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WO2006002064A2 - Anticorps inhibant l'activite du facteur des cellules souches et utilisation de ces derniers dans le traitement de l'asthme - Google Patents

Anticorps inhibant l'activite du facteur des cellules souches et utilisation de ces derniers dans le traitement de l'asthme Download PDF

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Publication number
WO2006002064A2
WO2006002064A2 PCT/US2005/021043 US2005021043W WO2006002064A2 WO 2006002064 A2 WO2006002064 A2 WO 2006002064A2 US 2005021043 W US2005021043 W US 2005021043W WO 2006002064 A2 WO2006002064 A2 WO 2006002064A2
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Prior art keywords
antibody
seq
chain variable
human
stem cell
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PCT/US2005/021043
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English (en)
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WO2006002064A3 (fr
Inventor
Steven Neben
Toshihiko Takeuchi
Adrian Tomkinson
Kathy Delaria
Kelly Yan
Teresa Wong
Malinda Longphre
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Aerovance, Inc.
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Priority claimed from US10/867,506 external-priority patent/US20050112698A1/en
Application filed by Aerovance, Inc. filed Critical Aerovance, Inc.
Publication of WO2006002064A2 publication Critical patent/WO2006002064A2/fr
Publication of WO2006002064A3 publication Critical patent/WO2006002064A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD

Definitions

  • the present invention relates to antibodies and treatments for asthma and other disorders in which stem cell factor protein is expressed in certain cells. More specifically, the present invention relates to human antibodies that inhibit stem cell factor activity, polynucleotide sequences encoding the antibodies, and use of the antibodies in the treatment of asthma and other disorders in which stem cell factor is expressed in certain cells.
  • Allergic asthma is characterized by variable reversible airway obstruction, and airway hyperresponsiveness associated with airway inflammation.
  • the inflammation is itself characterized by the presence of activated mast cells and eosinophils, which through the generation of proinflammatory mediators and cytokines play a fundamental role in the pathogenesis of the disease.
  • the eosinophil is thought to be a major effector cell in the development of airway hyperresponsiveness, through the release of cytotoxic granule proteins.
  • the initial induction of IgE-mediated mast cell activation/degranulation constitutes the primary mechanism that drives the allergic airway response and changes in airway physiology.
  • mast cells by cross linking of IgE leads to the release of histamine and generation of leukotrienes that appear to contribute to the early bronchoconstriction occurring within minutes of allergen exposure.
  • cytokines TNF, IL-4, IL-5
  • angiogenic and fibrotic factors VEGF, IL-4, IL-5
  • chemokines RANTES, eotaxin, MCP-I, TARC
  • the relationship between mast cell activation and eosinophil recruitment has substantial consequences on the pathogenesis of asthma.
  • Stem Cell Factor also known as Mast Cell Growth Factor, Steel Factor or c-kit ligand
  • SCF Stem Cell Factor
  • SCF Stem Cell Factor
  • SCF is an important hematopoietic factor that drives the differentiation of mast cells in the bone marrow.
  • SCF as a result of alternate splicing exists as a membrane bound and soluble form and is produced by bone marrow stromal cells.
  • SCF is also produced by several cell types found in peripheral tissues including fibroblasts, endothelial cells, epithelial cells, mast cells and eosinophils, and is thought to be the primary cytokine regulating the survival, activation, and degranulation of mature mast cells in the lung microenvironment.
  • SCF can directly induce mast cell activation and degranulation resulting in the release of inflammatory mediators, cytokines and chemokines as discussed above.
  • SCF strongly "augments the IgE-mediated activation of mast cells.
  • the prolonged activation of local airway mast cell populations by SCF after initial IgE-mediated activation may play a significant role in persistent activation leading to prolonged impairment of lung function.
  • SCF also induces mast cell adhesion to extra cellular matrix proteins as well as their chemotaxis.
  • SCF has also demonstrated a direct role on eosinophil adhesion by altering avidity of VLA-4 on the surface of eosinophils which has significant consequences for eosinophil migration to the lung.
  • the invention provides human antibodies that bind to stem cell factor. These antibodies are useful for a variety of therapeutic and diagnostic purposes, including the treatment of asthma.
  • the invention provides a purified human antibody which binds to stem cell factor protein, the antibody comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 19-24 or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-18, the antibody being optionally bound to a cytotoxic molecule or detectable label.
  • the antibody comprises heavy and light chain variable regions comprising a pair of amino acid sequences selected from the group consisting of SEQ ID NOS: 13 and 19, SEQ ID NOS: 14 and 20, SEQ ID NOS: 15 and 24, SEQ ID NOS: 16 and 21, SEQ ID NOS: 17 and 22 and SEQ ID NOS: 18 and 23.
  • the antibody comprises heavy and light chain variable regions comprising SEQ ID NOS: 14 and 20.
  • the antibody can also comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 57, 59, 61, 63 and 65 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 67, 69, 71, 73 and 75.
  • the antibody comprises a heavy chain variable region comprising SEQ ID NO: 14, a light chain variable region comprising SEQ ID NO: 20, a light chain variable region comprising SEQ ID NO: 59, and a light chain variable region comprising SEQ ID NO: 69.
  • the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 86-92, the antibody being optionally bound to a cytotoxic molecule or detectable label. More preferably, the light chain variable region further comprises SEQ ID NO: 59 and SEQ ID NO: 69.
  • the antibody comprises a heavy chain variable region comprising SEQ ID NO: 14. More preferably, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 14, a light chain variable region comprising SEQ ID NO: 59, a light chain variable region comprising SEQ ID NO: 69, and a light chain variable region selected from the group consisting of SEQ ID NO: 86-92.
  • the antibody comprises heavy chain variable and constant regions having the amino acid sequence of SEQ ID NO: 79, and light chain variable and constant regions having the amino acid sequence of SEQ ID NO: 77 wherein the amino acid subsequence at residues 87-94 is replaced with a sequence selected from the group consisting of SEQ ID NO: 86-92.
  • the heavy and light chain variable regions of the antibody can comprise framework residues from "native human antibodies, but preferably the heavy and light chain variable region framework are comprised of consensus framework residues. More preferably, the heavy chain variable region comprises human VH3 consensus framework residues.
  • the light chain variable region can comprise either human V ⁇ l consensus framework residues or human V ⁇ l consensus framework residues.
  • the invention also provides a preparation comprising the antibodies of the invention.
  • the purified preparations can contain one or more different antibodies of the invention.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier.
  • a further aspect of the invention provides an isolated polynucleotide or nucleotides encoding the antibodies.
  • the antibodies of the invention typically contain light and heavy chains, and the sequences encoding the light and heavy chains can be contained in one polynucleotide or in two or more polynucleotides.
  • An additional aspect of the invention provides an expression vector comprising the purified polynucleotide or polynucleotides encoding the antibodies, and host cells comprising the expression vector.
  • the invention further provides a method of producing a human antibody, comprising the steps of: culturing a host cell comprising an expression vector of the invention under conditions whereby the antibody is expressed; and purifying the antibody from the host cell or host cell culture medium.
  • Yet another aspect of the invention provides a method of treating a human disorder in which stem cell factor protein is expressed in certain cells, preferably asthma, comprising the steps of administering to a mammal, preferably a human, in need of such treatment an effective amount of a human stem cell factor antibody of the invention wherein the antibody is bound to a cytotoxic molecule, which cytotoxic molecule is capable of inducing apoptosis in the stem cell factor expressing cells.
  • a further aspect of the invention provides a method of treating a human disorder in which stem cell factor protein is expressed in certain cells, preferably asthma comprising the steps of administering to a mammal, preferably a human, in need of such treatment an effective amount of a human stem cell factor antibody of the invention.
  • FIG. 1 Vector map of pMORPHx9_Fabl_FS.
  • Naturally occurring antibodies comprise two heavy chains linked together by disulfide bonds and two light chains, one light chain being linked to each of the heavy chains by disulfide bonds.
  • Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • Each light chain has a variable domain (V L ) at one end and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains, see e.g. Chothia et al., J. MoI. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985).
  • the constant domains are not involved directly in binding the antibody to an antigen, but are involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.
  • the variable domains of each pair of light and heavy chains are involved directly in binding the antibody to the antigen.
  • the domains of natural light and heavy chains have the same general structure, and each domain comprises four framework (FR) regions, whose sequences are somewhat conserved, connected by three hyper-variable or complementarity determining regions (CDRs) (see Kabat, E. A. et a., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)).
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.
  • Antibody as used herein includes intact immunoglobulin molecules ⁇ e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA), as well as fragments thereof, such as Fab, F(ab') 2 , scFv, and Fv, which are capable of specific binding to an epitope of a human Stem Cell Factor protein.
  • immunoglobulin molecules ⁇ e.g., Ig., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA
  • fragments thereof such as Fab, F(ab') 2 , scFv, and Fv, which are capable of specific binding to an epitope of a human Stem Cell Factor protein.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab 1 , F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Antibodies that specifically bind to Stem Cell Factor provide a detection signal at least 5- ,10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies that specifically bind to human Stem Cell Factor do not detect other proteins in immunochemical assays and can immunoprecipitate the Stem Cell Factor from solution.
  • the antibodies of the invention will comprise substantially all of at least one, and typically two, variable domains (such as Fab, Fab', F(ab')-sub.2, Fabc, Fv) in which one or more of the CDR regions are synthetic amino acid sequences that bind with Stem Cell Factor, and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the framework regions can also be those of a native human immunoglobulin sequence.
  • Other CDR regions in the antibody can be selected to have human immunoglobulin consensus sequences for such CDRs or the sequence of a native human antibody.
  • the antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHl, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the antibody will be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and lgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgG 1 . Where such cytotoxic activity is not desirable, the constant domain may be of the IgG 2 class.
  • the antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the sequences of human immunoglobulin heavy and light chains are known in the art.
  • variable regions can also be selected from human immunoglobulin consensus sequences known in the art, such as the sequences disclosed in Knappik et ah, J. MoI. Biol. 296, 57-86 (2000) and U.S. Patent 6,300,064.
  • the dissociation constant (K d ) of human antibody binding to Stem Cell Factor can be assayed using any method known in the art, including technologies such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et ah, Curr. Opin. Struct. Biol. 5, 699-705, 1995).
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • human antibodies of the present invention specifically bind to human Stem Cell Factor with a K d in the range from about 1 nM (1 x 10 "9 M) to about 70 nM (70 x 10 "8 M). More preferred human antibodies of the present invention specifically bind to human Stem Cell Factor with a K d of approximately 4 nM to about 50 nM, with the most preferred antibodies of this invention binding human Stem Cell Factor protein with a K d of approximately 1 nM.
  • human antibodies of the present invention specifically bind to human Stem Cell Factor with a K d of approximately 0.8-1.7 nM (I x IQ- 9 M).
  • human antibodies of this invention will preferably bind Stem Cell Factor and neutralize its biological activity with an IC 50 ranging from about lO ⁇ g/ml to 30 ⁇ g/ml. More preferred human antibodies bind Stem Cell Factor and neutralize its biological activity with an IC 5O ranging from approximately 1 ⁇ g/ml to 3 ⁇ g/ml, with the most preferred antibodies of this invention neutralizing activity of human Stem Cell Factor protein with an IC 50 of approximately 0.1 ⁇ g/ml to 0.3 ⁇ g/ml.
  • ICs 0 is the dose that effectively neutralizes 50% of the biological response induced by human stem cell factor. (IC 50 can also refer to the inhibitory or effective concentration causing 50% of the maximum response induced by stem cell factor.)
  • This invention uses Morphosys phage-antibody technology to generate fully human antibodies against the Stem Cell Factor protein in accordance with the method of Knappik et al., J. MoI. Biol. 296, 57-86 (2000) and U.S. Patent 6,300,064; 6,696,242; and 6,706,248 .
  • a fully synthetic human combinatorial antibody library (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides is screened to identify antibodies that bind to stem cell factor.
  • the library is based upon human backbones, greatly reducing the probability of immunogenicity.
  • a number of human antibodies having the Stem Cell Factor binding characteristics described above have been identified by screening the MorphoSys HuCAL Fab library.
  • the randomized CDR cassettes assembled for the HuCAL library were designed to achieve a length distribution ranging from 5 to 28 amino acid residues, covering the stretch from position 95 to 102.
  • a number of human antibodies having the Stem Cell Factor binding characteristics described above have been identified by screening the MorphoSys HuCAL Fab library.
  • the CDR3 region is first randomized, and antibodies identified as specific for the test protein are then optimized by randomizing the CDRl and/or CDR2 regions of such antibodies to identify additional antibodies with more desirable binding profiles.
  • Human antibodies with the Stem Cell Factor binding activity described above can be identified from the MorphoSys HuCAL library as follows. Human Stem Cell Factor is coated on a microtiter plate and incubated with the MorphoSys HuCAL-Fab phage library (see Example 1). Those phage-linked Fabs not binding to Stem Cell Factor can be washed away from the plate, leaving only phage that tightly bind to Stem Cell Factor. The bound phage can be eluted by a change in pH and amplified by infection of E. coli hosts. This panning process can be repeated once or twice to enrich for a population of phage-linked antibodies that tightly bind to Stem Cell Factor.
  • the Fabs from the enriched pool are then expressed, purified, and screened in an ELISA assay.
  • the identified hits are then tested for binding using a BIAcoreTM assay, and these hits can be further screened in the cell adhesion assay as described above.
  • the initial panning of the HuCAL-Fab library also can be performed with Stem Cell Factor as the antigen in round one, followed in round 2 by Stem Cell Factor peptides fused to carrier proteins, such as BSA or transferrin, and in round 3 by Stem Cell Factor antigen again.
  • Human Stem Cell Factor peptides that can be used for panning include the amino acid shown in SEQ LD. NO: 25.
  • the CDR3 of the heavy chain which heavy chain preferably comprises a VH3 consensus heavy chain, has an amino acid sequence shown in SEQ ID NOS: 13-18.
  • the CDR3 region of the light chain has an amino acid sequence shown in SEQ ID NOS: 19- 23.
  • the light chain variable region preferably comprises a VLKI consensus variable region.
  • the CDR3 region of the light chain has the amino acid sequence shown in SEQ ID NO: 24, and the light chain is preferably comprised of a VL ⁇ l consensus variable region.
  • Human antibodies that have Stem Cell Factor binding activity are as shown in Table 3.
  • the consensus variable regions for antibodies with Stem Cell Factor binding activity are shown in Tables 1 and 2.
  • the antibody comprises heavy and light chain variable regions comprising a pair of amino acid sequences selected from the group consisting of SEQ ID NOS: 13 and 19, SEQ ID NOS: 14 and 20, SEQ ID NOS: 15 and 24, SEQ ID NOS: 16 and 21, SEQ ID NOS: 17 and 22 and SEQ ID NOS: 18 and 23.
  • the first sequence is preferably in the heavy chain CDR3 region and the second sequence is preferably in the light chain CDR3 region.
  • the antibody comprises heavy and light chain variable regions comprising the pair of amino acid sequences SEQ ID NOS: 14 and 20, wherein SEQ ID NO: 14 is in the heavy chain CDR3 region and SEQ ID NO: 20 is in the light chain CDR3 region.
  • Such preferred antibodies can also contain a light chain variable region, corresponding to the CDRl region, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 57, 59, 61, 63 or 65, and a light chain variable region, corresponding to the CDR2 region, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 67, 69, 71, 73 and 75.
  • the antibody comprises a heavy chain variable region comprising SEQ ID NO: 14, a light chain variable region comprising SEQ ID NO: 20, a light chain variable region comprising SEQ ID NO: 59, and a light chain variable region comprising SEQ ID NO: 69.
  • a preferred antibody of the invention comprises light chain variable and constant regions as shown in SEQ ID NO: 77, and heavy chain variable and constant regions as shown in SEQ ID NO: 79.
  • the antibodies of the invention comprise a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 86-92, and a heavy chain variable region comprising SEQ ID NO: 14.
  • the light chain variable region preferably comprises a VLKI consensus variable region
  • the heavy chain variable region preferably comprises a VH3 consensus heavy chain.
  • the antibodies comprise a light chain variable region comprising SEQ ID NO: 59, SEQ ID NO: 69 and a sequence selected from the group consisting of SEQ ID NO: 86-92, and a heavy chain variable region comprising SEQ ID NO: 14.
  • SEQ ID NO: 59 is in the CDRl position of the light chain variable region
  • SEQ ID NO: 69 is in the CDR2 position of the light chain variable region
  • the sequence selected from the group consisting of SEQ ID NO: 86-92 is in the CDR3 position of the light chain variable region.
  • the antibodies comprise a heavy chain having the sequence of SEQ ID NO: 79 and a light chain having the sequence of SEQ ID NO: 77 wherein the amino acid subsequence at residues 87-94 is replaced with a sequence selected from the group consisting of SEQ ID NO: 86-92.
  • Human antibodies with the characteristics described above also can be purified from any cell that expresses the antibodies, including host cells that have been transfected with antibody-encoding expression constructs.
  • the host cells are cultured under conditions whereby the human antibodies are expressed.
  • the antibodies can then be purified from the host cells or cell culture medium. Suitable methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a purified antibody is one which has been separated from other compounds that normally associate with the antibody in the cell, such as certain proteins, carbohydrates, or lipids or separated from reagents used for chemical synthesis of the antibody, using methods well known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • the antibody will be purified (1) to at least 70% by weight of antibody as determined by the Lowry method, more preferably 80%, 90%, 95% or 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS- PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • a preparation of the antibodies comprises at least one type of antibody of the invention in a purified form, wherein the antibodies preferably comprise at least 70% by weight of the total protein in the preparation, more preferably, 80%, 90%, 95% or 99% by weight of the total protein in the preparation. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • a preparation of purified human antibodies of the invention can contain more than one type of human antibody with the Stem Cell Factor binding and neutralizing characteristics described above.
  • human antibodies can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et ah, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer).
  • fragments of human antibodies can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized molecules can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983) or other methods known in the art.
  • the composition of a synthetic polypeptide can be confirmed by amino acid analysis or sequencing (e.g., using Edman degradation).
  • the invention also provides polynucleotides encoding human stem cell factor antibodies. These polynucleotides can be used, for example, to produce quantities of the antibodies for therapeutic or diagnostic use.
  • the antibodies of the invention typically contain light and heavy chains, and the sequences encoding the light and heavy chains can be contained in one polynucleotide or in two or more polynucleotides.
  • the polynucleotides can be DNA or RNA.
  • Polynucleotides that can be used to encode the CDR3 regions of SEQ ID NOS: 13-24, and SEQ ID NOS: 86-92 are shown in SEQ ID NOS: 1-13 and SEQ ID NO: 93-99, respectively.
  • Polynucleotides that encode consensus heavy chains and light chains of human antibodies that have been isolated from the Morphosys HuCAL library are shown in Table 1 (SEQ ID NOS: 26-39).
  • Polynucleotides that can be used to encode the CDRl and CDR2 regions are shown in Table 5.
  • Polynucleotides encoding human immunoglobulin light and heavy chains are known in the art. Any combination of native human immunoglobulin light and heavy chains or consensus sequences can be used in the production of the antibodies of the invention.
  • Polynucleotides of the invention present in a host cell can be isolated from other cellular components such as membrane components, proteins, and lipids.
  • the polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated polynucleotides encoding antibodies of the invention. For example, restriction enzymes and probes can be used to isolate polynucleotides which encode the antibodies.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human antibody-encoding cDNA molecules of the invention can be made with standard molecular biology techniques, using mRNA as a template. Thereafter, cDNA molecules can be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989). An amplification technique, such as PCR, can be used to obtain additional copies of the polynucleotides.
  • synthetic chemistry techniques can be used to synthesize polynucleotides encoding antibodies of the invention.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to those depicted in SEQ ID NOS: 1-6, 7-11, 12, or 93-99 to be synthesized that will encode an antibody having, for example, one of the VH3- CDR3, VL ⁇ -CDR3, VL ⁇ -CDR3 amino acid sequences shown in SEQ ID NOS: 13-18, 19- 23, 24 or 86-92, respectively.
  • a polynucleotide encoding an antibody of the invention can comprise all or a portion of the antibody.
  • the polynucleotide can encode the light chain or heavy chain or both, or any portion of the light chain or heavy chain, or any portion of the light and heavy chains.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Expression vectors of the invention thus comprise a polynucleotide or polynucleotides encoding the antibody or portion thereof and at least one element necessary for the transcription and translation of the coding sequence, such as a promoter operably linked to the coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding human antibodies and appropriate transcriptional and translational control elements.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems, such as CHO-Kl cells or HKBI l cells described in U.S. Patent 6,136,599 and the vectors described therein. HKBI l cells and vectors described in U.S. Patent 6,136,599 are a preferred expression vector/host cell system.
  • control elements or regulatory sequences are those non-translated regions of the vector, including enhancers, promoters, and 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human antibody, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • the antibody can be tested in vitro in TF-I proliferation and eosoniphil survival assays as detailed in Examples 7 & 8, respectively.
  • the ability of Stem Cell Factor antibodies to inhibit Stem Cell Factor activity can be measured in vivo with a cynomologous monkey model.
  • Any of the human Stem Cell Factor antibodies described above can be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier preferably is non-pyrogenic.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine, and the like.
  • These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • the concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1 % to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. See U.S. Patent 5,851,525. If desired, more than one type of human antibody, for example with different K d for Stem Cell Factor binding, can be included in a pharmaceutical composition.
  • compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones, hi addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intratracheal, inhalation, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • the invention also provides methods of ameliorating symptoms of a disorder in which Stem Cell Factor is elevated. These disorders include, without limitation, asthma and other immunological or allergic disorders.
  • a therapeutically effective dose of a human antibody of the invention is administered to a patient having a disorder in which Stem Cell Factor activity is elevated, such as those disorders above.
  • a therapeutically effective dose refers to the amount of human antibody that is used to effectively treat asthma or other disorder in which stem cell factor is elevated compared with the efficacy that is evident in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially in animal models, usually rats, mice, rabbits, dogs, pigs or non-human primates.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • An experimental mouse model of allergic asthma is described in Example 9.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population) of a human antibody, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration are adjusted to provide sufficient levels of the human antibody or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Polynucleotides encoding human antibodies of the invention can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the mode of administration of human antibody-containing pharmaceutical compositions of the invention can be any suitable route which delivers the antibody to the host.
  • Pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular, intravenous, or intranasal administration.
  • the antibodies of the invention can also be bound to a cytotoxic molecule such as a chemotherapeutic agent, a toxin (such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioco ⁇ jugate).
  • a cytotoxic molecule such as a chemotherapeutic agent, a toxin (such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioco ⁇ jugate).
  • the cytotoxic molecule is capable of inducing apoptosis in stem cell factor expressing cells upon administration of the antibody with bound cytotoxic molecule.
  • Antibodies of the invention bound to a cytotoxic molecule can be used in the same manner as antibodies without a bound cytotoxic molecule, with adjustments to the dose, route of administration and dosing regimen
  • Suitable chemotherapeutic agents include adriamycin, doxorubicin, 5- fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxotere (docetaxel), busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincreistine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins, taxane and maytansin derivatives.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • a variety of radionuclides are available for the production of radioco ⁇ jugated antibodies including 212 Bi, 131 1, 131 In, 90 Y and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as 1 dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidy
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon-14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the invention also provides diagnostic methods, with which human Stem Cell Factor can be detected in a test preparation, including without limitation a sample of serum, broncheoalveolar lavage fluid, lung, a cell culture system, or a cell-free system (e.g., a tissue homogenate). Such diagnostic methods can be used, for example, to diagnose disorders in which Stem Cell Factor is altered.
  • diagnostic methods can be used, for example, to diagnose disorders in which Stem Cell Factor is altered.
  • detection of an amount of the antibody-Stem Cell Factor complex in a test sample from a patient which varied with that of an amount of the complex in a normal sample identifies the patient as likely to have the disorder.
  • the test preparation is contacted with a human antibody of the invention, and the test preparation is then assayed for the presence of an antibody-Stem Cell Factor complex.
  • Suitable assay formats include those described herein for detecting Stem Cell Factor or antibodies of the invention.
  • the invention also provides methods for detecting Stem Cell Factor or antibodies to Stem Cell Factor.
  • the methods comprise the steps of adding an antibody of the invention to a test sample suspected of containing Stem Cell Factor and detecting antibody bound to Stem Cell Factor, whereby the presence of bound antibody indicates the presence of Stem Cell Factor in the test sample.
  • the invention provides methods for detecting antibodies of the invention comprising adding Stem Cell Factor to a test sample suspected of containing antibodies of the invention and detecting Stem Cell Factor bound to antibodies of the invention, whereby the presence of bound Stem Cell Factor indicates the presence of antibodies of the invention in the test sample.
  • the human antibody can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • the antibody can be bound to a solid support, which can accommodate automation of the assay.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the antibody to the solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached to the antibody and the solid support. Binding of Stem Cell Factor and the antibody can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • Suitable radioisotopic labels include radioisotopes, such as 35 S, 14 C, 125 I, 3 H, and 131 I.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley- Interscience, New York, N.Y., Pubs. (1991) for example and radioactivity can be measured using scintillation counting.
  • Suitable fluorescent labels include rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • Suitable enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, .beta.- galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocydic .oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases e.g., firefly luciferase and bacterial luciferase, lucifer
  • enzyme-substrate combinations include, for example: Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and beta.-D- galactosidase (.beta.
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • AP alkaline phosphatase
  • beta.-D- galactosidase .beta.
  • chromogenic substrate e.g., p-nitrophenyl-.beta.-D- galactosidase
  • fluorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
  • the detectable label can be indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten e.g., digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • the antibody of the invention need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the antibody of the invention.
  • tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin.
  • the antibodies may also be used for in vivo diagnostic assays.
  • the antibody is labeled with a radio nuclide (such as 111 In, 99 Tc, 14 C, 131 1, 125 1, 3 H, 32 P or 35 S) so that the Stem Cell Factor can be localized using immunoscintiography.
  • a radio nuclide such as 111 In, 99 Tc, 14 C, 131 1, 125 1, 3 H, 32 P or 35 S
  • the antibodies of the invention may be used as affinity purification agents.
  • the antibodies are immobilized on a solid phase such as glass or plastic slides, tissue culture plates, rriicrotiter wells, tubes, silicon chips, or particles such as beads, using methods well known in the art.
  • the immobilized antibody is contacted with a sample containing the Stem Cell Factor to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except Stem Cell Factor protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as a buffer at a pH that will release the Stem Cell Factor protein from the antibody.
  • the antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • HuCAL-Fab 1 is a fully synthetic, modular human antibody library in the Fab antibody fragment format.
  • HuCAL-Fab 1 was assembled starting from an antibody library in the single-chain format (HuCAL-scFv) in accordance with the method of Knappik et ah, J. MoI. Biol. 296: 55-86 (2000)).
  • the heavy and light chain variable region sequences in the library are shown in Tables 1 and 2.
  • HuCAL-Fab 1 was cloned into a phagemid expression vector pMORPHl ⁇ Fabl ( Figure 1).
  • This vector comprises the Fd fragment with a phoA signal sequence fused at the C-terminus to a truncated gene III protein of filamentous phage, and further comprises the light chain VL-CL with an ompA signal sequence. Both chains are under the control of the lac operon.
  • the constant domains C ⁇ , Ck, and CH are synthetic genes fully compatible with the modular system of HuCAL (Knappik et al., 2000).
  • V ⁇ and VK libraries were isolated from HuCAL-scFv by restriction digest using EcoRV/Dralll and EcoRV/BsiWI, respectively. These V ⁇ and VK libraries were cloned into ⁇ MORPH18 Fabl cut with EcoRV/Dralll and EcoRV/BsiWI, respectively. After ligation and transformation in E. coli TG-I, library sizes of 4.14 x 10 8 and 1.6 x 10 8 , respectively, were obtained, in both cases exceeding the VL diversity of HuCAL-scFv.
  • VH library was isolated from HuCAL-scFv by restriction digest using Styl/Munl. This VH library was cloned into the pMORPH18-V ⁇ and VK libraries cut with Styl/Munl. After ligation and transformation in E. coli TG-I, a total library size of 2.09 x 10 10 was obtained, with 67% correct clones (as identified by sequencing of 207 clones).
  • HuCAL-Fab was amplified in 2 x TY medium containing 34 ⁇ g/ml chloramphenicol and 1 % glucose (2 x TY-CG). After helper phage infection (VCSMl 3) at 37°C at an OD600 of about 0.5, centrifugation and resuspension in 2 x TY / 34 ⁇ g/ml chloramphenicol/ 50 ⁇ g/ml kanamycin, cells were grown overnight at 30°C. Phage were PEG-precipitated from the supernatant (Ausubel et al., 1998), resuspended in PBS/20% glycerol, and stored at -80°C.
  • VCSMl 3 helper phage infection
  • Phage amplification between two panning rounds was conducted as follows: mid-log phase TGl -cells were infected with eluted phage and plated onto LB-agar supplemented with 1% of glucose and 34 ⁇ g/ml of chloramphenicol. After overnight incubation at 30°C, colonies were scraped off and adjusted to an OD600 of 0.5. Helper phage were added as described above. AERO1220-3WO 30
  • position 85 of VK 1 and V ⁇ 3 can be either T or V.
  • position 85 of V ⁇ l and V ⁇ 3 was varied as follows: Vfcl original, 85T (codon ACC); V ⁇ l library, 85T or 85V (TRIM codons ACT or GTT); V ⁇ 3 original, 85V (codon GTG); V ⁇ 3 library, 85T or 85V (TRIM codons ACT or GTT); the same applies to HuCAL Fabl.
  • the designed CDR3 length distribution is as follows. Residues which were varied are shown in brackets (x) in TABLE 1. VK CDR3, 8 amino acid residues (position 89 to 96) (occasionally 7 residues), with Q90 fixed; V lambda CDR3, 8 to 10 amino acid residues (position 89 to 96) (occasionally 7-10 residues), with Q89, S90, and D92 fixed; and VH CDR3, 5 to 28 amino acid residues (position 95 to 102) (occasionally 4-28), with DlOl fixed.
  • the Fab-encoding inserts of the selected HuCAL Fab fragments were subcloned into the expression vector pMORPHx7_FS to facilitate rapid expression of soluble Fab.
  • the DNA preparation of the selected HuCAL Fab clones was digested with Xbal / EcoRI, thus cutting out the Fab encoding insert (ompA-VL and pho A-Fd).
  • Fabs expressed in this vector carry two C-terminal tags (FLAG and Strep) for detection and purification.
  • VAAPSVFIFPPSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC SEQ ID NO: 83
  • the constant regions of human IgGl (PIR J00228), IgG4 (EMBL K01316), and serum IgAl (EMBL J00220) were dissected into overlapping oligonucleotides with lengths of about 70 bases. Silent mutations were introduced to remove restriction sites non-compatible with the HuCAL design. The oligonucleotides were spliced by overlap extension-PCR.
  • the multiple cloning site of ⁇ cDNA3.1/Zeo+ was replaced by two different sniffers.
  • the ⁇ -stuffer provided restriction sites for insertion of a ⁇ -leader (Nhel / EcoRV), HuCAL-scFv Vk-domains (EcoRV / BsiWI,) and the ⁇ -chain constant region (BsiWI / Apal).
  • the corresponding restriction sites in the ⁇ - stuffer were Nhel / EcoRV (1-leader), EcoRV / Hpal (Vl- domains), and Hpal / Apal ( ⁇ - chain constant region).
  • the ⁇ -leader (EMBL Z00022) as well as the ⁇ -leader (EMBL L27692) were both equipped with Kozak sequences.
  • the constant regions of the human K- (EMBL J00241) and ⁇ -chain (EMBL Ml 8645) were assembled by overlap extension- PCR as described above.
  • CHO-Kl cells were co-transfected with an equimolar mixture of IgG heavy and light chain expression vectors. Double-resistant transfectants were selected with 600 ⁇ g/ml G418 and 300 ⁇ g/ml Zeocin (Invitrogen) followed by limiting dilution. The supernatant of single clones was assessed for IgG expression by capture-ELISA (see below). Positive clones were expanded in RPMI-1640 medium supplemented with 10% ultra-low IgG-FCS (Life Technologies, Rockville, MD, USA). After adjusting the pH of the supernatant to 8.0 and sterile filtration, the solution was subjected to standard protein A column chromatography (Poros 20 A, PE Biosystems, Foster City, CA, USA) AERO1220-3WO 36
  • TFl human hematopoietic cell proliferation assay The capacity of stem cell factor antibodies to inhibit Stem Cell Factor growth factor activity was measured utilizing a TFl human hematopoietic cell proliferation assay.
  • TFl cells American Type Culture Collection, Rockville, Maryland, USA
  • RPMI + 10% serum with Stem Cell Factor human from R&D Systems, rat and cyno from Bayer
  • anti-human Stem Cell Factor murine polyclonal antibody R&D Systems, catalog #AF-255-NA, Minneapolis, MN, USA.
  • GM-CSF(R&D Systems) treatment was used as a positive control.
  • This assay was used to measure the bioactivity of the Fabs and IgGs, species cross-reactivity of the Fabs and IgGs, and for other preformulation activities.
  • Stem cell factor was shown to stimulate TF-I cell proliferation with an IC 5O of 2ng/ml while the addition of polyclonal Stem Cell Factor antibodies neutralized this activity with an IC 50 of 8 ⁇ g/ml.
  • the IC 50 values for inhibition of cyno SCF were between 20 and 60 ug/ml for all three Fabs tested.
  • the IC 50 values for inhibition of rat SCF were between 20 and 60 ug/ml for Al and A2 and between 6.1 and 20 ug/ml for E6.
  • the observation that rat SCF could be inhibited by any of the Fabs was unexpected due to the lack of strong binding between the Fabs and rat SCF as determined by BIAcore assay.
  • the reason for an inhibitory effect in the TF-I proliferation assays is still unclear but may be due to low affinity binding of the Fabs to rat SCF that is adequate enough to block its interaction with c-kit on the TF-I cells.
  • Fab protein More of the Fab protein was expressed for all 5 Fab clones.
  • the five Fab clones were assayed for their ability to inhibit TF-I proliferation with 10 ng/ml SCF or GM-CSF. All five clones were capable of inhibiting SCF-induced TF-I proliferation up to 96.6-139.7% at 200 ug/ml.
  • D5 and E6 had the most favorable IC 5O values (10-12ug/ml) compared to the other clones, which had IC 50 S of 29- 30 ug/ml. None of the clones had an effect on GM-CSF-induced TF-I proliferation at up to 200 ug/ml suggesting little to no non-specific or cytotoxic effects of the Fab clones on the TF-I cells. PBS dilution controls indicated no significant effect on proliferation compared to SCF alone. However, there was a trend to lower proliferation with the 1 :5 dilution (that used for the 200 ug/ml Fab clone treatment) which may have contributed to AERO 1220-3 WO 38
  • TF-I cells were stimulated with recombinant SCF from rat and cynomologous monkeys. It was determined that both recombinant cyno and rat SCF were capable of stimulating human TF-I proliferation in a dose responsive manner.
  • the approximate IC 50 for human, cyno and rat SCF in the assay were 2, 25 and 17 ng/ml, respectively
  • the human primary eosinophil survival and activation assays involved isolation of peripheral blood eosinophils and long term culture (i.e. up to 3 days) with or without growth and survival factors such as IL-5 or SCF. These assays were performed to confirm the bioactivity of Fabs and IgGs and evaluate species cross-reactivity in primary cells.
  • stem cell factor antibodies to inhibit Stem Cell Factor-mediated eosinophil survival and activation was determined in cells harvested from human and cynomolgus monkey blood.
  • eosinophils are isolated from peripheral blood by negative selection of CD 16 using a steel VarioMacs column protocol (Miltenyi Biotec, Auburn, CA). This protocol typically returns eosinophils at >95% purity and works equally well for human and cynomolgus monkey blood. Purified eosinophils are then plated at 1x10 5 cells/well in a 96 well plate with varying concentrations of Stem Cell Factor (R&D Systems) or anti-Stem Cell Factor antibodies.
  • R&D Systems Stem Cell Factor
  • SCF promotes eosinophil survival in a dose dependent manner and that survival can be inhibited by anti-SCF (10 ug/ml).
  • anti-SCF 10 ug/ml
  • the anti-apoptotic effect of anti-SCF was also dose dependent.
  • mice were immunized intraperitoneally (i.p.) with cockroach allergen (CRA) in phosphate buffered saline (PBS) on day 0.
  • CRA cockroach allergen
  • PBS phosphate buffered saline
  • Mice were then rechallenged on day 20 and 22 by intratracheal (i.t.) administration of CRA.
  • the second i.t. challenge is given at a time when there is a considerable amount of inflammation found within and around the airway, including eosinophils.
  • Anti-mouse Stem Cell Factor polyclonal antibody (20 mg/kg) was administered in saline buffer at the time of i.t. CRA challenge. Airway responsiveness to methacholine (AHR) was measured 24 hours after the last CRA challenge with a mouse plethysmograph (Buxco, Trot NY). Airway eosinophil counts were determined 24h after the last CRA challenge. Anti-mouse Stem Cell Factor polyclonal antibody yielded a 6-fold reduction in the development of AHR versus animals receiving no antibodies. Anti-mouse Stem Cell Factor polyclonal antibody also produced a 2-fold reduction in airway tissue eosinophilia.
  • Fabs A2 and D5 were optimized for higher affinity binding to human SCF.
  • the CDR3 loops from A2 and D5 were maintained, while the CDRl and CDR2 loops from both the light and heavy chain were randomized and approximately ten million A2 variants and ten million D5 variants were expressed on the surface of M 13 bacteriophage (Morphosys).
  • the CDRl and CDR2 loops were simultaneously excised from the A2 and D5 kappa light chain variable region with the restriction enzymes Pstl and SanDI. Randomized cassettes prepared according to the method in Knappik et al. (2000) containing randomized CDRl and CDR2 with the intervening Framework 2 region were then inserted to provide A2 and D5 kappa light chain variable regions with randomized CDRl and CDR2.
  • the CDRl region of the A2 and D5 heavy chain variable region VH3 was excised using BspEl and BssHII and randomized using cassettes prepared according to the method in Knappik et al. (2000).
  • the CDR2 region of the A2 and D5 heavy chain variable region H3 was excised with Xhol and BssHII and randomized using the same type of cassettes.
  • the phagemid expression vector pMORPHl ⁇ Fabl containing the CDRl and CDR2 libraries was amplified as described in Example 1.
  • the Fab phage libraries were panned for three rounds against human SCF as described in Example 3.
  • the Fab-encoding inserts of selected HuCAL Fab 1 clones were subcloned into the expression vector pMORPHx7_FS as described in Example 4 to create the Fab expression vector designated pMORPHx9_Fabl_FS.
  • the clones were expressed in E. coli and purified using StrepTagII via StrepTactin affinity chromatography (Sigma- Genosys, Woodland, TX).
  • Strep-tagged proteins readily bind StrepTactin with high specificity and the protein was eluted from the column upon competition with desthiobiotin.
  • a lL culture typically yielded 1-3 mg of purified Fab.
  • the expressed Fabs were tested in BiaCORE assays (Uppsala, Sweden). The Fabs with the highest affinity were selected for testing in the TF-I proliferation assay.
  • immunoglobulin expression vectors were constructed in accordance with the method of Example 6.
  • the scFv portion of the Fab heavy chain was inserted into the expression vector pcDNA3.1+ with IgGl heavy chain constant regions.
  • the scFv portion of the Fab light chain was inserted into the vector pcDNA3.1/Zeo+ with kappa light chain constant regions.
  • an expression vector containing both heavy and light chains was constructed.
  • the scFv portions of the Fab heavy and light chains were inserted into an expression vector of the type described in U.S. Patent 6,136,599 that contains heavy and light IgG chains.
  • the expression vector also contained the drug resistance gene hph.
  • the sequence of the CDRl region is AGAGCGAGCCAGGGCAT TCGGGGGTACCTGGGG.
  • the sequence of the CDR2 region is TCGGCCAGCAGCTTGCAAAGC.
  • the sequence of the CDR3 region is CAGCAGTATTCTGGTATGCCTTAT.
  • the sequence of the CDRl region is RASQGIRGYLG.
  • the sequence of the CDR2 region is SASSLQS:
  • the sequence of the CDR3 region is QQYSGMPY.
  • SCF antibody A2-G8 heavy chain variable region (DNA sequence): TTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGA GCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGC CAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCG GCAGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGAT AATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATA CGGCCGTGTATTATTGCGCGCGTCGTGATTTTTTTGCTCACTTTGATGTTTGGG GCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 78) AERO 1220-3 WO 44
  • the sequence of the CDRl region is GGATTTACCTTTAGCAGCTATG CGATGAGC.
  • the sequence of the CDR2 region is GCGATTAGCGGTAGCGGCGGC AGCACCTATTATGCGGATAGCGTGAAAGGC.
  • the sequence of the CDR3 region is CGTGATTTTTTTGCTCACTTTGATGTT.
  • the sequence of the CDRl region is GFTFSSY AMS.
  • the sequence of the CDR2 region is AISGSGGSTYY ADSVKG.
  • the sequence of the CDR3 region is RDFFAHFDV.
  • SCF antibody A2-G8 heavy chain constant region (DNA sequence): GCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGT GCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT TCCTCTTCCCCAAAACCCAAGGAACCCTCATGATCTCCCGGACCCCTGAG GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
  • SCF antibody A2-G8 heavy chain constant region protein sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 81)
  • SCF antibody A2-G8 light chain constant region (DNA sequence) is SEQ ID NO: 82.
  • SCF antibody A2-G8 light chain constant region is SEQ ID NO: 83.
  • HKBI l cells (as described in U.S. Patent 6,136,599) were co-transfected with an equimolar mixture of IgG heavy and light chain expression vectors. The antibody was expressed without selection.
  • IgG-heavy and light chain were cloned in a single expression vector also possessing a drug resistant gene (e.g. hph).
  • a drug resistant gene e.g. hph.
  • Hygromycin B 50 ⁇ g/ml
  • HygB-resistant cells were obtained ⁇ 3 weeks post transfection.
  • stable levels of IgG expression were obtained (5 - 6 weeks post transfection)
  • cells were scaled up (7 weeks post transfection) and large volume of cells (10 liters) were utilized for production of IgG on a weekly-basis using a Wave Bioreactor to produce smaller amounts of IgG until the required amount of IgG ( ⁇ 100 mg) was obtained.
  • the scaled-up culture had to be inoculated in a fermentor to produce larger volumes of material which took ⁇ 4 weeks. An additional three weeks were required for affinity purification.
  • the first screen was a cell-free binding assay using the BIAcore system to identify Fabs that bound purified SCF. Antibodies showing such activity proceeded to cell-based assays.
  • the second screen was a TF-I human hematopoietic cell line proliferation assay. SCF stimulates TF-I cells to proliferate with an IC 50 of approximately 1 ng/ml. Antibodies that were capable of inhibiting SCF-induced TF-I proliferation with an IC 50 of 100 ug/ml or less were tested on primary eosinophils and mast cells in vitro. In vitro survival of eosinophils harvested from either human or Cynomologus monkey peripheral blood can be prolonged by treatment with SCF. Antibodies capable of inhibiting this prolonged survival with an IC 50 of 100 ug/ml or better proceeded to in vivo screening.
  • Soluble human, cynomologous (also referred to as "cyno"), and rat SCF were immobilized on a BIAcore sensor chip through amine coupling. Binding to all three forms of SCF was measured for Fabs that were shown to bind to SCF to a level that was at least two-fold over background in a direct ELISA assay. Those antibodies that showed binding to human SCF were scaled up, purified, and decontaminated of endotoxins. These antibodies were further studied in cell-based assays described below.
  • Fabs were expressed at small scale and tested for binding using the BIAcore. Six anti-SCF Fabs with binding to human SCF were expressed and tested in the TF-I AERO 1220-3 WO 47
  • the lead candidates from this assay were Fabs A2 and D5.
  • the lead candidates from this assay were Fabs A2 and D5.
  • A2-G8 had the best inhibitory activity of all the clones and were chosen for conversion
  • VH primer Xhol 5' - CAC TCG AGA CCC TTC CAG -3' (SEQ ID NO: 100)
  • the amplified PCR product was gel purified and digested with Bbsl and Xhol.
  • the pMORPHl ⁇ phagemid vector with the Fab A2-G8 insert was similarly digested with Bbsl/Xhol and gel purified to obtain the remaining fragment of the A2-G8 Fab.
  • the purified PCR product was ligated into the Bbsl/Xhol digested vector and electroporated into electrocompetent TGl E. coli cells.
  • the resultant library contained 5 x 10 5 clones.
  • Cynomolgus Monkey SCF (cSCF) was expressed in HKBl 1 cells and purified using a NiNTA affinity resin (Process Sciences, Berkeley CA). The randomized CDR3 phage library was panned for three rounds against cSCF using 4 different panning strategies. All panning strategies were performed with 300 ⁇ l of 10 ⁇ g/ml cSCF, in PBS, coated in triplicate to wells of Maxisorp ELISA plates (Corning, ) in the first round of panning. The second strategy used 10 ⁇ g/ml cSCF in the second round of panning followed by 2 ⁇ g/ml cSCF in the third round, both coated to a single well.
  • Strategy 3 used 2 ⁇ g/ml cSCF coated to a single well of an ELISA plate in the second round of panning and 0.2 ⁇ g/ml cSCF in the third round.
  • the fourth strategy used 2 ⁇ g/ml cSCF in the second round and 0.05 ⁇ g/ml in the third.
  • the extracts containing Fab were also tested for the ability to inhibit SCF binding to the SCF receptor.
  • ELISA plates were coated overnight with 2 ⁇ g/ml human SCFR (R&D Systems, Minneapolis, MN) in PBS at 4°C. After washing with TBS-T, the plate was blocked with 5 mg/ml BSA in PBS. The extract was diluted 1/5 in PBS buffer containing 12.5 ng/ml cSCF and 1 mg/ml BSA. After a 1 hr incubation at room temperature, 100 ⁇ l was added to the blocked ELISA plate and incubated an additional hour.
  • the plate was washed 5 times with TBS-T, and 100 ⁇ l of a 0.4 ⁇ g/ml solution of anti-SCF (R&D Systems) in 1 mg/ml BSA in PBS was added. After 1 hour the plate was washed and incubated with anti-Mouse-HRP (Sigma, St. Louis, MO). The plate was developed with BM Blue POD substrate (Roche Diagnostics, Chicago, IL) and read at 370 mi. AERO1220-3WO 50
  • high affinity phage antibodies can be
  • the off-rates were determined using a high density surface CM5 chip (5000
  • the above transformed cells were plated onto agar plates containing 2YT/1% glucose/34 ⁇ g/ml chloramphenicol. A total of 188 clones were picked and expressed in 96-well deep well blocks containing 1.5 ml 2YT/0.1% glucose/34 ⁇ g/ml chloramphenicol. The cells were centrifuged and resuspended in BBS buffer (0.2 M boric acid, pH 8.0, 0.15 M NaCl, 2.5 mM EDTA ) containing 2.5 mg/ml lysozyme. After 1 hr at RT, the solution was centrifuged and filtered using a 96 well 0.45 ⁇ m plate (Millipore, Bedford, MA).
  • Fab binding to cSCF was performed by surface plasmon resonance using a BIAcore 2000 (Biacore, Piscataway, New Jersey), according to the manufacturer's instructions and a CM5 chip (Biacore) coated with cSCF.
  • a CM5 chip (Biacore) coated with cSCF.
  • a research grade CM5 chip was activated using the standard NHS/EDC procedure.
  • Cynomologus monkey SCF was diluted to 20 ⁇ g/ml in 10 mM NaAcetate, pH 5.0, and a total of 5000 RU's were coupled to channel 2 of the sensor surface. The remaining active sites were quenched by the addition of IM ethanolamine, pH 8.0.
  • Channel 1 was subjected to the coupling procedure in the absence of protein and was used as a reference surface. Extracts were diluted 1/5 in HBS-EP running buffer and injected for 2 min using a flow rate of 20 ⁇ l/min. The surface was regenerated by two consecutive injections of 25 ⁇ l, 10 mM glycine, pH 2.5, using a flow rate of 100 ⁇ l/min. The k of j was calculated from a single concentration of Fab using BIAevaluation software (Biacore).
  • the cells were induced by addition of IPTG to a final concentration of 0.6 mM. Growth continued at 30°C overnight. Cells were harvested by centrifugation (15 min at 5000 RPM in GS3 rotor) and periplasmic extracts prepared. Cells were resuspended in 40 ml buffer containing 30 mM Tris, pH 8.0, 1 mM EDTA, 20% sucrose, and protease inhibitor cocktail. After incubation for 1 hr at 4°C, the solution was centrifuged for 10 min at 5000 x g.
  • the supernatant was kept at 4°C and the remaining pellet resuspended in 40 ml buffer containing 0.2 M boric acid, pH 8.0, 0.15 M NaCl, 2.5 mM EDTA, and a protease inhibitor cocktail. The solution was left overnight at 4°C.
  • the periplasmic extract was applied to the column, washed with 20 mis running buffer and Fab eluted with 5 mis of 5 mM desthiobiotin in running buffer. Elutions were concentrated 10-fold using a Millipore Ultrafree Biomax 5K NMWL centrifugal devise.
  • the concentrated Fab was loaded onto a Vivapure Mini Q High Load spin column (Vivascience, Carlsbad, CA), which had been equilibrated in running buffer. The column was centrifuged for 5 min at 5000 rpm and flow through collected. The spin column was rinsed with 100 ⁇ l of running buffer, combined with original flow thru, and applied to an additional column. The resultant solution was dialyzed into RPMI media, sterile filtered, and stored at 4°C until needed. Protein concentration was determined using Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) using BSA as standard. Endotoxin levels were determined using QCLlOOO assay kit (Biowhittaker, Walkersville, MD).
  • Fabs were selected for large scale purification based on BIAcore k off ranking and the ability to inhibit receptor binding (Table 12). Affinity purification using StrepTactin Sepharose yielded between 0.5 and 2.0 mg of Fab from a 1 L of culture. The endotoxin in the preparations was removed using ion-exchange chromatography with a AERO 1220-3 WO 54
  • Fabs A12 and F5L were chosen to test the feasibility of purifying untagged versions using Protein L chromatography. Expression in E. coli TGl F- cells and preparation of periplasmic extracts were performed as described above. The extracts were dialyzed overnight in binding buffer containing 0.1 M NaPhosphate, pH 7.2, and 0.15 M NaCl. After equilibrating a 1 ml Protein L Plus column (Pierce, Rockford, IL) in binding buffer, the dialyzed extract was loaded at a flow rate of approximately 3 ml/min. The column was washed with 20 column volumes of binding buffer and eluted with low pH elution buffer (Pierce, Rockford, IL). Fractions (1 ml) were collected in tubes containing 100 ⁇ l of 1 M Tris, pH 7.5. Protein concentration was determined using Coomassie Plus Protein Assay Reagent using BSA as standard.
  • Protein L is an immunoglobulin-binding protein that came originally from the bacteria Peptostreptococcus magnus. Unlike Protein A and G, Protein L has the ability to bind immunoglobulins through the kappa light chain and hence has the unique ability to bind both ScFv and Fab fragments. Affinity purification using Protein L yielded 1.9 and 3.7 mg of the Fabs Al 2 and F5L, respectively, from a 1 L culture. This yield was 2-4 fold higher than the same clones purified via StrepTactin Sepharose. The activity of the Fab F5L purified via Protein L was comparable to that purified via StrepTactin Sepharose (data not shown), as judged by affinity characterization.
  • TFl cell proliferation assay was used to measure the ability of anti-SCF Fab' s and IgG to inhibit the proliferative effect of SCF on TFl cells.
  • TFl cells American Type Culture Collection, Rockville, MD
  • 96 well plates (1 x AERO1220-3WO 55
  • Fabs D6L, F5L, D5, Bl, and A12 had at least 6-fold lower IC 50 5 S than the A2-G8 Mab (Table 12). These Fabs were equal in potency when inhibiting the TF-I proliferation response to either human or cyno SCF (Table 12).
  • Affinity measurements were carried out on a BIAcore 2000 (Biacore) at 25°C.
  • a BIAcore CM5 chip (Biacore) was used to amine couple 185 RU's of cSCF and 150 RU's of hSCF as described above.
  • Purified Fab' s were diluted in HBS-EP running buffer at concentrations ranging from 1.95 to 125 nM. A volume of 100 ⁇ l was injected using a flow rate of 50 ⁇ l/min in Kinject mode.
  • the affinity constants for Fab binding to human SCF were slightly lower than those K d 's for cynomologus monkey SCF.
  • the affinity of the A2-G8 clone in an IgG vector was measured using the IgG immobilized to the surface of the CM5 chip, hi this orientation, the avidity affects of the antibody are not factored into the K d calculation and therefore represents a true measure of the affinity for the binding site.
  • the K d value was approximately equal to the A2-G8 in the Fab vector.
  • An improvement in the k Off was offset by a reduction in the on-rate.

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Abstract

La présente invention concerne des anticorps humains qui sont dirigés spécifiquement contre le facteur des cellules souches contenant au moins un CDR provenant d'une bibliothèque combinatoire d'anticorps. Cette invention se rapporte également à des compositions pharmaceutiques comprenant les anticorps et des méthodes de traitement de l'asthme, ainsi que des méthodes de détection du facteur des cellules souches au moyen des anticorps.
PCT/US2005/021043 2004-06-14 2005-06-14 Anticorps inhibant l'activite du facteur des cellules souches et utilisation de ces derniers dans le traitement de l'asthme WO2006002064A2 (fr)

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US57946204P 2004-06-14 2004-06-14
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US10/867,506 US20050112698A1 (en) 2001-12-17 2004-06-14 Antibody inhibiting stem cell factor activity and use for treatment of asthma

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10111966B2 (en) 2016-06-17 2018-10-30 Magenta Therapeutics, Inc. Methods for the depletion of CD117+ cells
WO2022197914A3 (fr) * 2021-03-17 2022-10-27 Opsidio, LLC Anticorps anti-facteur de cellules souches et leurs méthodes d'utilisation

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* Cited by examiner, † Cited by third party
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AR037756A1 (es) * 2001-12-17 2004-12-01 Bayer Corp Anticuerpo que inhibe la actividad del factor de las celulas precursoras y su uso para el tratamiento del asma.

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10111966B2 (en) 2016-06-17 2018-10-30 Magenta Therapeutics, Inc. Methods for the depletion of CD117+ cells
WO2022197914A3 (fr) * 2021-03-17 2022-10-27 Opsidio, LLC Anticorps anti-facteur de cellules souches et leurs méthodes d'utilisation

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