity, or one or more symptoms thereof. In a specific embodiment, the antibody (including antibody fragments thereof) contained in the liquid formulation is 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com - 3H5 or 7F3com-3D4, or an antigen-binding fragment thereof. In another embodiment, the antibody (including antibody fragments thereof) contained in the liquid formulation is not 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com- 2H2, 7F3com-3H5 or 7F3com-3D4 or an antigen-binding fragment thereof. Preferably, the kit also includes instructions for preventing, treating and/or controlling diseases (for example, using the liquid preparation of the present invention alone or in combination with another preventive or therapeutic agent), as well as instructions for dosage information of side effects and administration methods.

5.10. Products

The invention also includes packaged and labeled finished pharmaceutical products. Unit dosage forms are prepared in suitable vessels or containers, such as hermetically sealed glass pharmaceutical or other containers. In the case of dosage forms suitable for parenteral administration, the active ingredient, such as an antibody of the invention that immunospecifically binds an antigen of interest (such as an IL-9 polypeptide), is sterile and suitable for administration as a particle-free solution . In other words, the invention includes sterile parenteral solutions and lyophilized powders suitable for reconstitution prior to injection. Alternatively, the unit dosage form can be a solid suitable for oral, transdermal, intranasal or topical delivery.

In one embodiment, the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery. Accordingly, the present invention includes solutions, preferably sterile solutions, suitable for each route of delivery.

As with any pharmaceutical product, packaging materials and containers are designed to protect the product's stability during storage and transport. In addition, the products of the invention include instructions for use or other instructional material instructing physicians, technicians or patients how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instructional material indicating or suggesting a dosing regimen, including but not limited to: actual doses, monitoring procedures, total lymphocytes, mast cell counts, T cell counts, IgE production, and other monitoring information.

In particular, the present invention provides articles of manufacture comprising packaging materials including, for example, boxes, jars, tubes, vials, containers, nebulizers, insufflators, intravenous (i.v.) administration packs, envelopes, and the like; The packaging material contains at least one unit dosage form of the medicament, wherein the medicament comprises a liquid formulation comprising the antibody. The packaging material includes indications that the antibody can be used to prevent, treat and/or control diseases related to abnormal expression and/or activity of IL-9 polypeptides, etc. One or more symptoms of a disease, autoimmune disease, inflammatory disease, proliferative disease, infection (such as a respiratory infection) associated with abnormal expression and/or activity of IL-9R or one or more subunits thereof, or Descriptive material for one or more of their symptoms.

The invention also provides articles of manufacture comprising packaging material, such as a box, jar, tube, vial, container, nebulizer, insufflator, intravenous (i.v.) administration pack, envelope, etc.; said packaging material containing at least one unit dosage form Each of the medicaments, wherein one of the medicaments comprises a liquid preparation comprising an antibody of interest, such as an antibody that immunospecifically binds to an IL-9 polypeptide, and the other medicament comprises a second different antibody that immunospecifically binds to an IL-9 polypeptide, wherein The packaging material includes instructions indicating that the medicament can be used to prevent, treat and/or control diseases related to abnormal expression and/or activity of IL-9 polypeptide, and Diseases related to abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative diseases, infections (such as respiratory tract infections) or one or more thereof Descriptive material for symptoms.

The invention also provides articles of manufacture comprising packaging material, such as a box, jar, tube, vial, container, nebulizer, insufflator, intravenous (i.v.) administration pack, envelope, etc.; said packaging material containing at least one unit dosage form Each of the medicaments, wherein one of the medicaments includes a liquid preparation containing an antibody of interest, such as an antibody that immunospecifically binds to an IL-9 polypeptide, and the other medicament includes a prophylactic agent other than an antibody that immunospecifically binds to an IL-9 polypeptide or Therapeutic agent, wherein the packaging material includes an indication that the agent can be used to prevent, treat and/or control the abnormal expression and/or activity of the IL-9 polypeptide by administering a specific dose and using a specific dosing regimen as described herein. diseases associated with abnormal expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative diseases, infections (such as respiratory tract infections), or Descriptive material for multiple symptoms or one or more of them.

The invention provides that an article of manufacture for the prevention, treatment and/or management of one or more symptoms associated with an autoimmune disease, an inflammatory disease or an infection includes instructional material demonstrating that adverse effects can be mitigated or avoided by the methods of the invention . Adverse effects that may be mitigated or avoided by the methods of the present invention include, but are not limited to: abnormalities in vital signs (fever, tachycardia, bradycardia, hypertension, hypotension), blood problems (anemia, lymphopenia, leukopenia, thrombocytopenia) reduction), headache, chills, dizziness, nausea, weakness, back pain, chest pain (chest tenderness), diarrhea, myalgia, pain, pruritus, psoriasis, rhinitis, diaphoresis, injection site reactions, and vasodilation.

In addition, for the prevention, treatment and/or control of diseases or disorders, such as diseases or disorders related to or characterized by abnormal expression and/or activity of IL-9 polypeptides, IL-9R or one or more of its subtypes A disease, inflammatory disease, autoimmune disease, proliferative disease or infection (such as a respiratory infection) associated with or characterized by the abnormal expression and/or activity of the unit, or one or more symptoms thereof Guidance material may indicate that foreign proteins can also cause allergic reactions, including anaphylaxis or cytosine release syndrome. The guidance material should state that an allergic reaction may manifest as only a mild pruritic rash, or it may be a serious reaction such as erythroderma, Stevens-Johnson syndrome, vasculitis, or anaphylaxis. The guidance material should also state that anaphylaxis (anaphylaxis) is a serious, sometimes fatal, hypersensitivity reaction. When any foreign protein is injected into the human body, it may cause allergic reactions, including anaphylaxis. They can range from mild manifestations, such as hives or rashes, to fatal systemic reactions. Anaphylaxis occurs rapidly (usually within 10 minutes) after exposure. Patients may develop paresthesias, hypotension, laryngeal edema, altered mental status, facial or pharyngeal angioedema, airway obstruction, bronchospasm, urticaria and pruritus, serum sickness, arthritis, allergic nephritis, glomerular Nephritis, transient arthritis, or eosinophilia.

6. Specific implementation

CLAIMS 1. An antibody preparation comprising an aqueous carrier, phosphate, and an antibody or antibody fragment at a concentration of 50 mg/ml or greater, said antibody preparation being formulated for administration to a human subject.

2. An antibody preparation formulated to be suitable for administration to a human subject, said preparation comprising an aqueous carrier, a phosphate salt, and an antibody or antibody fragment at a concentration of 10 mg/ml or higher, wherein the antibody is administered at a pH lower than that of said antibody in the presence of a salt One or more of the following phase properties of the antibody or antibody fragment is reduced when formulated in phosphate buffer at the pI compared to the antibody formulated in histidine buffer at the pH in the presence of the same concentration of salt :

a) forming an unfolded intermediate;

b) colloidal instability;

c) soluble conjugation of antibody molecules; or

d) precipitation of antibody molecules;

Wherein said at least one or more phase properties are determined by a technique selected from the group consisting of: High Performance Size Exclusion Chromatography (HPSEC) technique, Tangential Flow Filtration (TFF) technique, Static Light Scattering (SLS) technique, Fu Leaf transform infrared spectroscopy (FTIR) technology, circular dichroism (CD) technology, urea-induced protein unfolding technology, tryptophan intrinsic fluorescence technology, differential scanning calorimetry (DSC) and 1-anilino- 8-naphthalenesulfonic acid (ANS) protein binding technology.

3. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment immunospecifically binds to the IL-9 polypeptide.

4. The formulation of embodiment 1 or 2, wherein the aqueous carrier is distilled water.

5. The formulation of embodiment 1 or 2, wherein the pH of the formulation is in the range of 4.0 to 8.0.

6. The formulation of embodiment 5, wherein the pH range is about 6.0 to 6.5.

7. The formulation of embodiment 1 or 2, further comprising a salt at a concentration of no more than about 200 mM.

8. The formulation of embodiment 7, wherein the concentration of the salt ranges from about 100 mM to about 200 mM.

9. The formulation of embodiment 6, further comprising a salt in a concentration ranging from about 100 to about 200 mM.

10. The formulation of embodiment 1 or 2, further comprising sugar.

11. The formulation of embodiment 6, further comprising sugar.

12. The formulation of embodiment 10, wherein the sugar is sucrose.

13. The formulation of embodiment 11, wherein the sugar is sucrose.

14. The formulation of embodiment 10, wherein the sugar is trehalose.

15. The formulation of embodiment 11, wherein the sugar is trehalose.

16. The formulation of embodiment 12, wherein the sucrose concentration is up to 10%.

17. The formulation of embodiment 13, wherein the sucrose concentration is up to 10%.

18. The formulation of embodiment 14, wherein the concentration of trehalose is up to 10%.

19. The formulation of embodiment 15, wherein the concentration of trehalose is up to 10%.

20. The formulation of embodiment 1 or 2, further comprising a surfactant.

21. The formulation of embodiment 6, further comprising a surfactant.

22. The formulation of embodiment 9, further comprising a surfactant.

23. The formulation of embodiment 21, wherein the surfactant is Tween-20 or Tween-80.

24. The formulation of embodiment 22, wherein the surfactant is Tween-20 or Tween-80.

25. The formulation of embodiment 23, wherein the concentration of the surfactant is up to 0.1%.

26. The formulation of embodiment 24, wherein the concentration of the surfactant is up to 0.1%.

27. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment is at a concentration of at least 100 mg/ml.

28. The formulation of embodiment 1 or 2, wherein the concentration of the antibody or antibody fragment ranges from about 50 mg/ml to about 150 mg/ml.

29. The formulation of embodiment 1 or 2, wherein the concentration of phosphate ranges from about 10 mM to about 100 mM.

30. The formulation of embodiment 29, wherein the concentration of phosphate ranges from about 25 mM to about 75 mM.

31. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment has a stability of at least 15 days during storage at 40°C as determined by high performance size exclusion chromatography (HPSEC).

32. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment has a stability of at least 6 months during storage at room temperature as determined by HPSEC.

33. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment has a stability of at least 1.5 years during storage at 4°C as determined by HPSEC.

34. The formulation of embodiment 1 or 2, wherein less than 5% of the antibody or antibody fragment forms aggregates during storage as determined by HPSEC.

35. The formulation of embodiment 6, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

36. The formulation of embodiment 9, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

37. The formulation of embodiment 1 or 2, wherein less than 2% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

38. The formulation of embodiment 6, wherein less than 2% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

39. The formulation of embodiment 9, wherein less than 2% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

40. The formulation of embodiment 1 or 2, wherein less than 1% of the antibody or antibody fragment forms aggregates during storage as determined by HPSEC.

41. The formulation of embodiment 6, wherein less than 1% of the antibody or antibody fragment forms aggregates during storage as determined by HPSEC.

42. The formulation of embodiment 9, wherein less than 1% of the antibody or antibody fragment forms aggregates during storage as determined by HPSEC.

43. The formulation of embodiment 1 or 2, wherein the antibody or fragment thereof retains at least 80% of its binding capacity compared to a reference antibody representing the pre-storage antibody.

44. The formulation of embodiment 6, wherein the antibody or fragment thereof retains at least 80% of its binding capacity compared to a reference antibody representing the pre-storage antibody.

45. The formulation of embodiment 9, wherein the antibody or fragment thereof retains at least 80% of its binding capacity compared to a reference antibody representing the pre-storage antibody.

46. The formulation of embodiment 1 or 2, wherein the antibody or fragment thereof retains at least 85% of its binding capacity compared to a reference antibody.

47. The formulation of embodiment 6, wherein the antibody or fragment thereof retains at least 85% of its binding capacity compared to a reference antibody.

48. The formulation of embodiment 9, wherein the antibody or fragment thereof retains at least 85% of its binding capacity compared to a reference antibody.

49. The formulation of embodiment 1 or 2, wherein the antibody or fragment thereof retains at least 90% of its binding capacity compared to a reference antibody.

50. The formulation of embodiment 6, wherein the antibody or fragment thereof retains at least 90% binding capacity compared to a reference antibody.

51. The formulation of embodiment 9, wherein the antibody or fragment thereof retains at least 90% of its binding capacity compared to a reference antibody.

52. The formulation of embodiment 1 or 2, wherein the antibody or fragment thereof retains at least 95% of its binding capacity compared to a reference antibody.

53. The formulation of embodiment 6, wherein the antibody or fragment thereof retains at least 95% of binding capacity compared to a reference antibody.

54. The formulation of embodiment 9, wherein the antibody or fragment thereof retains at least 95% of binding capacity compared to a reference antibody.

55. The formulation of embodiment 1 or 2, wherein the antibody or antibody fragment is 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com - 3H5 or 7F3com-3D4, or an antigen-binding fragment thereof.

56. The formulation of embodiment 6, wherein the antibody or antibody fragment is 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4, or an antigen-binding fragment thereof.

57. The formulation of embodiment 9, wherein the antibody or antibody fragment is 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com- 3H5 or 7F3com-3D4, or an antigen-binding fragment thereof.

58. The formulation of embodiment 57, wherein the antibody or antibody fragment is 7F3com-2H2.

59. A pharmaceutical unit dosage form suitable for parenteral administration to a human comprising the antibody formulation of embodiment 1 or 2 in a suitable container.

60. The pharmaceutical unit dosage form of embodiment 59, wherein said antibody formulation is for intravenous, subcutaneous or intramuscular injection.

61. A pharmaceutical unit dosage form suitable for aerosol administration to a human comprising the antibody formulation of embodiment 1 or 2 in a suitable container.

62. The pharmaceutical unit dosage form of embodiment 61, wherein the antibody formulation is administered intranasally.

63. An antibody formulation produced by lyophilization of the aqueous antibody formulation of embodiment 1 or 2.

64. A sealed container containing the formulation of embodiment 1 or 2.

65. A sealed container containing the formulation of embodiment 9.

66. The sealed container of embodiment 64, which has a volume sufficient for reconstitution with a pharmaceutically acceptable carrier.

67. The sealed container of embodiment 66, wherein the carrier is water for injection, USP, 5% dextrose in water (D5W) or saline.

68. The sealed container of embodiment 67, wherein the container is capable of maintaining a sterile environment and reconstituting the formulation without loss of sterility.

69. A kit comprising an antibody preparation comprising an aqueous carrier, phosphate and an antibody or fragment thereof at a concentration of 50 mg/ml or greater in one or more containers and instructions for use of the preparation , and is formulated suitable for administration to a human subject.

70. The kit of embodiment 69, wherein the formulation is a sterile formulation.

71. The kit of embodiment 69, wherein the aqueous carrier is distilled water.

72. The kit of embodiment 70, wherein the antibody or antibody fragment is 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com- 3H5 or 7F3com-3D4, or an antigen-binding fragment thereof.

73. The kit of embodiment 72, wherein the antibody or antibody fragment is 7F3com-2H2.

74. The kit of embodiment 70, 72 or 73, wherein said formulation is produced by lyophilization of an aqueous antibody formulation.

75. The kit of embodiment 70 or 73, wherein the antibody or antibody fragment is present at a concentration of about 50 mg/ml to about 150 mg/ml.

76. A method for preventing, controlling, treating or improving inflammatory diseases, autoimmune diseases, diseases related to abnormal expression and/or activity of IL-9 polypeptide, related to or related to abnormal expression and/or activity of IL-9 polypeptide Diseases or disorders characterized by, associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative A method for a disease or infection (preferably a respiratory infection), or one or more symptoms thereof, comprising administering a prophylactically or therapeutically effective amount of the antibody preparation of embodiment 56 to a subject in need thereof.

77. A disease or disorder that prevents, controls, treats or improves the abnormal expression and/or activity of IL-9 polypeptide or is characterized by it, and the abnormal expression and A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (preferably a respiratory tract infection) associated with or characterized by the activity, or a method for one or more symptoms thereof comprising administering A prophylactically or therapeutically effective amount of the antibody preparation described in Embodiment 57 for a subject in need.

78. A disease or disorder that prevents, controls, treats or improves the abnormal expression and/or activity of IL-9 polypeptide or is characterized by it, and the abnormal expression and A disease or disorder, autoimmune disease, inflammatory disease, proliferative disease or infection (preferably a respiratory tract infection) associated with or characterized by the activity, or a method for one or more symptoms thereof comprising administering A prophylactically or therapeutically effective amount of the antibody preparation according to Embodiment 58 for a subject in need.

79. The method of embodiment 76, wherein the antibody or antibody fragment polypeptide thereof has a stability of at least 15 days during storage at 40° C. as determined by HPSEC.

80. The method of embodiment 77, wherein the antibody or antibody fragment polypeptide thereof has a stability of at least 15 days during storage at 40° C. as determined by HPSEC.

81. The method of embodiment 78, wherein the antibody or antibody fragment polypeptide thereof has a stability of at least 15 days during storage at 40° C. as determined by HPSEC.

82. The method of embodiment 76, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

83. The method of embodiment 77, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

84. The method of embodiment 78, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

85. The method of embodiment 76, wherein the antibody or fragment thereof retains at least 80% of its ability to bind the IL-9 polypeptide compared to a reference antibody representing the pre-storage antibody or antibody fragment.

86. The method of embodiment 77, wherein the antibody or fragment thereof retains at least 80% of its ability to bind the IL-9 polypeptide compared to a reference antibody representing the pre-storage antibody or antibody fragment.

87. The method of embodiment 78, wherein the antibody or fragment thereof retains at least 80% of its ability to bind the IL-9 polypeptide compared to a reference antibody representing the pre-storage antibody or antibody fragment.

88. The method of embodiment 76, wherein the formulation is administered parenterally.

89. The method of embodiment 77, wherein the formulation is administered parenterally.

90. The method of embodiment 78, wherein the formulation is administered parenterally.

91. The method of embodiment 76, wherein the formulation is administered subcutaneously, orally, or intranasally.

92. The method of embodiment 77, wherein the formulation is administered subcutaneously, orally, or intranasally.

93. The method of embodiment 78, wherein the formulation is administered subcutaneously, orally, or intranasally.

94. The method of embodiment 76, wherein the inflammatory disease is asthma or allergy.

95. The method of embodiment 77, wherein the inflammatory disease is asthma or allergy.

96. The method of embodiment 78, wherein the inflammatory disease is asthma or allergy.

97. An antibody formulation for administration to a subject, said formulation comprising an aqueous carrier, phosphate, and 7F3com-2H2 or an antigen-binding fragment thereof at a concentration of 50 mg/ml or greater.

98. The formulation of embodiment 97, wherein the formulation is sterile.

99. The formulation of embodiment 97, wherein the aqueous carrier is distilled water.

100. The formulation of embodiment 97 or 98, wherein the pH of the formulation is in the range of 4.0 to 8.0.

101. The formulation of embodiment 100, wherein the pH range is about 6.0 to 6.5.

102. The formulation of embodiment 97 or 98, further comprising a salt at a concentration of not more than about 200 mM.

103. The formulation of embodiment 102, wherein the concentration of the salt ranges from about 100 mM to about 200 mM.

104. The formulation of embodiment 101, wherein the concentration of the salt ranges from about 100 mM to about 200 mM.

105. The formulation of embodiment 97 or 98, further comprising a sugar.

106. The formulation of embodiment 101, further comprising a sugar.

107. The formulation of embodiment 105, wherein the sugar is sucrose.

108. The formulation of embodiment 106, wherein the sugar is sucrose.

109. The formulation of embodiment 105, wherein the sugar is trehalose.

110. The formulation of embodiment 106, wherein the sugar is trehalose.

111. The formulation of embodiment 107, wherein the concentration of sucrose is up to 10%.

112. The formulation of embodiment 108, wherein the sucrose concentration is up to 10%.

113. The formulation of embodiment 109, wherein the concentration of trehalose is up to 10%.

114. The formulation of embodiment 110, wherein the concentration of trehalose is up to 10%.

115. The formulation of embodiment 97 or 98, further comprising a surfactant.

116. The formulation of embodiment 101, further comprising a surfactant.

117. The formulation of embodiment 104, further comprising a surfactant.

118. The formulation of embodiment 116, wherein the surfactant is Tween-20 or Tween-80.

119. The formulation of embodiment 117, wherein the surfactant is Tween-20 or Tween-80.

120. The formulation of embodiment 118, wherein the concentration of Tween-20 or Tween-80 is up to 0.1%.

121. The formulation of embodiment 119, wherein the concentration of Tween-20 or Tween-80 is up to 0.1%.

122. The formulation of embodiment 97 or 98, wherein the antibody or antibody fragment is at a concentration of at least 100 mg/ml.

123. The formulation of embodiment 97 or 98, wherein the concentration of the antibody or antibody fragment ranges from about 50 mg/ml to about 150 mg/ml.

124. The formulation of embodiment 97 or 98, wherein the concentration of phosphate ranges from about 10 mM to about 100 mM.

125. The formulation of embodiment 123, wherein the concentration of phosphate ranges from about 25 mM to about 75 mM.

126. The formulation of embodiment 124, wherein the concentration of phosphate ranges from about 25 mM to about 75 mM.

127. The formulation of embodiment 97 or 98, wherein the antibody or antibody fragment has a stability of at least 15 days during storage at 40°C, as determined by high performance size exclusion chromatography (HPSEC).

128. The formulation of embodiment 97 or 98, wherein the antibody or antibody fragment has a stability of at least 6 months during storage at room temperature as determined by HPSEC.

129. The formulation of embodiment 97 or 98, wherein the antibody or antibody fragment has a stability of at least 1.5 years during storage at 4°C as determined by HPSEC.

130. The formulation of embodiment 97 or 98, wherein less than 5% of the antibody or antibody fragment forms aggregates during storage as determined by HPSEC.

131. The formulation of embodiment 101, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

132. The formulation of embodiment 104, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

133. A method of preventing, treating or controlling a disease or disorder, said method comprising administering a prophylactically or therapeutically effective amount of the antibody preparation of embodiment 1 or 2 to a subject in need thereof.

134. An antibody formulation administered to a subject, said antibody formulation comprising an aqueous carrier, about 50 mg/ml to 150 mg/ml 7F3com-2H2 antibody, about 10 mM to about 75 mM phosphate, about 100 mM to 200 mM NaCl, wherein said formulation The pH is about 5.5 to 6.5.

135. The formulation of embodiment 134, wherein the formulation comprises an aqueous carrier, about 100 mg/ml 7F3com-2H2 antibody, about 25 mM phosphate, about 150 mM NaCl, wherein the pH of the formulation is about 6.0.

136. The formulation of embodiment 135, wherein the formulation is isotonic.

137. The formulation of embodiment 135, wherein the antibody or antibody fragment has a stability of at least 15 days during storage at 40°C, as determined by high performance size exclusion chromatography (HPSEC).

138. The formulation of embodiment 135, wherein the antibody or antibody fragment has a stability of at least 6 months during storage at room temperature as determined by HPSEC.

139. The formulation of embodiment 135, wherein the antibody or antibody fragment has a stability of at least 1.5 years during storage at 4°C as determined by HPSEC.

140. The formulation of embodiment 135, wherein less than 5% of the antibodies or antibody fragments form aggregates during storage as determined by HPSEC.

141. A method for preventing, controlling, treating or improving inflammatory diseases, autoimmune diseases, diseases related to abnormal expression and/or activity of IL-9 polypeptide, related to abnormal expression and/or activity of IL-9 polypeptide or Diseases or disorders characterized by, associated with or characterized by aberrant expression and/or activity of IL-9R or one or more subunits thereof, autoimmune diseases, inflammatory diseases, proliferative A method for a disease or infection (preferably a respiratory infection), or one or more symptoms thereof, comprising administering a prophylactically or therapeutically effective amount of the antibody preparation of embodiment 135 to a subject in need thereof.

7. Example

7.1. Antibody purification and antibody preparation

The following sections describe methods for purifying antibodies for use in formulations of the invention (see Figure 16).

7.1.1. Buffer components and equipment

Test buffers for bioburden and endotoxin.

Buffers and Processing Solutions

0.1M citric acid, 10mM sodium citrate, 80mM NaCl, pH 4.6

25mM sodium phosphate, pH 6.5

20mM Tris-HCl, 40mM NaCl, pH 7.5

0.5M sodium phosphate, pH 6.5

5mM sodium phosphate, 40mM NaCl, pH 6.5

50mM Glycine-HCl, 30mM NaCl, pH 2.5

50mM Glycine-HC, pH 2.35

1.0M Tris base

Cleaning and Storage Solutions

Water for Injection (WFI)

1.0N NaOH

0.1N NaOH

20% (v/v) ethanol

0.5N NaOH, 400ppm sodium hypochlorite

formulation buffer

≥10mg/mL antibody of interest

10-100mM phosphate buffer, pH 4-8

0-200mM NaCl, or 0-10% sucrose or trehalose

0-0.1% Tween-20 or Tween-80

Equipment (replacement with equivalent performing material is acceptable)

300kg balance

conductivity meter

stirring plate

pH meter

Containers: Stedim ^TM bags of appropriate size, buffer tanks, PETG bottles

Watson Marlow 1700 Peristaltic Pump

Wedgewood UV, pH, Conduction Unit

Amersham Pharmacia Chromatography Controller

Filled POROS HS50 Cation Exchange Gel

Packed Pharmaciar Protein A Affinity Gels

Packed POROS HQ Anion Exchange Gel

Sterile, depyrogenated silicone tubing

Integritest Filter Integrity Tester II

Sterile Asahi Planova 20N Membrane Virus Removal Filters

Millipore 0.2 micron Durapore filter

Millipore Multimedia Filter

CUNO 60LP, 10/60SP filter

CUNO filter housing

Class 100 Fume Hood

7.1.2. Antibody purification and preparation

Figure 16 outlines the process steps for purifying and formulating antibodies of the invention, including antibody fragments thereof. The purification method consisted of three chromatography steps, a nanofiltration step, a low pH treatment step and formulation steps. These steps are designed to remove host cell proteins, DNA, and cell culture components such as BSA and transferrin. Additionally, the method includes steps to control bioburden and endotoxins as well as remove and inactivate viruses.

7.1.2.1. Conditioned Media (Steps 1-6)

Purify conditioned medium from a single cell culture batch or pooled from multiple cell culture batches into a single batch. The purpose of combining multiple cell culture batches into one purification batch is to take advantage of downstream processing steps that are sized to a single batch and reduce the number of purification batches. For example, since the working volumes of the 130L and 250L cell culture bioreactors are approximately 100L and 200L, respectively, the two cell culture batches can be pooled and run as one 300L purification batch. Analyze DNA from processed product samples using PicoGreen or quantitative PCR assays to detect DNA. Protein concentration can be determined by protein A combined HPLC or by UV absorbance at 280 nm. Monitoring of endotoxins and bioburden in product-containing industrial process streams. Monitor the column effluent for endotoxin. A description of each step is summarized below.

7.1.2.2. Conditioned medium adjustment and filtration (step 7)

Conditioned medium was adjusted to pH 4.6 ± 0.2 with 0.1 M citric acid. The adjusted conditioned medium was then filtered using a CUNO filter and a Millipore 0.2 micron Durapore filter in series.

7.1.2.3. Cation exchange chromatography step (step 8)

Conditioned and filtered conditioned medium was loaded onto a cation exchange column equilibrated with 10 mM sodium phosphate, 80 mM sodium chloride, pH 4.6. Bound antibodies were washed with the same buffer. The column was then washed with 25 mM sodium phosphate pH 6.5 to remove processing impurities, especially BSA. The product was eluted using 20 mM Tris-HCl buffer, 40 mM NaCl, pH 7.5. After product elution, the column was cleaned with 1.0N NaOH and stored in 0.1N NaOH at room temperature.

7.1.2.4.r Protein A Chromatography (Step 9)

The cation-exchange product was directly loaded onto an r-protein A column equilibrated with 20 mM Tris-HCl buffer, 40 mM NaCl, pH 7.5. After loading, the column was washed with equilibration buffer and the product was eluted with 50 mM glycine, 30 mM NaCl, pH 3.2. The r-protein A product was neutralized to pH 6.5 ± 0.2 with 1.0 M Tris base. This chromatographic step removes additional processing-related impurities. At the end of this step, the column was washed with equilibration buffer, cleaned with 0.1 N NaOH, washed with equilibration buffer and stored in 20% (v/v) ethanol at room temperature.

7.1.2.5. Anion exchange chromatography (step 10)

This chromatographic step is the final step designed to remove any traces of processing-related impurities. The column was equilibrated with 0.5M sodium phosphate, pH 6.5, then with 5mM sodium phosphate, 40mM sodium chloride, pH 6.5. Under these conditions, the neutralized r-protein A product is loaded onto an equilibrated anion-exchange column, and under these conditions, the product is recovered in the unbound fraction while processing-related impurities remain on the column. The column was cleaned with 1.0N NaOH and kept in 0.1N NaOH at room temperature.

7.1.2.6. Nanofiltration (step 11)

The anion exchange product was filtered through a sterile Planova ^™ 20N membrane (pore size = 20 nm) prepared by rinsing with WFI followed by 5 mM sodium phosphate, 40 mM sodium chloride pH 6.5. After filtering the product, the filter was chased with a small volume of 5 mM sodium phosphate, 40 mM sodium chloride, pH 6.5, to maximize product recovery. After filtration, test the integrity of the nanofilter.

7.1.2.7 Low pH treatment (step 12)

The pH of the nanofiltration product was adjusted to 3.4 ± 0.1 with 50 mM glycine, pH 2.35, and maintained at this pH for 30 ± 10 minutes. After low pH treatment, the product pH was adjusted to 6.5±0.2 with 1.0M Tris base.

7.1.2.8. Preparation of anti-IL-9 antibody

储存袋中。通过大小排阻色谱(SEC)确认样品纯度。Sodium chloride (150 mM) was added to the drug intermediate via 4M NaCl stock solution. Then, 0.2 micron filtration was performed and tangential flow filtration was performed through a 30 kD membrane to concentrate to 20 g/L. The product was then diafiltered into formulation buffer with at least five buffer changes. Then, the product was concentrated to 100 g/L and filtered through a 1.2/0.2 micron membrane to sterile

in the storage bag. Sample purity was confirmed by size exclusion chromatography (SEC).

7.2. Buffer ion-induced intermediate formation

This example demonstrates that the interaction of histidine with full-length mAbs leads to a reduction in domain-domain interactions and destabilization of some domains. Due to this action of histidine, the protein molecule gradually unfolds through the formation of intermediate substances. In contrast, under the conditions described, the interaction of phosphate with full-length monoclonal antibodies did not result in the formation of intermediate species.

7.2.1. Materials and methods

Urea-induced unfolding assay

Urea-induced unfolding of 7F3com-2H2 was performed in 10 mM histidine (pH 6.0) or 10 mM phosphate buffer (pH 6.0) in the absence of salt or in the presence of 150 mM NaCl. Unfolding of 7F3com-2H2 was determined by monitoring the spectral center of mass (CSM) change with urea concentration by intrinsic tryptophan fluorescence. The protein solution was excited at 293 nm and the emission spectrum was recorded from 310 to 400 nm. A spectral bandwidth of 4 nm was established for both excitation and emission. In unfolding and refolding experiments, samples were incubated at 23°C for 12 hours prior to assay. This incubation time is optimal for the protein to reach unfolding equilibrium. The data were normalized by (Y _U −Y _obs )/(Y _U −Y _F ), where Y _obs is the observed spectral value at a certain urea concentration and Y _F and Y _U are the spectral values of folded and unfolded 7F3com-2H2, respectively . All experiments were carried out at 23°C with a protein concentration of 0.67 μM.

Differential Scanning Calorimetry (DSC):

DSC was performed on 7F3com-2H2 under the following conditions: (a) 10 mM histidine, pH 6.0, and the absence of salt, 25 mM NaCl, or the presence of 150 mM NaCl; (b) 10 mM phosphate buffer, pH 6.0, and the absence of salt or in the presence of 150 mM NaCl. DSC experiments were performed on a VP DSC ultrasensitive calorimeter from Microcal. Completely dialyze the protein solution against the corresponding buffer for at least 12 hours. Use the final dialysate in the reference cell. Degas all protein solutions and buffers by gentle agitation under vacuum before loading into the calorimeter to ensure no air bubbles during heating. Experiments were performed at a scan rate of 1.5°C/min in the range of 30-100°C. Buffer baselines were corrected using normalized heat capacity (Cp) data. DSC experiments were performed with a protein concentration of 67 μM.

Potassium iodide (KI) quenching experiment

SV plots of native and intermediate 7F3com-2H2 in 10 mM histidine, 150 mM NaCl, pH 6.0, and SV plots of native and unfolded 7F3com-2H2 in 10 mM phosphate, 150 mM NaCl, pH 6.0 were generated. Stepwise aliquots of KI from the 6M stock solutions were added to 0.67 μM protein solutions in each buffer. Prepare the KI solution with 0.2 mM sodium thiosulfate to prevent the formation of free iodine. After excitation of 7F3com-2H2 at 293 nm, the fluorescence intensity at the emission maximum (native 331 nm, intermediate 348, unfolded 352 nm) was measured.

KI-induced tryptophan fluorescence quenching was analyzed according to the Stern-Volmer and Lehrer equation. The quenching process can be described by the classical SV relationship: F ₀ /F=1+K _SV [Q], where F ₀ and F are the fluorescence intensities in the absence or presence of a quencher, [Q] is the quencher concentration, _KSV is the SV quenching constant. The fraction of all fluorophores accessible to the quencher can be calculated from a modified SV curve, also known as the Lehrer curve: F ₀ /ΔF=1/K _Q f _a Q]+1/f _a , where F ₀ and [Q] In the same sense as previously defined, ΔF is the quenching-induced independent change in fluorescence, _KQ is the SV quenching constant for exposed tryptophan residues, and _f is the fraction of initial fluorescence accessible to the quencher. From this equation, the F ₀ /ΔF=f(1/[Q]) curve produces a straight line which, when extrapolated to 1/[Q]=0, yields the value 1/f _a on the F ₀ /ΔF axis.

1-anilino-8-naphthalenesulfonic acid (ANS) binding experiment

Urea-induced 7F3com-2H2 unfolding at pH 8.1 in 10 mM histidine or 10 mM phosphate was determined by tryptophan fluorescence and ANS binding assays. In all experiments, the protein concentration used was 0.67 [mu]M and the data were normalized using the formula (Y _U -Y _obs )/(Y _U -Y _F ) previously described. For ANS binding studies, 7F3com-2H2 was equilibrated overnight at 23°C with different concentrations of urea. As such, proteins were equilibrated with varying concentrations of denaturants and then treated with freshly prepared ANs. The ratio of protein to ANS was maintained at 1:100 so that there was a large excess of ANS in solution. The mixture was incubated at 23°C for 2 hours. Fluorescence intensity at 400–600 nm was measured after excitation at 350 nm to monitor the binding of the hydrophobic dye ANS to the protein.

7.2.2. Results

Urea-induced folding experiments demonstrated that antibody unfolding was a simple 2-step process in the presence of histidine alone at pH 6.0, suggesting that all domains unfold cooperatively, whereas upon addition of salt, the 7F3com-2H2 domain Unfolding is a stepwise process through the formation of a population of intermediates (see Figure 17A). In contrast, in the presence of phosphate, pH 6.0, this intermediate was not formed regardless of the presence of salt (see Figure 17B).

DSC experiments revealed that in the absence of salt, at pH 6, the DSC thermogram was composited with 3 partially overlapping transition peaks (see Figure 18). Overlap of the 3 transition peaks indicates strong domain-domain interactions. Addition of 150 mM NaCl destabilizes the first transition peak as indicated by a decrease in T _m1 of about 3.5°C.

KI quenching studies showed that in the presence of 7.5M urea, when the intermediate state was observed (see Figure 17), f _a values of 0.82 ± 0.02 were obtained (see Figure 19A). This revealed that only 82% of the tryptophan fluorescence emission could be quenched by iodine in the intermediate state, suggesting a partially folded structure (ie, containing buried tryptophan residues) of the intermediate. In contrast, the f _a value obtained from the Lehrer curve under the condition of 7.5M urea and 10mM phosphate is 1.07±0.02, which indicates that all tryptophan fluorescence emission is quenched by iodine, so it is exposed to the solvent, which indicates that the protein molecule Fully unfolded (see Figure 19B).

ANS binding studies showed that at pH 8.1 (pI), the unfolding transition in histidine was a multistep process through the formation of stable intermediate species even in the absence of salt (see Figure 20A). However, under similar conditions in tris or phosphate buffer at this pH, no significant population of intermediates was observed (see Figure 20B). Moreover, these studies showed that the ANS in the samples had increased binding to a large number of intermediate species, which indicated exposure of the hydrophobic region.

7.3 Phase Properties of Antibodies: Effect of Histidine on Stability

This example shows that certain mAbs can have different phase properties. These studies also demonstrated that certain buffer types, such as histidine buffer, interact with 7F3com-2H2 under certain conditions, thereby disrupting domain-domain interactions, leading to intermolecular attraction and binding. This results in reduced solubility of 7F3com-2H2 during histidine processing.

7.3.1. Materials and methods

static light scattering

Determine the second virial coefficient of promising precipitation conditions using static light scattering techniques. Electro-Optics laser model 1145AP (Hsintien City, Taiwan), Brookhaven Instruments goniometer, and BI-200SM and BI-APD cascaded photodiode detection (Holtsville, NY) were used to determine the excess Raleigh ratio (scattering by proteins only) at a 90° angle to the 633 nm incident beam, respectively. To examine Raleigh scattering, the intensity at 60° and 120° angles was also measured. If the observed particle is a Raleigh scattered wave (<λ/20), the intensity is independent of angle. The relationship used to determine the second virial coefficient is given here, which is derived from the virial unfolding of the ideal osmotic pressure equation.

$\frac{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="90"\; label="k\; R"\; filenames="A20078004363702561.png,A20078004363702611.png"\; state="\{\{state\}\}">k</figure-callout></span><figure-callout\; id="90"\; label="k\; R"\; filenames="A20078004363702561.png,A20078004363702611.png"\; state="\{\{state\}\}">\; </figure-callout>}}{{\mathrm{<span\; class="notranslate">\; </span>}}_{}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 2</span>}{\mathrm{<span\; class="notranslate">\; B</span>}}_{\mathrm{<span\; class="notranslate">\; twenty\; two</span>}}\mathrm{<span\; class="notranslate">\; c</span>}$

in,

c = protein concentration (g/mL)

M = mass average protein molecular weight (grams/mole).

R ₉₀ = Excess Raleigh Ratio of 90°

Equation 2 describes the optical constant K.

$\mathrm{<span\; class="notranslate">\; K</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 4</span>}{\mathrm{<span\; class="notranslate">\; \&pi;</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; dn</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; dc</span>}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; N</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}{\mathrm{<span\; class="notranslate">\; \&lambda;</span>}}^{\mathrm{<span\; class="notranslate">\; 4</span>}}}$

in,

n ₀ = Refractive index of the solvent

dn/dc = Refractive index increment of protein-solvent pair

N _A = Avogadro's constant

λ = incident beam wavelength (in vacuum)

Equation 3 gives the Raleigh ratio.

$\mathrm{<span\; class="notranslate">\; R</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; \&theta;</span>}}{\mathrm{<span\; class="notranslate">\; r</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; inc</span>}}{\mathrm{<span\; class="notranslate">\; V</span>}}_{\mathrm{<span\; class="notranslate">\; obs</span>}}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; \&theta;</span>}}\mathrm{<span\; class="notranslate">\; c</span>}$

in,

r = distance from the observation volume to the detector

I _inc = incident intensity of the laser beam

I _θ = measured intensity of scattered light

V _obs = observation volume

The constant c can be determined by systems known from Raleigh, eg toluene at 633 nm (14*10 ⁻⁶ cm ⁻¹ ). Once the constant c is determined, the raw intensity measurements can be converted to the Raleigh ratio, the excess Raleigh ratio being simply the Raleigh ratio of the sample minus the Raleigh ratio of the pure solvent.

The slope of the Kc/R ₉₀ vs. concentration curve is equal to twice the second virial coefficient (SVC or B ₂₂ ). The concentration of each solution sample for light scattering was determined by absorbance measurement at 280 nm. All solutions were filtered with 0.1 μm Anotop filters (Whatman Inc., England) to remove dust and protein concentrations were varied from 0.5 to 10 mg/mL as SVC is a dilute solution parameter. All salts and buffers used for light scattering were HPLC grade from Sigma Aldrich or Fischer. A positive SVC indicates net pairwise repulsion between protein molecules, while a negative SVC indicates net attraction between protein pairs. Such pairwise or dilute solution parameters have been shown to be useful in predicting crystallization and estimating solubility, even for proteins with high solubility.

Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transform infrared (FTIR) spectra were measured with a Bomem ^™ IR spectrometer (Quebec, Canada) and a dTGS detector. Fixed path length _CaF2 units and variable path length _CaF2 units with 7.5 μm polyester film (Mylar) spacers were used for liquid and solid measurements, respectively. 128-scan interferograms were acquired by singular means at 4 cm ^- 1 resolution. Subtract background water vapor scans from solvent and protein samples. Then, the solvent spectrum was subtracted from each protein spectrum and a 7-point Savitsky-Golay smooth function was fitted to obtain the second derivative spectrum. This spectrum is then baseline corrected to establish a zero baseline. The curve areas were then integrated and normalized so that the area of the amide I region (1705-1600 cm ⁻¹ ) equals unity in all spectra. This enables direct comparison of samples of different concentrations, such as solids and liquids. With the exception of baseline correction and area normalization, all subtractions and curve fitting were performed with GRAMS ^™ software (Thermo Electron Corp., Waltham, MA). A protein concentration of 10 to 12 mg/mL was used in all liquid IR scans.

Due to the carbon-oxygen stretching and nitrogen-hydrogen stretching caused by hydrogen bonds along the peptide backbone of the protein molecule, the amide I region produces a peak in the range of about 1700-1600 cm ^-1 , thereby determining the secondary structure. The general peak positions of specific types of protein secondary structure are assigned empirically from proteins of known structure, therefore, FTIR is primarily used as a qualitative technique.

7.3.2. Results

The mass average molecular weight (MW) determined by SLS indicated that 7F3com-2H2 was less bound at 10 mM histidine and 150 mM NaCl or 10 mM phosphate, pI, compared to histidine buffer alone (see below Table 8).

Table 8: Mass average molecular weight (MW) determined by static light scattering (SLS) measurements.

Monoclonal antibody sample (pI=8.1) B ₂₂ Up to 95% Lower limit 95% MW Up to 95% Lower limit 95% 10mM His, 150mMNaCl, pH 6 (mother solution) 3.5E-05 8.8E-05 -1.8E-05 153 161 145 BX to 10mM His pH 6 -1.0E-04 -5.2E-05 -1.5E-04 127 131 122 BX to 10mM His pH 8.1 -5.5E-05 -2.5E-05 -9.2E-05 282 299 262 BX to 10mM His, 150mM NaCl, pH 8.1 2.0E-05 6.3E-05 -3.7E-05 192 208 175 BX to 10mM Tris, pH 8.1 -8.9E-05 -4.9E-05 -1.3E-04 147 143 152 BX to 10mM sodium phosphate, pH 8.1 -1.5E-04 -1.4E-04 -1.7E-04 125 126 124

FTIR results showed that pH alone was unable to induce 7F3com-2H2 binding in solution (pI = 8.1 ) as observed by SLS (see Figure 21 ). Moreover, the ability of 7F3com-2H2 to bind strongly depends on the buffer type. Specifically, samples containing Tris and phosphate buffer had a higher proportion of monomeric protein than aggregated protein. In contrast, samples containing histidine buffer had a higher proportion of proteins in the aggregated state than in the monomeric state.

6.4 Effect of histidine on antibody domain stability and domain-domain interactions

In this example, the effects of different buffer substances on the domain stability and domain-domain interactions of antibody 7F3com-2H2 were determined by intrinsic tryptophan fluorescence spectroscopy and differential scanning calorimetry.

6.4.1 Materials and methods

f _c and F _ab generation of fragments

_Fc and _Fab fragments were generated by papain digestion and purified by chromatography.

Urea Unfolding Studies

Unfolding of 7F3com-2H2 was determined by intrinsic tryptophan fluorescence. All experiments were carried out at 23°C with a protein concentration of 0.67 μM.

Differential Scanning Calorimetry

DSC experiments were performed using a protein concentration of 6.7 μΜ; all measurements were performed at a scan rate of 1.5°C/min.

6.4.2 Results

Results from the full-length 7Fcom3-2H2 antibody as well as the isolated F _ab fragment and the isolated F _c fragment of this antibody indicated that the interaction of histidine with mAbs at pH 6 appears to require the charge-shielding effect of sodium chloride. Urea unfolding studies showed that the interactions lead to destabilization of some domains or domain-domain interactions in the full-length molecule. See Figure 22. In addition, DSC showed that the CH ₂ domain of the _Fc fragment was the most unstable domain, and after interacting with histidine, the T _m of the CH ₂ region decreased by 5°C compared with the control value of 70°C. See Figure 23.

Taken together, the fluorescence (Fig. 22) and DSC (Fig. 23) data indicate that strong domain-domain interactions occur in full-length 7Fcom3-2H2 at pH 6; NaCl modulates the interaction of histidine with the _Fab region effect; the unfolding properties of 7Fcom3-2H2 in the presence of histidine and salts indicated reduced domain-domain interactions and preferentially affected the stability of some domains; the _CH2 domain of the _Fc fragment was the least stable domain; the interaction between the _CH2 and _CH3 regions in the _Fc fragment is weak; there is a strong subdomain interaction in the _Fab ; in conclusion, histidine appears to affect the _CH2 domain in the presence of salt stability and interaction.

6.5 Protein-Protein Interactions, Viscosity, and Opalescence at High Antibody Concentrations

This example describes the measurement of the colloidal properties of 7Fcom3-2H2 over a concentration range of 1-100 g/L using viscometry, membrane osmometry and light scattering.

6.5.1 Materials and methods

Antibody preparation

The 7Fcom3-2H2 antibody is a liquid formulation. All buffer conditions were obtained by complete dialysis at pH 6, and protein concentration was determined by UV absorbance at 280 nm with an extinction coefficient of 1.61 L/g-cm.

viscometry

A Brookfield LVDV-II+Pro cone/plate viscometer (Middleboro, MA) with external pc control was used with a CPE 40 spindle to Measure the viscosity of the mAb solution. A shear rate constant is applied and a reading is taken after the system has stabilized (approximately 30 seconds). A constant temperature of 23.0 ± 0.1 °C was maintained using an external water bath.

Turbidity

Turbidity was measured on a spectrofluorometer (SLM AMINCO, Urbana, IL, US) equipped with a temperature control unit (temperature maintained at 23.0 ± 0.1 °C). The incident light beam was set at 510 nm, and the scattered light intensity at 90° was measured at the same wavelength. AMCO clarified turbidity standards from GFS Chemicals, Inc., Columbus, Ohio, a set of 0-100 Nephelometric Turbidity Units (NTU) (Catalog #8255 and 8256) A calibration curve was generated to convert the intensity of the protein solution to NTU.

Osmotic pressure

Osmotic pressure was measured on a Wescor Model 4420 Colloid Osmometer (Logan, Utah) equipped with a PM Series membrane (Catalog #SS-030) with a molecular weight cut-off of 30,000 to determine Second virial coefficient and number average molecular weight of monoclonal antibody solution. The corresponding final dialyzate was used as reference solution. The osmometer/membrane system was also calibrated using a BSA standard solution of known osmolarity (Catalog #SS-025) from Wescor.

6.5.2 Results

It was shown that ionic strength negatively affects the solution viscosity and opalescent properties of highly concentrated solutions. The combined results show that charge repulsion at low ionic strength produces high viscosity. The negative second virial coefficient observed at higher ionic strengths is associated with large apparent molecular weights and increased light scattering at high concentrations (>20 g/L). Increased light scattering produces an opalescent appearance. In addition, the second virial coefficient also suggested that the opalescent appearance may be related to the phase transition.

Taken together, viscosity (Figure 25), turbidity (Figure 26), and osmolarity (Figures 27 and 28) suggest that charge repulsion is the source of high solution viscosity at low ionic strength; A negative virial coefficient is related, which leads to increased opalescence; a positive second virial coefficient results in a smaller apparent molecular weight and thus a less opalescent appearance; ionic strength can inversely regulate opalescence and viscosity; observations It shows that the viscosity and opalescence can be balanced by the intermediate salt concentration; as determined by Pellicane et al., the second virial coefficient of the high opalescence solution is close to the critical value of phase separation, indicating that the increase of opalescence may be associated with the phase transition.

6.6 Formulation Development

Formulation studies are performed to assess the chemical and physical stability and solubility of proteins. The goal is to determine the most suitable conditions for long-term storage of the product. The research was divided into three main areas: formulation design, concentration and stability. Formulation screening studies were used to assess the effect of pH, buffer concentration, and other excipients on the chemical and physical stability of proteins. These studies subject proteins to a range of buffers, other excipients, and storage temperature ranges (usually 2-8°C, 23-27°C, and 38-42°C) and pH conditions, using techniques such as high-pressure size-exclusion chromatography (HPSEC) and high performance ion exchange chromatography (HPIEC) to assess chemical and physical stability. Concentration studies will provide information on the original protein-protein interactions during the concentration step and on the effects of high concentrations on long-term storage. Additionally, solution pH studies are performed to test buffers over a physiologically useful pH range for optimal solubilization of proteins at high concentrations.

The stability of four antibody preparations containing 5g/L 7F3com-2H2 anti-IL-9 antibody was tested under conditions of accelerated antibody degradation. The four formulations listed below were prepared using standard laboratory procedures:

1) 10mM histidine, 150mM NaCl, pH6.0

2) 2.2mM sodium phosphate, 150mM NaCl, pH6.0

3) 10mM histidine, pH 6.0

4) 2.2mM sodium phosphate, pH 6.0

Small aliquots of each formulation were sealed in glass vials. The glass bottles were stored at 40°C in an upright state. At different time points, individual glass vials were analyzed by HPSEC. Figure 30 shows overlay elution profiles of 2.2 mM sodium phosphate, pH 6.0 formulations recorded at different time points. The last data point was obtained after 75 days of incubation. Figure 29 provides a graph of the total monomer concentration and antibody fragment concentration versus time for each of the four formulations described above.

8. Equivalence

Those skilled in the art will recognize, or be able to ascertain using routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference herein. The contents of US Provisional Application Nos. 60/847,239, filed September 25, 2006 and 60/949,999, filed July 16, 2007 are hereby incorporated by reference in their entirety for all purposes.

Citation or discussion of references described herein should not be construed as an admission that such references are prior art to the present invention.

sequence listing

<110> MedImmune, Inc.

J. Carpenter (Carpenter, John)

H. Suthers (Sathish, Hasige)

T. Randolph (Randolph, Theodore W.)

C. Allan (Allan, Christian)

S. Bishop (Bishop, Steven)

B. Salinas (Salinas, Branden)

<120> Stabilized antibody preparations and applications thereof

<130>AE709PCT

<150>60/847,239

<151>2006-09-25

<150>60/949,999

<151>2007-07-16

<160>65

<170> FastSEQ version 4.0 for Windows

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR1

<400>1

Gly Tyr Thr Phe Thr Gly Tyr Trp Ile Glu

1 5 5 10

<210>2

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR2

<400>2

Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Pro Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210>3

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223>VH CDR3

<400>3

Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr

1 5 10

<210>4

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR1

<400>4

Lys Ala Ser Gln His Val Gly Thr His Val Thr

1 5 5 10

<210>5

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR2

<400>5

Ser Thr Ser Tyr Arg Tyr Ser

1 5

<210>6

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR3

<400>6

Gln His Phe Tyr Ser Tyr Pro Leu Thr

1 5

<210>7

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 4D4

<400>7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu VaI Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>8

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 4D4 and 4D4 H2-1 D11

<400>8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Tyr Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>9

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 4D4 H2-1 D11

<400>9

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Trp Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 70 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>10

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR2

<400>10

Glu Trp Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210>11

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR1

<400>11

Gly Tyr Thr Phe Thr Tyr Tyr Trp Ile Glu

1 5 5 10

<210>12

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223>VH CDR3

<400>12

Ala Asp Tyr Tyr Gly Ser Asp His Val Lys Phe Asp Tyr

1 5 5 10

<210>13

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR1

<400>13

Leu Ala Ser Gln His Val Gly Thr His Val Thr

1 5 5 10

<210>14

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR2

<400>14

Gly Thr Ser Tyr Arg Tyr Ser

1 5

<210>15

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 4D4com-XF-9

<400>15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Trp Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp His Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>16

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 4D4com-XF-9

<400>16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Tyr Asp Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>17

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 4D4com-2F9

<400>17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Trp Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp His Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>18

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 4D4com-2F9

<400>18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>19

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR1

<400>19

Gly Gly Thr Phe Ser Gly Tyr Trp Ile Glu

1 5 5 10

<210>20

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR3

<400>20

Gln Gln Phe Tyr Glu Tyr Pro Leu Thr

1 5

<210>21

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 7F3 and 7F3 22D3

<400>21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>22

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 7F3

<400>22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>23

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 71A10

<400>23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Pro Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>24

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 71A10

<400>24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>25

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 7F3 22D3

<400>25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>26

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR1

<400>26

Gly Gly Thr Phe Ser Tyr Tyr Trp Ile Glu

1 5 5 10

<210>27

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 7F3com-2H2

<400>27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Pro Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>28

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 7F3com-2H2

<400>28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Ile Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Ser Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>29

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 7F3com-3H5

<400>29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Pro Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>30

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 7F3com-3H5

<400>30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>31

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> VH region of antibody 7F3com-3D4

<400>31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Pro Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Tyr Tyr Gly Ser Asp Tyr Val Lys Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>32

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> VL region of antibody 7F3com-3D4

<400>32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Ile Thr His

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>33

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 1

<400>33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210>34

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 2

<400>34

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 5 10

<210>35

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 3

<400>35

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>36

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 4

<400>36

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 5 10

<210>37

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 1

<400>37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210>38

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 3

<400>38

Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>39

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> VL framework region 1

<400>39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210>40

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 2

<400>40

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>41

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 3

<400>41

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210>42

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> VH framework region 4

<400>42

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 5 10

<210>43

<211>591

<212>DNA

<213> Artificial sequence

<220>

<223> VH region of 7F3com-2H2

<400>43

ccgctgtcaa gatgcttctg gccatggtcc ttacctctgc cctgctcctg tgctccgtgg 60

caggccaggg gtgtccaacc ttggcgggga tcctggacat caacttcctc atcaacaaga 120

tgcaggaaga tccagcttcc aagtgccact gcagtgctaa tgtgaccagt tgtctctgtt 180

tgggcattcc ctctgacaac tgcaccagac catgcttcag tgagagactg tctcagatga 240

ccaataccac catgcaaaca agatacccac tgattttcag tcgggtgaaa aaatcagttg 300

aagtactaaa gaacaacaag tgtccatatt tttcctgtga acagccatgc aaccaaacca 360

cggcaggcaa cgcgctgaca tttctgaaga gtcttctgga aattttccag aaagaaaaga 420

tgagagggat gagaggcaag atatgaagat gaaatattat ttatcctatt tattaaattt 480

aaaaagcttt ctctttaagt tgctacaatt taaaaatcaa gtaagctact ctaaatcagt 540

atcagttgtg attatttgtt taacattgta tgtctttatttgaaataaa t 591

<210>44

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> VH CDR1

<400>44

ggaggcacct tcagctatta ctggatagag 30

<210>45

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> VH CDR2

<400>45

gagattttac ctggaagtgg tactactaac ccgaatgaga agttcaaggg c 51

<210>46

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223>VH CDR3

<400>46

gcggattact acggtagtga ttacgtcaag tttgactac 39

<210>47

<211>321

<212>DNA

<213> Artificial sequence

<220>

<223> VL area of 7F3com-2H2

<400>47

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgca aggcaagtca gcatgtgatt actcatgtaa cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatggg acatcctaca gctacagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttatta ctgtcagcaa ttttacgagt atcctctcac gttcggcgga 300

gggaccaagg tggagatcaa a 321

<210>48

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> VL CDR1

<400>48

aaggcaagtc agcatgtgat tactcatgta acc 33

<210>49

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> VL CDR2

<400>49

gggacatcct acagc 15

<210>50

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> VL CDR3

<400>50

cagcaatttt acgagtatcc tctcacg 27

<210>51

<211>591

<212>DNA

<213> Homo sapiens

<400>51

ccgctgtcaa gatgcttctg gccatggtcc ttacctctgc cctgctcctg tgctccgtgg 60

caggccaggg gtgtccaacc ttggcgggga tcctggacat caacttcctc atcaacaaga 120

tgcaggaaga tccagcttcc aagtgccact gcagtgctaa tgtgaccagt tgtctctgtt 180

tgggcattcc ctctgacaac tgcaccagac catgcttcag tgagagactg tctcagatga 240

ccaataccac catgcaaaca agatacccac tgattttcag tcgggtgaaa aaatcagttg 300

aagtactaaa gaacaacaag tgtccatatt tttcctgtga acagccatgc aaccaaacca 360

cggcaggcaa cgcgctgaca tttctgaaga gtcttctgga aattttccag aaagaaaaga 420

tgagagggat gagaggcaag atatgaagat gaaatattat ttatcctatt tattaaattt 480

aaaaagcttt ctctttaagt tgctacaatt taaaaatcaa gtaagctact ctaaatcagt 540

atcagttgtg attatttgtt taacattgta tgtctttatttgaaataaa t 591

<210>52

<211>144

<212>PRT

<213> Homo sapiens

<400>52

Met Leu Leu Ala Met Val Leu Thr Ser Ala Leu Leu Leu Leu Cys Ser Val

1 5 10 15

Ala Gly Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe

20 25 30

Leu Ile Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser

35 40 45

Ala Asn Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys

50 55 60

Thr Arg Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr

65 70 75 80

Met Gln Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val

85 90 95

Glu Val Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro

100 105 110

Cys Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu

115 120 125

Leu Glu Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile

130 135 140

<210>53

<211>808

<212>PRT

<213> Homo sapiens

<400>53

Met Ala Glu Leu Leu Ala Ser Ala Gly Ser Ala Cys Ser Trp Asp Phe

1 5 10 15

Pro Arg Ala Pro Pro Ser Phe Pro Pro Pro Ala Ala Ser Arg Gly Gly

20 25 30

Leu Gly Gly Thr Arg Ser Phe Arg Pro His Arg Gly Ala Glu Ser Pro

35 40 45

Arg Pro Gly Arg Asp Arg Asp Gly Val Arg Val Pro Met Ala Ser Ser

50 55 60

Arg Cys Pro Ala Pro Arg Gly Cys Arg Cys Leu Pro Gly Ala Ser Leu

65 70 75 80

Ala Trp Leu Gly Thr Val Leu Leu Leu Leu Ala Asp Trp Val Leu Leu

85 90 95

Arg Thr Ala Leu Pro Arg Ile Phe Ser Leu Leu Val Pro Thr Ala Leu

100 105 110

Pro Leu Leu Arg Val Trp Ala Val Gly Leu Ser Arg Trp Ala Val Leu

115 120 125

Trp Leu Gly Ala Cys Gly Val Leu Arg Ala Thr Val Gly Ser Lys Ser

130 135 140

Glu Asn Ala Gly Ala Gln Gly Trp Leu Ala Ala Leu Lys Pro Leu Ala

145 150 155 160

Ala Ala Leu Gly Leu Ala Leu Pro Gly Leu Ala Leu Phe Arg Glu Leu

165 170 175

Ile Ser Trp Gly Ala Pro Gly Ser Ala Asp Ser Thr Arg Leu Leu His

180 185 190

Trp Gly Ser His Pro Thr Ala Phe Val Val Ser Tyr Ala Ala Ala Leu

195 200 205

Pro Ala Ala Ala Leu Trp His Lys Leu Gly Ser Leu Trp Val Pro Gly

210 215 220

Gly Gln Gly Gly Ser Gly Asn Pro Val Arg Arg Leu Leu Gly Cys Leu

225 230 235 240

Gly Ser Glu Thr Arg Arg Leu Ser Leu Phe Leu Val Leu Val Val Leu

245 250 255

Ser Ser Leu Gly Glu Met Ala Ile Pro Phe Phe Thr Gly Arg Leu Thr

260 265 270

Asp Trp Ile Leu Gln Asp Gly Ser Ala Asp Thr Phe Thr Arg Asn Leu

275 280 285

Thr Leu Met Ser Ile Leu Thr Ile Ala Ser Ala Val Leu Glu Phe Val

290 295 300

Gly Asp Gly Ile Tyr Asn Asn Thr Met Gly His Val His Ser His Leu

305 310 315 320

Gln Gly Glu Val Phe Gly Ala Val Leu Arg Gln Glu Thr Glu Phe Phe

325 330 335

Gln Gln Asn Gln Thr Gly Asn Ile Met Ser Arg Val Thr Glu Asp Thr

340 345 350

Ser Thr Leu Ser Asp Ser Leu Ser Glu Asn Leu Ser Leu Phe Leu Trp

355 360 365

Tyr Leu Val Arg Gly Leu Cys Leu Leu Gly Ile Met Leu Trp Gly Ser

370 375 380

Val Ser Leu Thr Met Val Thr Leu Ile Thr Leu Pro Leu Leu Phe Leu

385 390 395 400

Leu Pro Lys Lys Val Gly Lys Trp Tyr Gln Leu Leu Glu Val Gln Val

405 410 415

Arg Glu Ser Leu Ala Lys Ser Ser Gln Val Ala Ile Glu Ala Leu Ser

420 425 430

Ala Met Pro Thr Val Arg Ser Phe Ala Asn Glu Glu Gly Glu Ala Gln

435 440 445

Lys Phe Arg Glu Lys Leu Gln Glu Ile Lys Thr Leu Asn Gln Lys Glu

450 455 460

Ala Val Ala Tyr Ala Val Asn Ser Trp Thr Thr Ser Ile Ser Gly Met

465 470 475 480

Leu Leu Lys Val Gly Ile Leu Tyr Ile Gly Gly Gln Leu Val Thr Ser

485 490 495

Gly Ala Val Ser Ser Gly Asn Leu Val Thr Phe Val Leu Tyr Gln Met

500 505 510

Gln Phe Thr Gln Ala Val Glu Val Leu Leu Ser Ile Tyr Pro Arg Val

515 520 525

Gln Lys Ala Val Gly Ser Ser Glu Lys Ile Phe Glu Tyr Leu Asp Arg

530 535 540

Thr Pro Arg Cys Pro Pro Ser Gly Leu Leu Thr Pro Leu His Leu Glu

545 550 555 560

Gly Leu Val Gln Phe Gln Asp Val Ser Phe Ala Tyr Pro Asn Arg Pro

565 570 575

Asp Val Leu Val Leu Gln Gly Leu Thr Phe Thr Leu Arg Pro Gly Glu

580 585 590

Val Thr Ala Leu Val Gly Pro Asn Gly Ser Gly Lys Ser Thr Val Ala

595 600 605

Ala Leu Leu Gln Asn Leu Tyr Gln Pro Thr Gly Gly Gln Leu Leu Leu

610 615 620

Asp Gly Lys Pro Leu Pro Gln Tyr Glu His Arg Tyr Leu His Arg Gln

625 630 635 640

Val Ala Ala Val Gly Gln Glu Pro Gln Val Phe Gly Arg Ser Leu Gln

645 650 655

Glu Asn Ile Ala Tyr Gly Leu Thr Gln Lys Pro Thr Met Glu Glu Ile

660 665 670

Thr Ala Ala Ala Val Lys Ser Gly Ala His Ser Phe Ile Ser Gly Leu

675 680 685

Pro Gln Gly Tyr Asp Thr Glu Val Asp Glu Ala Gly Ser Gln Leu Ser

690 695 700

Gly Gly Gln Arg Gln Ala Val Ala Leu Ala Arg Ala Leu Ile Arg Lys

705 710 715 720

Pro Cys Val Leu Ile Leu Asp Asp Ala Thr Ser Ala Leu Asp Ala Asn

725 730 735

Ser Gln Leu Gln Val Glu Gln Leu Leu Tyr Glu Ser Pro Glu Arg Tyr

740 745 750

Ser Arg Ser Val Leu Leu Ile Thr Gln His Leu Ser Leu Val Glu Gln

755 760 765

Ala Asp His Ile Leu Phe Leu Glu Gly Gly Ala Ile Arg Glu Gly Gly

770 775 780

Thr His Gln Gln Leu Met Glu Lys Lys Gly Cys Tyr Trp Ala Met Val

785 790 795 800

Gln Ala Pro Ala Asp Ala Pro Glu

805

<210>54

<211>140

<212>PRT

<213> Homo sapiens

<400>54

Met Val Leu Thr Ser Ala Leu Leu Leu Cys Ser Val Ala Gly Gln Gly

1 5 10 15

Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys

20 25 30

Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser Ala Asn Val Thr

35 40 45

Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg Pro Cys

50 55 60

Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr Met Gln Thr Arg

65 70 75 80

Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys

85 90 95

Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn Gln Thr

100 105 110

Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe

115 120 125

Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile

130 135 140

<210>55

<211>2171

<212>DNA

<213> Homo sapiens

<400>55

agcagctctg taatgcgctt gtggtttcag atgtgggcgg cctgtgtgaa cctgtcgtgc 60

aaagctcacg tcaccaactg ctgcagttat ctcctgaatc aggctgaggg tctttgctgt 120

gcacccagag atagttgggt gacaaatcac ctccaggttg gggatgcctc agacttgtga 180

tgggactggg cagatgcatc tgggaaggct ggaccttgga gagtgaggcc ctgaggcgag 240

acatgggcac ctggctcctg gcctgcatct gcatctgcac ctgtgtctgc ttgggagtct 300

ctgtcacagg ggaaggacaa gggccaaggt ctagaacctt cacctgcctc accaacaaca 360

ttctcaggat cgattgccac tggtctgccc cagagctggg acagggctcc agcccctggc 420

tcctcttcac cagcaaccag gctcctggcg gcacacataa gtgcatcttg cggggcagtg 480

agtgcaccgt cgtgctgcca cctgaggcag tgctcgtgcc atctgacaat ttcaccatca 540

ctttccacca ctgcatgtct gggagggagc aggtcagcct ggtggacccg gagtacctgc 600

cccggagaca cgttaagctg gacccgccct ctgacttgca gagcaacatc agttctggcc 660

actgcatcct gacctggagc atcagtcctg ccttggagcc aatgaccaca cttctcagct 720

atgagctggc cttcaagaag caggaagagg cctgggagca ggcccagcac agggatcaca 780

ttgtcggggt gacctggctt atacttgaag cctttgagct ggaccctggc tttatccatg 840

aggccaggct gcgtgtccag atggccaacac tggaggatga tgtggtagag gaggagcgtt 900

atacaggcca gtggagtgag tggagccagc ctgtgtgctt ccaggctccc cagagacaag 960

gccctctgat cccaccctgg gggtggccag gcaacaccct tgttgctgtg tccatctttc 1020

tcctgctgac tggcccgacc tacctcctgt tcaagctgtc gcccagggtg aagagaatct 1080

tctaccagaa cgtgccctct ccagcgatgt tcttccagcc cctctacagt gtacacaatg 1140

ggaacttcca gacttggatg ggggcccacg gggccggtgt gctgttgagc caggactgtg 1200

ctggcacccc acaggagcc ttggagccct gcgtccagga ggccactgca ctgctcactt 1260

gtggcccagc gcgtccttgg aaatctgtgg ccctggagga ggaacaggag ggccctggga 1320

ccaggctccc ggggaacctg agctcagagg atgtgctgcc agcagggtgt acggagtgga 1380

gggtacagac gcttgcctat ctgccacagg aggactgggc ccccacgtcc ctgactaggc 1440

cggctccccc agactcagag ggcagcagga gcagcagcag cagcagcagc agcaacaaca 1500

acaactactg tgccttgggc tgctatgggg gatggcacct ctcagccctc ccaggaaaca 1560

cacagagctc tgggcccatc ccagccctgg cctgtggcct ttcttgtgac catcagggcc 1620

tggagaccca gcaaggagtt gcctgggtgc tggctggtca ctgccagagg cctgggctgc 1680

atgaggacct ccagggcatg ttgctccctt ctgtcctcag caaggctcgg tcctggacat 1740

tctaggtccc tgactcgcca gatgcatcat gtccattttg ggaaaatgga ctgaagtttc 1800

tggagccctt gtctgagact gaacctcctg agaaggggcc cctagcagcg gtcagaggtc 1860

ctgtctggat ggaggctgga ggctcccccc tcaacccctc tgctcagtgc ctgtggggag 1920

cagcctctac cctcagcatc ctggccacaa gttcttcctt ccattgtccc ttttctttat 1980

ccctgacctc tctgagaagt ggggtgtggt ctctcagctg ttctgccctc atacccttaa 2040

agggccagcc tgggcccagt ggacacaggt aaggcaccat gaccacctgg tgtgacctct 2100

ctgtgcctta ctgaggcacc tttctagaga ttaaaagggg cttgatggct gttaaaaaaa 2160

aaaaaaaaaa a 2171

<210>56

<211>2175

<212>DNA

<213> Homo sapiens

<400>56

agcagctctg taatgcgctt gtggtttcag atgtgggcgg cctgtgtgaa cctgtcgtgc 60

aaagctcacg tcaccaactg ctgcagttat ctcctgaatc aggctgaggg tctttgctgt 120

gcacccagag atagttgggt gacaaatcac ctccaggttg gggatgcctc agacttgtga 180

tgggactggg cagatgcatc tgggaagtaa ctgctgcaag aacggacaga cactgctgca 240

gagaacttgc cacggtgttt catgctgtgg ctggtggttc caggctgcac gctccattct 300

aggaaagggg ccctcagccc agtcccttgc aggctggacc ttggagagtg aggccctgag 360

gcgagacatg ggcacctggc tcctggcctg catctgcatc tgcacctgtg tctgcttggg 420

agtctctgtc acagggggaag gacaagggcc aaggtctaga accttcacct gcctcaccaa 480

caacattctc aggatcgatt gccactggtc tgccccagag ctgggacagg gctccagccc 540

ctggctcctc ttcaccaggc tcctggcggc acacataagt gcatcttgcg gggcagtgag 600

tgcaccgtcg tgctgccacc tgaggcagtg ctcgtgccat ctgacaattt caccatcact 660

ttccaccact gcatgtctgg gagggagcag gtcagcctgg tggacccgga gtacctgccc 720

cggagacacg agcaacatca gttctggcca ctgcatcctg acctggagca tcagtcctgc 780

cttggagcca atgaccacac ttctcagcta tgagctggcc ttcaagaagc aggaagaggc 840

ctgggagcag gcccagcaca gggatcacat tgtcggggtg acctggctta tacttgaagc 900

ctttgagctg gaccctggct ttatccatga ggccaggctg cgtgtccaga tggccaacact 960

ggaggatgat gtggtagagg aggagcgtta tacaggccag tggagtgagt ggagccagcc 1020

tgtgtgcttc caggctcccc agagacaagg ccctctgatc ccaccctggg ggtggccagg 1080

caacaccctt gttgctgtgt ccatctttct cctgctgact ggcccgacct acctcctgtt 1140

caagctgtcg cccagacttg gatgggggcc cacggggccg gtgtgctgtt gagccaggac 1200

tgtgctggca ccccacagggg agccttggag ccctgcgtcc aggaggccac tgcactgctc 1260

acttgtggcc cagcgcgtcc ttggaaatct gtggccctgg aggaggaaca ggagggccct 1320

gggaccaggc tcccggggaa cctgagctca gaggatgtgc tgccagcagg gtgtacggag 1380

tggagggtac agacgcttgc ctatctgcca caggaggact gggcccccac gtccctgact 1440

aggccggctc ccccagactc aggggcagc aggagcagca gcagcagcag cagcagcaac 1500

aacaacaact actgtgcctt gggctgctat ggggatggc acctctcagc cctcccagga 1560

aacacacaga gctctgggcc catcccagcc ctggcctgtg gcctttcttg tgaccatcag 1620

ggcctggaga cccagcaagg agttgcctgg gtgctggctg gtcactgcca gaggcctggg 1680

ctgcatgagg acctccaggg catgttgctc ccttctgtcc tcagcaaggc tcggtcctgg 1740

acattctagg tccctgactc gccagatgca tcatgtccat tttgggaaaa tggactgaag 1800

tttctggagc ccttgtctga gactgaacct cctgagaagg ggcccctagc agcggtcaga 1860

ggtcctgtct ggatggaggc tggaggctcc cccctcaacc cctctgctca gtgcctgtgg 1920

ggagcagcct ctaccctcag catcctggcc acaagttctt ccttccattg tcccttttct 1980

ttatccctga cctctctgag aagtggggtg tggtctctca gctgttctgc cctcataccc 2040

ttaaagggcc agcctgggcc cagtggacac aggtaaggca ccatgaccac ctggtgtgac 2100

ctctctgtgc cttactgagg cacctttcta gagattaaaa ggggcttgat ggctgttaaa 2160

aaaaaaaaaa aaaaa 2175

<210>57

<211>1451

<212>DNA

<213> Homo sapiens

<400>57

gaagagcaag cgccatgttg aagccatcat taccattcac atccctctta ttcctgcagc 60

tgcccctgct gggagtgggg ctgaacacga caattctgac gcccaatggg aatgaagaca 120

ccacagctga tttcttcctg accactatgc ccactgactc cctcagtgtt tccactctgc 180

ccctccccaga ggttcagtgt tttgtgttca atgtcgagta catgaattgc acttggaaca 240

gcagctctga gccccagcct accaacctca ctctgcatta ttggtacaag aactcggata 300

atgataaagt ccagaagtgc agccactatc tattctctga agaaatcact tctggctgtc 360

agttgcaaaa aaaggagatc cacctctacc aaacatttgt tgttcagctc caggacccac 420

gggaacccag gagacaggcc acagatgc taaaactgca gaatctggtg atcccctggg 480

ctccagagaa cctaacactt cacaaactga gtgaatccca gctagaactg aactggaaca 540

acagattctt gaaccactgt ttggagcact tggtgcagta ccggactgac tgggaccaca 600

gctggactga acaatcagtg gattatagac ataagttctc cttgcctagt gtggatgggc 660

agaaacgcta cacgtttcgt gttcggagcc gctttaaccc actctgtgga agtgctcagc 720

attggagtga atggagccac ccaatccact gggggagcaa tacttcaaaa gagaatcctt 780

tcctgtttgc attggaagcc gtggttatct ctgttggctc catgggattg attatcagcc 840

ttctctgtgt gtatttctgg ctggaacgga cgatgccccg aattcccacc ctgaagaacc 900

tagaggatct tgttactgaa taccacggga acttttcggc ctggagtggt gtgtctaagg 960

gactggctga gagtctgcag ccagactaca gtgaacgact ctgcctcgtc agtgagattc 1020

ccccaaaagg aggggccctt ggggaggggc ctggggcctc cccatgcaac cagcatagcc 1080

cctactgggc ccccccatgt tacaccttaa agcctgaaac ctgaacccca atcctctgac 1140

agaagaaccc cagggtcctg tagccctaag tggtactaac tttccttcat tcaacccacc 1200

tgcgtctcat actcacctca ccccactgtg gctgatttgg aattttgtgc ccccatgtaa 1260

gcacccccttc atttggcatt ccccacttga gaattaccct tttgccccga acatgttttt 1320

cttctccctc agtctggccc ttccttttcg caggattctt cctccctccc tctttccctc 1380

ccttcctctt tccatctacc ctccgattgt tcctgaaccg atgagaaata aagtttctgt 1440

tgataatcat c 1451

<210>58

<211>521

<212>PRT

<213> Homo sapiens

<400>58

Met Gly Leu Gly Arg Cys Ile Trp Glu Gly Trp Thr Leu Glu Ser Glu

1 5 10 15

Ala Leu Arg Arg Asp Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile

20 25 30

Cys Thr Cys Val Cys Leu Gly Val Ser Val Thr Gly Glu Gly Gln Gly

35 40 45

Pro Arg Ser Arg Thr Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile

50 55 60

Asp Cys His Trp Ser Ala Pro Glu Leu Gly Gln Gly Ser Ser Pro Trp

65 70 75 80

Leu Leu Phe Thr Ser Asn Gln Ala Pro Gly Gly Thr His Lys Cys Ile

85 90 95

Leu Arg Gly Ser Glu Cys Thr Val Val Leu Pro Pro Glu Ala Val Leu

100 105 110

Val Pro Ser Asp Asn Phe Thr Ile Thr Phe His His Cys Met Ser Gly

115 120 125

Arg Glu Gln Val Ser Leu Val Asp Pro Glu Tyr Leu Pro Arg Arg His

130 135 140

Val Lys Leu Asp Pro Pro Ser Asp Leu Gln Ser Asn Ile Ser Ser Gly

145 150 155 160

His Cys Ile Leu Thr Trp Ser Ile Ser Pro Ala Leu Glu Pro Met Thr

165 170 175

Thr Leu Leu Ser Tyr Glu Leu Ala Phe Lys Lys Gln Glu Glu Ala Trp

180 185 190

Glu Gln Ala Gln His Arg Asp His Ile Val Gly Val Thr Trp Leu Ile

195 200 205

Leu Glu Ala Phe Glu Leu Asp Pro Gly Phe Ile His Glu Ala Arg Leu

210 215 220

Arg Val Gln Met Ala Thr Leu Glu Asp Asp Val Val Glu Glu Glu Arg

225 230 235 240

Tyr Thr Gly Gln Trp Ser Glu Trp Ser Gln Pro Val Cys Phe Gln Ala

245 250 255

Pro Gln Arg Gln Gly Pro Leu Ile Pro Pro Trp Gly Trp Pro Gly Asn

260 265 270

Thr Leu Val Ala Val Ser Ile Phe Leu Leu Leu Thr Gly Pro Thr Tyr

275 280 285

Leu Leu Phe Lys Leu Ser Pro Arg Val Lys Arg Ile Phe Tyr Gln Asn

290 295 300

Val Pro Ser Pro Ala Met Phe Phe Gln Pro Leu Tyr Ser Val His Asn

305 310 315 320

Gly Asn Phe Gln Thr Trp Met Gly Ala His Gly Ala Gly Val Leu Leu

325 330 335

Ser Gln Asp Cys Ala Gly Thr Pro Gln Gly Ala Leu Glu Pro Cys Val

340 345 350

Gln Glu Ala Thr Ala Leu Leu Thr Cys Gly Pro Ala Arg Pro Trp Lys

355 360 365

Ser Val Ala Leu Glu Glu Glu Gln Glu Gly Pro Gly Thr Arg Leu Pro

370 375 380

Gly Asn Leu Ser Ser Glu Asp Val Leu Pro Ala Gly Cys Thr Glu Trp

385 390 395 400

Arg Val Gln Thr Leu Ala Tyr Leu Pro Gln Glu Asp Trp Ala Pro Thr

405 410 415

Ser Leu Thr Arg Pro Ala Pro Pro Asp Ser Glu Gly Ser Arg Ser Ser

420 425 430

Ser Ser Ser Ser Ser Ser Ser Asn Asn Asn Asn Asn Tyr Cys Ala Leu Gly Cys

435 440 445

Tyr Gly Gly Trp His Leu Ser Ala Leu Pro Gly Asn Thr Gln Ser Ser

450 455 460

Gly Pro Ile Pro Ala Leu Ala Cys Gly Leu Ser Cys Asp His Gln Gly

465 470 475 480

Leu Glu Thr Gln Gln Gly Val Ala Trp Val Leu Ala Gly His Cys Gln

485 490 495

Arg Pro Gly Leu His Glu Asp Leu Gln Gly Met Leu Leu Pro Ser Val

500 505 510

Leu Ser Lys Ala Arg Ser Trp Thr Phe

515 520

<210>59

<211>332

<212>PRT

<213> Homo sapiens

<400>59

Met His Leu Gly Ser Asn Cys Cys Lys Asn Gly Gln Thr Leu Leu Gln

1 5 10 15

Arg Thr Cys His Gly Val Ser Cys Cys Gly Trp Trp Phe Gln Ala Ala

20 25 30

Arg Ser Ile Leu Gly Lys Gly Pro Ser Ala Gln Ser Leu Ala Gly Trp

35 40 45

Thr Leu Glu Ser Glu Ala Leu Arg Arg Asp Met Gly Thr Trp Leu Leu

50 55 60

Ala Cys Ile Cys Ile Cys Thr Cys Val Cys Leu Gly Val Ser Val Thr

65 70 75 80

Gly Glu Gly Gln Gly Pro Arg Ser Arg Thr Phe Thr Cys Leu Thr Asn

85 90 95

Asn Ile Leu Arg Ile Asp Cys His Trp Ser Ala Pro Glu Leu Gly Gln

100 105 110

Gly Ser Ser Pro Trp Leu Leu Phe Thr Arg Leu Leu Ala Ala His Ile

115 120 125

Ser Ala Ser Cys Gly Ala Val Ser Ala Pro Ser Cys Cys His Leu Arg

130 135 140

Gln Cys Ser Cys His Leu Thr Ile Ser Pro Ser Leu Ser Thr Thr Ala

145 150 155 160

Cys Leu Gly Gly Ser Arg Ser Ala Trp Trp Thr Arg Ser Thr Cys Pro

165 170 175

Gly Asp Thr Ser Asn Ile Ser Ser Ser Gly His Cys Ile Leu Thr Trp Ser

180 185 190

Ile Ser Pro Ala Leu Glu Pro Met Thr Thr Leu Leu Ser Tyr Glu Leu

195 200 205

Ala Phe Lys Lys Gln Glu Glu Ala Trp Glu Gln Ala Gln His Arg Asp

210 215 220

His Ile Val Gly Val Thr Trp Leu Ile Leu Glu Ala Phe Glu Leu Asp

225 230 235 240

Pro Gly Phe Ile His Glu Ala Arg Leu Arg Val Gln Met Ala Thr Leu

245 250 255

Glu Asp Asp Val Val Glu Glu Glu Arg Tyr Thr Gly Gln Trp Ser Glu

260 265 270

Trp Ser Gln Pro Val Cys Phe Gln Ala Pro Gln Arg Gln Gly Pro Leu

275 280 285

Ile Pro Pro Trp Gly Trp Pro Gly Asn Thr Leu Val Ala Val Ser Ile

290 295 300

Phe Leu Leu Leu Thr Gly Pro Thr Tyr Leu Leu Phe Lys Leu Ser Pro

305 310 315 320

Arg Leu Gly Trp Gly Pro Thr Gly Pro Val Cys Cys

325 330

<210>60

<211>369

<212>PRT

<213> Homo sapiens

<400>60

Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu

1 5 10 15

Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly

20 25 30

Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp

35 40 45

Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val

50 55 60

Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro

65 70 75 80

Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn

85 90 95

Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr

100 105 110

Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe

115 120 125

Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln

130 135 140

Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu

145 150 155 160

Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn

165 170 175

Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp

180 185 190

Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe

195 200 205

Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg

210 215 220

Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp

225 230 235 240

Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe

245 250 255

Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu

260 265 270

Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro

275 280 285

Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His

290 295 300

Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser

305 310 315 320

Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro

325 330 335

Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn

340 345 350

Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu

355 360 365

Thr

<210>61

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> VH CDR2

<400>61

Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210>62

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR1

<400>62

Lys Ala Ser Gln His Val Ile Thr His Val Thr

1 5 5 10

<210>63

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR3

<400>63

Gln His Phe Tyr Asp Tyr Pro Leu Thr

1 5

<210>64

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR3

<400>64

Gln His Phe Tyr Glu Tyr Pro Leu Thr

1 5

<210>65

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> VL CDR2

<400>65

Gly Thr Ser Tyr Ser Tyr Ser

1 5

Claims (141)

1. antibody preparation, it comprises the antibody or the antibody fragment of aqueous carrier, phosphate and 50mg/ml or higher concentration, and described antibody preparation is fit to the administration of human object through preparing.

2. one kind through preparing the antibody preparation that is fit to the administration of human object, described preparation comprises the antibody or the antibody fragment of aqueous carrier, phosphate and 10mg/ml or higher concentration, wherein, in the presence of salt, be lower than phosphate buffer when preparation of described antibody pI with pH, exist the described antibody of preparing with the histidine buffering liquid of described pH down to compare with the salt of same concentrations, the following phase behaviour of one or more of described antibody or antibody fragment reduces:

(a) folding intermediate is separated in formation;

(b) colloid unstability;

(c) the solubility combination of antibody molecule; Or

(d) precipitation of antibody molecule;

Wherein said at least a or multiple phase behaviour is by being selected from down the technical measurement of group: efficient size exclusion chromatograph (HPSEC) technology, tangential flow filtration (TFF) technology, static light scattering (SLS) technology, Fourier transform infrared spectroscopy (FTIR) technology, circular dichroism (CD) technology, carbamide-inductive protein unfolding technology, tryptophan primary fluorescence technology, differential scanning calorimetry (DSC) and 1-anilino--8-LOMAR PWA EINECS 246-676-2 (ANS) protein bound technology.

3. preparation as claimed in claim 1 or 2 is characterized in that, described antibody or antibody fragment immunologic opsonin are in conjunction with the IL-9 polypeptide.

4. preparation as claimed in claim 1 or 2 is characterized in that described aqueous carrier is a distilled water.

5. preparation as claimed in claim 1 or 2 is characterized in that, the pH scope of described preparation is 4.0 to 8.0.

6. preparation as claimed in claim 5 is characterized in that, described pH scope is about 6.0 to 6.5.

7. preparation as claimed in claim 1 or 2 also comprises the salt that concentration is no more than about 200mM.

8. preparation as claimed in claim 7 is characterized in that, the concentration range of described salt is that about 100mM is to about 200mM.

9. preparation as claimed in claim 6, also comprising concentration range is about salt of 100 to about 200mM.

10. preparation as claimed in claim 1 or 2 also comprises sugar.

11. preparation as claimed in claim 6 also comprises sugar.

12. preparation as claimed in claim 10 is characterized in that, described sugar is sucrose.

13. preparation as claimed in claim 11 is characterized in that, described sugar is sucrose.

14. preparation as claimed in claim 10 is characterized in that, described sugar is trehalose.

15. preparation as claimed in claim 11 is characterized in that, described sugar is trehalose.

16. preparation as claimed in claim 12 is characterized in that, described concentration of sucrose is up to 10%.

17. preparation as claimed in claim 13 is characterized in that, described concentration of sucrose is up to 10%.

18. preparation as claimed in claim 14 is characterized in that, the concentration of described trehalose is up to 10%.

19. preparation as claimed in claim 15 is characterized in that, the concentration of described trehalose is up to 10%.

20. preparation as claimed in claim 1 or 2, also comprises surfactant.

21. preparation as claimed in claim 6, also comprises surfactant.

22. preparation as claimed in claim 9, also comprises surfactant.

23. preparation as claimed in claim 21 is characterized in that, described surfactant is tween 20 or tween 80.

24. preparation as claimed in claim 22 is characterized in that, described surfactant is tween 20 or tween 80.

25. preparation as claimed in claim 23 is characterized in that, described surfactant concentrations is up to 0.1%.

26. preparation as claimed in claim 24 is characterized in that, described surfactant concentrations is up to 0.1%.

27. preparation as claimed in claim 1 or 2 is characterized in that, the concentration of described antibody or antibody fragment is 100mg/ml at least.

28. preparation as claimed in claim 1 or 2 is characterized in that, the concentration range of described antibody or antibody fragment is that about 50mg/ml is to about 150mg/ml.

29. preparation as claimed in claim 1 or 2 is characterized in that, phosphatic concentration range is that about 10mM is to about 100mM.

30. preparation as claimed in claim 29 is characterized in that, phosphatic concentration range is that about 25mM is to about 75mM.

31. preparation as claimed in claim 1 or 2 is characterized in that, measures through efficient size exclusion chromatograph (HPSEC), described antibody or antibody fragment are stable during at least 15 days 40 ℃ of storages.

32. preparation as claimed in claim 1 or 2 is characterized in that, measures through HPSEC, described antibody or antibody fragment are stable during at least 6 months in room temperature storage.

33. preparation as claimed in claim 1 or 2 is characterized in that, measures through HPSEC, described antibody or antibody fragment are stable during at least 1.5 years 4 ℃ of storages.

34. preparation as claimed in claim 1 or 2 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

35. preparation as claimed in claim 6 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

36. preparation as claimed in claim 9 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

37. preparation as claimed in claim 1 or 2 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 2% between the storage life form aggregation.

38. preparation as claimed in claim 6 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 2% between the storage life form aggregation.

39. preparation as claimed in claim 9 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 2% between the storage life form aggregation.

40. preparation as claimed in claim 1 or 2 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 1% between the storage life form aggregation.

41. preparation as claimed in claim 6 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 1% between the storage life form aggregation.

42. preparation as claimed in claim 9 is characterized in that, measures through HPSEC, described antibody or antibody fragment less than 1% between the storage life form aggregation.

43. preparation as claimed in claim 1 or 2 is characterized in that, the reference antibody that stores preceding antibody with representative is compared, and described antibody or its fragment keep at least 80% binding ability.

44. preparation as claimed in claim 6 is characterized in that, the reference antibody that stores preceding antibody with representative is compared, and described antibody or its fragment keep at least 80% binding ability.

45. preparation as claimed in claim 9 is characterized in that, the reference antibody that stores preceding antibody with representative is compared, and described antibody or its fragment keep at least 80% binding ability.

46. preparation as claimed in claim 1 or 2 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 85% binding ability.

47. preparation as claimed in claim 6 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 85% binding ability.

48. preparation as claimed in claim 9 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 85% binding ability.

49. preparation as claimed in claim 1 or 2 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 90% binding ability.

50. preparation as claimed in claim 6 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 90% binding ability.

51. preparation as claimed in claim 9 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 90% binding ability.

52. preparation as claimed in claim 1 or 2 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 95% binding ability.

53. preparation as claimed in claim 6 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 95% binding ability.

54. preparation as claimed in claim 9 is characterized in that, compares with reference antibody, described antibody or its fragment keep at least 95% binding ability.

55. preparation as claimed in claim 1 or 2, it is characterized in that, described antibody or antibody fragment are 4D4,4D4H2-1 D11,4D4com-XF-9,4D4com-2F9,7F3,71A10,7F3 22D3,7F3com-2H2,7F3com-3H5 or 7F3com-3D4, or its Fab.

56. preparation as claimed in claim 6, it is characterized in that, described antibody or antibody fragment are 4D4,4D4H2-1 D11,4D4com-XF-9,4D4com-2F9,7F3,71A10,7F3 22D3,7F3com-2H2,7F3com-3H5 or 7F3com-3D4, or its Fab.

57. preparation as claimed in claim 9, it is characterized in that, described antibody or antibody fragment are 4D4,4D4H2-1 D11,4D4com-XF-9,4D4com-2F9,7F3,71A10,7F3 22D3,7F3com-2H2,7F3com-3H5 or 7F3com-3D4, or its Fab.

58. preparation as claimed in claim 57 is characterized in that, described antibody or antibody fragment are 7F3com-2H2.

59. a suitable parenteral administration people pharmaceutical unit dosage forms, it comprises claim 1 or the 2 described antibody preparations that are contained in the suitable vessel.

60. pharmaceutical unit dosage forms as claimed in claim 59 is characterized in that, described antibody preparation is used for intravenous, subcutaneous or intramuscular injection.

61. a pharmaceutical unit dosage forms that is fit to the aerosol administration of human, it comprises claim 1 or the 2 described antibody preparations that are contained in the suitable vessel.

62. pharmaceutical unit dosage forms as claimed in claim 61 is characterized in that, described antibody preparation is an intranasal administration.

63. an antibody preparation, it produces by lyophilizing claim 1 or 2 described aqueous antibody preparations.

64. a sealed container, it contains claim 1 or 2 described preparations.

65. a sealed container, it contains the described preparation of claim 9.

66. as the described sealed container of claim 64, it has is enough to the volume rebuild with acceptable carrier pharmaceutically.

67., it is characterized in that described carrier is the aqueous solution (D5W) or the saline of water for injection, USP, 5% glucose as the described sealed container of claim 66.

68., it is characterized in that described container is kept gnotobasis as the described sealed container of claim 67, and allow under the condition of not losing aseptic, to rebuild described preparation.

69. medicine box, it comprises the antibody preparation that is contained in one or more containers and the operation instructions of said preparation, described antibody preparation contains antibody or its fragment of aqueous carrier, phosphate and 50mg/ml or higher concentration, and is fit to the administration of human object through preparing.

70., it is characterized in that described preparation is a sterile preparation as the described medicine box of claim 69.

71., it is characterized in that described aqueous carrier is a distilled water as the described medicine box of claim 69.

72. as the described medicine box of claim 70, it is characterized in that, described antibody or antibody fragment are 4D4,4D4H2-1 D11,4D4com-XF-9,4D4com-2F9,7F3,71A10,7F3 22D3,7F3com-2H2,7F3com-3H5 or 7F3com-3D4, or its Fab.

73., it is characterized in that described antibody or antibody fragment are 7F3com-2H2 as the described medicine box of claim 72.

74., it is characterized in that described preparation produces by lyophilizing aqueous antibody preparation as claim 70,72 or 73 described medicine boxs.

75., it is characterized in that the concentration of described antibody or antibody fragment is that about 50mg/ml is to about 150mg/ml as claim 70 or 73 described medicine boxs.

76. prevention, control, treatment or improve inflammatory diseases, autoimmune disease, unconventionality expression and/or active diseases associated with the IL-9 polypeptide, with the unconventionality expression of IL-9 polypeptide and/or active relevant or be the disease or the imbalance of feature with it, with the unconventionality expression of IL-9R or its one or more subunits and/or active relevant or be the disease or the imbalance of feature with it, autoimmune disease, inflammatory diseases, proliferative disease or infection (preferred respiratory tract infection), or the method for its one or more symptoms, described method comprises object prevention that needs or the described antibody preparation of claim 56 for the treatment of effective dose.

77. the unconventionality expression of a prevention, control, treatment or improvement and IL-9 polypeptide and/or active relevant or with its be the disease of feature or imbalance, with the unconventionality expression of IL-9R or its one or more subunits and/or active relevant or be disease or imbalance, autoimmune disease, inflammatory diseases, proliferative disease or the infection (preferably respiratory tract infection) of feature with it, or the method for its one or more symptoms, described method comprises object prevention that needs or the described antibody preparation of claim 57 for the treatment of effective dose.

78. the unconventionality expression of a prevention, control, treatment or improvement and IL-9 polypeptide and/or active relevant or with its be the disease of feature or imbalance, with the unconventionality expression of IL-9R or its one or more subunits and/or active relevant or be disease or imbalance, autoimmune disease, inflammatory diseases, proliferative disease or the infection (preferably respiratory tract infection) of feature with it, or the method for its one or more symptoms, described method comprises object prevention that needs or the described antibody preparation of claim 58 for the treatment of effective dose.

79., it is characterized in that measure through HPSEC, described antibody or its antibody fragment polypeptide are stable during at least 15 days 40 ℃ of storages as the described method of claim 76.

80., it is characterized in that measure through HPSEC, described antibody or its antibody fragment polypeptide are stable during at least 15 days 40 ℃ of storages as the described method of claim 77.

81., it is characterized in that measure through HPSEC, described antibody or its antibody fragment polypeptide are stable during at least 15 days 40 ℃ of storages as the described method of claim 78.

82., it is characterized in that as the described method of claim 76, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

83., it is characterized in that as the described method of claim 77, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

84., it is characterized in that as the described method of claim 78, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

85., it is characterized in that the reference antibody that stores preceding antibody or antibody fragment with representative is compared as the described method of claim 76, the binding ability of described antibody or maintenance at least 80% of its fragment and IL-9 polypeptide.

86., it is characterized in that the reference antibody that stores preceding antibody or antibody fragment with representative is compared as the described method of claim 77, the binding ability of described antibody or maintenance at least 80% of its fragment and IL-9 polypeptide.

87., it is characterized in that the reference antibody that stores preceding antibody or antibody fragment with representative is compared as the described method of claim 78, the binding ability of described antibody or maintenance at least 80% of its fragment and IL-9 polypeptide.

88., it is characterized in that described preparation is through parenteral administration as the described method of claim 76.

89., it is characterized in that described preparation is through parenteral administration as the described method of claim 77.

90., it is characterized in that described preparation is through parenteral administration as the described method of claim 78.

91., it is characterized in that described preparation gives through subcutaneous, oral or intranasal as the described method of claim 76.

92., it is characterized in that described preparation gives through subcutaneous, oral or intranasal as the described method of claim 77.

93., it is characterized in that described preparation gives through subcutaneous, oral or intranasal as the described method of claim 78.

94., it is characterized in that described inflammatory diseases is asthma or allergy as the described method of claim 76.

95., it is characterized in that described inflammatory diseases is asthma or allergy as the described method of claim 77.

96., it is characterized in that described inflammatory diseases is asthma or allergy as the described method of claim 78.

97. an antibody preparation that gives object, described preparation comprise 7F3com-2H2 or its Fab of aqueous carrier, phosphate and 50mg/ml or higher concentration.

98., it is characterized in that described preparation is aseptic as the described preparation of claim 97.

99., it is characterized in that described aqueous carrier is a distilled water as the described preparation of claim 97.

100., it is characterized in that the pH scope of described preparation is 4.0 to 8.0 as claim 97 or 98 described preparations.

101., it is characterized in that described pH scope is about 6.0 to 6.5 as the described preparation of claim 100.

102., also comprise the salt that concentration is no more than about 200mM as claim 97 or 98 described preparations.

103., it is characterized in that the concentration range of described salt is that about 100mM is to about 200mM as the described preparation of claim 102.

104., it is characterized in that the concentration range of described salt is that about 100mM is to about 200mM as the described preparation of claim 101.

105., also comprise sugar as claim 97 or 98 described preparations.

106., also comprise sugar as the described preparation of claim 101.

107., it is characterized in that described sugar is sucrose as the described preparation of claim 105.

108., it is characterized in that described sugar is sucrose as the described preparation of claim 106.

109., it is characterized in that described sugar is trehalose as the described preparation of claim 105.

110., it is characterized in that described sugar is trehalose as the described preparation of claim 106.

111., it is characterized in that described concentration of sucrose is up to 10% as the described preparation of claim 107.

112., it is characterized in that described concentration of sucrose is up to 10% as the described preparation of claim 108.

113., it is characterized in that the concentration of described trehalose is up to 10% as the described preparation of claim 109.

114., it is characterized in that the concentration of described trehalose is up to 10% as the described preparation of claim 110.

115. as claim 97 or 98 described preparations, also comprises surfactant.

116. as the described preparation of claim 101, also comprises surfactant.

117. as the described preparation of claim 104, also comprises surfactant.

118., it is characterized in that described surfactant is tween 20 or tween 80 as the described preparation of claim 116.

119., it is characterized in that described surfactant is tween 20 or tween 80 as the described preparation of claim 117.

120., it is characterized in that the concentration of described tween 20 or tween 80 is up to 0.1% as the described preparation of claim 118.

121., it is characterized in that the concentration of described tween 20 or tween 80 is up to 0.1% as the described preparation of claim 119.

122., it is characterized in that the concentration of described antibody or antibody fragment is 100mg/ml at least as claim 97 or 98 described preparations.

123., it is characterized in that the concentration range of described antibody or antibody fragment is that about 50mg/ml is to about 150mg/ml as claim 97 or 98 described preparations.

124., it is characterized in that phosphatic concentration range is that about 10mM is to about 100mM as claim 97 or 98 described preparations.

125., it is characterized in that phosphatic concentration range is that about 25mM is to about 75mM as the described preparation of claim 123.

126., it is characterized in that phosphatic concentration range is that about 25mM is to about 75mM as the described preparation of claim 124.

127., it is characterized in that measure through efficient size exclusion chromatograph (HPSEC), described antibody or antibody fragment are stable during at least 15 days 40 ℃ of storages as claim 97 or 98 described preparations.

128., it is characterized in that measure through HPSEC, described antibody or antibody fragment are stable during at least 6 months in room temperature storage as claim 97 or 98 described preparations.

129., it is characterized in that measure through HPSEC, described antibody or antibody fragment are stable during at least 1.5 years 4 ℃ of storages as claim 97 or 98 described preparations.

130., it is characterized in that as claim 97 or 98 described preparations, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

131., it is characterized in that as the described preparation of claim 101, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

132., it is characterized in that as the described preparation of claim 104, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

133. the method for a prevention, treatment or control disease or imbalance, described method comprise the object prevention that needs or the claim 1 or the 2 described antibody preparations of treatment effective dose.

134. antibody preparation that gives object, described antibody preparation comprises aqueous carrier, about 50mg/ml to 150mg/ml 7F3com-2H2 antibody, about 10mM to about 75mM phosphate, about 100mM to 200mM NaCl, and the pH of wherein said preparation is about 5.5 to 6.5.

135., it is characterized in that described preparation comprises aqueous carrier, about 100mg/ml 7F3com-2H2 antibody, about 25mM phosphate, about 150mM NaCl as the described preparation of claim 134, the pH of wherein said preparation is about 6.0.

136., it is characterized in that described preparation is to wait to open as the described preparation of claim 135.

137., it is characterized in that measure through efficient size exclusion chromatograph (HPSEC), described antibody or antibody fragment are stable during at least 15 days 40 ℃ of storages as the described preparation of claim 135.

138., it is characterized in that measure through HPSEC, described antibody or antibody fragment are stable during at least 6 months in room temperature storage as the described preparation of claim 135.

139., it is characterized in that measure through HPSEC, described antibody or antibody fragment are stable during at least 1.5 years 4 ℃ of storages as the described preparation of claim 135.

140., it is characterized in that as the described preparation of claim 135, to measure through HPSEC, described antibody or antibody fragment less than 5% between the storage life form aggregation.

141. prevention, control, treatment or improve inflammatory diseases, autoimmune disease, unconventionality expression and/or active diseases associated with the IL-9 polypeptide, with the unconventionality expression of IL-9 polypeptide and/or active relevant or be the disease or the imbalance of feature with it, with the unconventionality expression of IL-9R or its one or more subunits and/or active relevant or be the disease or the imbalance of feature with it, autoimmune disease, inflammatory diseases, proliferative disease or infection (preferred respiratory tract infection), or the method for its one or more symptoms, described method comprises object prevention that needs or the described antibody preparation of claim 135 for the treatment of effective dose.

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