| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 05.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 02.05.00 |
| PATENT TITLE |
CD40 antigen antibody complex |
| PATENT ABSTRACT |
Methods for preventing or treating an antibody-mediated diease in a patient are presented, the methods comprising administration of a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell. Monoclonal antibodies useful in these methods, and epitopes immunoreactive with such monoclonal antibodies are also presented. -------------------------------------------------------------------------------- |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 05.06.95 |
| PATENT REFERENCES CITED |
M. Den Hartog et al., "Generation of a humanized anti-CD40 mab for treatment of autoimmune diseases," Immunotechnology, 2(4):299 (Nov. 1996). W. Fanslow et al., "CD40 mAbs M2 and M3 inhibit CD40L binding and function," Tissue Antigens, 42(3):304 (Oct. 1993). E. Padlan, "A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand binding properties," Molecular Immunology, 28(4/5):489-498 (Apr. 1991). Paul, ed. "Fundamental Immunology" Second Edition, chapters 8 and 12, pp. 176-181 and 315-352, 1989. Stamenkovic et al., EMBO Journal, 8(5):1403-1410 "A B-lymphocyte Activation Molecule Related to the Nerve Growth Factor Receptor and Induced by Cytokines in Carcinomas," (1989). Armitage et al., "Molecular and Biological Characterization of a Murine Ligand for CD40", Nature, 357:80-82 (May 7, 1992). Banchereau et al., "Growing Human B Lymphocytes in the CD40 System", Nature, 353:678-679 (Oct. 17, 1991). Banchereau et al., "Long-Term Human B Cell Lines Dependent on Interleukin-4 and Antibody to CD40", Science, 251:70-72 (Jan. 4, 1991). Chothia, et al., "Canonical Structures for the Hpervariable Regions of Immunoglobulins," J. Mol. Biol. 196, 901-917 (1987). Clark and Shu, "Association Between IL-6 and CD40 Signaling IL-6 Induces Phosphorylation of CD40 Receptors", J. Immunol, 145(5):1400-1406 (Sep. 1, 1990). Clark et al., "Activation of Human B Cells Mediated Through Two Distinct Cell Surface Differentiation Antigens, Bp35 and Bp50", Proc. Natl. Acad. Sci. USA, 83:4494-4498 (Jun 1986). Cosimi, et al., "Use of Monoclonal Antibodies To T-Cell Subsets for Immunologic Monitoring and Treatment in Recipients of Renal Allografats," N. Eng. J. Med., 305:308-313 (Aug. 6, 19881). de Boer et al., "Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins", J. Immunol. Meth., 152:15-23 (1992). DeFranco et al., "Separate Control of B Lymphocyte Early Activation and Proliferation in Response to Anti-IgM Antibodies", J. Immunol., 135(1):87-94 (Jul. 1985). DiSanto et al., "Generation of Anti-human CD8.beta.-Specific Antibodies Using Transfectants Expressing Mixed CD8 Heterodimers," J. Immunol. Methods, 141:123-1312 (1991). Freedman et al., "B7, A B Cell-Restricted Antigen that Identifies Preactivated B Cells", J. Immunol., 139(10):3260-3267 (Nov. 15, 1987). Gascan et al., "Anti-CD40 Monoclonal Antibodies or CD4.sup.+ T Cell Clones and IL-4 Induce IgG4 and IgE Switching in Purified Human B Cells Via Different Signaling Pathways", J. Immunol., 147(1):8-13 (Jul. 1, 1991). Gauchat et al., "Modulation of IL-4 Induced Germline .epsilon. RNA Synthesis in Human B Cells by Tumor Necrosis Factor-.alpha., Anti-CD40 Monoclonal Antibodies or Transfroming Growth Factor-.beta. Correlates with Levels of IgE Production", Int. Immunol., 4(3):397-406 (1991). Golub, Immunology A Synthesis, published by Sinauer Assoc. Inc., Sunderland, MA, pp. 19-20 (1987). Gordon et al., "Resting B Lymphocytes Can Be Triggered Directly Through the CDw40 (BP50) Antigen", J. Immunol., 140(5):1425-1430 (Mar. 1, 1988). Gruber et al., "Anti-CD45 Inhibition of Human B Cell Proliferation Depends on the Nature of Activation Signals and the State of B Cell Activation", J. Immunol., 142(12):4144-4152 (Jun. 15, 1989). Harris et al., "Therapeutic antibodies--the coming of age", TIBTECH, 11(2):42-44 (Feb. 1993). Jabara et al., "CD4 and IgE: Synergism between Anti-CD40 Monoclonal Antibody and Interleukin 4 in the Induction of IgE Synthesis by Highly Purified Human B Cells", J. Exp. Med., 172:1861-1864 (Dec. 1990). June et al., "Role of the CD28 Receptor in T-Cell Activation", Immunology Today, 11(6):211-216 (1990). Jung and Fu, "Selective Inhibition of Growth Factor-Dependent Human B Cell Proliferation by Monoclonal Antibody ABI to an Antigen Expressed by Activated B Cells", J. Exp. Med., 160:1919-1924 (Dec. 1984). Kabat et al., "Sequences of Proteins of Immunological Interest,Tabulation and Analysis of Amino Acid and Nucleic Acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, .beta..sub.2 -Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Post-gamma Globulin, and .sub.2 -Macroglobulin," sponsored through Contract N01-RR-8-2118 by components of the National Institutes of Health, Bethesda, MD 20205 (1983). Kriegler et al., "A Novel Form of TNF/Cachectin Is a Cell Surface Cytotoxic Transmembrane Protein: Ramifications for the Complex Physiology of TNF," Cell 53:45-53 (1988). Kwekkenboom et al., "CD40 plays an essential role in the activation of human B cells by murine EL4B5 cells", Immunology, 79:439-444 (1993). Lane et al., "Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participated in T cell-dependent activation of B lymphocytes", Eur. J, Immunol., 22:2573-2578 (1992). Ledman et al., "Anti-CD40 Monoclonal Antibody Blocks the Contact Dependent T Helper Signal Mediated by 5C8 Ag.", Clinical Research, 40:154A (1992). Linsley et al., "CTLA-4 is a Second Receptor for the B Cell Activation Antigen B7", J. Exp. Med., 174:561-569 (Sep. 1991). Muraguchi et al., "Sequential Requirements for Cell Cycle Progression of Resting Human B Cells after Activation by Anti-Ig", J. Immunol., 132(1):176-180 (1984). Noelle and Snow, "T helper cells", Current Opinion in Immunology, 4:333-337 (1992). Paulie et al., "The Human B Lymphocyte and Carcinoma Antigen, CDw40, Is a Phosphoprotein Involved in Growth Signal Transduction", J. Immunol., 142(2):590-595 (Jan. 15, 1989). Ross et al., "Characterization of nerve growth factor in neural crest tumors using monoclonal antibodies", Proc. Natl. Acad. Sci. USA, 81:6681-6685 (Nov. 1984). Rousset et al., "Cytokine-induced Proliferation and Immunoglobulin Production of Human B Lymphocytes Triggered Through Their CD40 Antigen", J. Exp. Med., 173:705-710 (Mar. 1991). Sato et al., "Biological Effects in Vitro of Monoclonal Antibodies to Human Epidermal Growth Factor Receptors", Mol. Biol. Med., 1:511-529 (1983). Splawski et al., "Immunoregulatory Role of CD40 in Human B Cell Differentiation", J. Immunol., 150(4):1276-1285 (Feb. 15, 1993). Tanaka et al., "Distinct Reactivities of Four Monoclonal Antibodies with Human Interleukin 2 Receptor", Microbiol. Immunol., 29(10):959-972 (1985). Uckun et al., "Temporal Association of CD40 Antigen Expression with Discreet Stages of Human B-Cell Ontogeny and the Efficacy of Anti-CD40 Immunotoxins Against Clonogenic B-Lineage Acute Lymphoblastic Leukemia as well as B-Lineage Non-Hodgkin's Lymphoma Cells", Blood, 76(12):2449-2456 (Dec. 15, 1990). Valle et al., "mAb 104, A New Monoclonal Antibody, Recognizes the B7 antigen that is Expressed on Activated B Cells HTLV-1-Transformed T Cells", Immunology, 69:531-535 (1990). Webb, "Cell-surface Expression And Proliferation of Human CD4 Produced in Baculovirus-Infested Insect Cells", Proc. Natl. Acad. Sci. USA, 86:7731-7735 (Oct. 1989). Wetzel et al., "Evidence for Two Distinct Activation States Available to B Lymphocytes", J. Immunol., 133(5):2327-2332 (Nov. 1984). Yellin et al., "CD40 Molecules Induce Down-Modulation and Endocytosis of T Cell Surface T Cell-B Cell Activating Molecule/CD40-L", J. Immunol., 152:598-608 (1994). Zhang et al., "CD40 Stimulation Provides and IFN-.gamma. Independent and IL-4-Dependent Differentiation Signal Directly to Human B Cells For IgE Production", J. Immunol., 146(6):1836-1842 (Mar. 15, 1991). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A complex comprising an epitope on a human CD40 antigen on the surface of a human B cell, and an anti-CD40 monoclonal antibody or an antigen binding fragment thereof immunologically bound to said epitope, said epitope characterized by being immunoreactive with an anti-CD40 monoclonal antibody that is 5D12, 3A8 or 3C6 as secreted by a hybridoma having ATCC Accession numbers HB 11339, HB 12024 and HB 11340, respectively. 2. The complex of claim 1, wherein the binding of said anti-CD40 monoclonal antibody or antigen binding fragment thereof to said epitope on the surface of a normal human B cell prevents or inhibits the growth or differentiation of said normal human B cell. 3. The complex of claim 1, wherein said epitope is characterized by being immunoreactive with said monoclonal antibody 5D12. 4. The complex of claim 1, wherein said epitope is characterized by being immunoreactive with said monoclonal antibody 3A8. 5. The complex of claim 1, wherein said epitope is characterized by being immunoreactive with said monoclonal antibody 3C6. 6. The complex of claim 1, wherein said epitope is bound to said antigen binding fragment. 7. The complex of claim 6, wherein said antigen binding fragment is F(ab').sub.2, Fab or F.sub.v. 8. The complex of claim 1, wherein said anti-CD40 monoclonal antibody or antigen binding fragment thereof is humanized. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to novel methods of treating diseases of the immune system. In particular, this invention relates to methods of preventing or treating antibody-mediated diseases such as IgE-mediated disease (allergies) and autoimmune diseases including systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC), and idiopathic thrombocytopenic purpura (ITP). BACKGROUND OF THE INVENTION I. B-Cell Activation B cells play an important role during the normal in vivo immune response. A foreign antigen will bind to surface immunoglobulins on specific B cells, triggering a chain of events including endocytosis, processing, presentation of processed peptides on MHC-class II molecules, and up-regulation of the B7 antigen on the B-cell surface. A specific T cell then binds to the B cell via T-cell receptor (TCR) recognition of processed antigen presented on the MHC-class II molecule. Stimulation through the TCR begins to activate the T cell and initiates T-cell cytokine production. Interaction between the CD28 antigen on T cells and the B7 antigen on B cells can provide a second signal further activating the T cell, resulting in high level cytokine secretion. Additionally, the CD40 ligand, which is not expressed on resting human T cells, is up-regulated on the T-cell surface when the above-mentioned signals are received. The B cell is then stimulated by the CD40 ligand through the CD40 antigen on the B-cell surface, and also by soluble cytokines, causing the B cell to mature into a plasma cell secreting high levels of soluble immunoglobulin. II. The EL4B5 Cell Line A few years ago, Zubler et al., J. Immunol. (1985) 134:3662, observed that a mutant subclone of the mouse thymoma EL-4 line, known as EL4B5, could strongly stimulate B cells of both murine and human origin to proliferate and differentiate into immunoglobulin-secreting plasma cells in vitro. This activation was found to be antigen-independent and not MHC restricted. For optimal stimulation of human B cells, the presence of supernatant from activated human T cells was needed, but a B-cell response also occurred when EL4B5 cells were preactivated with phorbol-12-myristate 13-acetate (PMA) or IL-1. Zubler et al., Immunological Reviews (1987) 99:281; and Zhang et al., J. Immunol. (1990) 144:2955. B-cell activation in this culture system is efficient--limiting dilution experiments have shown that the majority of human B cells can be activated to proliferate and differentiate into antibody-secreting cells. Wen et al. Eur. J. Immunol. (1987) 17:887. The mechanism by which these mutant EL-4 cells activate both murine and human B cells has not been elucidated previously. It is, however, clear that cell-cell contact is required for EL4B5-induced B-cell activation. First, B cells do not proliferate in the presence of supernatant from PMA-stimulated EL4B5 cells. Zubler et al. (1985) supra. Second, B cells do not proliferate when they are separated from PMA-treated EL4B5 cells by a semipermeable filter membrane. Zhang et al., supra. Antibodies against mouse LFA-1, human LFA-1 or human LFA-3 and antibodies against mouse or human MHC class II molecules do not inhibit EL4B5-induced proliferation of human or murine B cells. Zubler et al. (1987) and Zhang et al., supra. III. The CD40 Antigen, the CD40 Antigen Ligand, and Anti-CD40 Antibodies The CD40 antigen is a glycoprotein expressed on the cell surface of B cells. During B-cell differentiation the molecule is first expressed on pre-B cells and then disappears from the cell surface when the B cell becomes a plasma cell. Crosslinking of the CD40 molecules with anti-CD40 antibodies mediates a variety of effects on B cells. The CD40 antigen is known to be related to the human nerve growth factor (NGF) receptor and tumor necrosis factor-alpha (TNF-.alpha.) receptor, suggesting that CD40 is a receptor for a ligand with important functions in B-cell activation. A ligand for CD40 has been identified on the cell surface of activated T cells. Fenslow et al., J. Immunol. (1992) 149:655; Lane et al., Eur. J. Immunol. (1992) 22:2573; Noelle et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:6550. cDNA cloning of the CD40 ligand revealed a molecule with characteristics of a type-II transmembrane glycoprotein with homology to TNF-.alpha.. Armitage et al., Nature (1992) 357:80 and Spriggs et al., J. Exp. Med. (1992) 176:1543. The extracellular domain of the CD40 ligand contains two arginine residues proximal to the transmembrane region, providing a potential proteolytic cleavage site that could give rise to a soluble form of the ligand. Expression of recombinant CD40 ligand has demonstrated that this molecule can stimulate the proliferation of purified B cells and, in combination with IL-4, mediate the secretion of IgE. Armitage et al. and Spriggs et al., supra. It has been reported that abnormalities in the gene for the CD40 ligand, resulting in the absence of a functional molecule on activated T cells, is responsible for the occurrence of X-linked hyper-IgM syndrome, a rare disorder characterized by the inability of these patients to produce normal levels of antibody isotypes other than IgM. Allen et al., Science (1993) 259:990; and Korthauer et al., Nature (1993) 361:539. All anti-CD40 antibodies known in the art have a stimulatory effect on human B cells. Cross-linking of the CD40 molecule on the B-cell surface using known anti-CD40 antibodies mediates a variety of effects on B cells. Anti-CD40 monoclonal antibodies (mAbs) can induce intercellular adhesion, proliferation and, in combination with certain cytokines, maturation to antibody secreting cells. For example, known anti-CD40 mAbs have been shown to mimic the effects of T helper cells in B-cell activation. When presented on adherent cells expressing Fc.gamma.RII, these antibodies induce B-cell proliferation. J. Banchereau et al., Science (1989) 251:70. Moreover, the known anti-CD40 mAbs can replace the T helper signal for secretion of IgM, IgG and IgE in the presence of IL-4. H. Gascan et al., J. Immunol. (1991) 147:8. Furthermore, known anti-CD40 mAbs can prevent programmed cell death (apoptosis) of B cells isolated from lymph nodes. However, the anti-CD40 antibodies known in the art stimulate B cells but are incapable of inhibiting the B-cell response. Furthermore, no anti-CD40 antibodies are known that are (1) capable of inhibiting the B-cell response and (2) can be used to prevent or treat antibody-mediated disease. SUMMARY OF THE INVENTION The current invention is based on the discovery of anti-CD40 antibodies that do not stimulate the growth and differentiation of human B cells. In contrast, these antibodies can inhibit human B-cell responses at relatively low concentrations. Accordingly, these antibodies can be used to prevent or treat diseases or conditions that are mediated by antibodies produced by the human B-cell response. These antibodies also recognize novel epitopes on the CD40 antigen useful in modulating the B-cell response. Accordingly, it is a primary object of this invention to provide a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell. It is a further object of this invention to provide a method for preventing or treating an antibody-mediated disease in a patient, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell, in a pharmaceutically acceptable excipient. It is another object of this invention to provide a method for preventing or treating an IgE-mediated disease such as an allergy in a patient, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell, in a pharmaceutically acceptable excipient. It is yet another object of this invention to provide a method for preventing or treating an antibody-mediated autoimmune disease in a patient, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a monoclonal antibody capable of binding to a human CD40 antigen located on the surface of a human B cell, wherein the binding of the antibody to the CD40 antigen prevents the growth or differentiation of the B cell, in a pharmaceutically acceptable excipient. Particular autoimmune diseases contemplated for treatment by this method include sytematic lupus erythematosus (SLE), primary biliary cirrhosis (PBC), and idiopathic thrombocytopenic purpura (ITP). It is a further object of this invention to provide a CD40 antigen epitope capable of competing with the binding of a CD40 antigen to an anti-CD40 monoclonal antibody wherein the binding of that antibody to a human CD40 antigen located on the surface of a human B cell prevents the growth or differentiation of the B cell. In more preferred embodiments of the above objects, the monoclonal antibody is either 5D12, 3A8 or 3C6. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows a schematic representation of the baculoviral transfer vector pAcC8 and the sequence of the multiple cloning site (SEQ ID NO: 1). As shown, the multiple cloning site was inserted between nucleotide numbers +37 and +176 of the polyhedrin gene. FIG. 1B shows a schematic representation of the generation of Sf9 cells which express human CD40 or B7 antigen. FIG. 2 shows the sequences of polymerase chain reaction primers used in the preparation of coding regions for human CD40 and human B7 antigens. These primers were constructed on the basis of the published complete DNA coding sequences for antigens B7 and CD40. Specifically shown are the primers for full length B7, which are designated as MR67 (SEQ ID NO: 2) and MR68 (SEQ ID NO: 3); the primers for soluble B7, which are designated as MR67 (SEQ ID NO: 2) and MR145 (SEQ ID NO: 4); the primers for full length CD40, which are designated as MR108 (SEQ ID NO: 5) and MR112 (SEQ ID NO: 6); and the primers for soluble CD40, which are designated as MR108 (SEQ ID NO: 5) and MR150 (SEQ ID NO: 7). FIG. 3 shows the results of ELISA assays examining the reaction of anti-CD40 monoclonal antibody 52C6 with Sf9 cells expressing CD40 and with Sf9 cells expressing B7. FIGS. 4A-4C show the results of the fluorescent cell staining of EBV-transformed B-cell line ARC cells expressing CD40. FIG. 4A shows the results of staining ARC EBV transformed cells with serum from a mouse immunized with B7 expressing Sf9 cells (solid line) or with normal mouse serum (dotted line). FIG. 4B shows the results of staining ARC EBV transformed cells with serum from a mouse immunized with CD40 expressing Sf9 cells (solid line) or with normal mouse serum (dotted line). FIG. 4C shows the results of staining ARC EBV transformed cells with serum from a mouse immunized with control Sf9 cells (solid line) or with normal mouse serum (dotted line). FIG. 5A compares the ability of three new and one old anti-CD40 mAbs to co-stimulate anti-IgM induced human B-cell proliferation. FIG. 5B repeats the experiment of FIG. 5A in the presence of recombinant interleukin-2 (rIL-2). FIG. 6 shows the ability of three new anti-CD40 mAbs to inhibit human B-cell proliferation induced by costimulation with immobilized anti-IgM and anti-CD40 mAb 52C6. FIG. 7 shows the effect of three new anti-CD40 mAbs on EL4B5-induced human B-cell proliferation. FIG. 8 shows the effect of soluble CD40 (hCD40..mu.) on EL4B5-induced human B-cell proliferation. FIGS. 9A and 9B show the effect of one new anti-CD40 mAb 5D12 on human T-cell induced immunoglobulin production by human B cells. DETAILED DESCRIPTION OF THE INVENTION The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference. Definitions: As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as F.sub.ab, F.sub.(ab')2, F.sub.v, and other fragments which retain the antigen binding function of the parent antibody. As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as F.sub.ab, F.sub.(ab')2, F.sub.v, and others which retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rat or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies. As used herein, the term "humanized antibodies" means that at least a portion of the framework regions of an immunoglobulin are derived from human immunoglobulin sequences. As used herein, the term "single chain antibodies" refer to antibodies prepared by determining the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only that part of the variable domain necessary for binding to the antigen. Determination and construction of single chain antibodies are described in U.S. Pat. No. 4,946,778 to Ladner et al. The term "CD40 antigen epitope" as used herein refers to a molecule which is capable of immunoreactivity with the anti-CD40 monoclonal antibodies of this invention, excluding the CD40 antigen itself. CD40 antigen epitopes may comprise proteins, protein fragments, peptides, carbohydrates, lipids, and other molecules, but for the purposes of the present invention are most commonly proteins, short oligopeptides, oligopeptide mimics (i.e., organic compounds which mimic the antibody binding properties of the CD40 antigen), or combinations thereof. Suitable oligopeptide mimics are described, inter alia, in PCT application US91/04282. The Antibody The antibodies of the current invention bind to a human CD40 antigen on the surface of a human B cell and do not stimulate the growth or differentiation of the B cell. These antibodies may be polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof. 1. Antibody Preparation Monoclonal antibodies 5D12, 3A8 and 3C6 are prepared as described in Example 1 herein. Other monoclonal antibodies of the invention may be prepared similarly, or as follows. First, polyclonal antibodies are raised against the CD40 antigen. Second, monoclonal antibodies specific for CD40 are selected. a) Polyclonal Sera Polyclonal sera may be prepared by conventional methods. In general, a solution containing the CD40 antigen is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 .mu.g/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera are obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25.degree. C. for one hour, followed by incubating at 4.degree. C. for 2-18 hours. The serum is recovered by centrifugation (e.g., 1,000.times. g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits. b) Monoclonal Antibodies Monoclonal antibodies are prepared using the method of Kohler and Milstein, Nature (1975) 256:495-96, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) are removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the desired immunizing cell-surface antigen (and which do not bind to unrelated antigens). The selected mAb-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly .sup.32 P and .sup.125 I, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, .sup.125 I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a mAb. Further, one may combine various labels for desired effect. For example, mAbs and avidin also require labels in the practice of this invention: thus, one might label a mAb with biotin, and detect its presence with avidin labeled with .sup.125 I, or with an anti-biotin mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention. CD40 Antigen Epitopes The CD40 antigen epitopes of this invention are molecules that are immunoreactive with anti-CD40 monoclonal antibodies whose binding to a human CD40 antigen located on the surface of a human B cell prevents the growth or differentiation of the B cell. That is, such epitopes compete with the binding of said antibodies to the CD40 antigen. Systematic techniques for identifying these epiotpes are known in the art, as described by H. M. Geysen in U.S. Pat. No. 4,708,871, which is incorporated herein by reference. Typically these epitopes are short amino acid sequences. These sequences may be embedded in the sequence of longer peptides or proteins, as long as they are accessible. The epitopes of the invention may be prepared by standard peptide synthesis techniques, such as solid-phase synthesis. Alternatively, the sequences of the invention may be incorporated into larger peptides or proteins by recombinant methods. This is most easily accomplished by preparing a DNA cassette which encodes the sequence of interest, and ligating the cassette into DNA encoding the protein to be modified at the appropriate site. The sequence DNA may be synthesized by standard synthetic techniques, or may be excised from the phage pIII gene using the appropriate restriction enzymes. Epitopes identified herein may be prepared by simple solid-phase techniques. The minimum binding sequence may be determined systematically for each epitope by standard methods, for example, employing the method described by H. M. Geysen, U.S. Pat. No. 4,708,871. Briefly, one may synthesize a set of overlapping oligopeptides derived from the CD40 antigen bound to a solid phase array of pins, with a unique oligopeptide on each pin. The pins are arranged to match the format of a 96-well microtiter plate, permitting one to assay all pins simultaneously, e.g., for binding to an anti-CD40 monoclonal antibody. Using this method, one may readily determine the binding affinity for every possible subset of consecutive amino acids. Analogs of the invention are also prepared by standard solid-phase methods, and those methods described in PCT application US91/04282. Formulations and Methods of Administration The antibodies of this invention are administered at a concentration that is therapeutically effective to prevent or treat antibody-mediated diseases such as allergies, SLE, PBC and ITP. To accomplish this goal, the antibodies may be formulated using a variety of acceptable excipients known in the art. Typically, the antibodies are administered by injection, either intravenously or intraperitoneally. Methods to accomplish this administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes. Before administration to patients, formulants may be added to the antibodies. A liquid formulation is preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono, di, or polysaccharides, or water soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is most preferred. "Sugar alcohol" is defined as a C.sub.4 to C.sub.8 hydrocarbon having an --OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0 w/v% and 7.0 w/v%, more preferable between 2.0 and 6.0 w/v%. Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110. Additionally, antibodies can be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546 which are all hereby incorporated by reference in their entireties. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R(O--CH.sub.2 --CH.sub.2).sub.n O--R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention. Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and a discussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106, both of which are hereby incorporated by reference in their entireties. Another drug delivery system for increasing circulatory half-life is the liposome. Methods of preparing liposome delivery systems are discussed in Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, Biochem Biophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980) 9:467. Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al., DRUG DELIVERY SYSTEMS (R. L. Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, Pharm Revs (1984) 36:277. After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art. As stated above, the antibodies and compositions of this invention are used to treat human patients to prevent or treat antibody-mediated diseases such as allergies, SLE, PBC and ITP. The preferred route of administration is parenterally. In parenteral administration, the compositions of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are saline, Ringer's solution, dextrose solution, and Hanks' solution. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% dextrose in saline. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. The dosage and mode of administration will depend on the individual. Generally, the compositions are administered so that antibodies are given at a dose between 1 .mu.g/kg and 20 mg/kg, more preferably between 20 .mu.g/kg and 10 mg/kg, most preferably between 1 and 7 mg/kg. Preferably, it is given as a bolus dose, to increase circulating levels by 10-20 fold and for 4-6 hours after the bolus dose. Continuous infusion may also be used after the bolus dose. If so, the antibodies may be infused at a dose between 5 and 20 .mu.g/kg/minute, more preferably between 7 and 15 .mu.g/kg/minute. The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way. |
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