Anti-CD154 antibodies with improved binding, functional and safety properties and uses in human immunotherapy - Patents.com

Abstract

To provide use of an anti-human CD154 antibody with improved efficacy, safety, and pharmacokinetics in the manufacture of a medicament.SOLUTION: Provided is the use of an anti-human CD154 antibody comprising a variable heavy chain polypeptide and a variable light chain polypeptide having a specific amino acid sequence, and further comprising a heavy chain and a light chain constant region having a specific amino acid sequence in the manufacture of a medicament for use in the treatment of lupus, optionally systemic lupus erythematosus (SLE), in a human subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 192,269, filed July 14, 2015; U.S. Provisional Patent Application No. 62 / 197,966, filed July 28, 2015; and U.S. Provisional Patent Application No. 62 / 277,201, filed January 11, 2016, the contents of which are incorporated by reference in their entireties.

[0002] The present invention relates to improved anti-CD154 (CD154) antibodies with reduced toxicity and functional properties and their use in immunotherapy, particularly in the treatment of inflammatory disorders, allergies, autoimmunity, transplantation, and cancer. In particular, the present invention provides high-affinity anti-CD154 antibodies with improved affinity and functional activity that do not induce thrombogenic or coagulation responses in vivo and that induce desirable therapeutic properties, such as induction of immune tolerance and blockade of humoral immunity.

[0003] The present invention relates to improved anti-CD154 (CD154) antibodies with reduced toxicity and functional properties and their use in immunotherapy, particularly in the treatment of inflammatory disorders, allergies, autoimmunity, transplantation, and cancer. In particular, the present invention provides high-affinity anti-CD154 antibodies with improved affinity and functional activity that do not induce thrombogenic or coagulation responses in vivo and that induce desirable therapeutic properties, such as induction of immune tolerance and blockade of humoral immunity. [Background technology]

[0004] CD40L (CD154) is a highly validated and valuable therapeutic target for autoimmune, graft rejection, and other immune-mediated diseases in mice, nonhuman primates (NHPs), and humans. Numerous phase II clinical trials have demonstrated that αCD154 effectively blocks CD154 activity in vivo and ameliorates disease. αCD154 is distinct from all other therapeutic agents in its effects on the immune response, and it is one of the few therapeutic agents capable of inducing functional immune tolerance, as demonstrated in both mice and monkeys. In mice, nearly all autoimmune disease models can be effectively ameliorated with αCD154 therapy (NPL 1; NPL 2; NPL 3; and NPL 4), with long-term remissions observed.

[0005] In NHPs, permanent alloimmune tolerance can be achieved using short-term treatments containing αCD154 (Non-Patent Document 5; Non-Patent Document 6).

[0006] Furthermore, phase II human clinical trials have shown that αCD154 is effective in SLE (Non-Patent Document 7), multiple sclerosis (see preliminary data), and idiopathic thrombocytopenia (Non-Patent Document 7). Thus, αCD154 is a unique drug that allows for short-term therapeutic intervention with long-term clinical benefit. Its failure was attributed to unexpected toxicity, not efficacy.

[0007] In the early 1990s, IDEC Pharmaceuticals and Biogen Inc. (now Biogen Idec) launched two different αCD154 mAbs into multiple Phase I / II clinical trials. The antibody developed by IDEC (IDEC-131) was a humanized IgG1 derived from a murine anti-human CD154 antibody developed at Dartmouth College. This antibody and humanized variants are disclosed in U.S. Patent Nos. 6,233,999; 6,233,999; 6,233,999; 6,233,999; and 6,233,999, the contents of which are all incorporated herein by reference. Early indications demonstrated that the drugs were highly effective. However, continued clinical development was hindered by the toxicity of other anti-CD154 antibodies. In this study, observed toxicities included the induction of thromboembolic events in patients. Based on toxicity concerns, all trials were halted, and efforts were directed toward redesigning the mAb to maintain efficacy and reduce toxicity.

[0008] Although reduced toxicity was achieved, the efficacy and tolerance-inducing ability of αCD154 mAb were significantly reduced (Non-Patent Document 8).

[0009] Biogen-Idec and UCB recently reported the development of an anti-CD154 antibody with reportedly improved safety. Specifically, they reported the production of a pegylated anti-human CD154 Fab, designated CDP-7657 or ruplizumab. Because Fabs typically have extremely short in vivo half-lives (i.e., approximately 4–8 hours), this Fab improved their pharmacokinetic properties. However, while reportedly clinically effective, this antibody reportedly exhibited low potency. Additionally, Bristol Meyers Squib reported the development of a domain antibody-Fc fusion protein containing specificity for CD154. This fusion protein reportedly demonstrated efficacy in mouse disease models, particularly in the KLH challenge model and the NZB × NZW SLE (lupus) model. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 6,001,358 [Patent Document 2] U.S. Patent No. 6,440,418 [Patent Document 3] U.S. Patent No. 6,506,383 [Patent Document 4] U.S. Patent No. 7,074,406 [Patent Document 5] U.S. Patent No. 7,122,187 [Non-patent literature]

[0011] [Non-licensed Document 1] Noelle, RJ, Mackey, M., Foy, T., Buhlmann, J. and Burns, C., "CD40 and its ligand in autoimmunity", Ann NY Acad Sci 1997, pp. 815:384~391 [Non-licensed Document 2] Mackey, MF, Barth, RJ, Jr. and Noelle, RJ, "The role of CD40 / CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells", J Leukoc Biol 1998. 63: 418~428 pages [Non-licensed Document 3] Noelle, RJ, "CD40 and its ligand in cell-mediated immunity", Agents Actions Suppl 1998. Pages 49:17~22 [Non-licensed Document 4] Quezada, SA, Jarvinen, LZ, Lind, EF and Noelle, RJ, "CD40 / CD154 Interactions at the Interface of Tolerance and Immunity", Annu Rev Immunol 2004. 22:307~328 pages [Non-licensed Document 5] Kenyon, N.S., Chatzipetrou, M., Masetti, M., Ranuncoli, A., Oliveira, M., Wagner, J.L., Kirk, A.D., Harlan, D.M., Burkly, L.C. and Ricordi, C., "Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154", Proc Natl Acad Sci., USA 1999, 96: 8132 - 8137 [Non-Patent Document 6] Kirk, A.D., Burkly, L.C., Batty, D.S., Baumgartner, R.E., Berning, J.D., Buchanan, K., Fechner, J.H., Jr., Germond, R.L., Kapen, R.L., Patterson, N.B., Swanson, S.J., Tadaki, D.K., TenHoor, C.N., White, L., Knechtle, S.J. and Harlan, D.M., "Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates", Nat Med 1999, 5: 686 - 693 [Non-Patent Document 7] Sidiropoulos, P.I. and Boumpas, D.T., "Lessons learned from anti-CD154 treatment in systemic lupus erythematosus patients", Lupus 2004, 13: 391 - 397 [Non-Patent Document 8] Ferrant, JL, Benjamin, CD, Cutler, AH, Kalled, SL, Hsu, YM, Garber, EA, Hess, DM, Shapiro, RI, Kenyon, NS, Harlan, DM, Kirk, AD, Burkly, LCand Taylor, FR, "The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge", Int Immunol. 2004 16:1583~1594 Summary of the Invention [Problem to be solved by the invention]

[0012] Notwithstanding the foregoing, there remains a significant need in the art for improved anti-CD154 antibodies, i.e., anti-CD154 antibodies that are safe and effective and contain superior potency and pharmacokinetic properties. The present invention achieves these goals. [Means for solving the problem]

[0013] It is an object of the present invention to provide anti-CD154 antibodies with improved efficacy, safety and pharmacokinetics for use in human therapy.

[0014] More specifically, it is an object of the present invention to provide a humanized anti-human CD154 antibody comprising (i) a variable heavy polypeptide comprising or consisting of a polypeptide having a sequence selected from SEQ ID NOs: 15, 17 and 18 as shown in Figures 2A-2C, and (ii) a variable light polypeptide comprising or consisting of a polypeptide having a sequence selected from SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31 as shown in Figures 2A-2C, with the proviso that the humanized anti-human CD154 antibody does not comprise the variable heavy polypeptide of SEQ ID NO: 15 and the variable light polypeptide of SEQ ID NO: 20.

[0015] It is a further object of the present invention to provide a humanized anti-human CD154 antibody comprising (i) a variable heavy polypeptide comprising or consisting of a polypeptide having the sequence of SEQ ID NO: 1 shown in Figures 1A-C, and (ii) a variable light polypeptide comprising or consisting of a polypeptide having the sequence of SEQ ID NO: 2 shown in Figures 1A-C.

[0016] It is a further object of the present invention to provide an anti-human CD154 antibody as described in the previous three paragraphs that comprises an IgG1, IgG2, IgG3, or IgG4 constant region that lacks the ability to bind C1Q and to bind FcγR2 and / or FcγR3.

[0017] It is a further object of the present invention to provide an anti-human CD154 antibody as described in the previous four paragraphs that comprises a human IgG1 constant region that lacks the ability to bind C1Q and to bind FcγR2 and / or FcγR3.

[0018] It is a further object of the present invention to provide an anti-human CD154 antibody as described in the previous five paragraphs, which comprises a human IgG1 constant region comprising E269R and K322A mutations (numbering according to Kabat).

[0019] It is a further object of the invention to provide a humanized anti-human CD154 antibody according to any of the preceding paragraphs, comprising the IgG1 light and heavy constant regions of SEQ ID NO:3 and SEQ ID NO:2 as shown in Figures 1A-C.

[0020] It is a further object of the present invention to provide a humanized anti-human CD154 antibody according to any of the preceding paragraphs, wherein the antibody is of IgG1 isotype and wherein the Fc region of the antibody has been further mutated to introduce at least one other mutation that affects effector function, such as a mutation that impairs FcRn binding, a mutation that impairs or eliminates glycosylation, or another mutation that impairs the ability of the antibody to bind to an FcR such as FcγR2 or FcγR3 or another FcR or that impairs complement binding.

[0021] It is a further object of the invention to provide a humanized anti-human CD154 antibody according to any of the preceding paragraphs, wherein the antibody is of the IgG1 isotype and the Fc region of the antibody has been mutated to further introduce an E233P and / or a D265A mutation, or any of the Fc mutations identified in Tables 1, 2 or 3.

[0022] It is a further object of the present invention to provide a humanized anti-human CD154 antibody according to any of the preceding paragraphs, wherein the antibody is of IgG1 isotype and the Fc region of the antibody has been mutated to introduce the E233P mutation.

[0023] It is a further object of the present invention to provide a humanized anti-human CD154 antibody according to any of the preceding paragraphs, wherein the Fc region of the antibody has been further mutated to introduce one or more other mutations that improve in vivo half-life.

[0024] It is further an object of the present invention to provide a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of at least one humanized anti-human CD154 antibody according to any of the preceding paragraphs.

[0025] It is a further object of the present invention to provide a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of at least one humanized anti-human CD154 antibody according to any of the preceding paragraphs, said pharmaceutical composition further comprising an antigen, a cell, a tissue, or an organ.

[0026] Further provided is a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of at least one humanized anti-human CD154 antibody according to any of the preceding paragraphs, wherein the antibody is administered to a subject who has received treatment with autoantigens, allergens, inflammatory agents, drugs, or HLA-mismatched (allo) or xenogeneic (non-human) donor cells. It is an object of the present invention to provide said pharmaceutical composition further comprising:

[0027] It is further an object of the present invention to provide a method of immunosuppression or immunotherapy using an antibody or composition according to any of the preceding paragraphs.

[0028] It is further an object of the present invention to provide a method of treating a subject or transplant recipient with an allergic, inflammatory, or autoimmune disease by administering an antibody or composition according to any of the preceding paragraphs.

[0029] It is further an object of the present invention to provide a method of treating or preventing GVHD in a subject by administering an antibody or composition according to any of the preceding paragraphs before, concurrently with, or after transplantation of donor tissue, organs, or cells, e.g., CAR-T (chimeric antigen receptor T cells) or NK (natural killer) cells, which may optionally be genetically engineered.

[0030] It is further an object of the present invention to provide a method for inducing tolerance or prolonged antigen-specific immunosuppression by administering an antibody or composition according to any of the preceding paragraphs, optionally further comprising the administration of a cell, antigen, tissue, or organ containing the antigen to which tolerance or prolonged immunosuppression is sought to be induced.

[0031] It is further an object of the present invention to provide a method of gene, cell, tissue or organ therapy comprising administering an antibody or composition according to any of the preceding paragraphs.

[0032] It is further an object of the invention to provide a method of treating a condition selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, oophoritis, lupus or SLE, diabetes, IBD, Crohn's disease, ITP, thyroiditis, rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes mellitus, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, drug-induced autoimmune disease, or drug-induced lupus by administering an antibody or composition according to any of the preceding paragraphs.

[0033] It is further an object of the present invention to provide a method of treating bone marrow, organs or immune cells for use in cell therapy or organ or bone marrow transplantation, comprising incubating said bone marrow, organ, tissue or immune cells with an antibody or composition according to any of the preceding paragraphs, e.g., wherein the treated bone marrow, tissue, organ or immune cells comprise donor and / or recipient T cells.

[0034] It is a further object of the present invention to provide a method for inducing tolerance without inducing a thrombotic event in a patient in need thereof, comprising administering to the patient an effective amount of an antibody or composition according to any of the preceding paragraphs, and optionally further comprising administering an antigen, such as an autoantigen, an allergen, an inflammatory agent, a drug, or HLA-mismatched (allo)donor cells.

[0035] It is a further object of the present invention to provide a method for inducing tolerance, prolonged immunity, or T or B cell suppression in a patient who will receive or has received transplanted cells, organs, or tissues, wherein, for example, the drug is a biopharmaceutical, such as a therapeutic antibody, receptor, fusion protein, hormone, growth factor, or cytokine.

[0036] It is further an object of the present invention to provide a method of treating a T cell-mediated autoimmune disorder by administering an effective amount of an antibody or composition according to any of the preceding paragraphs.

[0037] It is a further object of the present invention to provide a method of treating a B-cell mediated autoimmune disorder by administering an effective amount of an antibody or composition according to any of the preceding paragraphs.

[0038] It is further an object of the present invention to provide a method of treating an autoimmune disease selected from rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Graves' disease, Huntington's disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes mellitus, Parkinson's disease, psoriasis, Addison's disease, multiple sclerosis, lupus, and drug-induced autoimmune diseases, e.g., drug-induced lupus, by administering an effective amount of an antibody or composition according to any of the preceding paragraphs.

[0039] It is further an object of the present invention to provide a method of treatment according to any of the preceding paragraphs, said method comprising administration of an antigen.

[0040] It is further an object of the present invention to provide a method of treating or preventing GVHD, bone marrow transplantation (BMT), multiple sclerosis, lupus, ITP, rheumatoid arthritis, asthma, IBD or another inflammatory bowel disorder by administering an effective amount of an antibody or composition according to any of the preceding paragraphs.

[0041] A method for treating a human condition, disorder, or disease mediated in whole or in part by CD40 signaling, or any of the symptoms mentioned above, comprising administering an effective amount of an antibody or composition according to any of the preceding paragraphs, e.g., the human condition, disorder, or disease is an inflammatory, allergic, or autoimmune response or fibrosis, or the human condition, disorder, or disease is selected from lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus, spondyloarthritis, drug-induced lupus erythematosus, inflammatory bowel disease, Crohn's disease, psoriasis, and multiple sclerosis. or the human condition, disorder or disease is selected from allergic contact dermatitis, alopecia universalis, anaphylactoid purpura asthma, severe asthma, metabolic asthma, allergic asthma, atopic dermatitis, dermatitis herpetiformis, erythema elevatum diutinum, erythema annulare, erythema multiforme; erythema nodosum, allergic granulomatosis, granuloma annulare, granulocytopenia, hypersensitivity pneumonitis, keratitis, nephrotic syndrome, overlap syndrome, pigeon breeder's disease, idiopathic polyneuropathy, urticaria, uveitis, juvenile dermatomyositis, and vitiligo, or the human condition, disorder or disease is allergic bronchopulmonary aspergillosis; autoimmune disease Immune hemolytic anemia;Acanthosis nigricans;Allergic contact dermatitis;Addison's disease;Atopic dermatitis;Alopecia areata;Alopecia universalis;Amyloidosis;Anaphylactoid purpura;Anaphylactoid reactions;Aplastic anemia;Angioedema, hereditary;Angioedema, idiopathic;Ankylosing spondylitis;Arteritis, cranial;Arteritis, giant cell;Arteritis, Takayasu's;Arteritis, temporal;Asthma;Ataxia-telangiectasia;Autoimmune oophoritis;Autoimmune orchitis;Autoimmune polyendocrine deficiency;Behçet's disease;Berger's disease;Buerger's disease;Bullous pemphigus;Chronic mucocutaneous candidiasis;Kaplan's syndrome; Post-myocardial infarction syndrome; Post-pericardiotomy syndrome; Carditis; Celiac sprue; Chagas disease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan syndrome; Cold agglutinin disease; Crest syndrome; Crohn's disease; Cryoglobulinemia; Fibrotic alveolitis of unknown etiology; Dermatitis herpetiformis; Dermatomyositis; Diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupus erythematosus; Eosinophilic fasciitis; Episcleritis; Erythema elevatum perstans; Erythema marginatum; Erythema multiforme; Erythema nodosum; Familial Mediterranean fever; Felty's syndrome; Pulmonary fibrosis; Glomerulonephritis, anaphylactoid;Glomerulonephritis, autoimmune; Glomerulonephritis, poststreptococcal; Glomerulonephritis, posttransplant; Glomerulopathy, membranous; Goodpasture's syndrome; Graft-versus-host disease; Agranulocytopenia, immune-mediated; Granulomatosis annulare; Granulomatous disease, allergic; Granulomatous myositis; Graves' disease; Hashimoto's thyroiditis; Hemolytic disease of the newborn; Hemochromatosis, idiopathic; Henoch-Schönlein purpura; Hepatitis, chronic active and chronic progressive; Histiocytosis x; Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura; Job's syndrome; Juvenile dermatomyositis; Juvenile rheumatoid arthritis (juvenile chronic arthritis); Kawasaki disease; Keratitis; Keratoconjunctivitis sicca; Landry-Guillain-Barré-Straull syndrome; Leprosy, et al. cystoid type;Löffler's syndrome;Lyell's syndrome;Lyme disease;Lymphomatoid granulomatosis;Mastocytosis, systemic;Mixed connective tissue disease;Mononeuritis multiplex;Muckle-Wells syndrome;Mucocutaneous lymph node syndrome;Mucocutaneous lymph node syndrome;Multicentric reticulohistiocytosis;Multiple sclerosis;Myasthenia gravis;Mycosis fungoides;Necrotizing vasculitis, systemic;Nephrotic syndrome;Overlap syndrome;Panniculitis;Paroxysmal cold hemoglobinuria;Paroxysmal nocturnal hemoglobinuria;Pemphigoid;Pemphigus erythematosus;Pemphigus foliaceus;Pemphigus vulgaris;Pigeon breeder's disease;Interstitial pneumonia, hypersensitivity;Polyarteritis nodosa;Polymyalgia rheumatica;Polymyositis;Polyneuritis, idiopathic;Portuguese familial polyneuropathy;Preeclampsia / eclampsia;Primary It is an object of the present invention to provide such a method, wherein the disease is selected from biliary cirrhosis; progressive systemic sclerosis (scleroderma); psoriasis; psoriatic arthritis; pulmonary alveolar proteinosis; pulmonary fibrosis, Raynaud's phenomenon / syndrome; Riedel's thyroiditis; Reiter's syndrome, relapsing polychondritis; rheumatic fever; rheumatoid arthritis; sarcoidosis; scleritis; sclerosing cholangitis; serum sickness; Sézary syndrome; Sjögren's syndrome; Stevens-Johnson syndrome; Still's disease; subacute sclerosing panencephalitis; sympathetic ophthalmia; systemic lupus erythematosus; transplant rejection; ulcerative colitis; undifferentiated connective tissue disease; urticaria, chronic; urticaria, cold; uveitis; vitiligo; Weber-Christian disease; Wegener's granulomatosis; and Wiskott-Aldrich syndrome. [Brief explanation of the drawings]

[0042] [Figure 1-1]1A-1C show the amino acid sequences of the anti-CD154 antibodies of the invention derived from IDEC-131, in which variable region residues in both the heavy and light chain polypeptides are shown in bold, CDRs are highlighted in yellow, affinity maturation mutation residues are underlined, and Fc mutations (E→R and K→A) are shown in red and underlined. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 2-1] 2A-2C are diagrams aligning different modified IgG1 constant regions and modified light and heavy chain sequences from IDEC-131 and its parent murine antibody. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 3] 3 shows the results of a BIAcore binding assay comparing the binding of a humanized anti-human CD154 antibody of the invention (referred to in the figure and in the present application as "INX021") to IDEC-131 for its ability to bind to CD154-expressing Jurkat cells. As shown, the antibody of the invention has an approximately 5-fold increased binding affinity for CD154 (expressed on Jurkat cells) compared to IDEC-131. [Figure 4] FIG. 4 depicts experimental results showing that an antibody of the present invention ("INX021") promotes T cell CD154-driven B cell activation more potently than IDEC-131. [Figure 5] FIG. 5 shows experimental results demonstrating that an FcR mutant of a human IgG anti-mouse CD154 antibody containing the same mutations as INX021 (K322A and E269R) induces tolerance in a skin tolerance model. [Figure 6] FIG. 6 depicts experimental results showing that INX021 immunoconjugates do not induce aggregation or activation of mouse or human platelets in vitro. [Figure 7]Figures 7A-7D are diagrams of experimental results demonstrating that INX021 does not induce platelet activation or other effects on platelets compared to a positive control antibody. Figure 7A shows that INX021 does not induce thrombotic stress, Figure 7B shows that INX021 does not cause platelet aggregation or coagulation as evidenced by the lack of observed pulmonary emboli, Figure 7C shows that INX021 does not affect the number of circulating platelets, and Figure 7D summarizes the observed benefits of this antibody observed in FcγRIIa Tg mice. [Figure 8] Figures 8A and 8B show that anti-mouse CD154 IgG1 antibodies containing Fc mutations that reduce or eliminate FcR binding and complement activity (the E269R and K322A mutations contained in INX021) are immunosuppressive in animal models. Figure 8A shows that anti-CD154 antibodies containing these mutations suppress EAE, and Figure 8B shows that anti-CD154 antibodies containing these mutations suppress humoral immunity and induce tolerance in response to myelin / oligodendrocyte glycoprotein ("MOG"). [Figure 9] Figure 9 compares the effect of different antibody variants of IDEC-131, including INX021, on T cell-induced B cell activation based on CD86 expression, demonstrating that the effects were variable and increased binding affinity does not necessarily correlate with greater potency, i.e., immunosuppressive activity. [Figure 10] Figures 10A and 10B compare the effects of INX021 and chimeric 5c8 antibody on IgG and IgM antibody production in a KLH assay: Figure 10A compares the effect of INX021 and chimeric 5c8 antibody on IgG, and Figure 10B compares the effect of INX021 and chimeric 5c8 antibody on IgM. [Figure 11]Figures 11A and 11B compare the effects of vehicle, INX021, and 5c8 antibody on germinal center scores. Figure 11A shows the germinal score comparison, and Figure 11B contains histograms comparing the number of germinal centers and the degree of cellularity in the spleens and lymph nodes (LNs) of treated animals. [Figure 12] FIG. 12 is a table summarizing the observed lesion frequencies compared with published results using another anti-CD154 antibody (PEGylated αCD154 Fab from Biogen / UCB). DETAILED DESCRIPTION OF THE INVENTION

[0043] Prior to disclosing the present invention in detail, the following definitions are provided: Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] The term "antibody" as used herein in connection with the present invention includes isolated, recombinant, or synthetic antibodies, antibody conjugates, or antibody derivatives. The term "antibody" is often intended to include antibody fragments, including antigen-binding fragments, unless otherwise indicated or understood from the context. Antigen-binding fragments compete with intact antibodies for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding fragments include Fab, F(ab)2, Fab', F(ab')2, F(ab')3, Fd, Fv, domain antibodies (dAbs), other monovalent and bivalent fragments, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv, scFab, and scFabδC), chimeric antibodies, diabodies, triabodies, minibodies, nanobodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen binding to a polypeptide, as well as fusions and derivatives of the foregoing. See, e.g., Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005) and Hust et al. See, al., BMC Biotech 7:14 (2007).

[0045] Unless otherwise stated or implied otherwise by context, "antibodies" of the present invention include whole antibodies and any antigen-binding fragments thereof, antibody conjugates (i.e., antibodies or antigen-binding fragments thereof conjugated or associated with a functional moiety), antibody derivatives or variants that may contain one or more modifications (e.g., amino acid insertions, deletions, substitutions, post-translational modifications or lack thereof, etc.). Antibody derivatives, including antibody conjugates, may be based on or include antigen-binding fragments of the present invention that specifically bind to CD154. Additionally, the antibody embodiments described above may include murine, hamster, goat, rabbit, chimeric, humanized, or fully human antibodies, fragments, derivatives, or clones. It is understood that in certain aspects of the invention, the term "antibody" may exclude one or more of the antibody embodiments listed above; such situations will be apparent to one of skill in the art.

[0046] The term "effector function" refers to the functional ability of an antibody's Fc or constant region to bind to proteins and / or cells of the immune system. Antibodies with reduced effector function and methods for engineering such antibodies are well known in the art (see, e.g., WO 05 / 18572, WO 05 / 03175, and U.S. Pat. No. 6,242,195) and are described in further detail herein. Exemplary effector functions include the ability to bind complement proteins (e.g., complement protein C1q) and / or Fc receptors (FcR) (e.g., FcγRI, FcγRII, FcγRIIa, FcγRIII, and / or FcγRIIIb). Functional implications of being able to bind one or more of the aforementioned molecules include, but are not limited to, opsonization, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or effector cell modulation. Reduced effector function refers to a reduction in one or more biochemical or cellular activities induced at least in part by Fc binding to its cognate receptor or to complement proteins or effector cells, while maintaining the antigen-binding activity of the antibody variable region (or fragment thereof), albeit with reduced, similar, identical, or increased binding affinity. Certain antibodies of the present invention exhibit reduced effector function. A reduction in effector function, e.g., Fc binding to an Fc receptor or complement protein, can be expressed in terms of a fold reduction (e.g., a 1.5-fold, 2-fold, etc. reduction), and may be calculated, for example, based on the percent reduction in binding activity determined using binding assays known in the art (see, e.g., WO 05 / 18572).

[0047] Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0048] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0049] The term "CD154" refers to a ligand expressed on activated T cells. CD154 is known by several other names in the art, including CD40 ligand (CD40L), CD40 counter-receptor (CD40CR), gp39, T-BAM, T-cell activation molecule, TRAF, TNF-related activation protein (TRAP), and tumor necrosis factor ligand superfamily member 5 (TNFSF5) (Gauchat et al., 1993 FEBS Lett. 315:259-266; Graf (Hollenbaugh et al., 1992, Europ. J. Immun. 22:3191-3194; Hollenbaugh et al., 1992, EMBO J. 11:4313-4321). These terms are used interchangeably throughout this application. CD154 binding proteins, including antibodies, of the present invention specifically bind to human CD154 and may cross-react and therefore specifically bind to CD154 of other species. In certain embodiments, CD154 binding proteins, including antibodies, of the present invention specifically bind to human CD154, mouse CD154, or non-human primate CD154.

[0050] The term "anti-CD154 antibody" also encompasses synthetic or recombinant antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by bacteriophage. The term "anti-CD154 antibody" also encompasses antibodies produced by synthesis of a DNA molecule encoding the antibody, as well as antibodies in which the DNA molecule expresses the antibody protein or amino acid sequence that specifies the antibody. The term "antibody" should be construed to include the DNA or amino acid sequences of the polypeptides, which DNA or amino acid sequences have been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0051] As used herein, "mutation(s) that eliminate or reduce FcR binding and eliminate toxicity" refers to mutation(s) that eliminate or reduce thrombocytopenia or thrombosis or coagulation in vitro and / or in vivo. Portions of the Fc or constant region of different human antibody types, including human IgG1, IgG2, IgG3, and IgG4, that may be modified to eliminate or reduce FcR binding or affect (impair or enhance) other Fc effector functions are well known in the art. Exemplary mutations that may be engineered in the human IgG1 Fc or constant region are identified in Tables 1-3 and in the Examples below.

[0052] "Mutation(s) that eliminate or reduce complement function and maintain tolerance-inducing properties" refers to mutation(s) in a human, chimeric, or humanized antibody containing a human constant region, preferably a human IgG1 or IgG3 constant region, whose Fc region has been mutated at one or more sites to eliminate or substantially reduce complement binding. Preferably, such mutations do not appreciably affect the antibody's ability to induce tolerance in vivo. This can be established using an appropriate tolerance model, such as a skin graft model. Sites in the Fc or constant regions of different human antibody types, including human IgG1, IgG2, IgG3, and IgG4, that may be modified to eliminate or reduce complement binding or affect (impair or enhance) other Fc effector functions are well known in the art. Exemplary sites that may be modified in IgG1 are identified in Tables 1-3 and in the Examples below.

[0053] "Patient" can refer to a human or non-human, preferably a mammal. In a preferred embodiment, the present invention produces anti-human CD154 antibodies suitable for human therapy that contain mutated IgG1 or IgG3 constant regions, where such mutations eliminate or substantially inhibit toxicities or safety concerns, such as thrombocytopenia or thrombosis or coagulation responses, or toxicities associated with complement responses, and preferably, such antibodies retain the ability to induce tolerance or prolonged humoral suppression in vivo.

[0054] As used herein, a "subject" refers to an organism or a cell, tissue, or organ sample derived therefrom, including, for example, a cultured cell line, a biopsy, a blood sample, or a cell-containing fluid sample. In many instances, a subject or a sample derived therefrom contains multiple cell types. In one embodiment, the sample contains, for example, a mixture of tumor and normal cells. In one embodiment, the sample contains at least 10%, 15%, 20%, cf. below, 90%, or 95% tumor cells. The organism may be an animal, including, but not limited to, animals such as cows, pigs, mice, rats, chickens, cats, dogs, etc., and is typically a mammal, such as a human.

[0055] The term "treating," in its various grammatical forms, in the context of this invention refers to preventing (i.e., chemopreventing), curing, reversing, attenuating, alleviating, minimizing, suppressing, or arresting the deleterious effects of a disease state, disease progression, disease-causing agent (e.g., bacteria or virus), or other abnormal condition. For example, treating may include alleviating a symptom (i.e., not necessarily every symptom) of a disease or attenuating the progression of a disease.

[0056] Anti-CD154 antibodies are potentially useful therapeutic agents for the treatment of autoimmune diseases. "Treating or preventing" or another disease state, such as cancer, infection, inflammation, allergy, transplantation, graft-versus-host disease, and other conditions, refers to partially or totally inhibiting, delaying, or preventing the progression of said disease. In the case of cancer, this means treating or inhibiting cancer metastasis in a mammal, e.g., a human; inhibiting, delaying, or preventing cancer recurrence, including cancer metastasis; or preventing the onset or development of cancer (chemoprotection). Furthermore, the methods of the present invention may be practiced for the treatment of human patients with cancer. However, the methods may also be effective in treating cancer in other mammals. In preferred embodiments, the subject antibodies are used to treat autoimmunity, allergy, inflammation, transplantation, GVHD, bone marrow transplant (BMT), and to induce antigen-specific tolerance or prolonged antigen-specific immune suppression, particularly the suppression of T-cell and / or B-cell differentiation, activation, and / or proliferation, in a subject in need thereof. Preferred indications include multiple sclerosis, lupus or SLE, ITP, IBD, Crohn's disease, inflammatory bowel disease, Crohn's disease, psoriasis, uveitis, rheumatoid arthritis, asthma, GVHD, bone marrow transplant, diabetes, rheumatoid arthritis, psoriatic arthritis, psoriasis, cell transplant, tissue transplant, organ transplant, bone marrow transplant, cell and gene therapy, oophoritis and thyroiditis.

[0057] As used herein, the term "therapeutically effective amount" is intended to qualify the amount of treatment in a therapeutic regimen necessary to treat a condition, e.g., autoimmunity, allergy, inflammation, GVHD or transplantation, or to reduce or eliminate an immune response, particularly a T cell or B cell antibody response, to an antigen, such as an autoantigen, an allergen, an inflammation-inducing drug, a transplanted cell, tissue, or organ, or to a drug, such as a biopharmaceutical, e.g., a therapeutic antibody, an Ig fusion protein, a hormone, a growth factor, or other therapeutic protein or polypeptide.

[0058] As used herein, the term "prophylactically effective amount" is intended to qualify the amount of treatment in a therapeutic regimen needed to prevent a condition, or disease progression and symptoms, such as an autoimmune, allergic, inflammatory, GVHD, or anti-transplant, drug, or rejection response.

[0059] The present invention provides improved anti-CD154 antibodies for use in human therapy derived from the humanized antibody IDEC-131 developed by IDEC Pharmaceuticals (now BiogenIDEC). IDEC-131 binds human CD154 with a K of 151 pm as detected by BIAcore at 25°C. D It is a high-affinity antibody that binds to

[0060] IDEC-131 is a humanized anti-CD154 antibody of the human IgG1 isotype derived from the murine anti-human CD154 antibody (24-31) disclosed in U.S. Patent No. 5,747,037. A commercially available hybridoma cell line secreting 24-31 has been deposited with the American Type Culture Collection, and the deposited cell line has been designated ATCC Accession No. HB1712.

[0061] The sequence of IDEC-131 and its use in treating various allergic, autoimmune, and inflammatory indications, and for suppressing T- and B-cell-mediated immune responses, are disclosed in U.S. Patent Nos. 6,001,358, 6,440,418, 6,506,383, 7,074,406, and 7,122,187. Based on its properties, including its potent immunosuppressive effect on T- and B-cell immunity and high affinity, IDEC-131 has been advanced into human clinical trials. In particular, IDEC-131 has been used in human clinical trials sponsored by IDEC to treat two autoimmune indications, namely, idiopathic thrombocytopenic purpura ("ITP") and remitting / relapsing multiple sclerosis ("RR-MS"). These clinical trials using IDEC-131 have been partially showed clinical efficacy (albeit very early on in a relatively small number of patients), but the clinical trials were halted after it was reported that another IgG1 anti-human CD154 antibody developed by Biogen, also derived from a murine anti-human CD154 antibody (the murine 5c8 antibody isolated by Seth Lederman at Columbia University), induced adverse effects in human patients. Notably, when the Biogen chimerized 5c8 antibody was used in clinical trials, this antibody led to adverse thrombotic events ("TE"), resulting in stroke and death in several patients in the clinical trials. The present invention addresses these issues while obtaining other pharmacokinetic advantages over IDEC-131.

[0062] In contrast, the present invention provides humanized mutant anti-human CD154 antibodies derived from IDEC-131, which, like IDEC-131, are of the human IgG1 isotype but whose Fc region has been engineered to eliminate both FcR binding and complement activation. In particular, their constant regions have been engineered to contain E269R and K322A mutations (these mutant residues are numbered according to the Kabat antibody numbering system). These mutations result in reduced FcγRIIa and C1q binding, respectively. Notably, the resulting modified antibodies have limited FcR / C1q binding; i.e., they do not bind to FcγR2 or FcγR3 on platelets, and they do not bind or activate complement. In contrast, their binding to FcRn is unaffected. As disclosed below and supported by the experimental data disclosed herein, anti-CD154 IgG1 antibodies containing these mutations do not exhibit any detectable prothrombotic activity on mouse or human platelets in vitro or in vivo when assayed in different experimental models.

[0063] Furthermore, the variable heavy and light sequences of IDEC-131 were further engineered by affinity maturation. Affinity maturation was performed in the hope of enhancing the binding affinity of the resulting anti-human CD154 antibody compared to IDEC-131 while still maintaining its desired immunosuppressive properties. However, as mentioned, this result was far from guaranteed, since IDEC-131 already has a strong binding affinity for human CD154 (161pm) and is potently immunosuppressive in different models (EAE, humoral immunosuppression, and allospecific tolerance models).

[0064] After many experiments and sequential screening procedures, mutant versions of the variable heavy and light chains of IDEC-131 were obtained that produced humanized antibodies (including INX02) with improved affinity compared to IDEC-131. Specifically, INX021 was obtained, as were 12 other variants. These humanized variants were evaluated, and INX021 was selected as the lead candidate for human therapy based on its favorable binding (approximately 5-fold higher affinity for human CD154 than IDEC-131), and INX021 also has superior functional (immunosuppressive) properties compared to IDEC-131 and compared to the other variants.

[0065] As shown in Figures 1A-C (which contain the sequence of the humanized IgG1 anti-human CD154 antibody of the present invention, INX021), this antibody contains two mutations in the heavy chain CDR3, three mutations in the light chain CDR1, and two mutations in the light chain CDR3 (compared to IDEC-131). Thus, a total of seven CDR residues were altered. Surprisingly, as shown below, the resulting humanized anti-human CD154 antibody binds to human CD154 with approximately five-fold higher affinity than the parent antibody, which, as previously noted, itself binds to human CD154 with very high affinity. This is surprising, since even a single CDR modification can have a dramatic effect on antibody binding affinity. Furthermore, it is not predictable that the affinity of an already relatively high-affinity antibody could be improved.

[0066] This engineered humanized antibody has highly potent immunosuppressive properties when assayed in different in vitro and in vivo models. Specifically, INX021 inhibits the immune system. It potently suppresses humoral immunity, EAE, and induces allospecific tolerance or prolonged antigen-specific T cell unresponsiveness. As shown below, INX021 induces a more potent inhibition of T cell CD154-driven B cell activation compared to IDEC-131.

[0067] Thus, the antibodies of the present invention (compared to IDEC-131) (i) has five times the binding affinity for human CD154 as IDEC-131; (ii) induces more potent immunosuppressive activity than IDEC-131; (iii) like IDEC-131, suppress T and B (humoral) immunity, e.g., suppress EAE, induce allospecific tolerance, and (iv) exhibits an improved safety profile compared to IDEC-131 due to mutations in the Fc region that substantially reduce FcR and C1q binding, and as a result does not exhibit any detectable prothrombotic activity on mouse or human platelets in vitro or in vivo.

[0068] Based on these combined properties, the subject anti-human CD154 antibody is well suited for use in human therapy, as it should exhibit an enhanced safety profile in patients and be more potent and therefore more effective than IDEC-131. The antibody's enhanced binding affinity is expected to facilitate its administration via subcutaneous administration, which is beneficial for its potential use in treating chronic human indications.

[0069] Furthermore, the subject antibodies are intact, i.e., contain full-length IGg1, and therefore may offer pharmacokinetic and safety advantages over the PEGylated Fabs being developed by Biogen / UCB and the CD154-specific diabodies being developed by Bristol Meyers Squibb.

[0070] Furthermore, the subject antibodies have higher potency, potentially allowing the antibodies to be administered less frequently, further enhancing dosing. This is important because the majority of applications for the subject antibodies are aimed at treating chronic human autoimmune, allergic, or inflammatory indications where any effective therapeutic regimen requires repeated dosing. In particular, the antibodies of the invention should be well suited for treatments where B- or T-cell immunosuppression is therapeutically desirable.

[0071] Thus, in this application, the applicant provides novel and improved humanized anti-human CD154 antibodies with improved affinity and other pharmacokinetic properties for use in human immunotherapy, which should not induce toxicity in human patients; in particular, these antibodies should not induce platelet aggregation or thromboembolic events in human subjects, and furthermore, based on their inability to bind and activate complement, should not mediate ADCC or CDC-mediated cytotoxicity. As shown in the examples, the antibodies of the present invention have significantly improved functional and pharmacokinetic properties compared to IDEC-131 and other CD154 antibodies previously used in human therapy. Thus, these antibodies should provide clinical benefits when used in human therapy.

[0072] While the antibody INX021 of the present invention contains two mutations that reduce FcγR2 and 3 binding, and C1q binding, respectively, it is further contemplated that other mutations in the Fc region may be introduced that may further reduce binding to other Fc receptors or complement or alter other effector functions. Examples of other possible mutations are known in the art, see, for example, Shields RL, Namenuk AK, Hong K, et al., ("High resolution mapping of the "Binding site on Human IgG1 for Fc for FcγRI, Fc for FcγRII, Fc for FcγRIII, and FcRn" reports the design of IgG1 mutants with impaired Fc binding for FcγR (J. Biol. Chem. 2001;276:6591-604). Furthermore, U.S. Patent Application Publication Nos. 2007 / 0237767 and 2010 / 0104564 describe Fc mutagenesis that eliminates FcR binding. Specific Fc mutations and their effects are listed in Tables 1-3 below. These mutations are exemplary and are not intended to encompass other mutations and combinations thereof that are known to enhance or impair Fc effector functions, such as FcR binding, complement fixation, glycosylation, ADCC activity, CDC activity, and FcRN binding, among others.

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3]

[0076] Based on this combination of safety, functional, and pharmacokinetic properties, the humanized anti-human CD154 IgG1 antibodies of the present invention can be used to treat or prevent conditions in which suppression of T and / or B cell activation, differentiation, and proliferation is therapeutically desirable, such as allergies, autoimmunity, transplants (allo- or xenogeneic organ, cell, or tissue transplants), and inflammatory conditions. Specific, non-limiting examples include, for example, multiple sclerosis, systemic lupus erythematosus (SLE), other forms of lupus, autoimmune thrombocytopenia (ITP), kidney transplants, skin and pancreatic islet transplants, rheumatoid arthritis, sarcoidosis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, Crohn's disease, COPD, asthma, diabetes, thyroiditis, graft-versus-host disease, bone marrow (BM) or hematopoietic stem cell transplants, and atherosclerosis. Similarly, some of these variants, including INX021, have increased efficacy, including the ability to induce tolerance in vivo.

[0077] As evidenced by experimental data, a particularly envisioned application of the antibodies of the invention, including INX021, is to reduce or prevent anti-drug reactions, particularly those elicited to biopharmaceuticals and other drugs such as therapeutic antibodies, fusion proteins, hormones, growth factors, enzymes, peptides, antibiotics, antivirals, and the like, which may allow these drugs to be administered for longer periods, potentially increasing the number of patients who respond to the drug and / or promoting efficacy in drug-naive patients.

[0078] As discussed herein, the Fc region of human IgG1 and other sites in other human constant regions are known to be involved in complement fixation and / or activation and FcR binding. Thus, the described mutations are examples of suitable mutations in the Fc region of IgG1 or IgG3 or other antibodies that result in the loss of one or both of complement fixation and FcR binding.

[0079] The tolerance-inducing or prolonged (T or B) immunosuppressive effects of complement-fixing mutant αCD154 variants or their variants may be evaluated in a well-studied model of haplo-mismatched skin allograft survival, in which long-term tolerance is induced by administration of αCD154 and alloantigen. However, other tolerance models may alternatively be used to evaluate the efficacy of αCD154 complement-deficient and / or Alternatively, the ability of FcR deletion variants (containing mutated Fc regions) to induce tolerance may be assessed.

[0080] The thromboembolic activity of the FcR mutant αCD154 may be tested in a mouse model expressing the human FcγRIIA receptor, which recapitulates events observed in NHPs (Ferrant, JL, Benjamin, CD, Cutler, AH, Kalled, SL, Hsu, YM, Garber, EA, Hess, DM, Shapiro, RI, Kenyon, NS, Harlan, DM, Kirk, AD, Burkly, LC and Taylor, FR. "The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge." Int Immunol 2004;16:1583-1594). In such mice, treatment with αCD154 (anti-mouse CD154) induces pulmonary thrombi, and thus abolishing FcR binding in anti-CD154 antibodies containing mutant human Fc regions (mutated IgG1 constant regions) eliminates or appreciably reduces thrombus formation. This or comparable models can be used to determine the effect of specific Fc mutations on the toxicity associated with αCD154 therapy.

[0081] Furthermore, mutant CD154-specific antibodies of the present invention derived from IDEC-131 may be used for the treatment and prevention of any condition in which attenuating the effects of CD154, including CD154 / CD40 signaling, or blocking or inhibiting CD154 binding to CD40, could be therapeutically beneficial and reduce disease symptoms. Examples include the treatment of allergy, autoimmune, cancer, transplantation, GVHD, inflammatory, and other conditions, particularly conditions in which tolerance induction and / or suppression of humoral or T-cell immunity is therapeutically desirable. Specific examples include multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, thyroiditis, lupus erythematosus, autoimmune thrombocytopenia, diabetes, graft-versus-host disease, cell therapy, organ and tissue transplantation, e.g., skin, kidney, pancreas, bone marrow, atherosclerosis, and other conditions in which humoral or T-cell suppression is desirable.

[0082] The subject antibodies targeting CD154 and having an improved safety profile have great therapeutic potential because CD154 is an extremely attractive target for immune intervention in a wide range of autoimmune and graft-related diseases. Virtually all mouse models of autoimmune disease tested to date have been therapeutically improved by αCD154 treatment. Beyond simply blocking CD154-CD40 interactions, αCD154 therapy induces immune tolerance. Prevention of graft rejection by blocking CD40-CD154 interactions has been shown to be beneficial in the skin (Gordon, EJ, Markees, TG, Phillips, NE, Noelle, RJ, Shultz, LD, Mordes, JP, Rossini, AA, and Greiner, DL, "Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody," Diabetes 1998; 47:1199-1206; Markees, TG, Phillips, NE, Noelle, RJ, Shultz, LD, Mordes, JP, Greiner, DL, and Rossini, AA, "Prolonged survival of mouse skin allografts"). in recipients treated with donor splenocytes and antibody to CD40 ligand', Transplantation 1997, 64:329-335; Jarvinen, LZ, Blazar, BR, Adeyi, OA, Strom, TBand. Noelle, RJ, “CD154 on the surface of CD4 + CD25 + regulatory T cells contributes to "skin transplant tolerance", Transplantation 2003, 76: 1375 - 1379; Quezada, S.A., Fuller, B., Jarvinen, L.Z., Gonzalez, M., Blazar, B.R., Rudensky, A.Y., Strom, T.B. and Noelle, R.J., "Mechanisms of donor - specific transfusion tolerance: preemptive induction of "clonal T-cell exhaustion via indirect presentation」Blood 2003, 102: 1920 - 1926; Frleta, D., Lin, J.T., Quezada, S.A., Wade, T.K., Barth, R.J., Noelle, R.J. and Wade, W.F., 「Distinctive maturation of in vitro versus in vivo anti-CD40 mAb-matured dendritic cells in mice」J Immunother 2003, 26: 72 - 84; Quezada, S., Eckert, M., Schned, A., Noelle, R.J. and Burns, C., 「Distinct mechanisms of action of anti-CD154 in early versus late treatment of murine lupus nephritis」Arth Rheum. 2003; Elster, E.A., Xu, H., Tadaki, D.K., Montgomery, S., Burkly, L.C., Berning, J.D., Baumgartner, R.E., Cruzata, F., Marx, R., Harlan, D.M. and Kirk, A.D., 「Treatment with the humanized CD154-specific monoclonal antibody, hu5C8, prevents acute rejection of primary skin allografts in nonhuman primates, Transplantation」, 2001, 72: 1473 - 1478), pancreas (Benda, B., Ljunggren, H.G., Peach, R., Sandberg, J.O. and Korsgren, O., 「Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatment in CD40 ligand-deficient mice」Cell Transplantation 2002, 11: 715 - 720), bone marrow (Wekerle, T.and Sykes, M., "Mixed chimerism and transplantation tolerance," Annual Review of Medicine 2001, 52:353-37019, and various other transplant organs (Camirand, G., Caron, NJ, Turgeon, NA, Rossini, AA and Tremblay, JP, "Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation," Transplantation 2002, 73:453-461; Tung, TH, Mackinnon, SE and. Mohanakumar, T., "Long-term limb allograft survival using anti-CD154 antibody in a murine model," Transplantation, 2003, 75:644-650). Furthermore, α-human CD154 in NHPs has been shown to induce long-term tolerance to allogeneic skin grafts.

[0083] Overview of the methods of the invention for producing antibodies of the invention Exemplary Methods for the Synthesis of Antibodies of the Invention Hamster alpha mouse CD154 or IDEC-131 or variants disclosed herein V H and V L DNA encoding these was cloned and fused to DNA encoding the human γ1 CH1, CH2, and CH3 regions or to the IgG1 variants disclosed herein. The nucleotide sequences were confirmed using a Megabace™ sequence analyzer. A plasmid expression vector, pEE12, containing both the heavy and light chains of each of the MR1 variants was transfected into NSO cells, and the products were purified by protein A chromatography.

[0084] Binding of the antibody of the present invention to CD154 The binding activity of CD154 antibody mutants was compared by their binding to CHO cells transfected with mouse CD154. CD154-expressing CHO cells were incubated with biotin-labeled αCD154 in the presence of unlabeled αCD154 heavy chain mutants or isotype-matched antibodies for 1 hour at 4°C. Binding of biotinylated MR1 was detected using a streptavidin-conjugated fluorescent dye and flow cytometry was performed. The percentage of inhibition by the mutants was determined by recording the decrease in mean fluorescence intensity of MR1-stained cells.

[0085] Antibody Half-Life Using ELISA ELISA or Biacore can be used to determine the in vivo half-life of the alpha human IgG1 variant. Serum concentrations of hIgG1 may be determined at a set time, for example, one month after administration.

[0086] Binding of mutants to FcR Binding of mutant IgG1 mAbs to FcRs may be determined by solid-phase assays. Briefly, Maxisorb ELISA plates can be coated with mouse or human FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA (R&D Systems). Biotinylated versions of the mutants can be made. Binding can be determined by colorimetric detection using enzyme-linked avidin. The reduction in binding is determined for the mutants compared to the WT molecule.

[0087] Binding of αCD154 mAb to human C1q This can be accomplished using methods known or described herein. Purified human C1q can be titrated into wells where the IgG1 variants of MR1 or IDEC-131 have been absorbed onto a Maxisorb ELISA plate. Bound C1q is detected using HRP-chicken anti-C1q. All mutants were analyzed as described (Ferrant, JL, Benjamin, CD, Cutler, AH, Kalled, SL, Hsu, YM, Garber, EA, Hess, DM, Shapiro, RI, Kenyon, NS, Harlan, DM, Kirk, AD, Burkly, LC, and Taylor, FR, "The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge," Int Immunol 2004;16:1583-1594; and Taylor, PA, Lees, CJ, Wilson, JM, Ehrhardt, MJ, Campbell, MT, Noelle, RJ, and Blazar, BR, "Combined effects of calcineurin inhibitors or sirolimus with anti-CD40L mAb on alloengraftment under nonmyeloablative conditions," Blood 2002; 100:3400-3407) compared to C1q binding to WT IgG1 MR1.

[0088] Induction of tolerance using mutant αCD154 mAb The ability of anti-CD154 antibodies to induce tolerance or prolonged immunosuppression can be determined by known and specifically described methods herein. MR1, a hamster anti-mouse CD154 produced in our laboratory, has been shown by the inventors to induce tolerance or prolonged immunosuppression (Quezada, SA, Fuller, B., Jarvinen, L.Z., Gonzalez, M., Blazar, B.R., Rudensky, A.Y., Strom, T.B. and Noelle, R.J., "Mechanisms of “donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation”, Blood 2003, 102:1920-1926; Quezada, S.A., Bennett, K., Blazar, B.R., Rudensky, AY, Sakaguchi, S. and Noelle, R.J., Analysis of the underlying cellular mechanisms of anti-CD154-induced graft tolerance: the interplay. of clonal energy and immune regulation,” J Immunol 2005;175:771-779; Rossini, AA, Parker, DC, Phillips, NE, Durie, FH, Noelle, RJ, Mordes, JP, and Greiner, DL, “Induction of immunological tolerance to islet allografts,” Cell Transplant 1996;5:49-52) routinely induce long-lived transplantation tolerance.

[0089] Tolerance is induced by coadministration of alloantigen (in the form of donor splenocytes) and αCD154. A humanized IgG1 form of MR1 has also been shown to induce graft tolerance (24); therefore, the WT γ1 mutant will serve as a positive control for tolerance induction. A mutated version of MR1 that has lost the ability to bind complement will be tested for its ability to induce graft tolerance.

[0090] Skin grafting is performed as a modified version of the technique used by Markees et al. (12). Briefly, age-matched male CB6F1 mice are used as donors of both splenocytes (DST) and skin grafts. Recipient C57BL / 6 mice are transplanted with 5 × 10 spleen cells in 500 μL of Hank's balanced salt solution on days −3, −5, and −7. 7 Mice are injected intravenously with or without DST cells via tail vein injection (intravenously) and intraperitoneally with 500 μg of wild-type or mutant αCD154 or control immunoglobulin, hamster or human (HIgG1), in phosphate-buffered saline (PBS). Mice are treated with the appropriate antibody (250 μg / injection) three times per week for the duration of the experiment. On day 0, recipient mice are anesthetized with 50 μg each of ketamine and xylazine per gram of body weight intraperitoneally (15 mg / mL in PBS), and CB6F1 skin grafts are prepared using established methods. Rejection is defined as the day when less than 20% of the skin graft remains. Animals are evaluated for skin graft rejection for 100 days. Additionally, for each tolerance group, skin grafts are harvested on day 100 and evaluated by histochemistry for leukocyte infiltrates and scored based on the measured cell count / area. Finally, as previously published in this system of humoral immunity using mutant αCD154 mAb (Markees, TG, Phillips, NE, Noelle, RJ, Shultz, LD, Mordes, JP, Greiner, DL and Rossini, AA, "Prolonged survival of mouse skin allografts in recipients"). ients treated with donor splenocytes and Transplantation 1997, pp. 64:329-335. Markees, T., Phillips, N., Gordon, E., Noelle, RJ, Mordes, JP, Greiner, DLand Rossini, AA, "Improved skin allograft tolerance induced by treatment with donor splenocytes and an extended (See Markees, TG, Appel, MC, Noelle, RJ, Mordes, JP, Greiner, DL, and Rossini, AA, "Tolerance to islet xenografts induced by dual manipulation of antigen presentation and co-stimulation," Transplantation Proceedings 1996, 28:814-815). To ensure that the induced tolerance is antigen-specific, a third-party graft (H-2K skin) is transplanted into the tolerized mice (selection group).

[0091] In addition to measuring the effects of complement and FcR mutations on tolerance, the effect of antibody treatment on the generation of primary and secondary humoral immune responses will also be assayed as previously described. Briefly, mice (4 / group) will be immunized with chicken ovalbumin (200 μg / mouse) in CFA and treated with the MR1 mutant (200 μg / mouse x 3 times / week). On days 7, 14, and 21, IgM and IgG anti-OVA will be measured by standardized anti-OVA ELISA, and serum concentrations of anti-OVA will be quantified.

[0092] Toxicity studies using the αCD154 mAb of the present invention The thrombogenic activity of αCD154 has been demonstrated in a mouse model using mice expressing human FcγRIIA. This model parallels toxicity findings in NHPs using both intact and aglycosylated forms of anti-human CD154. Briefly, mice are injected with preformed immunoconjugates (IC) of sCD154 (R&D Systems) and each variant of αCD154 (138 μg mAb and 50 μg Ag, approximately 500 nM IC at a 1:3 (mAb / Ag) stoichiometry). Following injection, if the mixture is thrombolytic, mice will exhibit prolonged disorientation, shallow breathing, and impaired mobility. Mice exhibiting this activity are expected to have significantly reduced platelet counts. After 60 minutes, lungs are harvested, fixed in formalin, sectioned, and stained with H&E. Lung sections from mice will be evaluated for evidence of thrombosis (measured by intravascular thrombus), and the number of thrombi / section will be counted. Ten sections will be counted per mouse, and the total number of thrombi will be compared across all groups treated with different variants of IgG1 MR1. Additionally, total platelet counts (harvested by cardiac puncture at the time of euthanasia) will be assessed by flow cytometry and are expected to be reduced by 80% with the use of a thrombogenic antibody. These findings will determine which of the MR1 variants (FcR binding (N325L, K326V, E269R)) are thrombogenic and whether altering FcR binding alters this activity.

[0093] Blocking the development of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE) Female C57BL / 6 mice, 5-8 weeks of age, are immunized subcutaneously with 200 μg of MOG35-55 peptide emulsified in CFA supplemented with 5 mg / ml Mycobacterium tuberculosis. Mice receive intraperitoneal injections with 250 ng of pertussis toxin at the time of immunization and 48 hours later. Seven days later, mice receive an identical booster with MOG / CFA without pertussis toxin. Clinical disease usually begins between days 16 and 20 after immunization. Mice are maintained for the duration of the experiment (50 days). R1 mutant, human IgG (as a control for the mutant), hamster Ig (as a control for MR1) or hamster MR1 (200 μg / mouse 3× / week), respectively.

[0094] Clinical evaluation. Mice are scored four times per week as follows: 0, no detectable signs of EAE; 0.5, limp distal tail; 1, completely limp tail; 1.5, limp tail and hind limb weakness; 2, unilateral partial hind limb paralysis; 2.5, bilateral partial hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete hind limb paralysis and unilateral forelimb paralysis; 4, complete paralysis of both forelimbs and hind limbs; and 5, death. Mice with a score greater than 4 but less than 5 are euthanized.

[0095] Determination of toxicity Desired antibodies according to the present invention exhibit significantly reduced or no toxicity in the disclosed animal thrombosis models. Using the above models, it is demonstrated that IgG1 anti-CD154 antibodies whose heavy chain constant regions contain E269R and K322A mutations (according to the Kabat numbering system) do not induce platelet aggregation.

[0096] Efficacy assessment Efficacy (tolerance induction) may be assessed in the skin graft tolerance model described above.

[0097] Pharmaceutical and Diagnostic Uses of Anti-hCD154 Antibodies of the Invention In one embodiment of the present invention, an anti-CD154 antibody or a pharmaceutical composition comprising the antibody can inhibit an immune response in a subject, and the antibody of the present invention or the pharmaceutical composition of the present invention is administered to a subject in an effective inhibitory amount.

[0098] In certain embodiments, an "effective inhibitory amount" of an anti-CD154 antibody, or a pharmaceutical composition comprising the antibody, is any amount that is effective to inhibit CD154-CD40 interaction in a subject to which it is administered. Methods for determining an "inhibitory amount" are well known to those of skill in the art and depend on factors including, but not limited to, the type of subject involved, the size and age of the subject, and the pharmacokinetic properties of the particular therapeutic agent being delivered.

[0099] In another specific embodiment of the invention, the anti-CD154 antibody of the invention, or a pharmaceutical composition comprising said antibody, is capable of inhibiting an immune response by inhibiting CD154-CD40 interaction.

[0100] In one embodiment of the present invention, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can inhibit inflammation. For purposes of the present invention, an inflammatory response is characterized by redness, swelling, heat, and pain resulting from capillary dilation resulting from edema and migration of phagocytic leukocytes. Some examples of inflammatory responses include arthritis, contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases. Idiopathic inflammatory diseases include, for example, psoriasis and lupus (e.g., systemic lupus erythematosus (SLE), drug-induced lupus erythematosus, and lupus nephritis). See, for example, Gallin 1989, Fundamental Immunology, Chapter 26, Raven Press, 2d Ed., pp. 721-733, New York. The invention provides methods for treating or preventing symptoms of systemic lupus erythematosus (SLE) in an individual, the method comprising administering to the individual an anti-CD154 antibody of the invention in an amount effective to treat or prevent the symptoms of SLE.

[0101] Some examples of arthritis include: rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis associated with Lyme disease, and inflammatory osteoarthritis. Some examples of idiopathic inflammatory diseases include: psoriasis and In one embodiment of the present invention, the anti-CD154 antibody, or a pharmaceutical composition comprising the antibody, is capable of inhibiting rejection of a transplanted organ by a subject.

[0102] In a more specific embodiment of the invention, an anti-CD154 antibody of the invention, or a pharmaceutical composition comprising said antibody, is capable of inhibiting rejection by a subject of a transplanted heart, kidney, liver, skin, pancreatic islet cells or bone marrow.

[0103] In one embodiment of the present invention, the anti-CD154 antibody of the invention, or a pharmaceutical composition comprising said antibody, is capable of inhibiting graft-versus-host disease in a subject.

[0104] In one embodiment of the present invention, an anti-CD154 antibody of the invention, or a pharmaceutical composition comprising the antibody, can inhibit an allergic response, eg, hay fever or allergy to penicillin or other drugs, in a subject.

[0105] In one embodiment, the subject anti-CD154 antibodies, including INX021, are useful for reducing or preventing anti-drug reactions, particularly to other drugs such as biopharmaceuticals and therapeutic antibodies, fusion proteins, hormones, growth factors, enzymes, peptides, antibiotics, antivirals, and the like. This may allow these drugs to be administered for longer periods, potentially increasing the number of patients who respond to the drug and / or promoting efficacy in drug-naive patients. Examples are known in the art and include Humira, Enbrel, Rituxan, Xolair, Herceptin, and Avastin.

[0106] Other examples of therapeutic antibodies against which the subject anti-CD154 antibodies can be used to prevent or reduce anti-drug responses include: 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Afutuzumab, Alacizumab pego, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab Mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab (= Tocilizumab), Atorolimumab, Avelumab, Bapineuzumab, Bacilizumab Riximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab Mertansinemertansine, bleselumab, blinatumomab, brontuvetmab, blosozumab, bococizumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontuzumab, brontictuzumab, calibarizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide , Carlumab, Carotuximab, Catumaxomab, CBR96-doxorubicin immunoconjugate, Cedelizumab, Cergutuzumab amunaleukin, Certolizumab begol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cizutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan, Codrituzumab, Coltuximab Ravtansine, Conatumumab, Concizumab, Crenezumab, Crotedumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dapirolizumab pegol pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, derlotuximab biotin, detumomab, dinutuximab, diridavumab, domagrozumab, dorlimomabaritox, Drozitumab, Durigotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab ab), Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Ensituximab, Epitumomab Cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, FBTA05, Felvizumab ), Fezakinumab, Fivatuzumab, Ficlatuzumab, Figitumumab, Filibumarb, Frivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fresolimumab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lanadelumab, Laprituximab Emtansine Emtansine, Lebrikizumab, Lendalizumab, Lenzilumab, Lerdelimumab, Lexatumumab, Ribivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetlaxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegolpegol, lumiliximab, lumretuzumab, mapatuzumab, margetuximab, maslimomab, mavrilimumab, matuzumab, mepolizumab, methimumab, milatuzumab, minretumomab, mirvetuximab soravtansine, mitumomab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narnatumab, natalizumab, navicixizumab, navivumab, nevacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentane merpentan, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratuzumab, olokizumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pancomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab lizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pinatuzumab vedotin vedotin, pintumomab, placulumab, plozalizumab, pogalizumab, polatuzumab vedotin Vedotin, Ponezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Rituximab, Rivabazumab pegol pegol, robatumumab, roledumab, romosozumab, rontalizumab, rovalpituzumab tesirine, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sapelizumab, sarilumab, satumomab pendetide pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, SGN-CD19A, SGN-CD33A, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotinvedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, timolumab, tisotumab vedotin, TNX-650, atlizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urutoxazumab, Ustekinumab, Vadastuximab talirine, Bundeltuzumab vedotin These include: ndortuzumab Vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Bepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Xentuzumab, Salutumumab, Zanolimumab, Zatuximab, Ziralimumab, and Zolimomab aritox.

[0107] In one embodiment of the present invention, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can inhibit an autoimmune response in a subject suffering from an autoimmune disease. In some embodiments, the autoimmune response is associated with or results from a condition selected from the group consisting of rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes mellitus, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, drug-induced autoimmune disease, or drug-induced lupus. In certain embodiments, the autoimmune response is associated with or results from systemic lupus erythematosus.

[0108] In one embodiment of the present invention, the anti-CD154 antibodies of the present invention, or pharmaceutical compositions comprising said antibodies, are capable of inhibiting an autoimmune response resulting from an infection in a subject suffering from an autoimmune response.

[0109] In one embodiment of the present invention, the anti-CD154 antibodies of the present invention, or pharmaceutical compositions comprising the antibodies, are capable of inhibiting an autoimmune response in a subject suffering from an autoimmune response resulting from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infection, syphilis, or tuberculosis.

[0110] In one embodiment of the present invention, the anti-CD154 antibody of the invention, or a pharmaceutical composition comprising said antibody, is capable of inhibiting fibrosis in a subject.

[0111] Some examples of fibrosis include: pulmonary fibrosis and fibrotic diseases. Some examples of pulmonary fibrosis include: pulmonary fibrosis secondary to adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis. Some examples of fibrotic diseases include: hepatitis C; hepatitis B; cirrhosis; cirrhosis of the liver secondary to toxic injury; cirrhosis of the liver secondary to drugs; cirrhosis of the liver secondary to viral infection; and cirrhosis of the liver secondary to autoimmune disease.

[0112] In one embodiment of the present invention, the anti-CD154 antibodies of the invention, or pharmaceutical compositions comprising the antibodies, are capable of inhibiting gastrointestinal diseases, some examples of which include: esophageal hypoperistalsis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), gastritis, collagenous colitis (including lymphocytic colitis and microscopic colitis), celiac disease (gluten enteropathy, celiac sprue, or gluten intolerance), and scleroderma.

[0113] In one embodiment of the present invention, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can inhibit vascular diseases. Some examples of vascular diseases include atherosclerosis, renal artery disease, lymphedema, ischemic injury, and reperfusion injury. Also included are collagen vascular / immune complex diseases such as systemic lupus erythematosus or cryoglobulinemia.

[0114] In one embodiment of the present invention, an anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can inhibit the proliferation of T cell tumor cells in a subject suffering from a T cell cancer, e.g., T cell leukemia or lymphoma. Such an anti-CD154 antibody, or a pharmaceutical composition comprising the antibody, can be administered in an amount effective to inhibit the proliferation of T cell tumor cells in the subject. It can be administered to a subject.

[0115] In one embodiment of the present invention, an anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can inhibit viral infection of a subject's T cells by human T-cell leukemia virus type 1 (HTLVI). Such an anti-CD154 antibody, or a pharmaceutical composition comprising the antibody, can be administered to a subject in an amount effective to inhibit viral infection.

[0116] In one embodiment of the present invention, an anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can be used to image tumor or neoplastic cells in a subject that express the CD154 protein to which the antibody of the present invention specifically binds. A method for imaging tumor or neoplastic cells in a subject comprises the steps of: administering to the subject an effective amount of an anti-CD154 antibody of the present invention, or a composition comprising the antibody, under conditions that allow the formation of a complex between the antibody and the protein on the surface of the tumor or neoplastic cells; and imaging any antibody / protein complexes formed, thereby imaging any tumor or neoplastic cells in the subject.

[0117] In one embodiment of the present invention, an anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, can detect the presence of tumor or neoplastic cells in a subject that express the CD154 protein to which the antibody of the present invention specifically binds. One such method for detecting the presence of tumor or neoplastic cells in a subject comprises the steps of: administering to the subject an effective amount of an anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising the antibody, under conditions that allow for the formation of a complex between the antibody and the protein; removing any unbound imaging agent from the subject; and detecting the presence of any antibody / protein complexes formed, wherein the presence of such complexes indicates the presence of tumor or neoplastic cells in the subject.

[0118] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising a CD154 binding protein, eg, an anti-CD154 antibody, described in the present invention.

[0119] In one embodiment of the invention, the pharmaceutical composition comprises at least one anti-CD154 antibody of the invention.

[0120] The anti-CD154 antibodies of the invention or pharmaceutical compositions comprising said antibodies do not cause thrombosis, including thromboembolic events, and are therefore well suited for human therapy.

[0121] Such pharmaceutical compositions may further comprise any one or more of a pharmaceutically acceptable carrier, adjuvant, delivery vehicle, buffer, and / or stabilizer. Exemplary techniques for formulating and administering the antibodies of the invention can be found, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0122] In more specific embodiments of the present invention, the pharmaceutically acceptable carrier is phosphate buffered saline, saline, water, citric acid / sucrose / Tween formulations and emulsions, such as oil / water emulsions.

[0123] In one embodiment of the present invention, the pharmaceutical composition can be delivered in a microencapsulation device to reduce or prevent a host immune response to the composition. A binding agent, such as an antibody or antibody fragment of the invention, can be delivered microencapsulated in a membrane, such as a liposome, or other encapsulating or immunoprotective delivery vehicle.

[0124] In one embodiment of the present invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, a sterile injectable aqueous or oleaginous suspension, which may be formulated according to techniques known in the art using suitable dispersing, wetting agents, and suspending agents.

[0125] In one embodiment of the present invention, the pharmaceutical composition may be delivered orally, topically, or intravenously. When administered systemically, the therapeutic composition should be sterile, substantially pyrogen-free, and a parenterally acceptable solution with due regard to pH, isotonicity, and stability. For example, the pharmaceutical preparation is substantially pyrogen-free so as to be suitable for administration as a human therapeutic. These conditions are known to those skilled in the art.

[0126] In a more specific embodiment of the present invention, for oral administration, the pharmaceutical composition is formulated into a suitable capsule, tablet, aqueous suspension, or solution. Solid formulations of the composition for oral administration can contain suitable carriers or excipients such as cornstarch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid. Disintegrants that can be used include, but are not limited to, microcrystalline cellulose, cornstarch, sodium starch glycolate, and alginic acid. Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone™), hydroxypropylmethylcellulose, sucrose, starch, and ethylcellulose. Lubricants that can be used include magnesium stearate, stearic acid, silicone oil, talc, waxes, oils, and colloidal silica.

[0127] In a more specific embodiment of the present invention, for topical application, the pharmaceutical composition may be formulated in a suitable ointment. Some examples of formulations of compositions for topical use include: drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various supports and vehicles.

[0128] In one embodiment of the present invention, topical semi-solid ointment formulations typically contain a concentration of about 1 to 20%, for example 5 to 10%, of the active ingredient in a carrier such as a pharmaceutical cream base.

[0129] In one embodiment of the present invention, pharmaceutical compositions for inhalation and transdermal compositions can also be readily prepared. Therapeutic compositions can be administered, for example, through the nose or lungs as a liquid or powder aerosol (lyophilized).

[0130] In one embodiment of the present invention, liquid formulations of pharmaceutical compositions for oral administration prepared with water or other aqueous solvents can contain various suspending agents such as methylcellulose, alginic acid, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. Liquid formulations of pharmaceutical compositions of the present invention can also include solutions, emulsions, syrups, and elixirs containing wetting agents, sweeteners, and coloring and flavoring agents together with the active compound(s). Various liquid and powder formulations of pharmaceutical compositions can be prepared by conventional methods for inhalation into the lungs of the mammal to be treated.

[0131] In one embodiment of the present invention, liquid formulations of injectable pharmaceutical compositions can include a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols, i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like. In some embodiments, the composition includes a citrate / sucrose / Tween carrier. For intravenous injection, a water-soluble version of the composition can be administered by infusion, whereby a pharmaceutical formulation containing an antifungal agent and a physiologically acceptable excipient is infused. The physiologically acceptable excipient Vehicles may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Suitable insoluble compositions may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long-chain fatty acid, e.g., ethyl oleate.

[0132] In one embodiment of the invention, the pharmaceutical composition comprises about 0.1 to 90% by weight (such as 1 to 20% or 1 to 10%) of an anti-CD154 antibody of the invention in a pharmaceutically acceptable carrier.

[0133] In one embodiment of the present invention, the optimal percentage of the anti-CD154 antibody of the present invention in each pharmaceutical composition varies according to the formulation itself and the desired therapeutic effect in a particular pathology and associated treatment regimen. Pharmaceutical formulations are well established in the art. Pharmaceutical compositions can be administered to subjects using conventional methods known to those skilled in the pharmaceutical arts.

[0134] Thus, the pharmaceutical compositions of the present invention relate to sustained-release formulations. Sustained-release, or controlled-release or sustained-release, refers to drug formulations that release active drugs, such as polypeptide or antibody drugs, over a period of time following administration to a subject. Sustained release of polypeptide drugs, which can occur over a range of desired periods (e.g., minutes, hours, days, weeks, or longer, depending on the drug formulation), differs from standard formulations in which substantially the entire dosage unit is available for immediate absorption or distribution through the bloodstream. In certain embodiments, sustained-release formulations may provide a level of circulating drug from a single administration that is sustained for, for example, 8 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, or even, for example, one week, two weeks or more, or, for example, one month or more. Sustained-release compositions may comprise the anti-CD154 antibodies of the present invention.

[0135] In some embodiments of the present invention, the pharmaceutical composition further comprises another immunosuppressive or immunomodulatory compound. For example, such an immunosuppressive or immunomodulatory compound may be one of the following: an agent that interferes with T cell costimulatory signaling via CD28; an agent that interferes with calcineurin signaling, a corticosteroid, an antiproliferative agent, and an antibody that specifically binds to a protein expressed on the surface of immune cells, including, but not limited to, CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LTβ, and VLA-4.

[0136] In some embodiments of the invention, the immunosuppressive or immunomodulatory compound is tacrolimus, sirolimus, mycophenolate mofetil or its active form mycophenolic acid, mizoribine, deoxyspergualin, brequinar sodium, leflunomide, rapamycin, or azaspirane.

[0137] In other embodiments of the invention, an antibody of the invention or a pharmaceutical composition comprising the antibody can be included in a container, package, or dispenser alone, or as part of a kit together with a label and instructions for administration.

[0138] Administration and Delivery Routes The anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention can be administered to a subject in any medically acceptable manner. For purposes of the present invention, "administration" means any of the standard methods of administering an antibody, antibody fragment, or pharmaceutical composition known to those of skill in the art, and are not intended to be limiting of the scope of the present specification. The present invention should not be limited to the examples provided in this document.

[0139] In some embodiments of the invention, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject by injection intravenously, subcutaneously, intraperitoneally, intramuscularly, intramedullary, intraventricularly, intraepidurally, intraarterially, intravascularly, intraarticularly, intrasynovially, intrasternally, intrathecally, intrahepatically, intraspinally, intratumorally, intracranially; by enteral, intrapulmonary, transmucosal, intrauterine or sublingual routes of administration, or locally, e.g., at a site of inflammation or tumor growth.

[0140] In some embodiments of the invention, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered orally or nasally, or by inhaled, ocular, rectal, or topical routes.

[0141] In more specific embodiments, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered orally to a subject in the form of a capsule, tablet, aqueous suspension, or solution.

[0142] In a more specific embodiment, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered topically to a subject by application of a cream, ointment, or the like.

[0143] In other embodiments of the invention, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered by inhalation through the use of a nebulizer, a dry powder inhaler, or a metered dose inhaler.

[0144] In further embodiments of the present invention, the anti-CD154 antibodies of the present invention and pharmaceutical compositions of the present invention may be administered to a subject by sustained release administration, by means such as depot injection of an erodible implant applied directly during surgery, or by implantation of an infusion pump or biocompatible sustained release implant into the subject.

[0145] In more specific embodiments, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject by an injectable depot route of administration, such as by using 1, 3, or 6 month depot injectable or biodegradable materials and methods.

[0146] In more specific embodiments, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject by applying a transdermal patch containing the antibody, antibody derivative, or pharmaceutical composition to the subject's skin and leaving the patch in contact with the subject's skin, generally for 1 to 5 hours per patch.

[0147] In other embodiments of the invention, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject at any dose per body weight and at any administration frequency that is medically acceptable, including a range of about 0.01 to 200 mg / kg of subject body weight.

[0148] The subject anti-CD154 antibodies are administered to a subject in an amount effective to induce the desired immune effect, i.e., suppression of T or B cell immunity, or to prevent, treat, or ameliorate the symptoms of a disease in which suppression of T or B cell immunity is therapeutically desirable. As noted, these conditions include certain autoimmune, inflammatory, and allergic indications.

[0149] The total administered dose of antibody may alternatively contain 0.01, 0.1, 1, 5, 10, 15, 20, 25, 50, 100 or more mg, or any intermediate value between the aforementioned mg values. In a preferred embodiment of the present invention, the anti-CD154 antibodies or binding fragments thereof, and combinations of said antibody fragments described herein may be administered to a recipient subject at a frequency of once every 16 weeks or less, once every 8 weeks or less, or once every 26 weeks or less, such as once every 4 weeks, monthly, every other week or less.

[0150] In any of the methods of using the antibodies or pharmaceutical compositions of the invention, the antibodies or pharmaceutical compositions may be administered to a subject in single or multiple doses daily, every 2, 3, 4, 5, or 6 days, weekly, monthly, or any fraction or multiple thereof, and may also be administered repeatedly to a subject at intervals ranging from daily to bimonthly as determined by one of skill in the art.

[0151] In any of the methods of using the antibodies or pharmaceutical compositions of the invention, the antibodies or pharmaceutical compositions comprising the antibodies may be administered to a subject in need thereof at intervals as long as medically necessary, ranging from a few days or weeks to the subject's lifetime. In further embodiments, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject repeatedly at intervals ranging from daily to every other month.

[0152] In one embodiment of the present invention, the anti-CD154 antibodies of the present invention and pharmaceutical compositions of the present invention can be administered in multiple doses per day, as needed, to achieve the desired total daily dose. The effectiveness of the treatment method can be assessed by monitoring the subject for known signs or symptoms of the disorder.

[0153] In all embodiments of the present invention, the dosage and rate of administration of the anti-CD154 antibodies of the present invention and pharmaceutical compositions of the present invention that will be effective to produce the desired effect will depend on various factors, such as the nature of the disease being treated, the size and age of the subject, the goals of the treatment, the particular pharmaceutical composition used, the pharmacokinetics of the active agent, and the judgment of the treating physician.

[0154] It is understood that effective dosages may depend on recipient subject characteristics such as, for example, age, sex, pregnancy status, body mass index, lean body mass, the condition(s) to which the composition is given, other health conditions of the recipient subject that may affect metabolism or tolerance of the composition, the recipient subject's level of IL-6, and tolerance to the composition (e.g., resulting from the patient developing antibodies to the composition). Those skilled in the art will be able to determine effective dosages and frequency of administration through routine experimentation guided, for example, by the disclosure herein and the teachings of Goodman, LS, Gilman, A., Brunton, LL, Lazo, JS, & Parker, KL (2006), Goodman & Gilman's the pharmacological basis of therapeutics. New York: McGraw-Hill; Howland, RD, Mycek, MJ, Harvey, RA, Champe, PC, & Mycek, MJ (2006). Pharmacology. Lippincott's illustrated reviews. Philadelphia: Lippincott Williams & Wilkins; and Golan, DE (2008). Principles of pharmacology: the pathophysiologic basis of drug therapy. Philadelphia, Pa., [etc.]: Lippincott Williams & Wilkins.

[0155] Thus, the anti-CD154 antibodies of the present invention and pharmaceutical compositions of the present invention are administered in an amount effective to achieve their intended purpose. A therapeutically effective amount may refer to an amount of antibody effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of a subject undergoing treatment. A therapeutically effective amount may vary by varying the dosage and schedule of administration of the subject antibodies. This may be achieved by:

[0156] The anti-CD154 antibodies of the present invention and pharmaceutical compositions of the present invention may be administered as a single dose for certain indications, such as preventing an immune response to an antigen to which a subject is exposed for a short period of time, such as a foreign antigen administered in a one-day treatment. Examples of such treatments would include coadministering the antibody fragments of the present invention with a therapeutic agent, such as an antigenic pharmaceutical, an allergen or blood product, or a gene therapy vector. For indications in which an antigen is chronically present, such as controlling an immune response to transplanted tissue or a chronically administered antigenic pharmaceutical, the antibody fragments or pharmaceutical compositions of the present invention are administered at intervals as long as medically necessary, ranging from a few days or weeks to the subject's lifetime.

[0157] In any of the methods described herein, the antibody or pharmaceutical composition may be administered to the subject together with a second agent. In certain embodiments, the agent is a therapeutic agent, such as, for example, an immunomodulatory or immunosuppressive agent. The immunomodulatory or immunosuppressive agent may be one of the following: (a) Agents that interfere with CD28-mediated T cell costimulatory signaling; (b) drugs that interfere with calcineurin signaling; (c) corticosteroids, (d) antiproliferative agents; and (e) antibodies that specifically bind to proteins expressed on the surface of immune cells, including, but not limited to, CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LTβ, and VLA-4 Any of the above may be used.

[0158] The immunosuppressive or immunomodulatory compound may be, for example, tacrolimus, sirolimus, mycophenolate mofetil, mizoribine, deoxyspergualin, brequinar sodium, leflunomide, rapamycin, or azaspirane. The antibody and the secondary agent may be administered simultaneously or sequentially. In some cases, it may be advantageous to administer one or more nucleic acids of the present invention to a subject in need thereof. The therapeutic and diagnostic methods of the present invention, comprising administering at least one nucleic acid of the present invention according to known methods, are within the scope of the present invention.

[0159] In one embodiment of the present invention, the subject(s) that can be treated by the above-mentioned method are animals.Preferably, animals are mammals.The examples of mammals that can be treated include but are not limited to humans, non-human primates, rodents (including rats, mice, hamsters and guinea pigs), cows, horses, sheep, goats, pigs, dogs and cats.Preferably, mammals are humans.

[0160] The present invention may be better understood based on the following examples, although those skilled in the art will readily recognize that the specific methods and results discussed are only illustrative of the invention described herein.

[0161] The following examples are put forth so as to fully disclose and describe to those of ordinary skill in the art how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, concentrations, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0162] Example 1: Higher affinity (lower K) for human CD154 D Design of antibody mutants with In this application, we describe a novel humanized anti-human CD154 IgG1 antibody having the sequence contained in Figures 1A-C intended for use in human therapy, particularly in therapeutic indications where suppression of T and B cell immunity is therapeutically desirable. As previously mentioned, this antibody was derived from IDEC-131 by the combined use of affinity maturation and mutagenesis methods and by modification of the IgG1 Fc region of IDEC-131 to remove FcγRII and FcγRIIII and C1q (complement binding).

[0163] In particular, various combinations of CDR residues in both the heavy and light variable regions of IDEC-131 were mutated with the aim of obtaining variants of IDEC-131 with improved binding affinity to human CD154. These experiments yielded numerous variants. The overwhelming majority of these variants did not bind to human CD154 better than IDEC-131, but rather had the same or worse binding affinity to human CD154 (results not shown).

[0164] These experiments resulted in 11 humanized variants containing variable light and heavy chain sequences, which were identified and are shown in Figures 2A-2C and 3. The binding affinity of each of these variants to human CD154 was then screened by Biacore. Biacore assays were performed as follows:

[0165] BIACORE assay Equipment:Biacore3000 Assay buffer - 10 nM HEPES buffer (pH 7.4), 150 nM NaCl, 3 mM EDTA, 0.05% P20 (polyoxyethylene sorbitan) Renaturation buffer - 10 mM glycine HCl (pH 1.75) Conjugation buffer - 10 nM sodium acetate buffer (pH 5) Flow rate - 5 μl / min for ligand capture, 30 μl / min for kinetic analysis.

[0166] procedure Binding experiments were performed on a Biacore300 at 25°C. Antibodies were directly immobilized on flow cell 2 (1000 RU of combination #23) and flow cell 4 (1000 RU of EWT #131), and CD154 antigen was flowed over the chip. Binding of the antigen to the test antibody was monitored in real time. The observed k on From k for each mutant off and K. d It was decided.

[0167] Scoring analysis was performed using a specific analyte concentration. At the test concentration, binding should be observed even if the ligand binding affinity is relatively weak. Full kinetics may be performed by known methods. For molecular interactions with rapid off-rates, steady-state kinetics may be used to calculate the K D A value may be determined.

[0168] Chi-square (χ2) analysis may be performed to determine the accuracy of the analysis. A χ2 within 1-2 is considered significant, and less than 1 is considered highly significant. This screen identified several sequence variants with significantly improved binding affinity, i.e., 4-5 fold compared to IDEC-131. As previously mentioned, this work demonstrates that IDEC-131 has a binding affinity or K of 151 pm. D This result was even more surprising because IDEC-131 was selected as the lead clinical candidate from among several different candidate humanized sequences based on its strong binding affinity for CD154.

[0169] As shown below, the K from IDEC-131 ranges from just 26 pm to 57 pm. D The humanized anti-CD154 antibodies having the following values ​​were obtained. The amino acid sequences of the heavy and light variable sequences of these variants, identified as variant 22, variant 23, variant 24, variant 25, variant 26, variant 27, variant 28, variant 29, variant 30 and variant 31, are contained in Figure 2.

[0170] Table 4 summarizes the different binding affinities of these variants as determined by Biacore.

[0171] [Table 4]

[0172] Example 2: Higher affinity (lower K) for human CD154 for SC administration D Use of antibody variants with As shown in the table above, the mutant selected as lead candidate #21 has a K of 29 pM. D , which is 4.7-fold greater than IDEC-131. This improved affinity is significant because it should enable the antibody to be administered subcutaneously. Previous studies with IDEC-131 have demonstrated human clinical efficacy above 10 mg / kg, or an upper limit of 20 mg / kg for analytical purposes. Based on this, and assuming an average human body weight of approximately 80 kg, the effective dose can be calculated as follows: 10mg / kg×80kg=800mg dose

[0173] However, antibody preparations for use typically do not exceed 100 ng / ml (http: / / www.sciencedirect.com / science / article / pii / S0169409X06000895).

[0174] Furthermore, typical tolerated dose volumes for sq administration range from approximately 0.5 to 5 mL (volumes greater than 3 mL are considered "large"). Therefore, an effective dose of IDEC-131 would require approximately 8 mL (see calculation below). This dose volume is sq .Well above pharmaceutical standards. (10mg / kg x 80kg) / 100mg / ml = 8mL

[0175] In contrast, mutant #21 (INX021) has a K D(i.e., binds with greater affinity to CD154), this variant would require 5 times less drug to achieve the same efficacy. As shown below, this would translate to a dose volume of approximately 1.6 ml, which is well within medical practice for sq injection into patients. (2mg / kg x 80kg) / 100mg / ml = 1.6mL

[0176] Therefore, unlike IDEC-131, INX021 should be well suited for administration via the sq route.

[0177] Example 3: Comparison of the efficacy of anti-CD154 antibodies according to the present invention Figure 4 depicts the results of an experiment detecting inhibition of T cell CD154-driven B cell activation. The results contained in the figure demonstrate improved potency, i.e., greater inhibition of T cell CD154-driven B cell activation, with INX021 compared to IDEC-131.

[0178] Example 4: Ability of antibodies of the invention to induce tolerance Figure 5 shows experimental results demonstrating that anti-mouse CD154 IgG1 antibodies containing the same Fc mutations (E269R and K322A) contained in INX021 can induce tolerance in the skin test described above. Therefore, anti-human CD154 antibodies containing these mutated IgG1 constant regions should induce tolerance and block other effects of CD154 in human patients.

[0179] Example 6: INX021 does not induce platelet aggregation in vitro Figure 6 shows the results of experiments demonstrating that INX021 antibodies containing mutations that reduce or eliminate FcR binding do not induce platelet aggregation in vitro in assays using immune complexes of the subject INX021 antibodies and collagen. These in vitro experiments demonstrated that immune complexes of the FcR mutants over a 16 or 20 minute period did not induce the activation or aggregation of isolated mouse or human platelets. In contrast, the control 5c8 antibody caused platelet aggregation in the same assays.

[0180] Example 7: Anti-CD154 antibodies according to the invention do not induce platelet aggregation in vivo Figures 7A-D show experimental results demonstrating that INX021 does not activate platelets, induce platelet aggregation, or affect (reduce) platelet count in vivo. The experiment in Figure 7A shows that INX021 does not induce thrombotic stress or activate platelets when compared to a positive control. The experiment in Figure 7B shows that INX021 does not affect the number of circulating platelets when compared to a positive control. The experiment in Figure 7C shows that INX021 does not induce the formation of blood clots (emboli) in the lungs when compared to a positive control. Figure 7D summarizes the benefits of INX021 in vivo: INX021 does not reduce platelet count, activate platelets, cause platelet stress, or induce clot formation.

[0181] Example 8: Use of anti-CD154 antibodies according to the invention in an autoimmune model The experiments summarized in Figures 8A-B show that the anti-humanized anti-CD154 IgG1 antibody INX021 containing Fc mutations that reduce or eliminate FcR binding is active in different autoimmune or inflammatory models.

[0182] The experiment in Figure 8A shows that a human CD154 IgG1 antibody containing the Fc mutation in INX021 potently suppresses EAE for approximately 20 days, and the experiment in Figure 8B shows that a humanized anti-CD154 IgG1 antibody containing the Fc mutation in INX021 potently suppresses humoral immunity against myelin oligodendrocyte glycoprotein ("MOG") comparable to the parent antibody.

[0183] Example 9: Testing IDEC-131 variants, including INX021, for their ability to suppress T cell-induced B cell activation The experiments in Figure 9 compare the inhibitory effects of different antibody variants according to the invention, namely variants #21 (INX021) to #31, on T cell-induced B cell activation based on CD86 expression. In these experiments, the inhibitory effects of antibody variants according to the invention were compared in a mixed lymphocyte reaction. The readout was CD86 expression by activated B cells. As shown in the figure, better K D Antibody titers did not necessarily correlate with improved functional efficacy in MLR responses. INX021 had superior immunosuppressive properties compared to other variants.

[0184] Example 10: INX021 Toxicity Study and T Cell-Dependent Antibody Response (TDAR) Measurement To assess its suitability for use in human therapy, the antibody of the invention, INX021, was tested in an 8-week non-GLP toxicology study in rhesus monkeys at the CRL and Harvard facilities. The study design is shown in the table below. The goal of this study was to evaluate the safety and efficacy of INX921 in non-human primates (NHPs).

[0185] [Table 5]

[0186] [Table 6]

[0187] Figures 10A-B compare the effects of INX021 and chimeric 5c8 antibody on the production of IgG and IgM antibodies in a KLH assay, with Figure 10A comparing its effect on IgG and Figure 10B comparing its effect on IgM.

[0188] Figures 11A-B compare the effects of vehicle, INX021, and 5c8 antibody on germinal center scores. Figure 11A shows the germinal center score comparison, and Figure 11B contains histograms comparing the number and degree of cellularity of germinal centers in the spleens and lymph nodes (LNs) of treated animals.

[0189] Figure 12 summarizes the observed lesion frequencies compared with published results using another anti-CD154 antibody (PEGylated αCD154 Fab from Biogen / UCB).

[0190] These results indicate that INX021 should be safe and effective in human therapy.

[0191] Example 11: INX021 suppresses anti-drug antibody (ADA) responses in NHPs During the INX021 toxicity / TDAR study discussed in the previous example, the presence of ADAs was measured in all cohorts. TDAR responses were suppressed in all animals, suggesting that antibody responses to soluble antigens may be prevented in INX021-treated animals. To examine this, ADA responses were measured in rhesus monkeys following 8 weeks of INX021 or 5C8 injections. All but one animal in the lowest dose of INX021 (1 mg / kg) failed to develop any detectable ADAs.

[0192] method: Detection of antibodies against INX021 and ch5C8 in rhesus monkeys (ELISA) Preparation of 2N sulfuric acid (stop solution) For every 1000 mL of 2N sulfuric acid to be prepared, combine 500 mL of 4N sulfuric acid and 500 mL of distilled water and mix well. The expiration date is one year from the date of preparation or the manufacturer's expiration date for each individual ingredient, whichever comes first. The solution is stored at room temperature (17°C to 27°C).

[0193] Preparation of coated 96-well plates In these protocols, the test materials are samples containing rhesus or cynomolgus monkey serum, samples containing positive controls, and / or blanks.

[0194] protocol 1. Prepare a 1.0 pg / mL coating solution with INX021 or ch5C8 using coating buffer (0.2% carbonate-bicarbonate buffer) as the diluent. 2. Add 100 pL of coating solution to each well of a 96-well clear polystyrene plate(s) and cover. 3. Incubate the plate(s) at 2°C to 8°C for 12 to 24 hours. 4. Aspirate (remove) the coating solution from each well of the plate(s), then wash each well 3 x 300 pL with 1 x PBS-T. 5. Add 200 pL of blocker casein to each well of the plate(s) and cover. 6. Incubate the plate for 1 hour ± 5 minutes. 7. Aspirate the blocker casein from each well of the plate(s), then wash each well 3 x 300 pL with 1 x PBS-T. 8. Use the plate(s) immediately after washing (complete plating within 2 hours of completing washing). Date plate preparations are completed.

[0195] Assay Procedure for Detection of Antibodies to INX021 or ch5C8 In these experimental protocols, "test material" refers to serum from rhesus or cynomolgus monkeys. The samples are those containing the ELISA kit, the positive control, and / or the blank. The room / ambient temperature is 17°C to 27°C.

[0196] protocol 1. Add 100 pL of each test material to the appropriate well(s) of the INX021 or ch5C8 coated plate(s) and cover with plate sealant. 2. Use the plate template to record the placement of test materials. 3. Incubate test materials with INX021 or ch5C8 coated plate(s) at room temperature for 1 hour ± 5 minutes.

[0197] Analysis procedure 1. Aspirate the test material from each well of the plate(s), then wash each well of the plate(s) 3 x 300 pL with 1 x PBS-T. 2. Add 100 pL of 0.125 pg / mL INX021-BIOTIN or 1.0 pg / mL ch5C8-BIOTIN to each well of the plate(s) and cover with plate sealant. 3. Incubate the plate(s) at room temperature for 1 hour ± 5 minutes. 4. Aspirate (remove) the INX021-BIOTIN or ch5C8-BIOTIN from each well of the plate(s), then wash each well of the plate(s) 3 x 300 pL with 1 x PBS-T. 5. Add 100 pL of 0.1 pg / mL SA-HRP to each well of the plate(s) and cover. 6. Incubate the plate(s) at room temperature for 30 minutes ± 5 minutes. 7. Aspirate (remove) the SA-HRP from each well of the plate(s), then wash each well of the plate(s) 3 x 300 pL with 1 x PBS-T. 8. Add 100 pL of TMB to each well of the plate(s). 9. Incubate the plate(s) at room temperature for 20 minutes ± 5 minutes. 10. Add 100 pL of 2N sulfuric acid to each well of the plate(s). 11. Read the plate(s) at 450 nm using a microplate spectrophotometer within 30 minutes of analysis.

[0198] Overall data organization For all test materials, unless otherwise stated, calculate the mean A450, standard deviation, and % difference (if n=2) or CV% (if n>3) to assess variance. If % difference is calculated instead of CV%, all CV% acceptance criteria apply to the % difference value. If three or more wells of test material are analyzed, one well may be excluded if the CV% does not pass the outlined criteria. %CV: r standard deviation > mean value x 100 % difference: r value A-value Bv average value x 100

[0199] A Maximum Normed Residual (MNR) analysis is performed to assess variance at the 5% level above the blank TS, removing up to two outlier A450 blank test sample (TS) values. The mean A450 is calculated from all values ​​without removing outliers. Additional wells may be excluded due to technical errors.

[0200] The plate specific cut point (PSCP) is calculated as: global cut point (CP) x blank TS mean A450 (no outliers) = PSCP.

[0201] Overall judgment criteria If the blank TS has more than two outliers, the plate has failed and should be reanalyzed. For MNR analysis, at least five blank TS A450 must remain after concealing outliers and wells masked due to technical error. Calculate the MNR value using the following equation: MNR blank TS value - average blank TS.

[0202] Standard deviation xVn-1 If the MNR value is greater than the critical value, the value is determined to be an outlier. The critical values ​​are defined as: n=12=0.727, n=11=0.745, n=10=0.763, n=9=0.783, n=8=0.804, n=7=0.825, n=6=0.844, and n=5=0.858. All outliers are hidden.

[0203] After removing outliers, the mean A450 of the blocker casein blank and blank TS was measured using LPC. A should be less than 0. The plate specific cut point (PSCP) is: PSCP = CF * Calculated as blank TS global mean (no outliers). Correction factor (CF) is added to the study file. LPC mean A450 must exceed PSCP; if not, LPC at that setting fails.

[0204] The assay buffer blank CV% should be less than 30%. If the assay buffer blank does not pass the criteria listed above, the plate will be evaluated during scientific review to determine if the data can be used.

[0205] For each set of PCs, HPC > MPC > LPC for the average A450. At least two-thirds of the PC levels must meet the criteria, and at least one PC at each PC level must be within the criteria.

[0206] The test sample % difference between duplicate plates should be less than 25%. If a test sample does not pass this criterion, the sample is reanalyzed.

[0207] Preparation of positive control, blank TS and assay buffer blank Positive controls are prepared by spiking surrogate positive control antibodies into 100% rhesus monkey serum (RHS) or cynomolgus monkey serum. The positive controls in 100% matrix are diluted 10-fold with blocker casein to produce the appropriate working concentration and 10% matrix. Each set of PCs is prepared independently of the others and analyzed on its own plate. On the day of analysis, the PCs are analyzed on their own plate.

[0208] Each plate contains two sets of PCs: LPC (50 ng / mL), MPC (250 ng / mL), and HPC (1000 ng / mL). Blank TSs are also prepared at a final concentration of 10% matrix diluted with blocker casein. 12 wells of blank TSs are run on each plate. Blocker casein is run in at least two wells on each plate. All test materials are analyzed in a minimum of two wells unless otherwise stated.

[0209] Screening assays For screening, samples are diluted 1 / 10 in blocker casein and analyzed by the procedure described in Section 7.0. During sample analysis, the mean absorbance value of the study sample is compared to the PSCP, and a mean absorbance value greater than the PSCP is reported as a potential positive, and a mean absorbance value less than the PSCP is reported as a negative.

[0210] result TDAR responses were suppressed in all animals, suggesting that antibody responses to soluble antigens were prevented in INX021-treated animals. To investigate this, ADA responses were measured in rhesus monkeys following 8 weeks of INX021 or 5C8 injections. All but one animal in the lowest dose of INX021 (1 mg / kg) did not develop any detectable ADA. Thus, the antibody INX021 of the present invention can be used to suppress humoral immunity to immunogenic agents, such as biopharmaceuticals, e.g., therapeutic antibodies, fusion proteins, and the like.

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[0212] The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, handbooks, books, or other disclosures) cited in the Background, Detailed Description, and Examples of the present invention is incorporated herein by reference in its entirety.

[0213] These and other changes can be made to the invention in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification and the claims. Therefore, the invention is not limited by the disclosure; instead, the scope of the invention is limited by all of the following claims. This will be determined by the

[0214] The present invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

Claims

1. Use of an anti-human CD154 antibody comprising a variable heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively, in the manufacture of a medicament for use in treating lupus in a human subject in need thereof, wherein the medicament is for subcutaneous administration.

2. The use described in claim 1, wherein the lupus is systemic lupus erythematosus (SLE).

3. Use of an anti-human CD154 antibody comprising a variable heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively, in the manufacture of a medicament for use in treating multiple sclerosis in a human subject in need thereof, wherein the medicament is for subcutaneous administration.

4. Use of an anti-human CD154 antibody comprising a variable heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively, in the manufacture of a medicament for use in treating Sjogren's syndrome in a human subject in need thereof, wherein the medicament is for subcutaneous administration.

5. Use of a composition comprising an anti-human CD154 antibody comprising a variable heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively, in the manufacture of a medicament for use in treating lupus in a human subject in need thereof, wherein the medicament is for subcutaneous administration.

6. The use described in claim 5, wherein the lupus is systemic lupus erythematosus (SLE).

7. Medicament for use in the treatment of multiple sclerosis (MS) in a human subject in need thereof 1. Use of a composition comprising an anti-human CD154 antibody, comprising a variable heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a variable light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, in the manufacture of a pharmaceutical, wherein the pharmaceutical is for subcutaneous administration.

8. Use of a composition comprising an anti-human CD154 antibody comprising a variable heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, and further comprising heavy and light chain constant regions having the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively, in the manufacture of a medicament for use in treating Sjogren's syndrome in a human subject in need thereof, wherein the medicament is for subcutaneous administration.

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