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WO1998012330A1 - Cytokine mammalienne et reactifs associes - Google Patents

Cytokine mammalienne et reactifs associes Download PDF

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Publication number
WO1998012330A1
WO1998012330A1 PCT/IB1997/001140 IB9701140W WO9812330A1 WO 1998012330 A1 WO1998012330 A1 WO 1998012330A1 IB 9701140 W IB9701140 W IB 9701140W WO 9812330 A1 WO9812330 A1 WO 9812330A1
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Prior art keywords
leu
protein
lys
ser
gin
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PCT/IB1997/001140
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English (en)
Inventor
Andrea Knappe
Helmut Fickenscher
Bernard Fleckenstein
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Andrea Knappe
Helmut Fickenscher
Bernard Fleckenstein
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Application filed by Andrea Knappe, Helmut Fickenscher, Bernard Fleckenstein filed Critical Andrea Knappe
Priority to AU41334/97A priority Critical patent/AU4133497A/en
Publication of WO1998012330A1 publication Critical patent/WO1998012330A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention pertains to compositions related to proteins which function in controlling biology and physiology of mammalian cells, e.g., cells of a mammalian immune system.
  • mammalian cells e.g., cells of a mammalian immune system.
  • it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate activation, development, differentiation, and function of various cell types, including hematopoietic cells.
  • Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment.
  • the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • the carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • the carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
  • the mammalian immune response is based on a series of complex cellular
  • lymphokines soluble proteins
  • cytokines cytokines
  • monokines soluble proteins
  • lymphocytes which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal)
  • T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T-cells) making up the immune network.
  • mast cell which has not been positively identified in all mammalian species
  • mast cell is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts.
  • Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g. , histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis.
  • IL-10 Cytokine Synthesis Inhibitiory Factor (CSIF)
  • CSF Cytokine Synthesis Inhibitiory Factor
  • lymphokines e.g., related to IL-10
  • new lymphokines could contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve the immune system and/or hematopoietic cells.
  • lymphokines which enhance or potentiate the beneficial activities of known lymphokines would be highly advantageous .
  • the present invention provides new interleukin compositions and related compounds, and methods for their use.
  • the present invention is directed to mammalian, e.g., rodent, canine, feline, primate, interleukin-XX (IL-XX) and its biological activities. It includes nucleic acids coding for polypeptides themselves and methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein, and/or by functional assays for IL-10-like activities applied to the polypeptides, which are typically encoded by these nucleic acids. Methods for modulating or intervening in the control of an immune response are provided.
  • cDNA cloned complementary DNA
  • the present invention is based, in part, upon the discovery of a new cytokine exhibiting high secguence similarity to cellular IL-10.
  • it provides a gene encoding a protein whose mature size is about 150 a ino acids, which is expressed in virally transformed cells, and certain tissues, e.g., kidney, and possibly lung and liver.
  • Functional equivalents exhibiting significant sequence homology will be available from other mammalian, e.g., mouse and rat, and non-mammalian species.
  • the present invention provides a substantially pure or recombinant IL-XX protein or peptide fragment thereof.
  • Various embodiments include an antigenic protein or peptide selected from a protein or peptide from a warm blooded animal selected from the group of birds and mammals, including a primate; a protein or peptide comprising at least one polypeptide segment of SEQ ID NO: 2 ; a protein or peptide which exhibits a post-translational modification pattern distinct from natural IL-XX; or a protein or peptide which is capable of co-stimulating a T cell with another signal.
  • the protein or peptide can comprise a fusion protein.
  • Another embodiment is a composition comprising an IL-XX protein or peptide and a pharmaceutically acceptable carrier.
  • the invention also embraces an antibody which specifically binds a IL-XX protein or peptide, e.g., wherein the IL-XX is a mammalian protein, including a primate; the antibody is raised against a purified IL-XX peptide sequence of SEQ ID NO: 2; the antibody is a monoclonal antibody; or the antibody is labeled.
  • the antibodies also make available a method of purifying an IL- XX protein or peptide from other materials in a mixture comprising contacting the mixture to an anti-IL-XX antibody, and separating bound IL-XX from other materials.
  • nucleic acid capable of encoding a full length or mature IL-XX protein or peptide, including a nucleic acid which encodes a sequence of SEQ ID NO: 2; which includes a sequence of SEQ ID NO: 1; or which encodes a sequence from a natural IL-XX.
  • nucleic acid embodiments also include an expression or replicating vector.
  • the invention also provides a kit containing a substantially pure IL-XX or fragment; an antibody or receptor which specifically binds an IL-XX; or a nucleic acid, or its complement, encoding an IL-XX or peptide.
  • This kit also provides methods for detecting in a sample the presence of a nucleic acid, protein, or antibody, comprising testing said sample with such a kit.
  • the invention also supplies methods of modulating the physiology of a cell comprising contacting said cell with a substantially pure IL-XX or fragment; an antibody or binding partner which specifically binds an IL-XX; or a nucleic acid encoding an IL-XX or peptide.
  • Certain preferred embodiments include a method where the cell is a T cell and the modulating of physiology is activation of the T cell or apoptosis of the T cell; or where the cell is in a tissue and/or in an organism.
  • a method of expressing an IL-XX peptide by expressing a nucleic acid encoding an IL-XX polypeptide are also provided.
  • the invention also provides a cell, tissue, organ, or organism comprising a nucleic acid encoding an IL-XX peptide.
  • the invention also provides a recombinant nucleic acid comprising sequence at least about 70% identity over a stretch of at least about 30 nucleotides to an IL-XX nucleic acid sequence of SEQ ID NO: 1, useful, e.g., as a probe or PCR primer for a related gene.
  • Another embodiment further encodes a polypeptide comprising at least about 60% identity over a stretch of at least about 20 amino acids to an IL-XX sequence of SEQ ID NO: 2.
  • the invention further provides a method of treating a patient having an abnormal immune response by administering an effective dose of an antibody or binding partner specific for IL-XX; an IL-XX protein or polypeptide; or a nucleic acid encoding an IL-XX peptide.
  • the abnormal immune response is characterized by a T cell immune deficiency; chronic inflammation; or tissue rejection.
  • the present invention provides amino acid sequences and DNA sequences encoding various mammalian proteins which are cytokines, e.g., which are secreted molecules which can mediate a signal between immune or other cells. See, e.g., Paul (1994) Fundamental Immunology, Raven Press, N.Y. .
  • the full length cytokines, and fragments, or antagonists will be useful in physiological modulation of cells expressing a receptor. It is likely that IL-XX has either stimulatory or inhibitory effects on T-cells, B-cells, natural killer (NK) cells, macrophages, dentritic cells, hematopoietic progenitors, etc.
  • the proteins will also be useful as antigens, e.g., immunogens, for raising antibodies to various epitopes on the protein, both linear and conformational epitopes.
  • a cDNA encoding IL-XX was isolated from a virally infected cell.
  • the IL-XX cDNA contains a stretch of 510 bp in length and contained one large open reading frame encoding a small soluble cytokine-like protein.
  • Structural features include an N-terminal leader sequence of about 21 amino acids, though the natural cleavage site may vary with cell, and may be on either side by a few residues. See Table 1 and SEQ. ID. NO: 1 and 2.
  • IL-XX exhibits structural motifs characteristic of a member of the short chain cytokines.
  • Table 3 represents nucleotide sequences which encode the protein sequence.
  • Table 1 Human IL-XX nucleotide and predicted amino-acid sequence. Predicted leader sequence ends after about 21 amino acids, though natural boundaries may be different, also depending upon cell type.
  • the standard domain boundaries to helix A correspond to residues about 16-39; ⁇ l from about 47-55; helix B from about 81-100; ⁇ 2 from about 110-123; and helix D from about 125-150. See SEQ ID NO: 1 and 2.
  • CTGTGAGTGA CACACGCTGA GTGGGGTGAA GGGAA ATG CTG GTG AAT TTC ATT 53
  • AAAAAAAA 1076 Table 2: Comparison of various IL-10 embodiments compared to IL-XX. First group is signal sequences, which are not aligned. See SEQ ID NO: 2-6.
  • AREMKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKLLESSQ huIL-XX Table 3 : Reverse Translation of the amino acid sequence of human IL-XX, e.g., those nucleotide sequences which encode said protein. See SEQ ID NO: 7.
  • IL-XX is expressed in virus transformed T cell lines from primates, including humans .
  • RT PCR has indicated that IL-XX is also expressed in PHA activated PBMC, and in Jurkat and SupTil cell lines.
  • Hybridization to RNA indicates expression in human kidney, and is detected in lung and liver tissue.
  • the transcript size is about 1.0-1.2 kb, and the gene has been mapped to human chromosome 12ql5.
  • Transcripts for IL-XX have not been detected by Northern analysis in PHA activated PBMC, Jurkat cells, owl monkey kidney (OMK) cells, and human herpes infected OMK cells; and by RT PCR in HeLa cells, and the EBV-free B cell line BJA-B.
  • IL-XX as a small chain cytokine, likely mediates immune functions via a receptor of the class of cytokine receptors, possibly even sharing parts or all of the functional IL-10 receptor complex.
  • IL-XX agonists, or antagonists may also act as functional or receptor antagonists, e.g., which block IL-10 binding to its receptor, or mediating the opposite actions.
  • IL-XX, or its antagonists may be useful in the treatment of abnormal immune disorders, e.g., T cell immune deficencies, chronic inflammation, or tissue rejection.
  • the natural antigens are capable of mediating various biochemical responses which lead to biological or physiological responses in target cells.
  • the embodiment charcterized herein is from human, but other primate, or other species counterparts are expected to exist in nature. Additional sequences for proteins in other mammalian species, e.g., primates, canines, felines, and rodents, should also be available. See below. The descriptions below are directed, for exemplary purposes, to a human IL-XX, but are likewise applicable to related embodiments from other species.
  • the human IL-XX protein exhibits structural features characteristic of short chain cytokines.
  • Human IL-XX amino acid sequence is shown in SEQ ID NO: 2. These amino acid sequences, provided amino to carboxy, are important in providing sequence information in the cytokine allowing for distinguishing the protein antigen from other proteins and exemplifying numerous variants . Moreover, the peptide sequences allow preparation of peptides to generate antibodies to recognize such segments, and nucleotide sequences allow preparation of oligonucleotide probes, both of which are strategies for detection or isolation, e.g., cloning, of genes encoding such sequences .
  • human IL-XX shall encompass, when used in a protein context, a protein having amino acid sequence shown in SEQ ID NO: 2, or a significant fragment of such a protein, or another highly homologous protein derived from human, as distinguished from human IL-10.
  • Binding components e.g., antibodies, typically bind to an IL-XX with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM.
  • Homologous proteins would be found in mammalian species other than human, e.g., other primates or rodents.
  • Non-mammalian species should also possess structurally or functionally related genes and proteins, e.g., birds or amphibians.
  • polypeptide as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids, e.g., 35, 40, 45, 50, etc.
  • Such fragments may have ends which begin and/or end at virtually all positions, e.g., beginning at residues 1, 2, 3, etc., and ending at, e.g., 150, 149, 148, etc., in all combinations.
  • Particularly interesting peptides have ends corresponding to structural domain boundaries, e.g., helices A, B, C, and/or D. See Table 1. Note that the sequence of IL-XX exhibits particular identity to cellular IL-10 in the region from residue 126-137, and the other regions exhibit greater extents of IL-XX specific sequence. Seemingly important residues are those shared among all of the four entities in Table 3.
  • binding composition refers to molecules that bind with specificity to IL-XX, e.g., in an antibody-antigen interaction. It also includes compounds, e.g., proteins, which specifically associate with IL-XX, including in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent .
  • the molecule may be a polymer, or chemical reagent.
  • a functional analog may be a protein with structural modifications, or it may be a molecule which has a molecular shape which interacts with the appropriate binding determinants .
  • the compounds may serve as agonists or antagonists of a receptor binding interaction, see, e.g., Goodman, et al . (eds.) (1990) _
  • Substantially pure typically means that the protein is free from other contaminating proteins, nucleic acids, or other biologicals derived from the original source organism. Purity may be assayed by standard methods, typically by weight, and will ordinarily be at least about 40% pure, generally at least about 50% pure, often at least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in most preferred embodiments, at least about 95% pure. Carriers or excipients will often be added.
  • Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans and mice, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g. , to confer solubility, or associated with lipids or detergents.
  • the solvent and electrolytes will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological aqueous solvent.
  • the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • one or more detergents will be added, typically a mild non- denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-cholamidopropyl) dimethylammonio] -1-propane sulfonate) , or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • This invention also encompasses proteins or peptides having substantial amino acid sequence identity with the amino acid sequence of the IL-XX antigen.
  • the variants include species, polymorphic, or allelic variants.
  • Amino acid sequence homology, or sequence identity is determined by optimizing residue matches, if necessary, by introducing gaps as required. See also Needleham, et al . (1970) J. Mol. Biol. 48:443-453; Sankoff, et al . (1983) Chapter One in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, Addison- Wesley, Reading, MA; and software packages from
  • Typical homologous proteins or peptides will have from 25-100% identity (if gaps can be introduced), to 50-100% identity (if conservative substitutions are included) with the amino acid sequence of the IL-XX.
  • Identity measures will be at least about 35%, generally at least about 40%, often at least about 50%, typically at least about 60%, usually at least about 70%, preferably at least about 80%, and more preferably at least about 90% .
  • the isolated IL-XX DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, antigenic, or other functional activity. These modified sequences can be used to produce mutant antigens or to enhance expression. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms.
  • “Mutant IL-XX” encompasses a polypeptide otherwise falling within the sequence identity definition of the IL-XX as set forth above, but having an amino acid sequence which differs from that of IL-XX as normally found in nature, whether by way of deletion, substitution, or insertion. This generally includes proteins having significant identity with a protein having sequence of SEQ ID NO: 2, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most of the full length disclosed sequences. Full length sequences will typically be preferred, though truncated versions will also be useful, likewise, genes or proteins found from natural sources are typically most desired.
  • IL-XX mutagenesis can also be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins .
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • a similar concept applies to heterologous nucleic acid sequences.
  • new constructs may be made from combining similar functional domains from other proteins.
  • target-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Biol . Che . 263:15985-15992.
  • the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments.
  • a double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., PCR techniques.
  • IL-XX binding to IL-10 receptor may serve to induce signaling, e.g., send a signal similar to binding by IL-10.
  • IL-XX binding to IL-10 receptor may block IL-10 signaling.
  • An IL-XX antagonist would be expected to have the opposite effect as IL-XX.
  • In vitro assays of the present invention will often use isolated protein, soluble fragments comprising receptor binding segments of these proteins, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or cytokine mutations and modifications, e.g., IL-XX analogues.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to the cytokine, or receptor binding fragments compete with a test compound.
  • IL-XX antigens include amino acid sequence mutants from naturally occuring forms, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in IL-XX amino acid side chains or at the N- or C- termini, e.g., by standard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagents for Protein Modification, vols.
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versions of the peptides with the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyro ine, phosphoserine, or phosphothreonine . Fusion polypeptides between IL-XXs and other homologous or heterologous proteins are also provided.
  • cytokine receptors or other surface proteins are multimeric, e.g., homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a receptor-binding segment, so that the presence or location of the fused ligand may be easily determined. See, e.g., Dull, et al . , U.S. Patent No. 4,859,609.
  • gene fusion partners include bacterial ⁇ - galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, yeast alpha mating factor, and detection or purification tags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al . (1988) Science
  • Fusion peptides will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, e.g., in Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel , et al . (eds.) (1993) Current Protocols in Molecular Biology, Greene and Wiley, NY. Techniques for synthesis of polypeptides are described, e.g., in Merrifield (1963) J. Amer. Che . Soc .
  • This invention also contemplates the use of derivatives of IL-XX proteins other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties or protein carriers . Covalent or aggregative derivatives will be useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of binding partners, e.g., other antigens.
  • An IL-XX can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-IL-XX antibodies or an alternative binding composition.
  • the IL-XX proteins can also be labeled with a detectable group, e.g., for use in diagnostic assays.
  • Purification of IL-XX may be effected by an immobilized antibody or complementary binding partner, e.g., binding portion of a receptor.
  • a solubilized IL-XX or fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for binding.
  • Purified antigen can be used to screen monoclonal antibodies or antigen-binding fragments, encompassing antigen binding fragments of natural antibodies, e.g., Fab, Fab', F(ab)2, etc.
  • Purified IL-XX antigens can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the cytokine, which may be diagnostic of an abnormal or specific physiological or disease condition.
  • This invention contemplates antibodies raised against amino acid sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, or fragments of proteins containing it.
  • this invention contemplates antibodies having binding affinity to or being raised against specific domains, e.g., helices A, B, C, or D.
  • the present invention contemplates the isolation of additional closely related species variants. Southern and Northern blot analysis will establish that similar genetic entities exist in other mammals. It is likely that IL-XXs are widespread in species variants, e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and primates . _
  • the invention also provides means to isolate a group of related antigens displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the molecules will be greatly accelerated by the isolation and characterization of additional distinct species or polymorphic variants of them.
  • the present invention provides useful probes for identifying additional homologous genetic entities in different species.
  • the isolated genes will allow transformation of cells lacking expression of an IL-XX, e.g., either species types or cells which lack corresponding proteins and exhibit negative background activity. This should allow analysis of the function of IL-XX in comparison to untransformed control cells.
  • Specific segments of interaction of IL-XX with interacting components may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of signal transduction will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants. Further study of the expression and control of IL-XX will be pursued.
  • the controlling elements associated with the antigens should exhibit differential physiological, developmental, tissue specific, or other expression patterns. Upstream or downstream genetic regions, e.g., control elements, are of interest.
  • Antibodies can be raised to various epitopes of the IL-XX proteins, including species, polymorphic, or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to IL-XXs in either their active forms or in their inactive forms, including native or denatured versions. Anti-idiotypic antibodies are also contemplated.
  • Antibodies, including binding fragments and single chain versions, against predetermined fragments of the antigens can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective IL-XXs, or screened for agonistic or antagonistic activity, e.g., mediated through a receptor. Antibodies may be agonsitic or antagonistic, e.g., by sterically blocking binding to a receptor. These monoclonal antibodies will usually bind with at least a K- of about 1 M, more usually at least about 300 ⁇ M, typically at least about 100 ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better. The antibodies of this invention can also be useful in diagnostic applications.
  • capture or non-neutralizing antibodies they can be screened for ability to bind to the antigens without inhibiting binding to a receptor.
  • neutralizing antibodies they can be useful in competitive binding assays. They will also be useful in detecting or quantifying IL-XX protein or its receptors. See, e.g., Chan (ed. ) (1987) Immunology: A Practical Guide, Academic Press, Orlando, FLA; Price and Newman (eds.) (1991) Principles and Practice of Immunoassay, Stockton Press, N.Y.; and Ngo (ed. ) (1988) Nonisotopic Immunoassay, Plenum Press, N.Y.
  • the antibodies, including antigen binding fragments, of this invention can be potent antagonists that bind to the antigen and inhibit functional binding, e.g., to a receptor which may elicit a biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides so that when the antibody binds to antigen, a cell expressing it, e.g., on its surface, is killed. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting.
  • Antigen fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens .
  • An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York; Williams, et al. (1967) Methods in Immunology and Immunochemistry, vol. 1, Academic Press, New York; and
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc.
  • Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) , Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.),
  • polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos.
  • the antibodies of this invention can also be used for affinity chromatography in isolating the protein.
  • Columns can be prepared where the antibodies are linked to a solid support. See, e.g., Wilchek et al . (1984) Meth. Enzymol . 104:3-55.
  • Antibodies raised against each IL-XX will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • the described peptide sequences and the related reagents are useful in detecting, isolating, or identifying a DNA clone encoding IL-XX, e.g., from a natural source. Typically, it will be useful in isolating a gene from mammal, and similar procedures will be applied to isolate genes from other species, e.g., warm blooded animals, such as birds and mammals. Cross hybridization will allow isolation of IL-XX from the same, e.g., polymorphic variants, or other species. A number of different approaches should be available to successfully isolate a suitable nucleic acid clone.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above.
  • Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual , Cold Spring Harbor Press.
  • the specific binding composition could be used for screening of an expression library made from a cell line which expresses an IL-XX. Screening of intracellular expression can be performed by various staining or immunofluorescence procedures. Binding compositions could be used to affinity purify or sort out cells expressing a surface fusion protein.
  • the peptide segments can also be used to predict appropriate oligonucleotides to screen a library.
  • the genetic code can be used to select appropriate oligonucleotides useful as probes for screening. See, e.g. SEQ ID NO: 1 or 3.
  • synthetic oligonucleotides will be useful in selecting correct clones from a library.
  • Complementary sequences will also be used as probes, primers, or antisense strands.
  • Various fragments should be particularly useful, e.g., coupled with anchored vector or poly-A complementary PCR techniques or with complementary DNA of other peptides.
  • This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding IL-XX polypeptide.
  • this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide and which is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence disclosed in, e.g., SEQ ID NO: 2.
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which exhibit high identity to an IL-XX or which was isolated using cDNA encoding an IL-XX as a probe.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components which naturally accompany a native sequence, e.g., ribosomes, polymerases, and/or flanking genomic sequences from the originating species.
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule includes isolated forms of the molecule.
  • the nucleic acid will be in a vector or fragment less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • a "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
  • nucleic acid segments of desired functions are joined together to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms .
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • site specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., fusion, polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species or polymorphic variants.
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in particularly preferred embodiments will be at least about 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.
  • a DNA which codes for an IL-XX protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or similar proteins, as well as DNAs which code for homologous proteins from different species. There are likely homologs in other species, including primates, rodents, canines, felines, and birds. Various IL-XX proteins should be homologous and are encompassed herein. However, even proteins that have a more distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate IL- XX proteins are of particular interest.
  • Recombinant clones derived from the genomic sequences, e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals” in Roitt (ed. ) Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al .
  • Substantial homology in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 58%, ordinarily at least about 65%, often at least about 71%, typically at least about 77%, usually at least about 85%, preferably at least about 95 to 98% or more, and in particular embodiments, as high as about 99% or more of the nucleotides.
  • IL-XX e.g., in SEQ ID NO: 1 or 3.
  • selective hybridization will occur when there is at least about 55% identity over a stretch of at least about 30 nucleotides, preferably at least about 75% over a stretch of about 25 nucleotides, and most preferably at least about 90% over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • the length of identity comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 28 nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides.
  • Stringent conditions in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions.
  • Stringent temperature conditions will usually include temperatures in excess of about 30° C, usually in excess of about 37° C, typically in excess of about 55° C, preferably in excess of about 70° C.
  • Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 400 mM, typically less than about 250 mM, preferably less than about 150 mM, including about 100, 50, or even 20 mM.
  • the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) L_ Mol. Biol. 31:349-370.
  • IL-XX from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. Homology may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches .
  • DNA which encodes the IL-XX or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g., Okaya a and Berg (1982) Mol. Cell. Biol. 2:161-170; Gubler and Hoffman (1983) Gene 25:263-269; and Glover (ed. ) (1984) DNA Cloning: A Practical Approach, IRL Press, Oxford.
  • the sequences provided herein provide useful PCR primers or allow synthetic or other preparation of suitable genes encoding an IL-XX; including naturally occuring embodiments.
  • This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length IL-XX or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure/function studies.
  • Vectors as used herein, comprise plasmid ⁇ , viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. See, e.g., Pouwels, et al . (1985 and Supplements) Cloning Vectors: A Laboratory Manual.
  • DNA sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.
  • operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
  • Suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et al . (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev. Microbiol . 42:177-199.
  • IL-XX polypeptide in a system which provides a specific or defined glycosylation pattern. See, e.g., Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990) Meth. Enzvmol. 185:487-511.
  • the IL-XX may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C.
  • PI phosphatidyl inositol
  • a phosphatidyl inositol cleaving enzyme e.g., phosphatidyl inositol phospholipase-C.
  • IL-XX fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis. Springer-Verlag, New York; Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; and Villafranca (ed. ) (1991) Techniques in Protein Chemistry II , Academic Press, San Diego, Ca.
  • the present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in IL-XX mediated conditions, or below in the description of kits for diagnosis.
  • IL-XX naturally occurring or recombinant
  • fragments thereof, and antibodies thereto should be useful in the treatment of conditions associated with abnormal physiology or development, including inflammatory conditions, either acute or chronic.
  • a disease or disorder associated with abnormal expression or abnormal signaling by an IL-XX should be a likely target for an agonist or antagonist.
  • the new cytokine should play a role in regulation or development of hematopoietic cells, e.g., lymphoid or myeloid cells, which affect immunological responses, e.g., inflammation and/or autoimmune disorders.
  • the cytokine should mediate, in various contexts, cytokine synthesis by the cells, proliferation, etc .
  • antagonists of IL-XX may provide a selective and powerful way to block immune responses, e.g., in situations as inflammatory or autoimmune responses, including rheumatoid arthritis, systemic lupus erythematosis (SLE) , Hashimoto's autoimmune thyroiditis, as well as acute and chronic inflammatory responses, e.g., inflammatory bowel disease. See also Samter, et al . (eds) Immunological Diseases vols. 1 and 2, Little, Brown and Co.
  • Modulated cytokine release by the naturally occurring secreted form of IL-XX should be regulatable by reagents made available herein, e.g., in a transplantation rejection situation.
  • compositions would be useful, e.g., with other modulators of inflammation.
  • Such other molecules may include steroids, other versions of IL-10, including cellular species variants, or viral IL-lOs, e.g., EBV or EHV, and all of their respective antagonists.
  • IL-lOs e.g., EBV or EHV
  • Various abnormal conditions are known in each of the cell types shown to produce IL-XX mRNA by Northern blot analysis. See Berkow (ed. ) The Merck Manual of Diagnosis and Therapy. Merck & Co . , Rahway, N. J. ; Thorn, et al .
  • IL-XX antibodies can be purified and then administered to a patient, veterinary or human.
  • These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives.
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives.
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations .
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • Drug screening using IL-XX or fragments thereof can be performed to identify compounds having binding affinity to or other relevant biological effects on IL-XX functions, including isolation of associated components . Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the cytokine. Likewise, a compound having intrinsic stimulating activity can activate the signal pathway and is thus an agonist in that it simulates the activity of IL-XX. This invention further contemplates the therapeutic use of blocking antibodies to IL-XX as antagonists and of _
  • IL-XX may play a role in leukemogenesis or in viral infections by, e.g., HTLV or herpesviruses . It is induced by infection with herpesvirus saimiri .
  • the herpesvirus also encodes a homolog of the cytokine IL-17 (CTLA-8) .
  • CTLA-8 cytokine IL-17
  • the cytokine, or antagonists may be useful in anti-tumor therapy.
  • the viral correlation may suggest that the cytokine may be important in viral infection or proliferation processes, or oncology processes, e.g., oncogenic transformation and proliferative conditions, as cancers or leukemias. See, e.g., Thorn, et al .
  • the cytokine appears to be barely expressed in kidney cell, and may play a role in that organ's function, e.g., ion exchange or blood pressure regulation.
  • the cytokine may also have water balance functions.
  • the cytokine may have some detectable expression in kidney.
  • the quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co . , Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier.
  • Slow release formulations, or a slow release apparatus will often be utilized for continuous or long term administration. See, e.g., Langer (1990) Science 249:1527- 1533.
  • IL-XX may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, topical, or paren eral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al . (eds.) (1990) Goodman and Gilman' s: The Pharmacological Bases of
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins .
  • automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al .
  • Mutational analysis can be performed, e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558, to determine specific residues critical in the interaction and/or signaling. However, residues in the A and D helices are likely to be most important in receptor interaction.
  • antagonists can normally be found once the antigen has been structurally defined, e.g., by tertiary structure data. Testing of potential interacting analogues is now possible upon the development of highly automated assay methods using a purified IL-XX. In particular, new agonists and antagonists will be discovered by using screening techniques described herein. Of particular importance are compounds found to have a combined binding affinity for a spectrum of IL-XX molecules, e.g., compounds which can serve as antagonists for species variants of IL- XX.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing an IL-XX.
  • Cells may be isolated which express an IL-XX in isolation from other molecules.
  • Such cells either in viable or fixed form, can be used for standard binding partner binding assays. See also, Parce, et al . (1989) Science 246:243-247; and Owicki , et al. (1990) Proc. Nat ' 1 Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses .
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to an IL-XX and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface, see Fodor, et al . (1991) .
  • all the pins are reacted with solubilized, unpurified or solubilized, purified IL-XX, and washed.
  • the next step involves detecting bound IL-XX.
  • Rational drug design may also be based upon structural studies of the molecular shapes of the IL-XX and other effectors or analogues. Effectors may be other proteins which mediate other functions in response to binding, or other proteins which normally interact with IL-XX, e.g., a receptor.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions, as madeled, e.g., against cellular IL-10.
  • protein structural determination see, e.g., Blundell and Johnson (1976) Protein Crystallography. Academic Press, New York.
  • kits and methods for detecting the presence of another IL-XX or binding partner typically the kit will have a compartment containing either a defined IL- XX peptide or gene segment or a reagent which recognizes one or the other, e.g., IL-XX fragments or antibodies.
  • a kit for determining the binding affinity of a test compound to an IL-XX would typically comprise a test compound; a labeled compound, for example a binding partner or antibody having known binding affinity for IL-XX; a source of IL-XX (naturally occurring or recombinant) ; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the molecule.
  • a labeled compound for example a binding partner or antibody having known binding affinity for IL-XX
  • a source of IL-XX naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the molecule.
  • a preferred kit for determining the concentration of, e.g., an IL-XX in a sample would typically comprise a labeled compound, e.g., binding partner or antibody, having known binding affinity for the antigen, a source of cytokine
  • Antibodies including antigen binding fragments, specific for the IL-XX or fragments are useful in diagnostic applications to detect the presence of elevated levels of IL-XX and/or its fragments.
  • diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the antigen in serum, or the like.
  • Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-binding partner complex) or heterogeneous (with a separation step) .
  • Various commercial assays exist, such as radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , enzyme-multiplied immunoassay technique (EMIT) , substrate-labeled fluorescent immunoassay (SLFIA) , and the like. See, e.g., Van Vunakis, et al . (1980) Meth Enzv ol .
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against an IL-XX, as such may be diagnostic of various abnormal states. For example, overproduction of IL-XX may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal activation or differentiation .
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or unlabeled antibody or binding partner, or labeled IL-XX is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non- covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the binding partner, test compound, IL-XX, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125 I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups .
  • IL-XX can be immobilized on various matrixes followed by washing.
  • Suitable matrixes include plastic such as an ELISA plate, filters, and beads. See, e.g., Coligan, et al . (eds.) (1993) Current Protocols in Immunology, Vol. 1, Chapter 2, Greene and Wiley, NY.
  • Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al . (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
  • Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of an IL-XX. These sequences can be used as probes for detecting levels of the IL-XX message in samples from patients suspected of having an abnormal condition, e.g., inflammatory or autoimmune. Since the cytokine may be a marker or mediator for activation, it may be useful to determine the numbers of activated cells to determine, e.g., when additional therapy may be called for, e.g., in a preventative fashion before the effects become and progress to significance.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature.
  • kits which also test for the qualitative or quantitative expression of other molecules are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g.,
  • IL-XX cytokine without interfering with the binding to its receptor
  • an affinity label can be fused to either the amino- or carboxyl-terminus of the ligand, though based on IL-10, the amino-terminus is more likely to succeed.
  • Such label may be a FLAG epitpe tag, or, e.g., an Ig or Fc domain.
  • An expression library can be screened for specific binding of the cytokine, e.g., by cell sorting, or other screening to detect subpopulations which express such a binding component. See, e.g., Ho, et al .
  • Protein cross-linking techniques with label can be applied to isolate binding partners of the IL-XX cytookine . This would allow identification of proteins which specifically interact with the cytokine, e.g., in a ligand- receptor like manner.
  • IL-10R is involved in response (s) to IL-XX. It is also quite possible that the functional IL-10 receptor complex may share many or all components with an IL-XX receptor complex, either a specific receptor subunit or an accessory receptor subunit .
  • PBMC peripheral blood mononuclear cells
  • RNA from these PHA-blasts was used later to subtract the normally occurring cDNAs .
  • PBMC preparation was infected with herpesvirus saimiri C488. See Fickenscher and Fleckenstein , pp345-362, above; and Biesinger, et al . (1992) Proc. Nat ' 1 Acad. Sci. USA 89:3116-3119.
  • the infected cells were cultivated in the presence of IL-2 until growth transformation was established (several months) .
  • RNA was isolated from the transformed T-cell line, designated 3C (see Fickenscher, et al . (1996) The Immunologist 4:41-43), after the cells had been stimulated using 1 ng/ml TPA (Fickenscher, et al.(1996) J. Virol.
  • RNA was isolated according to Chomczynski and Sacchi (1987) Anal. Biochem. 162:156-159.
  • the subtractive cDNA library was prepared with a cDNA subtraction kit (from Clontech, Palo Alto, CA) .
  • PCR products were cloned using a TA cloning kit (Invitrogen) .
  • the resulting cDNA plasmids were sequenced from both termini on an automated sequencer (Applied Biosystems) .
  • Plasmid akl55 contains a cDNA fragment of 540 nt . There is a single large open reading frame found, starting at nucleotide 12, and ending at nucleotide 524. Termination signals are not found in this partial cDNA.
  • 5 ' and 3 ' RACE the remaining fragments of the entire cDNA were cloned.
  • the transcript size is approximately 1.0 to 1.2 kb.
  • Genomic structure analysis indicates that introns exist at or near to between nucleotides 206 and 207, of about 35 nucleotides; between 263 and 264, of about 60 nucleotides; between 398 and 399, of about 1.5 kb; and between 464 and 465, of about 86 nucleotides.
  • the sequences of the short introns have been determined.
  • a probe specific for cDNA encoding primate IL-XX is labeled, e.g., by random priming.
  • IL-XX/akl55 is strongly transcribed in various T-cell lines of human and non-human primates, which have been in- vitro transformed to stable IL-2 dependent growth by herpesvirus sai iri C488. This expression is analysed by Northern blotting.
  • Owl monkey kidney cells (OMK) which are a primate permissive system for the human virus, and virus- infected OMK were negative by Northern blotting.
  • TPA stimulation did not significantly increase IL-XX/akl55 transcript levels in virus-transformed T-cells; and cyclosporin A did not inhibit its expression. Transcription has been confirmed by RT-PCR from transformed human T-cells in 3C and CB15 cells (Biesinger, et al .
  • IL-XX is so strongly expressed in herpesvirus saimiri-transfomed T-cells, which suggests a role in the transformation mechanism. Expression was also detected in a monkey T cell line 93C488, a cell line from Saguinus fuscicollis monkeys that produces virus particles. By RT-PCR weak transcription was detected in human PHA- activated PBMC, and in T-cell tumor lines like Jurkat (Schneider, et al (1977) Int. J.
  • mRNA dot spot assay brain, amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, temporal lobe, thalamus, subthalamic nucleus, spinal cord, heart, aorta, sceletal muscle, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, mammary gland, small intestine, spleen, thymus , peripheral leukocytes, lymph node, bone marrow, appendix
  • Chromosome mapping of human IL-XX akl55 is neither transcribed during lytic infection of OMK cells with herpesvirus saimiri C488, nor can the akl55 sequence be amplified from purified virus DNA. Chromosome mapping is a standard technique. See, e.g., BIOS Laboratories (New Haven, CT) and methods for using a mouse somatic cell hybrid panel with PCR. The gene was mapped to the human chrosome 12ql5 region.
  • EXAMPLE 4 Purification of IL-XX Protein Multiple transfeeted cell lines are screened for one which expresses the cytokine at a high level compared with other cells. Various cell lines are screened and selected for their favorable properties in handling. Natural IL-XX can be isolated from natural sources, or by expression from a transformed cell using an appropriate expression vector. Early results suggest that the cytokine, after secretion, rebinds to the cell surface. Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants . FLAG or His6 segments can be used for such purification features.
  • the IL-XX cDNA can be used as a hybridization probe to screen a library from a desired source, e.g., a primate cell cDNA library. Many different species can be screened both for stringency necessary for easy hybridization, and for presence using a probe. Appropriate hybridization conditions will be used to select for clones exhibiting specificity of cross hybridization. Screening by hybridization using degenerate probes based upon the peptide sequences will also allow isolation of appropriate clones. Alternatively, use of appropriate primers for PCR screening will yield enrichment of appropriate nucleic acid clones. Similar methods are applicable to isolate either species, polymorphic, or allelic variants.
  • Species variants are isolated using cross-species hybridization techniques based upon isolation of a full length isolate or fragment from one species as a probe.
  • antibodies raised against human IL-XX will be used to screen for cells which express cross- reactive proteins from an appropriate, e.g., cDNA library.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above.
  • Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. The resulting antibodies are used for screening, purfication, or diagnosis, as described.
  • EXAMPLE 6 Preparation of antibodies specific for IL-XX Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual Cold Spring Harbor Press. Polyclonal serum, or hybridomas may be prepared. In appropriate situations, the binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods .
  • EXAMPLE 7 Evaluation of Breadth of Biological Functions
  • the native, recombinant, and fusion proteins would be tested for agonist and antagonist activity in many biological assay systems, e.g., on T-cells, B-cells, NK, macrophages, dentritic cells, hematopoietic progenitors, etc. Because of the IL-10 structural relationship, assays related to IL-10 activity would analysed
  • IL-XX is evaluated for agonist or antagonist activity on transfected cells expressing IL-10 receptor and controls. See, e.g., Ho, et al . (1993) Proc. Nat ' 1 Acad. Sci. USA 90, 11267-11271; Ho, et al . (1995) Mol. Cell. Biol. 15:5043- 5053;and Liu, et al . (1994). J. Immunol. 152:1821-1829.
  • the IL-XX is evaluated for effect in macrophage/dendritic cell activation and antigen presentation assays, T cell cytokine production and proliferation in response to antigen or allogeneic stimulus. See, e.g., de Waal Malefyt et al . (1991) J. EXP. Med. 174:1209-1220; de Waal Malefyt et al .
  • IL-XX will also be evaluated for effects on NK cell stimulation. Assays may be based, e.g., on Hsu, et al .
  • ADDRESSEE Schering-Plough Corporation
  • B STREET: 2000 Galloping Hill Road
  • CTGTGAGTGA CACACGCTGA GTGGGGTGAA GGGAA ATG CTG GTG AAT TTC ATT 53
  • MOLECULE TYPE protein
  • AARAAYATHM GNYTNYTNAA RAARAARACN AARAARCART TYATGAARAA YTGYCARTTY 240

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Gènes purifiés codant une nouvelle cytokine mammalienne, réactifs associés, y compris les protéines purifiées, les anticorps spécifiques et les acides nucléiques qui codent ce type de molécule. On décrit aussi des procédés relatifs à l'utilisation des réactifs, et des trousses incorporant les réactifs en question.
PCT/IB1997/001140 1996-09-23 1997-09-22 Cytokine mammalienne et reactifs associes WO1998012330A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41334/97A AU4133497A (en) 1996-09-23 1997-09-22 Mammalian cytokine, related reagents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US71875396A 1996-09-23 1996-09-23
US08/718,753 1996-09-23

Publications (1)

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WO1998012330A1 true WO1998012330A1 (fr) 1998-03-26

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PCT/IB1997/001140 WO1998012330A1 (fr) 1996-09-23 1997-09-22 Cytokine mammalienne et reactifs associes

Country Status (2)

Country Link
AU (1) AU4133497A (fr)
WO (1) WO1998012330A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5231012A (en) * 1989-06-28 1993-07-27 Schering Corporation Nucleic acids encoding cytokine synthesis inhibitory factor (interleukin-10)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5231012A (en) * 1989-06-28 1993-07-27 Schering Corporation Nucleic acids encoding cytokine synthesis inhibitory factor (interleukin-10)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MARTIN H M ET AL: "Cloning and characterisation of an ovine interleukin-10-encoding cDNA", GENE, vol. 159, no. 2, 4 July 1995 (1995-07-04), pages 187-191, XP004042204 *
MIRE-SLUIS A R ET AL: "Quantitative cell line based bioassays for human cytokines", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 187, no. 2, 1995, pages 191-199, XP004020949 *

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