Interleukin-1 Alpha Antibodies and Methods of Use

Abstract

Fully human monoclonal Abs includes (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1.alpha. and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is divisional application of U.S. nonprovisional patent application Ser. No. 12/455,458, filed on Jun. 1, 2009, which claims the priority of U.S. provisional patent application Ser. Nos. 61/057,586; 61/121,391; and 61/178,350 filed on May 30, 2008; Dec. 10, 2008; and May 14, 2009, respectively.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The invention relates generally to the fields of immunology, inflammation, cancer, vascular disorders, and medicine. More particularly, the invention relates to antibodies (Abs) which specifically bind interleukin-1.alpha. (IL-1.alpha.) and methods of using such Abs to treat, prevent, or detect a pathology associated with aberrant IL-1.alpha. expression.

BACKGROUND

[0004] IL-1.alpha. is pro-inflammatory cytokine that plays a role in a number of different activities including inflammation, immune responses, tumor metastasis, and hematopoiesis. IgG autoantibodies against IL-1.alpha. occur naturally in the general human population and are thought to be beneficial in diseases such as atherosclerosis.

SUMMARY

[0005] The invention is based on the development of fully human monoclonal Abs (mAbs) that include (i) an antigen-binding variable region that exhibits very high binding affinity for human IL-1.alpha. and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors. The IL-1.alpha. specific mAbs described herein was made by replacing the constant region of a human IgG4 mAb having a variable region specific for human IL-1.alpha. with the constant region of a human IgG1 mAb.

[0006] Accordingly, the invention features a purified human IgG1 mAb that specifically binds to human IL-1.alpha., the mAb including a heavy chain covalently joined to a light chain. The heavy chain can include the amino acid sequence of SEQ ID NO: 9 and the light chain can include the amino acid sequence of SEQ ID NO:11.

[0007] Also within the invention is a set of isolated nucleic acids including a first nucleic acid encoding the heavy chain of a human IgG1 mAb that specifically binds to IL-1.alpha., and a second nucleic acid encoding the light chain of the human IgG1 mAb that specifically binds to human IL-1.alpha.. The first nucleic acid can encode the amino acid sequence of SEQ ID NO: 9 and the second nucleic acid can encode the amino acid sequence of SEQ ID NO:11. The first nucleic acid can include the nucleotide sequence of SEQ ID NO: 10 and the second nucleic acid can include the nucleotide sequence of SEQ ID NO:12.

[0008] In another aspect, the invention features an expression vector including a nucleic acid encoding the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.

[0009] Another feature of the invention is an isolated host cell (e.g. a mammalian cell such as a CHO cell) including set of isolated nucleic acids including a first nucleic acid encoding the heavy chain of a human IgG1 mAb that specifically binds to IL-1.alpha., and a second nucleic acid encoding the light chain of the human IgG1 mAb that specifically binds to human IL-1.alpha.. The heavy chain can include the amino acid sequence of SEQ ID NO: 9 and a light chain can include the amino acid sequence of SEQ ID NO:11.

[0010] The invention further features a method of killing a cell expressing human IL-1.alpha.. This method can include the step of contacting the cell with a purified human IgG1 mAb that specifically binds to human IL-1.alpha..

[0011] A method of inhibiting migration of a human cell through a basement membrane matrix is also within the invention. This method can include the step of adding a purified mAb that specifically binds to human IL-1.alpha. to a mixture including a basement membrane matrix and the human cell.

[0012] Further within the invention is a method of inhibiting an IL-1.alpha.-induced increase in ICAM-1 and/or E-selectin expression on the surface of a human endothelial cell. This method can include the step of adding a purified mAb that specifically binds to human IL-1.alpha. to a mixture including the endothelial cell and IL-1.alpha..

[0013] The invention additionally includes a method of tracking inflammation in a human subject previously subjected to the steps of: obtaining from the subject a first sample of peripheral blood mononuclear cells at a first time; contacting the first sample with a purified mAb that specifically binds to human IL-1.alpha.; and determining the percent of cells in the first sample that bind the monoclonal Ab. This method can include the steps of: (a) obtaining from the subject a second sample of peripheral blood mononuclear cells at a second time; (b) contacting the second sample with the purified mAb that specifically binds to human IL-1.alpha.; (c) determining the percent of cells in the second sample that bind the monoclonal Ab; and (d) comparing the percent of cells in the first sample that bind the mAb to the percent of cells in the second sample that bind the monoclonal Ab.

[0014] In the foregoing methods, the purified mAb can be a human IgG1 mAb including a heavy chain covalently joined to a light chain, e.g., wherein the heavy chain includes the amino acid sequence of SEQ ID NO: 9 and the light chain includes the amino acid sequence of SEQ ID NO:11.

[0015] Another method within the invention features the steps of: (a) enriching a biological sample obtained from a human subject using a filter to separate molecules according to molecular weight into a first fraction including intact IgG complexed with IL-1.alpha. and second fraction including molecules less than 100 Kda; and (b) quantifying the amount of IL-1.alpha. in the first fraction.

[0016] Yet another method within the invention features the steps of: (a) enriching a sample of plasma obtained from a human subject using a filter that separates molecules according to molecular weight into a first fraction including intact IgG complexed with IL-1.alpha. and second fraction including molecule less than 100 Kda; (b) adding the first fraction to a substrate including immobilized anti-human IgG Abs under conditions that allow IgG in the first fraction to specifically bind the anti-human IgG Abs immobilized on the substrate; (c) washing the substrate to remove material in the first fraction that does not specifically bind the immobilized anti-human IgG Abs; (d) contacting the substrate washed in step (c) with an Ab that specifically binds human IL-1.alpha. under conditions that allows the Ab that specifically binds human IL-1.alpha. to specifically bind any human IL-1.alpha. bound to the substrate; (e) washing the substrate to remove any of the Ab that specifically binds human IL-1.alpha. that is not bound to the substrate; and (f) quantifying the amount of Ab that specifically binds human IL-1.alpha. remaining bound to the substrate after step (e).

[0017] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of biological terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

[0018] The term "specifically binds", as used herein, when referring to a polypeptide (including Abs) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under designated conditions (e.g. immunoassay conditions in the case of an Ab), the specified ligand or Ab binds to its particular "target" and does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or Ab may come in contact in an organism. Generally, a first molecule that "specifically binds" a second molecule has an equilibrium affinity constant greater than about 10.sup.5 (e.g., 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, and 10.sup.12 or more) liters/mole for that second molecule.

[0019] When referring to a protein molecule such as an Ab, "purified" means separated from components that naturally accompany such molecules. Typically, an Ab or protein is purified when it is at least about 10% (e.g., 9%, 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, and 100%), by weight, free from the non-Ab proteins or other naturally-occurring organic molecules with which it is naturally associated. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically-synthesized protein or other recombinant protein produced in a cell type other than the cell type in which it naturally occurs is "purified."

[0020] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION

[0021] The invention encompasses compositions and methods relating to fully human mAbs that include (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1.alpha. and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors. The below described preferred embodiments illustrate adaptation of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

[0022] Methods involving conventional immunological and molecular biological techniques are described herein. Immunological methods (for example, assays for detection and localization of antigen-Ab complexes, immunoprecipitation, immunoblotting, and the like) are generally known in the art and described in methodology treatises such as Current Protocols in Immunology, Coligan et al., ed., John Wiley & Sons, New York. Techniques of molecular biology are described in detail in treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. Ab methods are described in Handbook of Therapeutic Abs, Dubel, S., ed., Wiley-VCH, 2007. Cell culture techniques are generally known in the art and are described in detail in methodology treatises such as Culture of Animal Cells: A Manual of Basic Technique, 4th edition, by R Ian Freshney, Wiley-Liss, Hoboken, N.J., 2000; and General Techniques of Cell Culture, by Maureen A Harrison and Ian F Rae, Cambridge University Press, Cambridge, UK, 1994. Methods of protein purification are discussed in Guide to Protein Purification: Methods in Enzymology, Vol. 182, Deutscher M P, ed., Academic Press, San Diego, Calif., 1990.

[0023] In one aspect, the invention features a fully human mAb that includes (i) an antigen-binding variable region that exhibits very high binding affinity for human IL-1.alpha. and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors. The human Ab is preferably an IgG1. The Ka of the Ab is preferably at least 1.times.10.sup.9 M.sup.-1 or greater (e.g., greater than 9.times.10.sup.10 M.sup.-1, 8.times.10.sup.10 M.sup.-1, 7.times.10.sup.10 M.sup.-1, 6.times.10.sup.10 M.sup.-1, 5.times.10.sup.10 M.sup.-1, 4.times.10.sup.10 M.sup.-1, 3.times.10.sup.10 M.sup.-1, 2.times.10.sup.10 M.sup.-1, or 1.times.10.sup.10 M.sup.-1).

[0024] Because B lymphocytes which express Ig specific for human IL-1.alpha. occur naturally in human beings, a presently preferred method for raising mAbs is to first isolate such a B lymphocyte from a subject and then immortalize it so that it can be continuously replicated in culture. Subjects lacking large numbers of naturally occurring B lymphocytes which express Ig specific for human IL-1.alpha. may be immunized with one or more human IL-1.alpha. antigens to increase the number of such B lymphocytes. Human mAbs are prepared by immortalizing a human Ab secreting cell (e.g., a human plasma cell). See, e.g., U.S. Pat. No. 4,634,664.

[0025] In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, or more) human subjects (e.g., subjects not previously administered a human IL-1.alpha. vaccine) are screened for the presence of such human IL-1.alpha.-specific Ab in their blood. Those subjects that express the desired Ab can then be used as B lymphocyte donors. In one possible method, peripheral blood is obtained from a human donor that possesses B lymphocytes that express human IL-1.alpha.-specific Ab. Such B lymphocytes are then isolated from the blood sample, e.g., by cells sorting (e.g., fluorescence activated cell sorting, "FACS"; or magnetic bead cell sorting) to select B lymphocytes expressing human IL-1.alpha.-specific Ig. These cells can then be immortalized by viral transformation (e.g., using EBV) or by fusion to another immortalized cell such as a human myeloma according to known techniques. The B lymphocytes within this population that express Ig specific for human IL-1.alpha. can then be isolated by limiting dilution methods (e.g., cells in wells of a microtiter plate that are positive for Ig specific for human IL-1.alpha. are selected and subcultured, and the process repeated until a desired clonal line can be isolated). See, e.g., Goding, Monoclonal Abs: Principles and Practice, pp. 59-103, Academic Press, 1986. Those clonal cell lines that express Ig having at least nanomolar or picomolar binding affinities for human IL-1.alpha. are preferred. MAbs secreted by these clonal cell lines can be purified from the culture medium or a bodily fluid (e.g., ascites) by conventional Ig purification procedures such as salt cuts, size exclusion, ion exchange separation, and affinity chromatography.

[0026] Although immortalized B lymphocytes might be used in in vitro cultures to directly produce mAbs, in certain cases it might be desirable to use heterologous expression systems to produce mAbs. See, e.g., the methods described in U.S. patent application Ser. No. 11/754,899. For example, the genes encoding an mAb specific for human IL-1.alpha. might be cloned and introduced into an expression vector (e.g., a plasmid-based expression vector) for expression in a heterologous host cell (e.g., CHO cells, COS cells, myeloma cells, and E. coli cells). Because Igs include heavy (H) and light (L) chains in an H.sub.2L.sub.2 configuration, the genes encoding each may be separately isolated and expressed in different vectors.

[0027] Although generally less preferred, chimeric mAbs (e.g., "humanized" mAbs), which are antigen-binding molecules having different portions derived from different animal species (e.g., variable region of a mouse Ig fused to the constant region of a human Ig), might be used in the invention. Such chimeric Abs can be prepared by methods known in the art. E. G., Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984. Similarly, Abs can be humanized by methods known in the art. For example, monoclonal Abs with a desired binding specificity can be commercially humanized or as described in U.S. Pat. No. 5,693,762; 5,530,101; or 5,585,089.

[0028] The mAbs described herein might be affinity matured to enhance or otherwise alter their binding specificity by known methods such as VH and VL domain shuffling (Marks et al. Bio/Technology 10:779-783, 1992), random mutagenesis of the hypervariable regions (HVRs) and/or framework residues (Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994; Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol. 155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995; and Hawkins et al, J. Mol. Biol. 226:889-896, 1992. Amino acid sequence variants of an Ab may be prepared by introducing appropriate changes into the nucleotide sequence encoding the Ab. In addition, modifications to nucleic acid sequences encoding mAbs might be altered (e.g., without changing the amino acid sequence of the mAb) for enhancing production of the mAb in certain expression systems (e.g., intron elimination and/or codon optimization for a given expression system). The mAbs described herein can also be modified by conjugation to another protein (e.g., another mAb) or non-protein molecule. For example, a mAb might be conjugated to a water soluble polymer such as polyethylene glycol or a carbon nanotube (See, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605, 2005). See, U.S. patent application Ser. No. 11/754,899.

[0029] Preferably, to ensure that high titers of human IL-1.alpha.-specific mAb can be administered to a subject with minimal adverse effects, the mAb compositions of the invention are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.9 or more percent by weight pure (excluding any excipients). The mAb compositions of the invention might include only a single type of mAb (i.e., one produced from a single clonal B lymphocyte line) or might include a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different types of mAbs. In addition to human IL-1.alpha. mAbs, the Ab compositions of the invention might also include other mAbs that specifically bind antigens other than human IL-1.alpha..

[0030] To modify or enhance their function, the human IL-1.alpha. mAbs might be conjugated another molecule such as a cytotoxin or detectable label. A human IL-1.alpha. specific mAb might be conjugated with one or more cytotoxins to more effectively kill cells expressing IL-1.alpha.. Cytotoxins for use in the invention can be any cytotoxic agent (e.g., molecule that can kill a cell after contacting the cell) that can be conjugated to a human IL-1.alpha. specific mAb. Examples of cytotoxins include, without limitation, radionuclides (e.g., .sup.35S, .sup.14C, .sup.32P, .sup.125I, .sup.131I, .sup.90Y, .sup.89Zr, .sup.201Tl, .sup.186Re, .sup.188Re, .sup.57Cu, .sup.213Bi, and .sup.211At), conjugated radionuclides, and chemotherapeutic agents. Further examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouricil (5-FU), methotrexate (MTX), fludarabine, etc.), anti-microtubule agents (e.g., vincristine, vinblastine, colchicine, taxanes (such as paclitaxel and docetaxel), etc.), alkylating agents (e.g., cyclophasphamide, melphalan, bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g., cisplatin (also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide, tenoposide, and camptothecins), or other cytotoxic agents such as ricin, diptheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed viral protein, ethidium bromide, glucocorticoid, anthrax toxin and others. See, e.g., U.S. Pat. No. 5,932,188.

[0031] The human IL-1.alpha. specific mAb can also be conjugated to a detectable label. Useful detectable labels in the present invention include biotin or streptavidin, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S.sub., .sup.14C, .sup.32P, .sup.111In, .sup.97Ru, .sup.67Ga, .sup.68Ga, or .sup.72As), radioopaque substances such as metals for radioimaging, paramagnetic agents for magnetic resonance imaging, enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters. Fluorescent markers may also be used and can be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0032] The present invention also encompasses nucleic acid molecules encoding fully human mAbs specific for human IL-1.alpha.. Although the same nucleic acid molecule might encode both the heavy and light chains of a human IL-1.alpha.-specific mAb, two different nucleic acid molecules, one encoding the heavy chain and the other encoding the light chain might also be used. The amino acid sequences of three IgG1 mAbs specific for human IL-1.alpha. are presented herein. See SEQ ID NOs: 1, 3, 5, 7, 9, and 11. Exemplary nucleic acid molecules encoding these amino acid sequences are also described herein. See SEQ ID NOs: 2, 4, 6, 8, 10, and 12. Any other suitable nucleic acid that encodes the amino acid sequences of the two described IgG1 mAbs or other mAbs within the invention might also be used.

[0033] For production of mAbs, the nucleic acid molecules of the invention might be incorporated into an expression vector in an orientation wherein such nucleic acid molecules are operatively linked to expression control sequences such as transcriptional and translational control sequences. Examples of expression vectors include vectors derived from plasmids and vectors derived from viruses such as adenoviruses, adeno-associated viruses, and retroviruses. The nucleic acid molecules encoding a light chain and a heavy chain might be incorporated into a single vector or different vectors. The vectors of the invention might also include regulatory sequences such as promoters and/or enhancers (see, U.S. Pat. No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615), selectable markers, or sequences encoding affinity tags (for facilitating purification) or a detectable label.

[0034] For production of mAbs, the vectors of the invention can be introduced into a suitable host cell, e.g., a prokaryotic cell such as a bacteria or, preferably, a eukaryotic cell such as mammalian, plant, or yeast host cell. Examples of methods for introducing heterologous polynucleotides into host cells include use of viral vectors, electroporation, encapsulation of the polynucleotide(s) in liposomes, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, Agrobacterium-mediated transformation, biolistic transformation, and direct microinjection of the DNA into nuclei. Mammalian cell lines are presently preferred for expression of mAbs from vectors. Examples of mammalian host cells include Chinese hamster ovary (CHO) cells (e.g., the DG44 CHO cell line), HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells, and NIH-3T3 cells. The mAbs of the invention might also be expressed in transgenic animals or plants. See, e.g., U.S. Pat. Nos. 5,827,690; 5,756,687; 5,750,172; 5,741,957; 6,046,037; and 5,959,177.

[0035] The invention provides a method for detecting a human IL-1.alpha.-expressing cell in a sample by contacting the cell with a human IL-1.alpha.-specific mAb and detecting the mAb bound to the cell. The invention also provides a method for killing a human IL-1.alpha.-expressing cell by contacting the cell with a human IL-1.alpha.-specific mAb. Such killing can be accomplished by complement-mediated killing, Ab-dependent cell-mediated cytotoxicity, or Ab-mediated delivery of a cytotoxin. The Abs described herein have also been shown to be useful for other methods.

[0036] For example, MABp1 has been to reduce IL-1.alpha. induced ICAM1 and E-selectin expression on endothelial cells. MABp1 has also been shown to be used in immunoassays for detecting and quantifying IL-1.alpha. in a biological sample.

EXAMPLES

Example 1

Cloning of Anti-hIL-1.alpha. IgG1 and Kappa Chains

[0037] Variable region heavy chain (V-HC) and variable region light chain (V-LC) sequences were gene synthesized using amino acid sequence information provided in U.S. Pat. No. 5,959,085. V-HC was PCR amplified introducing HindIII/ClaI sites upstream of the ATG start codon and a NheI site at the 3' end. The human germline IgG1 constant region (including exons and introns) was PCR amplified modifying the two 5' triplets encoding for the first two amino acids Ala-Ser to an NheI site, and introducing a BamHI site at the 3' end. The human germline IgG1 constant region amino acid sequence corresponded to Swiss-Prot entry P01857, except for a K171Q and a V261L exchange. The V-HC and constant IgG1-HC sequence were ligated using the NheI site and cloned into pcDNA3 using HindIII and BamHI sites.

TABLE-US-00001 >hIL-1a-IgG1-HC (SEQ ID NO: 1) MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >hIL-1a-IgG1-HC (SEQ ID NO: 2) atggagttcgggctgagttgggtgttcctggtggctctgctgcggggcgtgcagtgccaggtgcagctggtgga- gagtgg gggtggcgtggtgcagcctggccggtctctgcgcctgtcttgcactgcctccggttttaccttttctatgtttg- gtgtgcactgg gtgcgccaggctcccggcaagggactggaatgggtggccgccgtgagttacgacgggtccaacaaatattacgc- tgaga gcgtgaaaggcagattcaccatcagcagagataattccaagaatattctgttcctgcagatggacagtctgaga- ctggagg acactgctgtgtactactgcgctcgtggacgccctaaggtggtcatccccgcccccctggcacattggggccag- ggaactc tggtgaccttttctagcgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctct- gggggca cagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg- accag cggcgtccacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccct- ccagcag cttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagc- ccaaa tcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctctt- ccccccaa aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac- cctga ggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcccagacaaagccgcgggaggagcagtaca- acag cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaagg- tctcca acaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac- accc tgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagc- gacatc gccctggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgg- ctcc ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat- gcatgag gctctgcacaaccactacacgcagaagagcctctccttaagtccgggaaaataa

[0038] The V-LC was PCR amplified introducing HindIII/ClaI sites upstream of the ATG start codon and a BsiWI site at the 3' end. The human constant Kappa-LC sequence was PCR amplified introducing a 5' BsiWI site encoding an additional Arg and the first amino acids Thr, and a BamHI site at the 3' end. The human constant Kappa-LC amino acid sequence corresponded to Swiss-Prot entry P01834. V-HC and constant Kappa-LC sequences were ligated using the BsiWI site and cloned into pcDNA3 using HindIII and BamHI sites.

TABLE-US-00002 >hIL-1a-K-LC [SEQ ID NO: 3] MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA TYYCQQTSSFLLSFGGGTKVEHRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC >hIL-1a-K-LC [SEQ ID NO: 4] atggacatgcgcgtgcccgcccagctgctggggctgctgctgctgtggttccctggatctaggtgcgacattca- gatgacc cagtcccccagctcagtgtcagcctccgtgggcgacagagtgacaatcacctgccgcgcctctcagggaatctc- tagttgg ctggcctggtaccagcagaagcctggaaaggcccccaagctgctgatctatgaagcctccaacctggagaccgg- cgtgc cctctcgcttcagcggctcaggctcaggcagtgattttactctgaccatcagctccctgcagccagaggatttc- gctacttact actgccagcagacctcttccttcctgctgtccttcgggggaggcacaaaggtggagcaccgtacggtggctgca- ccatctg tcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttc- tatcccaga ggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca- gcaa ggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcct- gcga agtcacccatcagggcctgagttcaccggtgacaaagagcttcaacaggggagagtgttag

Example 2

Generation of NATHMAB-hIL-1.alpha. IgG1 and Kappa Chain

[0039] The complete sequence encoding the NATHMAB-hIL-1.alpha./IgG1 heavy chain was gene synthesized. The V-HC sequence corresponded to the amino acid sequence described in U.S. Pat. No. 5,959,085. The human constant IgG1-HC sequence corresponded to Swiss-Prot entry P01857. The nucleotide sequence was codon optimized for expression in CHO cells. A Kozac sequence (gccacc) was added upstream of the start ATG.

TABLE-US-00003 >NATHMAB-hIL-1A-IGG1-HC [SEQ ID NO: 5] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >NATHMAB-hIL-1A-IGG1.sup.-HC [SEQ ID NO: 6] gccaccatggagtttggtctgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagct- ggtggagt ctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatg- tttggtgtgca ttgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatatt- acgctg agtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctg- aggctggaa gatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggaca- gggaac cctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctactt- ctggaggcac cgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctga- ctagcgg cgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctcca- gcagcctc ggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagaaggtcgaacctaa- atcttgcga taaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttccccccca- aacccaaa gataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaagt- caaattca actggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacatat- agggt ggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaagg- ccctgcc agccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccct- ctcgg gatgaactgaccaagaatcaggtgagcctgacatgtcttgtgaagggtttttatccctccgacattgccgtgga- gtgggaga gcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggttccttcttcctctac- tctaagctg acagtggataagtctaggtggcagcaggggaatgtgttctcctgctctgtgatgcacgaggcactgcacaatca- ttatacac aaaagtctctgtctctgtctccaggaaagtaa

[0040] The complete sequence encoding the NATHMAB-hIL-1.alpha./Kappa light chain was gene synthesized. The V-LC sequence corresponded to the amino acid sequence described in U.S. Pat. No. 5,959,085. The human constant Kappa-LC sequence corresponded to Swiss-Prot entry P01834. The nucleotide sequence was codon optimized for expression in CHO cells. A Kozac sequence (gccacc) was added upstream of ATG.

TABLE-US-00004 >NATHMAB-hIL-1A-K-LC [SEQ ID NO: 7] MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA TYYCQQTSSFLLSFGGGTKVEHTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC NATHMAB-hIL-1A-K-LC [SEQ ID NO: 8] gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtga- tattcag atgacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccaggg- gatctc tagttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcg- aaacag gcgtgccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgag- gattttgcc acatattactgtcagcaaacttcttcttttctgctctcctttggtgggggaactaaggtggagcacacagtggc- cgccccca gcgtctttatcttccccccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataat- ttttaccc tcgcgaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagc- aaga cagcaaggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgt- acgctt gtgaggtgactcaccagggactgagtagccctgtgacaaaatctttcaataggggagaatgctga

Example 3

Expression of NATHMAB-ILl-a (IgGl/k subtype)

[0041] NATHMAB-IL-1.alpha. was expressed and purified using a transient transfection method. Cell culture supernatant or protein G affinity purified Ab was subjected to further analysis as described below. Human embryonic kidney (HEK) 293T cells were cultured in DMEM containing 10% FCS, and transiently transfected using jetPEI reagent (Polyplus) according to manufacturer's protocol. Cells were seeded on 10 cm dishes (3.times.10.sup.6 cells per 10 cm dish) 24h prior to transfection to reach approximately 50% density at the time point of transfection. 5 .mu.g per dish of pcDNA3-anti-hIL-1.alpha.-IgG1-HC and a 2-fold molar excess of pcDNA3-anti-hIL-1.alpha.-Kappa were used for transfection. After recovery, medium was changed to DMEM containing 2% FCS (10 ml per dish) and Ab was collected for 5 to 6 days. The supernatant was collected, filtered, pH adjusted to 7.5-8, and stored at 4.degree. C. until further use.

[0042] Part of the supernatant (250 ml) was incubated with protein G sepharose (GE Healthcare) for 3 h at 4.degree. C. on a rotation wheel. Then, the protein G sepharose was loaded onto a gravity flow column and washed with PBS. Ab was eluted in 1 ml fractions using 100 mM glycine/150 mM NaCl into 100 .mu.l Tris (pH 8), followed by dialysis with PBS containing 10% glycerol. The total protein concentration of each fraction was measured using the BCA Protein Detection Kit (Pierce). Correct size of heavy and light chains, and of the assembled native Ab was confirmed by SDS-PAGE.

[0043] Supernatant containing NATHMAB-hIL-1.alpha. purified Ab and Triton X-100 cell lysates of producer HEK 293T cells were tested for antigen binding in a radioimmunoassay (RIA) using .sup.125I-hIL-1.alpha.. Binding was assayed by absorption to protein G. All samples bound .sup.1251I-hIL-1.alpha. with highest activity in the eluate. Binding of purified NATHMAB-hIL-1.alpha. in a concentration of 0.012% (half-max activity in RIA) to .sup.125I-hIL-1.alpha. was used for measuring the affinity coefficient. The Ka of NATHMAB-hIL-1.alpha. under these conditions was 3.03.times.10.sup.10 M.sup.-1. Back calculation revealed an estimated concentration of approximately 30 .mu.g/ml active anti-hIL-1.alpha.-IgG in the purified eluate.

[0044] Neutralizing activity of NATHMAB-hIL1.alpha. was tested in a bioassay using the murine EL4-6.1 subline which produces high levels of IL-2 when treated with murine or human IL-1.alpha. (Zubler et al., J. Immunol. 134:3662-3668, 1985). The indicated concentrations of NATHMAB-hIL-1.alpha. (eluate) were incubated for 30 min at 37.degree. C. with various concentrations of recombinant hIL-1.alpha. (eBioscience) in a final volume of 100 .mu.l/well in a 96-well culture plate (flat bottomed). Each point was carried out in triplicate and in culture medium (DMEM, 5% FCS). To each well were added 100 .mu.l of a suspension of EL4-6.1 cells (5.times.10.sup.5 cells/ml) in culture medium containing 0.2 .mu.g/ml ionomycin. After incubation for 24 h at 37.degree. C. in a 5% CO.sub.2 incubator, cell free supernatants were harvested and assayed for IL-2 concentrations using a commercially available ELISA (R&D Systems). The results showed that NATHMAB-IL-1.alpha. effectively neutralized hIL-1.alpha.-induced IL-2 secretion by EL-4 cells.

[0045] To test for neutralization of membrane-bound hIL-1.alpha., the same EL-4 cell-based assay as described above was used with following modifications. Different concentrations of NATHMAB-hIL-1.alpha. (eluate) were incubated with various numbers of human activated monocytes. For monocyte preparation, PBMC were isolated from buffy coat using Ficoll-Paque centrifugation. Monocytes were allowed to adhere for 1.5 h at 37.degree. C. in RPMI on plastic dishes. Non-adherent lymphocytes were washed away to yield a nearly pure monocyte culture. Monocytes were cultured in RPMI containing Gln, Pyr, and 10% FCS for 24 h with LPS (1 .mu.g/ml) at 37.degree. C. in a 5% CO.sub.2 incubator. Cells were detached with PBS/2 mM EDTA, carefully scraped from plates, and transferred into Falcon tubes. Cells were washed twice with PBS, resuspended in PBS/1% PFA and fixed for 10 min at 20.degree. C. Cells were washed with glycine buffer (150 mM glycine, 75 mM NaCl, pH 7.4), then with culture medium and counted. The results showed that NATHMAB-hIL-1.alpha. effectively neutralized IL-2 secretion by EL-4 cells induced by membrane-bound hIL-1.alpha.. In an experiment similar to that described above, NATHMAB-hIL-1.alpha. was tested for neutralization of murine IL-1.alpha.. Indicated amounts of NATHMAB-hIL-1.alpha. supernatant were incubated with recombinant human (h) or murine (m) IL-1.alpha. (eBioscience). The supernatant containing the Ab neutralized human, but not murine, IL-1.alpha..

Example 4

Ab-Mediated Killing of Cancer Cells

[0046] Human peripheral blood mononuclear cells (PBMC) isolated from the buffy coat by standard Ficoll Paque preparation were incubated in either RPMI-1640 CM or RPMI-1640-CM containing rhIL-2 (30 ng/ml, ebioscience) at 37.degree. C. and 5% CO2 overnight and used as effector cells (E). THP1 cells were used as the targets (T). The assay was carried out in 96-well plates with each point in triplicate. After 1.times.10.sup.4 targets that were incubated with different concentration of MABp1 for 15 mins, effector cells were added in an ET ratio of 25:1 and 50:1 to 1.times.10.sup.4 targets and incubated for another 4 hours. 75u1 of assay volume were transferred to a new 96-well plate and cytotoxicity was assayed using the LDH cytotoxicity detection kit (Roche) according to manufacturer's protocol. % specific lysis=(mean experimental release-mean spontaneous release without antibody).times.100/(mean maximal release from targets-mean spontaneous release from targets) A. untreated PBMC were used as effector cells. B. rhIL-2-treated PBMC were used as effector cells. In both cases, increasing concentrations (1.25 to 20 ug/ml) of MABp1 resulted in increased target cell killing (up to about 90%) at both ET ratios.

Example 5

Human Anti-IL1.alpha. Specific mAb Sequences

[0047] The complete sequence encoding for another human anti-hIL-1aIgGi.sub.1/Kappa light chain specific for human IL1.alpha. (MABp1) was synthesized and expressed as described above. In the nucleic acids encoding the heavy and light chains, a Kozac sequence (gccacc) was added upstream of the start ATG.

[0048] Heavy Chain

TABLE-US-00005 [SEQ ID NO: 9] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFG VHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMD SLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 10] gccaccatggagtttggtctgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagct- ggtggagt ctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatg- tttggtgtgca ttgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatatt- acgctg agtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctg- aggctggaa gatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggaca- gggaac cctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctactt- ctggaggcac cgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctga- ctagcgg cgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctcca- gcagcctc ggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagagggtcgaacctaa- atcttgcg ataaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttccccccc- aaacccaa agataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaag- tcaaattc aactggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacata- taggg tggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaag- gccctgc cagccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccc- tctcgg gaggaaatgaccaagaatcaggtgagcctgacatgtcttgtgaagggtttttatccctccgacattgccgtgga- gtgggaga gcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggttccttcttcctctac- tctaagctg acagtggataagtctaggtggcagcaggggaatgtgttctcctgctctgtgatgcacgaggcactgcacaatca- ttatacac aaaagtctctgtctctgtctccaggaaagtaa

[0049] Light Chain

TABLE-US-00006 [SEQ ID NO: 11] MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFA TYYCQQTSSFLLSFGGGTKVEHKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 12] gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtga- tattcagat gacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccagggga- tctctag ttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcgaaa- caggcgt gccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgaggatt- ttgccacatat tactgtcagcaaacttcttcttttctgctctcctttggtggaggaactaaggtggagcacaagcggacagttgc- tgctcctagc gtctttatcttccctccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataattt- ttaccctcgc gaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagcaaga- cagca aggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgtacgct- tgtgaggt gactcaccagggactgagtagccctgtgacaaaatctttcaataggggagaatgctga

Example 6

MABp1 Binding Affinity

[0050] The binding affinity of purified MABp1 was determined using surface plasmon resonanace (SPR) on a BIAcore 2000 instrument (GE Health Sciences). A mouse monoclonal anti-human IgG (Fc) Ab was covalently immobilized on the flow cells of a CM5 sensor chip using a human Ab capture kit and amine coupling kit (GE Health Sciences). Immobilization levels of 8000-14000 RU would typically be achieved. After immobilization of the mouse anti-human IgG (Fc) capture Ab, three start-up cycles with HBS-EP running buffer (GE Health Sciences) and two start-up cycles with MABp1 were run to stabilize the CM5 surface and to remove any non-covalently bound Ab. For analysis, MABp1 Ab was diluted into HBS-EP running buffer to a final concentration of 1 .mu.g/mL and immobilized to 700 RU on one flow cell of the CM5 sensor chip. Carrier-free human IL-1A cytokine (eBioscience, #34-8019) was serially diluted in HBS-EP running buffer over a test range from 100 nM to 0.05 nM. Flow rate was 30 .mu.l/min. Dissociation data for each cytokine dilution was recorded for 15 minutes. The CM5 surface was regenerated after each cycle using a single injection of 3 M MgC12 for 25 seconds at a flow rate of 30 .mu.l/min. BiaEvaluation software and a Langmuir binding model was used to fit the data. The K.sub.D for MABp1 was determined to be less than 2.0.times.10.sup.-10 M.

Example 7

MABp1 Inhibits Tumor Cell Invasion of a Basement Membrane Matrix

[0051] Matrigel (BD), a basement membrane matrix, was thawed at 40 C overnight and the dilute (5 mg/ml to 1 mg/ml) in serum free cold cell culture media. 100 ul of the diluted matrigel was placed into the upper chambers of a 24-well transwell (Costar) and the transwell was incubated at 370 C for at least 4 to 5 h for gelling. Tumor cells (MDA-MB-231 and THP-1) were harvested from tissue culture flasks by Trypsin/EDTA, washed with culture media, and resuspended in medium containing 1% FBS at a density of 1.times.10.sup.6 cells/ml. The gelled matrigel was gently washed with warmed serum free-culture media, and 100 ul of the cell suspension was added in each well. The lower chamber of the transwell was filled with 600 ul of culture media, and the plates was incubated at 37.degree. C. for 12 to 24 h. The cells that did not invade the matrigel were gently scraped off the top of each transwell with a cotton swab. The transwells were then removed from the 24-well plates and stained with crystal violet after fixing the invaded cells with 70% ethanol or methanol. The invaded cells were counted under a light microscope. The percent of cells invading the matrigel was significantly inhibited in the presence of MABp1.

Example 8

MABp1 Blocks Increase in ICAM1 Expression in Endothelial Cells

[0052] Human umbilical vein endothelial cells (HUVEC) (BD Biosciences) were seeded to 24-well plates at 5.times.10.sup.5 per well in 1 mL of M-200 medium supplemented with low-serum growth supplement (Invitrogen). Cells were allowed to settle for 3-4 hours. Medium was aspirated and a fresh 1 mL of M-200 was added per well. MABp1 was added directly to cells @ 4.26 vg/mL, co-incubated for 15 minutes at room temperature, and then recombinant human IL-1.alpha. (rhIL1A, eBioscience) was added to a final concentration of 40 pg/mL. Positive control wells received the addition of IL-1.alpha. only. HUVEC cells in the absence of IL-1.alpha. or the absence of MABp1 served as negative controls. After 17-20 hours incubation at 37.degree. C., 5% CO.sub.2, cells were lifted from the plates by a non-enzymatic treatment for 20 minutes using CellStripper reagent (Cellgro Mediatech) and then immediately assayed for CD54 (ICAM-1) expression using standard flow cytometry protocols. Staining buffer comprised Dulbecco's PBS supplemented with 2% heat-inactivated fetal bovine serum. PE-conjugated mouse anti-human CD54 (ICAM-1) mAb (eBioscience, clone HA58) or a PE-conjugated mouse IgG1k isotype control (eBiocience, #12-4714) were used per manufacturer's instructions to stain HUVEC cells in a 100 microliter staining volume for 20 minutes in the dark at room temperature. Two washes in staining buffer were subsequently performed and then samples were acquired on a FACSCalibur flow cytometer (BD Biosciences). Among several independent experiments (n=5) the upregulation of ICAM-1 adhesion molecules induced by rhIL1A on the surface of HUVEC cells was neutralized by MABp1 to baseline levels exhibited by the unstimulated HUVEC cells.

Example 9

MABp1 Blocks Increase in E-selectin Expression in Endothelial Cells

[0053] Similar to its effects on ICAM-1 induction, MABp1-mediated neutralization of induction of CD62E (E-selectin) on HUVEC cells was also observed. This effect was most pronounced when HUVEC cells were stimulated not by soluble rhIL-1.alpha. but by membranous IL-1.alpha. anchored by glycosyl-phosphatidylinositol to the surface of DG44 CHO cells (GP1-IL1A cells). In this experiment, confluent cultures of HUVEC cells in 6-well plates were co-cultured overnight with 5.times.10.sup.6 GPI-IL1A DG44 cells in M-200 medium, either alone, in the presence of 10 .mu.g/mL MABp1, or in the presence of 10 .mu.g/mL D5 isotype control Ab. After 17-20 hours, HUVEC monolayers were washed extensively with Dulbecco's PBS and then lifted by non-enzymatic treatment for 20 minutes with CellStripper reagent (Cellgro Mediatech) and then immediately assayed for CD62E (E-selectin) expression using standard flow-cytometry protocols. Staining buffer comprised Dulbecco's PBS supplemented with 2% heat-inactivated fetal bovine serum. PE-conjugated mouse anti-human CD62E mAb (eBioscience, clone P2H3) or a PE-conjugated mouse IgG1k isotype control (eBiocience, clone P3) were used per manufacturer's instructions to stain HUVEC cells in a 100 microliter staining volume for 20 minutes in the dark at room temperature. Two washes in staining buffer were subsequently performed and then samples were acquired on a FACSCalibur flow cytometer (BD Biosciences). Upregulated E-selectin expression on the surface of HUVEC cells induced by membranous GPI-IL-1.alpha. was neutralized by MABp1 to baseline levels exhibited by unstimulated HUVEC cells.

Example 10

MRC-5 Bioassay for MABp1 Potency (Neutralization of rhIL1A)

[0054] The MRC-5 cell line, derived from fetal human lung fibroblasts, was obtained from the ATCC collection (CCL-171). The IL-1 neutralizing potency of MABp1 was assayed by measuring IL-1A induced release of IL-6 from MRC-5 cells. MRC-5 cells were seeded at 5.times.10.sup.3 per well to a 96-well plate in 100 microliters of DMEM complete medium. Cells were cultured overnight at 37.degree. C. in a humidified 5% CO.sub.2 incubator. Conf.luent MRC-5 cells were subsequently cultured another 24 hours with 20 pg/mL of recombinant human IL-1A (rhIL1A, eBioscience) either alone or in the presence of increasing concentrations of MABp1. Negative control cells were not stimulated with rhIL1A. After the 24 hours, supernatants were collected and assayed for IL-6 release using and IL-6 ELISA kit from eBioscience. The IC.sub.50, or concentration of MABp1 required to inhibit 50% of the maximal IL-6 release, was in the range of 0.001-0.01 .mu.g/mL.

Example 11

MABp1 Identifies IL-1.alpha.+ Cells

[0055] One hundred microliters of sodium heparin anti-coagulated whole blood was aliquoted to polystyrene FACS tubes. Samples were incubated at room temperature for 15 minutes with 1 mg of human IgG (protein-A purified) plus 2 ml of heat-inactivated fetal bovine serum to block Fc receptors. Primary Abs were then added to the sample: Either 1 mg of Alexa-488 labeled MABp1, 1 mg of FITC-labeled monoclonal anti-membrane human IL1A Ab (FAB200F, R&D Systems), or 1 mg of a murine isotype control (IC002F, R&D Systems). Primary Abs were incubated with sample for 30 minutes at room temperature in the dark. Sample erythrocytes were then lysed (BD Biosciences PharmLyse solution) at room temperature for 15 minutes, centrifuged at 300.times.g for 5 minutes, and aspirated. Sample pellets were washed three times with 1 mL Hank's balanced salt solution (HBSS) containing 2% heat-inactivated fetal bovine serum. Sample was resuspended in 0.3 mL HBSS+2% FBS and data was acquired on a FACSCalibur flow cytometer and analyzed using CellQuest software. Flow cytometric analysis of human PBMC using MABp1 showed that only 0.2% of PBMC were positive for IL-1.alpha..

Example 12

MABp1 for Detecting and Tracking Infections and Inflammation

[0056] Flow cytometric analysis (as in Example 11) of human PBMC using MABp1 showed a 3.6-fold increase in the percent of PBMC positive for IL-1.alpha.+ in a subject with a sub-clinical infection compared to a normal control. Similarly, in a subject with an inflamed wisdom tooth, an increase in the percent of PBMC positive for IL-1.alpha..sup.+. A substantial decrease in the number of IL-1.alpha.+PBMC was observed from 14 to 45 days after removal of the wisdom tooth.

Example 13

Immunoassay for Detecting and/or Quantifying IL-1.alpha.

[0057] In general, very low levels of IL-1.alpha. are present in the plasma of human subjects. Because these levels are often beyond the detection threshold of conventional immunoassays, an ELISA with improved sensitivity was developed. In this ELISA, exogenous anti-IL-1.alpha. Ab (e.g., MABp1) can be added to a biological sample being tested (e.g., human plasma) under conditions that allow the Ab to bind IL-1.alpha. in the sample. Because, it was observed that almost all IL-1.alpha. in human plasma samples exists already bound to endogenous anti-IL-1.alpha. Ab, the latter step can often be omitted. The sample with IL-1.alpha.-Ab complexes is then applied to a filter (Amicon centrifugal device) with a molecular weight cutoff of about 100 kDa to separate the IL-1.alpha.-Ab complexes from molecules in the sample less than the molecular weight cutoff. In one experiment, this resulted in a 50-fold concentration. The processed sample (and dilutions thereof) was then added to wells of a microtiter plate coated with an anti-human IgG capture Ab (2 ug/ml mouse anti-human IgG, Fc-specific, Southern Biotech product code #9042-01). After allowing time to bind the IL-1.alpha.-Ab complexes in the sample, the wells were washed to remove non-binding material. A labeled anti-human IL-1.alpha. secondary Ab was then added to the wells (0.2 ug/ml biotin-conjugated monoclonal mouse anti-human IL-1A Ab, clone CRM6, eBioscience catalog #13-7017). After allowing time to bind the IL-1.alpha. in the wells, the plate was washed and the amount of labeled anti-human IL-1.alpha. in each well was quantified as an indication of the concentration of IL-1.alpha. in the sample being tested.

Other Embodiments

[0058] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

121471PRThomo sapiens 1Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Met Phe Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Ala Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75 80Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Ile Leu Phe Leu Gln Met Asp Ser Leu Arg Leu Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Arg Pro Lys Val Val Ile Pro Ala Pro Leu 115 120 125Ala His Trp Gly Gln Gly Thr Leu Val Thr Phe Ser Ser Ala Ser Thr 130 135 140Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser145 150 155 160Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu225 230 235 240Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300Gly Val Glu Val His Asn Ala Gln Thr Lys Pro Arg Glu Glu Gln Tyr305 310 315 320Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp385 390 395 400Ile Ala Leu Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Leu Ser Leu Ser Pro Gly Lys465 47021416DNAHomo sapiens 2atggagttcg ggctgagttg ggtgttcctg gtggctctgc tgcggggcgt gcagtgccag 60gtgcagctgg tggagagtgg gggtggcgtg gtgcagcctg gccggtctct gcgcctgtct 120tgcactgcct ccggttttac cttttctatg tttggtgtgc actgggtgcg ccaggctccc 180ggcaagggac tggaatgggt ggccgccgtg agttacgacg ggtccaacaa atattacgct 240gagagcgtga aaggcagatt caccatcagc agagataatt ccaagaatat tctgttcctg 300cagatggaca gtctgagact ggaggacact gctgtgtact actgcgctcg tggacgccct 360aaggtggtca tccccgcccc cctggcacat tggggccagg gaactctggt gaccttttct 420agcgctagca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 480gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540tcgtggaact caggcgccct gaccagcggc gtccacacct tcccggctgt cctacagtcc 600tcaggactct actccctcag cagcgtagtg accgtgccct ccagcagctt gggcacccag 660acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 720cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900tggtacgtgg acggcgtgga ggtgcataat gcccagacaa agccgcggga ggagcagtac 960aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200atcgccctgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380acgcagaaga gcctctcctt aagtccggga aaataa 14163235PRTHomo sapiens 3Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Asn Leu Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Thr Ser Ser Phe Leu Leu Ser Phe Gly Gly Gly Thr Lys Val Glu His 115 120 125Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe145 150 155 160Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 2354708DNAHomo sapiens 4atggacatgc gcgtgcccgc ccagctgctg gggctgctgc tgctgtggtt ccctggatct 60aggtgcgaca ttcagatgac ccagtccccc agctcagtgt cagcctccgt gggcgacaga 120gtgacaatca cctgccgcgc ctctcaggga atctctagtt ggctggcctg gtaccagcag 180aagcctggaa aggcccccaa gctgctgatc tatgaagcct ccaacctgga gaccggcgtg 240ccctctcgct tcagcggctc aggctcaggc agtgatttta ctctgaccat cagctccctg 300cagccagagg atttcgctac ttactactgc cagcagacct cttccttcct gctgtccttc 360gggggaggca caaaggtgga gcaccgtacg gtggctgcac catctgtctt catcttcccg 420ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660ggcctgagtt caccggtgac aaagagcttc aacaggggag agtgttag 7085471PRTHomo sapiens 5Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Met Phe Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Ala Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75 80Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Ile Leu Phe Leu Gln Met Asp Ser Leu Arg Leu Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Arg Pro Lys Val Val Ile Pro Ala Pro Leu 115 120 125Ala His Trp Gly Gln Gly Thr Leu Val Thr Phe Ser Ser Ala Ser Thr 130 135 140Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser145 150 155 160Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu225 230 235 240Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr305 310 315 320Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp385 390 395 400Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Leu Ser Leu Ser Pro Gly Lys465 47061422DNAHomo sapiens 6gccaccatgg agtttggtct gtcctgggtg ttcttggtgg ctctgctgag gggggtgcag 60tgccaggtcc agctggtgga gtctggtggg ggagtggtgc agcctgggag atctctgcgg 120ctgtcttgca ctgcctctgg tttcactttc tctatgtttg gtgtgcattg ggtcaggcaa 180gcaccaggca aaggactcga gtgggtcgca gctgtgagct atgacgggtc taacaaatat 240tacgctgagt ctgtcaaggg taggtttacc atcagccggg ataattccaa aaatatcctg 300ttcctgcaaa tggactctct gaggctggaa gatactgcag tctactattg tgcaaggggg 360aggccaaagg tggtgatccc cgctcccctc gctcactggg gacagggaac cctggtgact 420ttcagctctg ctagcaccaa gggccctagc gtgttcccat tggctccttc ctccaaatct 480acttctggag gcaccgccgc cctgggatgt ctcgtgaaag attattttcc tgagcccgtc 540accgtgagct ggaacagcgg cgccctgact agcggcgtgc acacctttcc cgcagtgctg 600caatctagcg ggctgtactc cctgagctct gtcgtgaccg tgccctccag cagcctcgga 660actcagacct acatctgcaa tgtcaatcat aaaccctcta ataccaaagt cgataagaag 720gtcgaaccta aatcttgcga taaaacccat acctgccccc cttgcccagc acccgaactg 780ctgggcggtc cctctgtgtt tctgttcccc cccaaaccca aagataccct gatgatctct 840aggacccccg aggtcacttg tgtcgtggtg gatgtgtccc acgaagatcc agaagtcaaa 900ttcaactggt atgtggacgg ggtcgaagtg cacaacgcaa agaccaagcc tagggaggaa 960cagtataata gcacatatag ggtggtcagc gtcctgaccg tcctgcatca ggactggctg 1020aatggcaaag aatataagtg taaagtgtcc aacaaggccc tgccagcccc aatcgaaaag 1080acaatctcta aagccaaggg gcaaccccgg gaacctcagg tctatacact gccaccctct 1140cgggatgaac tgaccaagaa tcaggtgagc ctgacatgtc ttgtgaaggg tttttatccc 1200tccgacattg ccgtggagtg ggagagcaat ggacaaccag aaaataacta caaaaccaca 1260ccccctgtgc tggactccga tggttccttc ttcctctact ctaagctgac agtggataag 1320tctaggtggc agcaggggaa tgtgttctcc tgctctgtga tgcacgaggc actgcacaat 1380cattatacac aaaagtctct gtctctgtct ccaggaaagt aa 14227234PRTHomo sapiens 7Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Asn Leu Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Thr Ser Ser Phe Leu Leu Ser Phe Gly Gly Gly Thr Lys Val Glu His 115 120 125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 2308711DNAHomo sapiens 8gccaccatgg acatgcgcgt tcctgcccag ctcctcggac tgctgctgct ttggttccca 60ggctcccggt gtgatattca gatgacacag tctccctcct ccgtatctgc atccgtgggc 120gacagggtca caatcacttg tagggccagc caggggatct ctagttggct cgcatggtac 180caacaaaagc caggtaaggc tccgaaactg ctcatttacg aagctagtaa cctcgaaaca 240ggcgtgccaa gccggtttag cggctccggt tccggttctg acttcaccct cactatttcc 300tccctgcaac ctgaggattt tgccacatat tactgtcagc aaacttcttc ttttctgctc 360tcctttggtg ggggaactaa ggtggagcac acagtggccg cccccagcgt ctttatcttc 420cccccaagcg atgaacagct gaagtcaggg accgccagcg tggtctgcct gctcaataat 480ttttaccctc gcgaggctaa ggtccaatgg aaagtggata acgccctcca gagcggtaac 540tctcaggagt ctgtcacaga gcaagacagc aaggatagca cctattccct ctccagcacc 600ctgacactgt ctaaggccga ctacgagaaa cacaaagtgt acgcttgtga ggtgactcac 660cagggactga gtagccctgt gacaaaatct ttcaataggg gagaatgctg a 7119471PRTHomo sapiens 9Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Met Phe Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Ala Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala65 70 75 80Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Ile Leu Phe Leu Gln Met Asp Ser Leu Arg Leu Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Gly Arg Pro Lys Val Val Ile Pro Ala Pro Leu 115 120 125Ala His Trp Gly Gln Gly Thr Leu Val Thr Phe Ser Ser Ala Ser Thr 130 135 140Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser145 150 155 160Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu225 230 235 240Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr305 310 315 320Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335Trp Leu Asn Gly

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp385 390 395 400Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Leu Ser Leu Ser Pro Gly Lys465 470101422DNAHomo sapiens 10gccaccatgg agtttggtct gtcctgggtg ttcttggtgg ctctgctgag gggggtgcag 60tgccaggtcc agctggtgga gtctggtggg ggagtggtgc agcctgggag atctctgcgg 120ctgtcttgca ctgcctctgg tttcactttc tctatgtttg gtgtgcattg ggtcaggcaa 180gcaccaggca aaggactcga gtgggtcgca gctgtgagct atgacgggtc taacaaatat 240tacgctgagt ctgtcaaggg taggtttacc atcagccggg ataattccaa aaatatcctg 300ttcctgcaaa tggactctct gaggctggaa gatactgcag tctactattg tgcaaggggg 360aggccaaagg tggtgatccc cgctcccctc gctcactggg gacagggaac cctggtgact 420ttcagctctg ctagcaccaa gggccctagc gtgttcccat tggctccttc ctccaaatct 480acttctggag gcaccgccgc cctgggatgt ctcgtgaaag attattttcc tgagcccgtc 540accgtgagct ggaacagcgg cgccctgact agcggcgtgc acacctttcc cgcagtgctg 600caatctagcg ggctgtactc cctgagctct gtcgtgaccg tgccctccag cagcctcgga 660actcagacct acatctgcaa tgtcaatcat aaaccctcta ataccaaagt cgataagagg 720gtcgaaccta aatcttgcga taaaacccat acctgccccc cttgcccagc acccgaactg 780ctgggcggtc cctctgtgtt tctgttcccc cccaaaccca aagataccct gatgatctct 840aggacccccg aggtcacttg tgtcgtggtg gatgtgtccc acgaagatcc agaagtcaaa 900ttcaactggt atgtggacgg ggtcgaagtg cacaacgcaa agaccaagcc tagggaggaa 960cagtataata gcacatatag ggtggtcagc gtcctgaccg tcctgcatca ggactggctg 1020aatggcaaag aatataagtg taaagtgtcc aacaaggccc tgccagcccc aatcgaaaag 1080acaatctcta aagccaaggg gcaaccccgg gaacctcagg tctatacact gccaccctct 1140cgggaggaaa tgaccaagaa tcaggtgagc ctgacatgtc ttgtgaaggg tttttatccc 1200tccgacattg ccgtggagtg ggagagcaat ggacaaccag aaaataacta caaaaccaca 1260ccccctgtgc tggactccga tggttccttc ttcctctact ctaagctgac agtggataag 1320tctaggtggc agcaggggaa tgtgttctcc tgctctgtga tgcacgaggc actgcacaat 1380cattatacac aaaagtctct gtctctgtct ccaggaaagt aa 142211236PRTHomo sapiens 11Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Asn Leu Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Thr Ser Ser Phe Leu Leu Ser Phe Gly Gly Gly Thr Lys Val Glu His 115 120 125Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 23512717DNAHomo sapiens 12gccaccatgg acatgcgcgt tcctgcccag ctcctcggac tgctgctgct ttggttccca 60ggctcccggt gtgatattca gatgacacag tctccctcct ccgtatctgc atccgtgggc 120gacagggtca caatcacttg tagggccagc caggggatct ctagttggct cgcatggtac 180caacaaaagc caggtaaggc tccgaaactg ctcatttacg aagctagtaa cctcgaaaca 240ggcgtgccaa gccggtttag cggctccggt tccggttctg acttcaccct cactatttcc 300tccctgcaac ctgaggattt tgccacatat tactgtcagc aaacttcttc ttttctgctc 360tcctttggtg gaggaactaa ggtggagcac aagcggacag ttgctgctcc tagcgtcttt 420atcttccctc caagcgatga acagctgaag tcagggaccg ccagcgtggt ctgcctgctc 480aataattttt accctcgcga ggctaaggtc caatggaaag tggataacgc cctccagagc 540ggtaactctc aggagtctgt cacagagcaa gacagcaagg atagcaccta ttccctctcc 600agcaccctga cactgtctaa ggccgactac gagaaacaca aagtgtacgc ttgtgaggtg 660actcaccagg gactgagtag ccctgtgaca aaatctttca ataggggaga atgctga 717

Claims

1. A method of inhibiting an IL-1.alpha. induced increase in expression of ICAM-1 or E-selectin on the surface of a human endothelial cell, the method comprising the step of adding a purified mAb that specifically binds to human IL-1.alpha. to a mixture comprising the endothelial cell and IL-1.alpha..

2. The method of claim 1, wherein the purified mAb is a human IgG1 mAb comprising a heavy chain covalently joined to a light chain.

3. The method of claim 2, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9 and a light chain comprises the amino acid sequence of SEQ ID NO:11.