Anti-CD154 antibody having improved binding, functional and safety characteristics and use in human immunotherapy

Abstract

To provide anti-CD154 antibodies which have improved therapeutic potency, in vivo half-life and ablated FcR binding and / or complement binding / activation.SOLUTION: Provided are a humanized anti-human CD154 antibody, comprising a variable heavy polypeptide having a specific sequence and a variable light polypeptide having a specific sequence; and a pharmaceutical composition comprising a prophylactically effective amount of the same. Use of these antibodies to induce tolerance and treat immune diseases including autoimmunity, inflammation, transplant recipients, fibrosis and allergic disorders is provided.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, especially in the treatment of inflammatory disorders, allergy, 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 thrombotic or coagulation responses in vivo and 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, especially in the treatment of inflammatory disorders, allergy, 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 thrombotic or coagulation responses in vivo and 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 in autoimmune, graft rejection and other immune-related diseases in mice, non-human 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 therapeutics in terms of its effect on the immune response, and αCD154 is one of the few therapeutics that can induce functional immune tolerance as demonstrated in both mice and monkeys. In mice, almost all autoimmune disease models can be effectively ameliorated with αCD154 therapy (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; and Non-Patent Document 4), with long-term remission observed.

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

[0006] Moreover, 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 short-term therapeutic intervention with long-term clinical utility. Its failure was due 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. 5,993, 6,133, 6,141, 6,152, 6,166, 6,171, 6,182, 6,193, 7,196, 8,197, and 9,203,101, all of which are incorporated herein by reference. Early indications demonstrated that the drug was highly effective. However, continued clinical development was hampered by other anti-CD154 toxicities. In this study, observed toxicities included 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 was 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 that they produced a pegylated anti-human CD154 Fab, called CDP-7657 or ruplizumab. Because Fabs typically have extremely short in vivo half-lives (i.e., approximately 4-8 hours), this Fab was to improve its pharmacokinetic properties. However, although clinically effective, this antibody reportedly exhibits low efficacy. Additionally, Bristol Meyers Squib has reported the development of a domain antibody-Fc fusion protein that contains specificity for CD154. This fusion protein has reportedly shown efficacy in mouse disease models, specifically 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. 6001358 [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-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patent document 5

[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 efficacy 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 as 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 as 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 invention to provide an anti-human CD154 antibody as described in the previous five paragraphs that comprises a human IgG1 constant region that includes E269R and K322A mutations (numbering according to Kabat).

[0019] It is further an 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 further an object of the 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 an 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 binding to complement.

[0021] It is further an object of the 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 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 previous 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 the 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 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, 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, the composition being capable of inhibiting 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 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 previous paragraphs before, simultaneously with or after transplantation of donor tissues, 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 immune suppression by administering an antibody or composition according to any of the preceding paragraphs, said method optionally further comprising administration of a cell, antigen, tissue or organ containing the antigen to which tolerance or prolonged immune suppression is to be induced.

[0031] It is further an object of the 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 previous paragraphs, e.g. wherein the treated bone marrow, tissue, organ or immune cells comprise donor and / or recipient T cells.

[0034] It is further an 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 previous paragraphs, and optionally further comprising administering an antigen, e.g. an autoantigen, an allergen, an inflammatory agent, a drug, or HLA-mismatched (allo)donor cells.

[0035] It is further an object of the present invention to provide a method of inducing tolerance, extended immunity or T or B cell suppression in a patient who is to 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 further an 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 aforementioned symptoms, 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 perstans, 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 selected from allergic bronchopulmonary aspergillosis; autoimmune diseases. 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;Candidosis, chronic mucocutaneous;Kaplan's syndrome; Post-myocardial infarction syndrome;Post-pericardiotomy syndrome;Cartritis;Celiac sprue;Chagas disease;Chediak-Higashi syndrome;Churg-Strauss disease;Cogan syndrome;Cold agglutinin disease;Crest syndrome;Crohn's disease;Cryoglobulinemia;Fibrinous alveolitis of unknown etiology;Dermatitis herpetiformis;Dermatomyositis;Diabetes mellitus;Diamond-Blackfan syndrome;DiGeorge syndrome;Discoid lupus erythematosus;Eosinophilic fasciitis;Episcleritis;Erythema elevatus 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;Granulocytosis, immune-mediated;Granuloma annulare;Granulomatosis, 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;Eosinophilic syndrome;Idiopathic thrombocytopenic purpura;Job's syndrome;Juvenile dermatomyositis;Juvenile rheumatoid arthritis (juvenile chronic arthritis);Kawasaki disease;Keratitis;Keratoconjunctivitis sicca;Landry-Guillain-Barré-Stroll syndrome;Hansen's disease, et al; fibroid 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;Pemphigus;Pemphigus erythematosus;Pemphigus foliaceus;Pemphigus vulgaris;Pigeon breeder's disease;Interstitial pneumonia, hypersensitive;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; Sjogren'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 description 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 the variable region residues of both the heavy and light chain polypeptides are shown in bold, the CDRs are highlighted in yellow, the affinity maturation mutation residues are underlined, and the 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 the 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. [Diagram 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 increase in binding affinity to CD154 (expressed on Jurkat cells) compared to IDEC-131. [Figure 4] FIG. 4 depicts experimental results showing that an antibody of the invention ("INX021") promotes T cell CD154-driven B cell activation more potently than IDEC-131. [Diagram 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 showing 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, showing that the effects were variable and increased binding affinity does not necessarily correlate with better potency, i.e., immunosuppressive activity. [Figure 10] Figures 10A and 10B compare the effect of INX021 and chimeric 5c8 antibodies on IgG and IgM antibody production in a KLH assay. Figure 10A compares the effect of INX021 and chimeric 5c8 antibodies on IgG, and Figure 10B compares the effect of INX021 and chimeric 5c8 antibodies on IgM. [Figure 11]Figures 11A and 11B compare the effect 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 frequency compared to published results using another anti-CD154 antibody (PEGylated αCD154 Fab from Biogen / UCB). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 relation to 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 that contain at least a portion of an antibody sufficient to confer specific antigen binding to the 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 by context, the "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), as well as 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 mouse, hamster, goat, rabbit, chimeric, humanized, or fully human antibodies, fragments, derivatives, or constructs. 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 the Fc or constant region of an antibody 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, for example, 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 to complement proteins (e.g., complement protein C1q), and / or Fc receptors (FcR) (e.g., FcγRI, FcγRII, FcγRIIa, FcγRIII, and / or FcγRIIIb). The functional significance of being able to bind to one or more of the aforementioned molecules includes, but is not limited to, opsonization, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or effector cell regulation. Reduced effector function refers to a reduction in one or more of the biochemical or cellular activities induced at least in part by the binding of Fc to its cognate receptor or to complement proteins or effector cells, while maintaining the antigen-binding activity of the variable region of the antibody (or a fragment thereof) albeit with a reduced, similar, identical, or increased binding affinity. Certain antibodies of the present invention exhibit reduced effector function. The reduction in effector function, e.g., Fc binding to Fc receptors or complement proteins, 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 the plural 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 integers or groups 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, such as 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 (Europ. J. Immun. 22:3191-3194; Hollenbaugh et al., 1992 EMBO J. 11:4313-4321). These terms are used interchangeably throughout this application. The 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, the 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" refers to antibodies produced by synthesis of a DNA molecule encoding the antibody, as well as antibodies in which the DNA molecule expresses an antibody protein or an amino acid sequence that specifies the antibody. The term "antibody" should be construed to include the DNA or amino acid sequences of the polypeptides described herein, which 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. 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 FcR binding and affect (impair or enhance) other Fc effector functions are well known in the art. Exemplary mutations that may be modified 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 a 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 ability of the antibody 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 and may 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 mean 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, such mutations eliminating or substantially inhibiting toxicities or safety concerns such as thrombocytopenia or thrombosis or coagulation reactions or toxicity associated with complement reactions, 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 liquid sample. In many instances, a subject or a sample derived therefrom includes multiple cell types. In one embodiment, the sample includes, for example, a mixture of tumor and normal cells. In one embodiment, the sample includes at least 10%, 15%, 20%, see 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., chemoprevention), curing, reversing, attenuating, mitigating, minimizing, suppressing, or arresting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or virus), or other abnormal condition. For example, treatment may include alleviating a symptom of a disease (i.e., not necessarily every symptom) or attenuating the progression of a disease.

[0056] Anti-CD154 antibodies are potentially useful therapeutic agents for use herein in the treatment of autoimmune disorders. "Treatment or prevention" 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 the recurrence of cancer, including cancer metastasis; or preventing the onset or development of cancer (chemoprevention). Furthermore, the method of the invention may be carried out for the treatment of human patients with cancer. However, said method may also be effective for the treatment of cancer in other mammals. In a preferred embodiment, the subject antibodies are used to treat autoimmunity, allergy, inflammation, transplantation, GVHD, bone marrow transplantation (BMT), and induce antigen-specific tolerance or prolonged antigen-specific immune suppression, particularly the inhibition 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 transplantation, diabetes, rheumatoid arthritis, psoriatic arthritis, psoriasis, cell transplantation, tissue transplantation, organ transplantation, bone marrow transplantation, 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 required 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 a self antigen, 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 required 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 at

[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 was deposited with the American Type Culture Collection and the deposited cell line was designated ATCC Accession No. HB1712.

[0061] The sequence of IDEC-131 and its use in treating various allergy, autoimmune and inflammatory indications, and for suppressing T and B cell mediated immune responses, are disclosed in US Patent No. 6,001,358, US Patent No. 6,440,418, US Patent No. 6,506,383, US Patent No. 7,074,406 and US Patent No. 7,122,187. Based on its properties, including its strong immunosuppressive effect and high affinity for T and B cell immunity, 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 with IDEC-131 have been to some extent showed clinical efficacy in trials (albeit very early on in a relatively small number of patients), but the clinical trials were halted after another IgG1 anti-human CD154 antibody developed by Biogen, also derived from a mouse anti-human CD154 antibody (the mouse 5c8 antibody isolated by Seth Lederman at Columbia University), was reported to have induced adverse effects in human patients. Notably, when the Biogen chimerized 5c8 antibody was used in clinical trials, the 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 a humanized mutant anti-human CD154 antibody derived from IDEC-131, which is of human IgG1 isotype like IDEC-131, but whose Fc region has been engineered to eliminate both FcR binding and complement activation. In particular, its constant region has 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. In particular, the resulting modified antibody has limited FcR / C1q binding, i.e., it does not bind to FcγR2 or FcγR3 on platelets, and does not bind or activate complement. In contrast, its 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 show any detectable prothrombotic activity on mouse or human platelets in vitro or in vivo when assayed in different experimental models.

[0063] Moreover, 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 successive screening methods, mutant versions of the variable heavy and light chains of IDEC-131 were obtained that produce humanized antibodies (including INX02) with improved affinity compared to IDEC-131. Specifically, INX021 was obtained as well as 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 Figure 1A-C (containing the sequence of the humanized IgG1 anti-human CD154 antibody of the 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 modified. Surprisingly, as shown below, the resulting humanized anti-human CD154 antibody binds to human CD154 with approximately five times the affinity of the parent antibody, which itself binds to human CD154 with very high affinity as previously described. This is surprising, since even a single CDR modification can have a dramatic effect on antibody binding affinity. Moreover, it is not predictable that the affinity of an already relatively high affinity antibody can be improved.

[0066] This engineered humanized antibody has highly potent immunosuppressive properties when assayed in different in vitro and in vivo models. 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 a binding affinity for human CD154 that is five times that of IDEC-131; (ii) induces more potent immunosuppressive activity than IDEC-131; (iii) like IDEC-131, suppresses T and B (humoral) immunity, e.g., suppresses EAE, induces 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, because it should show an enhanced safety profile in patients and be more potent and therefore more effective than IDEC-131. The enhanced binding affinity of this antibody 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, because the subject antibodies have a higher potency, the antibodies may potentially be administered less frequently, which may further enhance dosing. This is important since the majority of applications of 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 present 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 further 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, the antibodies should provide clinical benefits when used in human therapy.

[0072] Although 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, US Patent Application Publication No. 2007 / 0237767 and US Patent Application Publication No. 2010 / 0104564 describe Fc mutagenesis to eliminate FcR binding. Furthermore, 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 known to enhance or impair Fc effector functions such as FcR binding, complement binding, glycosylation, ADCC activity, CDC activity, 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 allergy, autoimmunity, transplantation (allo- or xenogeneic organ, cell, tissue transplantation) and inflammatory conditions. Specific non-limiting examples include, by way of example, multiple sclerosis, systemic lupus erythematosus (SLE), other forms of lupus, autoimmune thrombocytopenia (ITP), kidney transplantation, skin and pancreatic islet transplantation, 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 transplantation, 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 particular envisaged application of the antibodies of the invention, including INX021, is to reduce or prevent anti-drug reactions, particularly those induced against 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 of other human constant regions are known to be involved in complement binding 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 abolishment of one or both of complement binding 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 haplomismatched 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 and safety of αCD154 complement-deficient and / or alloantigen-inducing variants. Alternatively, the ability of FcR deletion mutants (containing mutated Fc regions) to induce tolerance may be assessed.

[0080] The thromboembolic activity of FcR mutant αCD154 may be tested in a mouse model expressing the human FcγRIIA receptor that 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 mutated human Fc regions (mutated IgG1 constant regions) eliminates or appreciably reduces thrombus formation. Using this or comparable models, the effect of specific Fc mutations on toxicity associated with αCD154 therapy can be determined.

[0081] Moreover, the mutant CD154-specific antibodies of the invention derived from IDEC-131 may be used for the treatment and prevention of any condition in which attenuating the effect of CD154, including CD154 / CD40 signaling, or blocking or inhibiting CD154 binding to CD40, may be therapeutically beneficial and reduce the symptoms of the disease. Examples include the treatment of allergy, autoimmunity, cancer, transplantation, GVHD, inflammatory and other conditions, particularly conditions in which induction of tolerance 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 transplants, 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 with 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 models of autoimmune disease in mice 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 associated with increased graft rejection 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, AAand 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, SEand. 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 the 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 H and V L DNA encoding the MR1 variants was cloned and fused to DNA encoding the human γ1 CH1, CH2, CH3 regions or to the IgG1 variants disclosed herein. The nucleotide sequence was 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 NS0 cells and the product was purified by Protein A chromatography.

[0084] Binding of the Antibody of the Present Invention to CD154 Comparison of the binding activity of CD154 antibody mutants was determined by their binding to CHO cells transfected with mouse CD154. CD154-expressing CHO cells are incubated with biotin-labeled αCD154 in the presence of unlabeled αCD154 heavy chain mutants or isotype-matched antibodies for 1 h at 4° C. Binding of biotinylated MR1 is detected using a streptavidin-conjugated fluorescent dye and flow cytometry is performed. The percentage of inhibition by the mutants can be determined by recording the decrease in the 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 variants. 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. Reduction of 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 MR1 or the IgG1 variants of IDEC-131 have been absorbed onto a Maxisorb ELISA plate. Bound C1q is detected using HRP-chicken anti-C1q. All mutants were assayed 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 with C1q binding to WT IgG1 MR1.

[0088] Induction of tolerance using mutant αCD154 mAb The ability of anti-CD154 antibodies to induce tolerance or extended immune suppression can be determined by methods known and specifically described herein. MR1, a hamster anti-mouse CD154 produced in our laboratory, inhibits tolerance to CD154 antibodies, as we have shown (Quezada, SA, Fuller, B., Jarvinen, LZ, Gonzalez, M., Blazar, BR, Rudensky, AY, Strom, TB and Noelle, RJ, "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 anergy 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 transplant immune tolerance.

[0089] Tolerance will be 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 grafts are 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 spleen cells (DST) and skin grafts. Recipient C57BL / 6 mice are transplanted with 5 × 10 7 DST cells are injected with or without 500 μg of WT or mutant αCD154 or control immunoglobulin, hamster or human, (HIgG1) in phosphate-buffered saline (PBS) via tail vein injection (intravenously) and intraperitoneally. Mice receive treatment with the appropriate antibody (250 μg / injection) three times per week for the duration of the experiment thereafter. On day 0, recipient mice are anesthetized with 50 μg each of ketamine and xylazine per gram 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 number of cells / area measured. Finally, in this system of humoral immunity using mutant αCD154 mAb, as previously published (Markees, TG, Phillips, NE, Noelle, RJ, Shultz, LD, Mordes, JP, Greiner, DL and Rossini, AA, "Prolonged survival of mouse skin allografts in recipients of grafts with αCD154 mAb"). 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 (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, third party grafts (H-2K skin) are transplanted into tolerized mice (in the selection group).

[0091] In addition to measuring the effect 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 thrombotic 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 the respective variants 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, the mice will show prolonged disorientation, shallow breathing, and impaired mobility. Mice showing this activity are expected to have significantly reduced platelet counts. After 60 min, lungs are harvested, fixed in formalin, sectioned, and stained with H&E. Lung sections of mice are evaluated for evidence of thrombosis (measured by intravascular clots) and the number of clots / section is counted. For each mouse, 10 sections are counted and the total number of clots is compared across all groups treated with different variants of IgG1 MR1. In addition, total platelet counts (harvested by cardiac puncture at the time of euthanasia) are evaluated by flow cytometry and are expected to be reduced by 80% with the use of antibodies that are thrombogenic. These findings will determine which of the MR1 variants (FcR binding (N325L, K326V, E269R)) are thrombogenic and whether alterations in FcR binding alter 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 an intraperitoneal injection with 250 ng of pertussis toxin at the time of immunization and 48 hours later. Seven days later, mice receive an identical boost 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 assessment. 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; 5, death. Mice with a score greater than 4 but less than 5 are euthanized.

[0095] Toxicity assessment The desired antibodies according to the invention have significantly reduced or no toxicity in the disclosed animal thrombotic models. Using the above models, it is shown that an IgG1 anti-CD154 antibody whose heavy chain constant region contains the E269R and K322A mutations (according to the Kabat numbering system) does not induce platelet aggregation.

[0096] Efficacy assessment Efficacy (induction of tolerance) 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 invention, the anti-CD154 antibody, or a pharmaceutical composition comprising said antibody, is capable of inhibiting an immune response in a subject. The antibody of the invention, or the pharmaceutical composition of the invention, is administered to the subject in an effective inhibitory amount.

[0098] In certain embodiments, an "effective inhibitory amount" of an anti-CD154 antibody, or a pharmaceutical composition comprising said 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 the 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 the purposes of the present invention, an inflammatory response is characterized by redness, swelling, heat and pain as a result of capillary dilation with 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 a method of 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 invention, the anti-CD154 antibody, or a pharmaceutical composition comprising said antibody, is capable of inhibiting rejection of a transplanted organ by a subject.

[0102] In a more specific embodiment of the invention, the 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 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 invention, the anti-CD154 antibodies of the invention, or pharmaceutical compositions comprising said antibodies, are capable of inhibiting an allergic response, eg, hay fever or allergies 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, increasing the number of patients who respond to the drug, and / or promoting efficacy in drug-naive patients.Examples of these 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 may be used to prevent or reduce anti-drug responses include the following: 3F8, 8H9, Abagogomab, 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, Liximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab Mertansinemertansine, Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Cabiralizumab, 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, Clivatuzumab 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, Elgemtum 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, Fibatuzumab, Ficlatuzumab, Figitumumab, Filibugumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, 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, Libivirumab, Rifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegolpegol, Lumiliximab, Lumretuzumab, Mapatuzumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Natuximab estafenatox, Naratuximab emtansine, Narnatumab, Natalizumab, Navicixizumab, Navivumab, Nevacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentane merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratuzumab, Olokizumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Orticumab, 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, rinucumab, 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, Tocilizumab (atlizumab), Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukinin celmoleukin, Tuvirumab, Ublituximab, Urocupulumab, Urelumab, Urutoxazumab, Ustekinumab, Vadastuximab talirine, Bundletuzumab vedotin These include: ndortuzumab Vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vepalimomab, 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 autoimmune response in a subject suffering from an autoimmune disease.In some embodiments, the autoimmune response is associated with or derived 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 derived from systemic lupus erythematosus.

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

[0109] In one embodiment of the present invention, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising said antibody, is 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 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 disorders; cirrhosis of the liver secondary to drugs; cirrhosis of the liver secondary to viral infections; and cirrhosis of the liver secondary to autoimmune diseases.

[0112] In one embodiment of the invention, the anti-CD154 antibody of the invention, or a pharmaceutical composition comprising said antibody, can inhibit 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 disease. Some examples of vascular disease 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, the 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., a 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 invention, the anti-CD154 antibody of the 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, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising said antibody, can image tumor or neoplastic cells in a subject expressing 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: administering to the subject an effective amount of the anti-CD154 antibody of the present invention, or a composition comprising said 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, the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising said antibody, can detect the presence of tumor or neoplastic cells in a subject expressing 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 the anti-CD154 antibody of the present invention, or a pharmaceutical composition comprising said antibody, under conditions that allow 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, where the presence of such complexes indicates the presence of tumor or neoplastic cells in the subject.

[0118] Pharmaceutical Compositions 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 pharma- ceutically 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 invention, the pharma- ceutically acceptable carrier is phosphate buffered saline, saline, water, citrate / sucrose / Tween formulations and emulsions, such as oil / water emulsions.

[0123] In one embodiment of the invention, the pharmaceutical composition can be delivered in a microencapsulation device to reduce or prevent a host immune response to the composition. Binding agents, such as antibodies or antibody fragments of the invention, may be delivered microencapsulated in a membrane, such as a liposome, or other encapsulating or immunoprotected delivery vehicle.

[0124] In one embodiment of the 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 a sterile, substantially pyrogen-free, 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 in a suitable capsule, tablet, aqueous suspension or solution. The solid formulation of the composition for oral administration can contain a suitable carrier or excipient, 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 invention, topical semisolid ointment formulations typically contain a concentration of active ingredient of about 1 to 20%, for example 5 to 10%, 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 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 the pharmaceutical composition for injection 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 drip infusion, whereby a pharmaceutical formulation containing an antifungal agent and a physiologically acceptable excipient is infused. The physiologically acceptable excipient can be used to inject the antifungal agent. 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 pharma- ceutically acceptable oil base, such as an ester of a long chain fatty acid, for example, 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 pharma- ceutically 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 specific disease state and the associated treatment regimen. Pharmaceutical formulations are well established in the art. The pharmaceutical composition can be administered to a subject using conventional methods known to those skilled in the art of medicine.

[0134] Thus, the pharmaceutical composition of the present invention relates 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 immediate distribution through the bloodstream. Sustained release formulations may, in certain embodiments, result in a level of circulating drug from a single dose that is sustained for, for example, 8 hours or longer, 12 hours or longer, 24 hours or longer, 36 hours or longer, 48 hours or longer, 60 hours or longer, 72 hours or longer, 84 hours or longer, 96 hours or longer, or even, for example, a week or 2 weeks or longer, for example, a month or longer. Sustained release compositions may include 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 immunosuppressive or immunomodulatory compound can 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 said 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 manner that is medically acceptable. For purposes of the present invention, "administration" refers to 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 invention. 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, intraspinal, intratumoral, 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 a more specific embodiment, 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 locally 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 invention, the anti-CD154 antibodies of the invention and pharmaceutical compositions of the invention may be administered to a subject by sustained release administration, by means such as depot injection of an erodible implant directly applied 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 present invention and pharmaceutical compositions of the present invention may be administered to a subject by an injectable depot administration route, such as by using a 1, 3, or 6 month depot injectable or biodegradable material and method.

[0146] In a more specific embodiment, the anti-CD154 antibodies 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, typically 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 dosage per body weight and at any frequency of administration that is medically acceptable, including ranges 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 effect on immunity, i.e., suppression of T or B cell immunity, or in an amount effective to prevent, treat or ameliorate the symptoms of a disease in which suppression of T or B cell immunity is therapeutically desirable. As mentioned, these conditions include certain autoimmune, inflammatory and allergic indications.

[0149] The total administered dose of the 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 invention, the anti-CD154 antibodies or binding fragments thereof, as well as 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, biweekly 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 said antibodies may be administered to a subject in need thereof at intervals as long as medically required, ranging from a few days or weeks to the life of the subject. 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 invention, the anti-CD154 antibodies of the invention and the pharmaceutical compositions of the 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 a variety of 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 an effective dosage may depend on recipient subject characteristics such as, for example, age, sex, pregnancy status, body mass index, lean body mass, condition(s) to which the composition is administered, other health conditions of the recipient subject that may affect metabolism or tolerance of the composition, the recipient subject's levels of IL-6, and tolerance to the composition (e.g., resulting from the patient developing antibodies to the composition). One of ordinary skill in the art would 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 invention and pharmaceutical compositions of the 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 a disease or prolong the survival of a subject being treated. A therapeutically effective amount may vary by varying the dosage and administration schedule of the subject antibodies. This may be achieved by:

[0156] The anti-CD154 antibody of the present invention and the pharmaceutical composition of the present invention may be administered as a single dose for certain indications, such as preventing immune responses to antigens to which the subject is exposed for a short period of time, such as foreign antigens administered in a one-day treatment. Examples of such treatments would include co-administration of the antibody fragment of the present invention together with therapeutic agents, such as antigenic pharmaceuticals, allergens or blood products, or gene therapy vectors. In indications where antigens are chronically present, such as controlling immune responses to transplanted tissues or chronically administered antigenic pharmaceuticals, the antibody fragment or pharmaceutical composition of the present invention is administered at intervals as long as medically required, ranging from a few days or weeks to the life of the subject.

[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 T cell costimulatory signaling through CD28; (b) agents 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 instances, 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 method are animals.Preferably, the animals are mammals.Examples of mammals that may 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, the mammals are humans.

[0160] The invention may be better understood based on the following examples, however, 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 set forth to fully disclose and explain 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 allowed 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. EXAMPLES

[0162] Example 1: Higher affinity (lower K 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 mentioned above, this antibody was derived from IDEC-131 by the combined use of affinity maturation and mutagenesis techniques 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 a large number of variants. The vast 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 identified and shown in Figures 2A-2C and 3. The binding affinity of each of these variants to human CD154 was then screened by Biacore. The Biacore assay was performed as follows:

[0165] BIACORE assay Equipment:Biacore3000 Assay buffer - 10 mM 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 - for ligand capture the flow rate was 5 μl / min. For kinetic analysis the flow rate was 30 μl / min.

[0166] procedure Binding experiments were performed on a Biacore300 at 25°C. Antibodies were directly immobilized on flow cell 2 (1000 RU combination #23) and flow cell 4 (1000 RU EWT #131) and CD154 antigen was flowed over the chip. Binding of antigen to the test antibodies 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 determine 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 indicates 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 antibody having the following values ​​was obtained. The amino acid sequences of the heavy and light variable sequences of these mutants, identified as mutant 22, mutant 23, mutant 24, mutant 25, mutant 26, mutant 27, mutant 28, mutant 29, mutant 30 and mutant 31, are contained in FIG. 2.

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

[0171] [Table 4]

[0172] Example 2: Higher affinity (lower K) antibodies to human CD154 for SC administration D Use of antibody variants having As shown in the table above, the mutant selected as lead candidate #21 had a K D , which is 4.7-fold higher than IDEC-131. This improved affinity is significant as it should allow for 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 volumes for sq administration range from about 0.5 to 5 mL (volumes larger than 3 mL are considered "large"). Therefore, an effective dose of IDEC-131 would require about 8 mL (see below for calculations). This dose volume is sq .Well above pharmaceutical standards. (10mg / kg x 80kg) / 100mg / ml = 8mL

[0175] In contrast, mutant #21 (INX021) had 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 dosage 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] Thus, unlike IDEC-131, INX021 should be well suited for administration via the sq route.

[0177] Example 3: Comparison of the potency of anti-CD154 antibodies according to the invention Figure 4 depicts the results of an experiment to detect inhibition of T cell CD154-driven B cell activation. The results contained in the figure show 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 for 16 or 20 minutes did not induce 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 7A-D show experimental results demonstrating that INX021 does not activate platelets, does not induce platelet aggregation, and does not affect (reduce) platelet count in vivo. The experiment in FIG. 7A shows that INX021 does not induce thrombotic stress or activate platelets when compared to a positive control. The experiment in FIG. 7B shows that INX021 does not affect the number of circulating platelets when compared to a positive control. The experiment in FIG. 7C shows that INX021 does not induce the formation of blood clots (emboli) in the lungs when compared to a positive control. FIG. 7D summarizes the advantages of INX021 in vivo, i.e., INX021 does not reduce platelet count, does not activate platelets, does not cause platelet stress, and does not induce the formation of blood clots.

[0181] Example 8: Use of anti-CD154 antibodies according to the invention in autoimmune models 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 Fc mutations in INX021 potently suppresses EAE for approximately 20 days, and the experiment in Figure 8B shows that a humanized anti-CD154 IgG1 antibody containing Fc mutations 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 FIG. 9 compare the inhibitory effect 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 effect of antibody variants according to the invention was compared in a mixed lymphocyte reaction. The readout was CD86 expression by activated B cells. As shown in the figure, the better the 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 INX021 of the present invention was tested in an 8-week non-GLP toxicity 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 effect of INX021 and the chimeric 5c8 antibody on the production of IgG and IgM antibodies in a KLH assay, Figure 10A compares its effect on IgG and Figure 10B compares its effect on IgM.

[0188] Figures 11A-B compare the effect 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 and degree of cellularity of germinal centers in the spleens and lymph nodes (LNs) of treated animals.

[0189] FIG. 12 summarizes the observed lesion frequency compared to 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 treatment.

[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 ADA was measured in all cohorts. The TDAR response in all animals was suppressed, 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 group (1 mg / kg) did not develop any detectable ADA.

[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 component, whichever comes first. The solution is stored at room temperature (17°C to 27°C).

[0193] 1. 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) and 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 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). Assign expiration dates for date plate preparations is complete.

[0195] Assay procedure for detection of antibodies to INX021 or ch5C8 In these experimental protocols, the "test material" refers to serum from rhesus or cynomolgus monkeys. The samples are samples containing the positive control, samples containing the positive control, and / or blanks. 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] Analytical procedures 1. Aspirate test material from each well of the plate(s) and 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) and 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) and 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, a 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 evaluate the variance at the 5% level above the blank TS and remove up to two outlier A450 blank test sample (TS) values. The mean A450 is calculated from all values ​​without removing the 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 is a failure and should be reanalyzed. For MNR analysis, at least 5 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 - Mean 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] Blocker casein blank and blank TS average A450 after outlier removal LPC A should be less than 0. The plate specific cut point (PSCP) is: PSCP=CF * Calculated as blank TS global average (no outliers). Correction factor (CF) is added to study file. LPC average A450 must be above PSCP, otherwise LPC at that setting has failed.

[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] HPC>MPC>LPC for the average A450 of each PC set. 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). A blank TS is also prepared with a final concentration of 10% matrix diluted in blocker casein. 12 wells of blank TS are run on each plate. Blocker casein is run in at least 2 wells on each plate. All test materials are analyzed in a minimum of 2 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 samples is compared to the PSCP and reported as potentially positive if the mean absorbance value is above the PSCP and as potentially negative if the mean absorbance value is below the PSCP.

[0210] result TDAR responses were suppressed in all animals, suggesting that antibody responses against soluble antigens could be 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 group (1 mg / kg) did not develop any detectable ADA. Thus, the antibody INX021 of the present invention can be used to suppress humoral immunity against immunogenic agents, such as, for example, biopharmaceuticals, e.g., therapeutic antibodies, fusion proteins, and the like.

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L., Hess, D. M., Garber, E. A., Taylor, F. R. and Burkly, L. C., FcR interactions do not play a major role in inhibition of experimental autoimmune encephalomyelitis by anti-CD154 monoclonal antibodies. J Immunol 2004. 173: 993-999. 50 Becher, B., Durell, B. G. and Noelle, R. J., Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12. J Clin Invest 2002. 110: 493-497. 51 Becher, B., Durell, B. G., Miga, A. V., Hickey, W. F. and Noelle, R. J., The clinical course of experimental autoimmune encephalomyelitis and inflammation is controlled by the expression of CD40 within the central nervous system. J Exp Med 2001. 193: 967-974. 52 Howard, L. M., Miga, A. J., Vanderlugt, C. L., Dal Canto, M. C., Laman, J. D., Noelle, R. J. and Miller, S. D., Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. J Clin Invest 1999. 103: 281-290.

[0212] The entire disclosure of each document cited in the Background, Detailed Description, and Examples of the present invention (including patents, patent applications, journal articles, abstracts, handbooks, books, or other disclosures) is hereby incorporated 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, but instead the scope of the invention is limited by all of the following claims. This will be determined by the

[0214] The 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. 2A-2C 。 1. 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 , provided that the humanized anti-human CD154 antibody does not comprise a variable heavy polypeptide of SEQ ID NO: 15 and a variable light polypeptide of SEQ ID NO:

20.

2. 1. 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 as 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 as shown in Figures 1A-C.

3. The anti-human CD154 antibody of claim 1 or 2, comprising an IgG1, IgG2, IgG3 or IgG4 constant region that lacks the ability to bind C1Q and to bind FcγR2 and / or FcγR3.

4. 3. The anti-human CD154 antibody of claim 1 or 2, comprising a human IgG1 constant region that lacks the ability to bind C1Q and to bind FcγR2 and / or FcγR3.

5. The humanized anti-human CD154 antibody of claim 4, wherein the human IgG1 constant region comprises E269R and K322A mutations (numbering according to Kabat).

6. The humanized anti-human CD154 antibody of any one of claims 1 to 5, comprising the IgG1 light and heavy constant regions of SEQ ID NO:3 and SEQ ID NO:2 shown in Figures 1A to 1C.

7. 7. The humanized anti-human CD154 antibody of any one of claims 1 to 6, wherein the antibody is of the IgG1 isotype and 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 binding to complement.

8. 8. The humanized anti-human CD154 antibody of any one of claims 1 to 7, 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.

9. A humanized anti-human CD154 antibody according to any one of claims 1 to 8, wherein the antibody is of the IgG1 isotype and the Fc region of the antibody has been mutated to introduce the E233P mutation.

10. A humanized anti-human CD154 antibody according to any one of claims 1 to 9, wherein the Fc region of the antibody has been further mutated to introduce one or more other mutations that improve in vivo half-life.

11. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of at least one humanized anti-human CD154 antibody according to any one of claims 1 to 10.

12. The composition of claim 11, further comprising an antigen, a cell, a tissue, or an organ.

13. 13. The composition of claim 12, wherein the antigen is an autoantigen, an allergen, an inflammatory agent, a drug, or an HLA-mismatched (allo) or xenogeneic (non-human) donor cell.

14. A method of immunosuppression or immunotherapy using an antibody or composition according to any one of claims 1 to 13.

15. 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 one of claims 1 to 13.

16. A method of treating or preventing GVHD in a subject by administering an antibody or composition according to any one of claims 1 to 13 prior to, simultaneously with or after transplantation of donor tissue, organ or cells, which may optionally be genetically engineered.

17. The method of claim 16, wherein the cell is a CAR-T or NK cell.

18. A method for inducing tolerance or prolonged antigen-specific immune suppression by administering an antibody or composition according to any one of claims 1 to 13.

19. 20. The method of claim 18, wherein the method further comprises administration of a cell, antigen, tissue, or organ containing the antigen to which tolerance or prolonged immunosuppression is sought to be induced.

20. A method of gene, cell, tissue or organ therapy comprising administering an antibody or composition according to any one of claims 1 to 13.

21. 16. The method of claim 15, wherein the condition being treated is 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.

22. 14. A method of treating bone marrow, organs or immune cells for use in cell therapy or organ or bone marrow transplantation, said method comprising incubating said bone marrow, organ, tissue or immune cells with an antibody or composition according to any one of claims 1 to 13.

23. 23. The method of claim 22, wherein the treated bone marrow, tissue, organ or immune cells comprise donor and / or recipient T cells.

24. A method for inducing tolerance without inducing a thrombotic event in a patient in need thereof, said method comprising administering to the patient an effective amount of an antibody or composition according to any one of claims 1 to 13.

25. 25. The method of claim 24, further comprising administration of an antigen.

26. 26. The method of claim 25, wherein the antigen is an autoantigen, an allergen, an inflammatory agent, a drug, or an HLA-mismatched (allo)donor cell.

27. The method according to claims 24 to 26, wherein the patient is to receive or has received transplanted cells, organs or tissues.

28. 27. The method of claim 26, wherein the drug is a biopharmaceutical such as a therapeutic antibody, receptor, fusion protein, hormone, growth factor or cytokine.

29. A method of treating a T cell mediated autoimmune disease by administering an effective amount of an antibody or composition according to any one of claims 1 to 13.

30. A method of treating a B cell mediated autoimmune disease by administering an effective amount of an antibody or composition according to any one of claims 1 to 13.

31. 30. The method of claim 29, wherein the disease is 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.

32. The method according to claims 29 to 31, comprising administration of an antigen.

33. A method of treating or preventing GVHD, bone marrow transplant (BMT) multiple sclerosis, lupus, ITP, rheumatoid arthritis, asthma, IBD or another inflammatory bowel disease by administering an effective amount of an antibody or composition of any one of claims 1 to 13.

34. 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 one of claims 1 to 13.

35. 35. The method of claim 34, wherein the human condition, disorder or disease is an inflammatory, allergic, or autoimmune response or fibrosis.

36. 36. The method of claim 34 or 35, wherein 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.

37. 35. The method of claim 34, wherein 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 diurnitum, 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.

38. The human condition, disorder or disease may be allergic bronchopulmonary aspergillosis; autoimmune 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; candidiasis, chronic mucocutaneous; Kaplan's syndrome; post-myocardial infarction syndrome; post-pericardiotomy syndrome; carditis; celiac sprue; Shah Gass disease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan syndrome; cold agglutinin disease; CREST syndrome; Crohn's disease; cryoglobulinemia; fibrosing alveolitis of unknown etiology; dermatitis herpetiformis; dermatomyositis; diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; discoid lupus erythematosus; eosinophilic fasciitis; episcleritis; erythema elevatus diurnalis; 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;Agranulocytosis, immune-mediated;Granuloma annulare;Granulomatosis, allergic;Granulomatous myositis;Graves' disease;Hashimoto's thyroiditis;Hemolytic disease of the newborn;Hemochromatosis, idiopathic;Henoch-Schonlein 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-Strole syndrome; leprosy, lepromatous 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; pemphigus erythematous; pemphigus foliaceus; pemphigus vulgaris; pigeon breeder's disease; Interstitial pneumonia, hypersensitive; polyarteritis nodosa; polymyalgia rheumatica; polymyositis; polyneuritis, idiopathic; Portuguese familial polyneuropathy; pre-eclampsia / eclampsia; primary 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; 35. The method of claim 34, wherein the condition is selected from undifferentiated connective tissue disease; urticaria, chronic; urticaria, cold; uveitis; vitiligo; Weber-Christian disease; Wegener's granulomatosis; and Wiskott-Aldrich syndrome.

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