WO2012138768A2 - Anti-cd154 antibodies having impaired fcr binding and/or complement binding properties and the use thereof in immune therapies - Google Patents

Abstract

Improved anti-CD154 antibodies are provided herein which have ablated FcR binding. The use of these antibodies for inducing tolerance and treating immune diseases including autoimmunity, inflammation and allergic disorders is disclosed herein.

Description

ANTI-CD154 ANTIBODIES HAVING IMPAIRED FcR BINDING AND/OR

COMPLEMENT BINDING PROPERTIES AND THE USE THEREOF IN

IMMUNE THERAPIES

1. Related Applications

[0001] The present invention claims priority to US provisional application

Serial No. 61/471,287 filed on April 4, 2011 .relates to improved anti-CD154 (CD40L) antibodies having reduced toxicity and their use in immune therapies, especially treatment of cancers, inflammatory disorders, allergy and autoimmunity. In particular the invention provides anti-CD 154 antibodies that are modified such that they do not elicit thrombogenic or clotting reactions in vivo, but which still retain desired therapeutic properties such as the induction of immune tolerance.

BACKGROUND

2. Field of the Invention

[0002] The present invention relates to improved anti-CD 154 (CD40L) antibodies having reduced toxicity and their use in immune therapies, especially treatment of cancers, inflammatory disorders, allergy and autoimmunity. In particular the invention provides anti-CD 154 antibodies that are modified such that they do not elicit thrombogenic or clotting reactions in vivo, but which still retain desired therapeutic properties such as the induction of immune tolerance.

[0003] Description of Related Art

[0004] CD40L (CD154) is a highly validated and valuable therapeutic target in autoimmunity, graft rejection and other immune-related diseases in mice, non-human primates (NHP) and humans. In numerous Phase II Clinical Trials, a-CD154 has been shown to effectively block the activities of CD 154 in vivo and ameliorate disease. CD154 is distinct from all other therapeutics in its impact on the immune response; it is one of the only therapeutics that can induce functional immunological tolerance, as demonstrated both in mice and monkeys. In mice, virtually all autoimmune disease models can be effectively ameliorated with CD154 therapy (Noelle, R. J., Mackey, M., Foy, T., Buhlmarm, J. and Burns, C, CD40 and its ligand in autoimmunity. Ann N Y Acad Sci 1997. 815: 384-391; Mackey, M. F., Barth, R. J., Jr. and Noelle, R. J., The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells. J Leukoc Biol 1998. 63: 418-428; Noelle, R. J., CD40 and its ligand in cell-mediated immunity. Agents Actions Suppl 1998. 49: 17-22; and Quezada, S. A., Jarvinen, L. Z., Lind, E. F. and Noelle, R. J., CD40/CD154 Interactions at the Interface of Tolerance and Immunity. Annu Rev Immunol 2004. 22: 307-328), with long-term remission observed.

[0005] In NHP, permanent allograft tolerance can be achieved using short courses of treatments comprised of ccCD154 (Kenyon, N. S., Chatzipetrou, M., Masetti, M., Ranuncoli, A., Oliveira, M., Wagner, J. L., Kirk, A. D., Harlan, D. M., Burkly, L. C. and Ricordi, C, Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD 154. Proc Natl Acad Sci U S A 1999. 96: 8132- 8137; Kirk, A. D., Burkly, L. C, Batty, D. S., Baumgartner, R. E., Berning, J. D., Buchanan, K., Fechner, J. H., Jr., Germond, R. L., Kampen, R. L., Patterson, N. B., Swanson, S. J., Tadaki, D. K., TenHoor, C. N., White, L., Knechtle, S. J. and Harlan, D. M., Treatment with humanized monoclonal antibody against CD 154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 1999. 5: 686-693).

[0006] Also, Phase II Clinical Trials in humans have indicated that aCDl 54 is effective in SLE (Sidiropoulos, P. I. and Boumpas, D. T., Lessons learned from anti- CD154 treatment in systemic lupus erythematosus patients. Lupus 2004. 13: 391-397), Multiple Sclerosis (see preliminary data) and idiopathic thrombocytopenia (Sidiropoulos, P. I. and Boumpas, D. T., Lessons learned from anti-CD 154 treatment in systemic lupus erythematosus patients. Lupus 2004. 13: 391-39). As such, aCD154 is a unique dmg that will allow for short-term intervention with long-term clinical benefit. Its failures have not been in efficacy, but due to an unanticipated toxicity.

[0007] Further, in the early 1990's IDEC Pharmaceuticals and Biogen Inc.

(now Biogen Idee) launched two different CD1 4 mAbs into multiple Phase Ι/Π Clinical Trials. The antibody developed by IDEC (IDEC- 131) was derived from a murine anti-hCD154 developed at Dartmouth College.

[0008] This antibody and humanized variants are disclosed in US Patent No.

6,440,418 the contents of which are incorporated by reference herein. While early indications demonstrated that the drug was highly effective, toxicity of the CD154 prohibited continued clinical development. In the trials, the observed toxicity included the induction of thromboembolic events in patients. Based on toxicity concerns, all trials were suspended and efforts were directed towards re-engineering the mAbs to sustain efficacy and reduce toxicity. While reduced toxicity has been achieved, there has been a substantial decrease in efficacy and the tolerance-inducing capacity of aCD154 mAbs (Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D. M._} Shapiro, R. I., Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly, L. C. and Taylor, F. R., The contribution of Fc effector mechanisms in the efficacy of anti-CD 154 immunotherapy depends on the nature of the immune challenge. Int Immunol 2004. 16: 1583-1594). None of the engineered mAb forms have progressed significantly into the clinic due to loss in efficacy.

[0009] Accordingly, there is a significant need in the art for improved anti-

CD 154 antibodies, i.e., those which are both safe and effective. This invention attains these goals.

DETAILED DESCRIPTION OF THE FIGURES

[0010] Figure 1 shows the impact of anti-CD154 therapy on relapse rate in RR

MS Patients. Patients with active disease were recruited into trial and were treated with 4 escalating doses of EDEC- 131 every week. Following treatment all patients were followed by EDSS scoring as well as assessment of gadolinium- enhanced lesions.

[0011] Figure 2 show the nucleotide sequence of hamster anti-murine CD 154.

Shown are the k and heavy chain sequence for the MR1 anti-human CD40L (CD 154) hamster IgGl.

[0012] Figure 3 shows the reduction in FcR binding in the E223PIgGl MR1 IgGl variant.

[0013] Figure 4 shows the effects of mutations in MR1 that ablate Clq binding.

[0014] Figure 5 shows that the loss of complement activation does not reduce the ability of anti-CD 154 to induce tolerance.

[0015] Figure 6 shows the thrombotic stress signs in all tested animals arranged by treatment groups.

[0016] Figure 7 shows platelet counts of all animals used in the study, arranged by treatment groups.

[0017] Figure 8: Average number of clots per field (200x original magnification) as observed microscopically, arranged by treatment groups.

[0018] Figure 9: Sample images of H&E stained lung sections from animals injected with PBS. Header values indicate original microscopic magnification. With these example images and with those below, the higher magnification images were acquired from within the field of the first (lOOx) image.

[0019] Figure 10: Sample images of H&E stained lung sections from animals injected with MRl-WT. Header values indicate original microscopic magnification. Blue aiTOw identifies thrombus. [0020] Figure 11: Sample images of H&E stained lung sections from animals injected with N325L. Header values indicate original microscopic magnification

[0021] Figure 12: Sample images of H&E stained lung sections from animals injected with 326V. Header values indicate original microscopic magnification

[0022] Figure 13: Sample images of H&E stained lung sections from animals injected with E269R. Header values indicate original microscopic magnification.

[0023] Figures 14-16 contain humanized sequences corresponding to IDEC-131.

[0024] Figures 17 and 18 contain the variable sequences for the parent chimeric antibody that IDEC-131 was derived.

DETAILED DESCRIPTION

[0025] Prior to disclosing the 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 to one of ordinary skill in the art to which this invention belongs.

[0026] As used herein, oligonucleotide sequences that are complementary to one or more of the genes described herein, refers to oligonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequence of said genes. Such hybridizable oligonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes.

[0027] "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.

[0028] The phrase "hybridizing specifically to" refers to the binding, duplexing or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0029] "Mutation or mutations that eliminate or reduces FcR binding and which eliminates toxicity" herein refers to a mutation or mutations shown to be effective (substantially or totally eliminate thrombocytopenia or thrombosis or clotting) in a murine thrombosis model disclosed infra that has been engineered to expresses human FcR.

[0030] "Mutation or mutations that eliminate or reduce complement function and which maintain tolerance inducing properties" refers to mutation or mutations that eliminate or reduce complement binding that maintain the ability of the antibody to induce tolerance in the skin transplant model disclosed herein.

[0031] A "patient" can mean either a human or non-human animal, preferably a mammal.

[0032] As used herein, "subject", as refers to an organism or to a cell sample, tissue sample or organ sample derived therefrom, including, for example, cultured cell lines, biopsy, blood sample, or fluid sample containing a cell. In many instances, the subject or sample derived therefrom, comprises a plurality of cell types. In one embodiment, the sample includes, for example, a mixture of tumor and normal cells. In one embodiment, the sample comprises at least 10%, 15%, 20%, et seq., 90%, or 95% tumor cells. The organism may be an animal, including but not limited to, an animal, such as a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, etc., and is usually a mammal, such as a human. [0033] The term "treating" in its various grammatical forms in relation to the present invention refers to preventing (i.e. chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing, or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g. bacteria or viruses), or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessarily all the symptoms) of a disease of attenuating the progression of a disease.

[0034] "Treatment of autoimmunity" or another disease condition," as used herein, refers to partially or totally inhibiting, delaying, or preventing the progression of the disease wherein antagonistic anti-CD40L antibodies have therapeutic application. . In the case of cancer this means treating or inhibiting cancer metastasis; inhibiting, delaying, or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (chemoprevention) in a mammal, for example, a human. In the preferred embodiments the subject antibodies are used to treat autoimmunity, allergy, inflammation, transplant, GVHD, bone marrow transplant (MMT), and to induce antigen specific tolerance in subjects in need thereof. Preferred indications are multiple sclerosis, lupus, ITP, IBD, Crohn's disease, psoriasis, uveitis, rheumatoid arthritis, asthma, GVHD, organ or graft transplant, bone marrow transplant, oophoritis and thyroiditis.

[0035] As used herein, the term "therapeutically effective amount" is intended to qualify the amount of the treatment in a therapeutic regimen, i.e., an anti-CD 154 antibody according to the invention, necessary to treat a condition e.g., autoimmunity.

[0036] The present invention provides novel and improved anti-CD 154 antibodies for use in therapies. These antibodies exhibit improved safety and efficacy compared to currently available anti-CD 154 antibodies.

[0037] It was initially thought that the therapeutic efficacy of an CD154 was due to its ability to simply block CD 154. However, later reports have suggested that complement binding is also required for such antibodies to actively induce tolerance. Also, it was unclear whether the ability of the antibody to bind to FcR had any impact on functionality, i.e., its ability to induce tolerance.

[0038] Notwithstanding the foregoing, the inventor proposed to develop mutated anti-CD 154 antibodies that do not bind FcR and/or complement with the hope that such antibodies would maintain the full tolerance-inducing capacity of aCD154, while eliminating its toxicity. Such an antibody will realize the full potential of this extraordinary target and prove to be an invaluable therapeutic agent for the treatment of an extremely broad spectrum of immune-related diseases wherein compounds that antagonize CD40L/CD40 signaling may be used to intervene in the disease process.

[0039] Previously studies in NHP using aglycosylated aCD 154 ( CD 154agly ) antibodies that do not effectively bind complement or FcR have suggested that the toxicities associated with aCD154 may have been eliminated. However, these same studies suggest that while aCD154agly reduces toxicity, it eliminates the ability of the antibody to induce tolerance (Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D. M, Shapiro, R. I, Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly, L. C. and Taylor, F. R., The contribution of Fc effector mechanisms in the efficacy of anti-CD 154 immunotherapy depends on the nature of the immune challenge. Int Immunol 2004. 16: 1583-1594) . This impairment of functionality (tolerance induction) suggested that complement binding and activation is essential for the ability of aCD154 to induce tolerance.

[0040] However, notwithstanding the foregoing, the present inventor hypothesized that complement binding may not be essential to the ability of a mutated anti-CD 154 antibody to induce tolerance may as tolerance may be assessed in different ways and using different models. Accordingly, it was theorized that the reported results as to the involvement of complement in tolerance may be erroneous or perhaps overstated. Also, it was theorized that the toxicity of aCD 154 which results in thrombosis may be addressed by introducing mutations which eliminate FcR binding and that such changes may not impair functionality, i.e., the antibody's ability to induce tolerance. Therefore, it was hoped that the disruption of FcR binding would eliminate thromboembolic events without causing adverse effects on antibody functionality.

However, this was not assured absent testing in an appropriate animal model.

[0041] In this regard, recently a rodent animal model for assaying

thrombocytopenia and thrombosis was developed by cloning human FcRs into a rodent. This animal model is disclosed in the experimental examples infra and confirmed the inventor's hope, i.e., that anti-CD154 antibodies may be mutagenized at specific sites to eliminate FcR binding and/or complement binding to eliminate toxicity without loss of functionality. .

[0042] Accordingly, based on the foregoing, mutated versions of a hlgGl ((γ-ι ,

ΤΓ°_> Yi^"FcR, yi^{"c FcR}) specific to CD154 with disruptions in complement binding and/or FcR binding are disclosed herein.

[0043] These mutants were tested in order to asses whether efficacy and toxicity of aCD154 are dependent on complement binding and FcR binding, respectively or are maintained in the absence of either or both. Each of the four engineered forms of aCD154 is tested for their ability to induce tolerance and their propensity to induce thromboembolic events in murine models.

[0044] The tolerance inducing effects of these aCD154 variants are evaluated in a well-studied model of haplo-mismatched skin allograft survival, where long-term tolerance is induced by the administration of aCD154 and alloantigen. The

thrombo embolic activities of otCD154 is tested in a murine model expressing the human FcocRIIA receptor that reproduces the events observed in NHP (Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D. M., Shapiro, R. I., Kenyon, N. S., Harlan, D. M._} Kirk, A. D., Burkly, L. C. and Taylor, F. R., The contribution of Fc effector mechanisms in the efficacy of anti-CD 154 immunotherapy depends on the nature of the immune challenge. Int Immunol 2004. 16: 1583-1594). In such mice, treatment with aCD154 induces pulmonary thrombi; therefore we evaluated therein the effect of the loss of FcR binding, as well as the loss of complement binding, and based thereon identify mutations that result in the eradication of the toxicity associated with aCD154 therapy.

[0045] As shown by the results in the experimental examples infra, the present inventor has surprisingly proven that complement binding is not required for the ability

of an aCD154 to induce T cell tolerance. Also, the results indicate that some, but not all mutations that have been suggested to impact FcR binding, eliminate the thromboembolic effects of aCD154. Based thereon, those antibodies containing appropriate mutations are well suited for use in anti-CD 154 therapies such as described below.

[0046] CD154 and aCD154 in experimental models of autoimmunity and graft rejection and its efficacy in human trials. Multiple sclerosis (EAE) Mouse, Human

CD 154 is a 39 kDa type II transmembrane protein Rheumatoid arthritis Mouse

Inflammatory bowel disease Mouse belonging to the Tumor Necrosis Factor (TNF) Thyroiditis Mouse

Systemic Lupus Erythematosa Mouse, Human superfamily, and binds to its receptor, CD40. CD 154 Autoimmune thrombocytopenia Human

Diabetes Mouse is transiently upregulated on the surface of activated Graft vs. host disease Mouse

Kidney transplantation Monkey mature CD4^÷ T lymphocytes. Its expression has Skin transplantation Mouse, Monkey

B transplantation Mouse

Atherosclerosis Mouse since been confirmed on ThO, Thl, Th2, Thl7, T^reg,

Table 1. Diseases and species that demonstrate CD8⁺, activated CD4^'CD8^' T cells expressing γ/δ or efficacy of aCD154.

α/β TCRs, as well as many other hematopoietic

cells. Expression of CD154 on the surface of activated T cells is critical for the

development of both humoral and cell-mediated immunity (U.S. Patent No. 6,444,018).

[0047] As such, CD154 is an extremely attractive target for immune

intervention in a wide spectrum of autoimmune, and graft-related diseases. Virtually all models of autoimmune disease in mice (see Table 1) are therapeutically ameliorated by

aCD154 treatment. Furthermore, the efficacy in mouse models has translated extremely well into man, as treatment of MS, Lupus and ITP all have documented efficacy of

ahuman CD 154 in clinical trials. [0048] Beyond simply blocking CD154-CD40 interactions, aCD154 therapy leads to the induction of immunologic tolerance (Prevention of transplant rejection by blocking CD40-CD154 interactions has been repeatedly documented for the induction of long-term tolerance to skin, Gordon, E. J., Markees, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P., Rossini, A. A. and Greiner, D. L., 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, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P., Greiner, D. L. and Rossini, A. A., Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand. Transplantation 1997. 64: 329-335; Jarvinen, L. Z., Blazar, B. R.₅ Adeyi, O. A., Strom, T. B. and Noelle, R. I, 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-CD 154 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., islets (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 a myriad of other transplanted organs (Camirand, G., Caron, N. J., Turgeon, N. A., Rossini, A. A. and Tremblay, J. P., Treatment with anti-CD 154 antibody and donor- specific transfusion prevents acute rejection of myoblast transplantation. Transplantation 2002. 73: 453-461; Tung, T. H., Mackinnon, S. E. and Mohanakumar, T., Long-term limb allograft survival using anti-CD154 antibody in a murine model. Transplantation 2003. 75: 644-650). Furthermore, ahuman CD 154 in NHP has been shown to induce long-term tolerance to allogeneic skin transplants.

[0049] As noted above, prior to the present invention it was thought that C was involved (required) for mediating graft tolerance. More specifically it was thought that CD154 must accomplish two things to induce tolerance, prevent inflammation and activate C\ Surprisingly, this is not the case.

[0050] This was theorized in part because previous aglycosylated antibodies that have resulted in complete disabling of the Fc region of CD154 have eradicated toxicity, but at a cost to tolerance inducing efficacy. Only exti'emely high levels in mice (50 mg kg x 3) of Fc disabled ocCD154 has been shown to induce tolerance, but lower doses (20 mg/kg) in monkeys clearly could not induce tolerance. (However, this is too high a dose to be clinically feasible).

[0051] Quite surprisingly the present inventor has discovered that neither C activating activity, nor binding to FcR, is necessary for an anti-CD 154 antibody to be therapeutically effective (induce tolerance) and that antibodies which comprise specific mutations that eliminate or reduce FcR binding do not elicit thrombolytic or

thrombocytopenia and therefore will be both effective and safe.

[0052] Assessing aCD154 toxicity in mice

[0053] Earlier studies clearly documented the tnrombogenic activities of anti-

CD154 mAbs in cynomolgus monkeys.

[0054] However, in evaluating engineered forms of anti-CD 154 mAbs, studies in NHP is a costly and cumbersome approach. Therefore, less cumbersome and costly methods would be desirable such as assays using transgenic rodents. [0055] With respect thereto, it is believed that the binding of anti-Cl 54- sCD154 (soluble (s) CD 154 is present in serum) immune complexes (IC) to platelets may be the basis for the thrombogenic activity of anti-D154 mAbs. Studies have shown that anti-CD 154 IC activate platelets in vitro via the IgG receptor (human FcyRUA) (Langer, F., Ingersoll, S. B., Amirkhosravi, A., Meyer, T., Siddiqui, F. A., Ahmad, S., Walker, J. M., Amaya, M., Desai, H. and Francis, J. L., The role of CD40 in CD40L- and antibody- mediated platelet activation. Thrombosis and homeostasis 2005. 93: 1137-1146.) on platelets and could cause thrombi formation. The prothrombotic effects of anti-CD 154 (using a human IgGl variant of MR1) also have been evaluated in vivo using hFcyRIIA transgenic mice(Robles-Carrillo, L., Meyer, T., Hatfield, M., Desai, H., Davila, M., Langer, F., Amaya, M., Garber, E., Francis, J. L., Hsu, Y. M. and Amirkhosravi, A., Anti-CD40L immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol 2010. 185: 1577-1583). These mice were produced because mice do not express FcyRIIA on platelets. Upon injection of hIgGl/D154-sCD154 IC, mice developed pulmonary thrombi consisting of platelet aggregates and fibrin, similar to that observed in NHP treated with anti- Use of aglycosylated anti-CD 154 (hlgGlMRlagly) did not induce pulmonary thrombi. We therefore elected to use this in vivo rodent assay to test the prothrombotic activity of different engineered human IgGi aCD154.

[0056] Therapeutic Applications of CD154 Antibodies of the Invention

[0057] As a category, there are nearly 50 million people in the US suffering from the 100+ known autoimmune diseases. Treatment costs are estimated to be over $100B/year and that figure is likely an underestimate. Costs for the 7 major autoimmune diseases (IBD, Lupus, MS, RA, psoriasis and scleroderma) alone are estimated to range between $ 1-70.6B/yr. In 2008, there were 23, 288 transplants performed in the US. With an average cost of $22, 350/yr, over $500M/yr is spent on immunosuppression post- transplant. [0058] ccCD 154 is potentially one of the most therapeutically valuable drugs for the treatment of autoimmunity and graft rejection. In addition to the demonstrated clinical efficacy seen in Lupus and ITP, we completed a Phase I Clinical Trial in remitting/relapsing (RR) MS. While only a small cohort of patients was treated (12), the results of the trial were striking. The conclusions of the trial were that 4 weekly treatments with IDEC-131 resulted in: 1) No significant changes in EDSS from baseline to 5 years for all doses; 2) Improved EDSS correlated with increased dose and 3) Long- term follow up demonstrated a profound reduction in clinical relapse rate that compares favorably to current IMD. As a result of this trial, we were awarded an NIH grant to execute a Phase II Clinical Trial in R/R MS but due to toxicity associated with aCD154 seen in other trials, the CD154 became unavailable (for a more complete description see³⁶). It is clear if toxicity can be resolved, and efficacy sustained, aCD154 is a viable and attractive therapeutic that will re-enter human Trials. MS will be our first indication that we target for commercial development.

[0059] There is a wealth of data indicating that complement activation is critical for the induction of tolerance by CD154. Studies in complement deficient mice clearly show that CD154 is completely ineffective at inducing tolerance. While this has been interpreted as resulting from complement-mediated elimination of activated T cells, this cannot be true. We believe that C activation at the cell surface by aCD154 facilitates the generation of adaptive T^re and explains the basis for why aglycosylated aCD154 mAbs in NHP are ineffective at inducing tolerance. Based thereon the subject antibodies will be safe and effective and these aCD154 mAbs useful for immune intervention.

[0060] Engineering safe, tolerance-inducing CD154.

[0061] To demonstrate efficacy a model antibody, MR1, was chimerized and engineered to eliminate or reduce FcR binding or complement binding. Studies have shown that the human IgGl version of MR1 is thrombogenic (Robles-Carrillo, L., Meyer, T., Hatfield, M., Desai, H., Davila, M., Langer, F., Amaya, M., Garber, E., Francis, J. L., Hsu, Y. M. and Amirkhosravi, A., Anti-CD154 immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol 2010. 185: 1577-1583) and that it can induce tolerance (Daley, S. R._} Cobbold, S. P. and Waldmann, H., Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance. Am J Transplant 2008. 8: 2265-2271).

[0062] We will therefore produce a chimeric hlgGl form of MR1 , and then engineer mutations in the hlgGl Fc region that disrupt CI q binding and/or FcR binding. If this is shown to be safe and effective, i.e., eliminates thrombotic properties while maintaining tolerogenic properties this demonstrates that other anti-CD154 antibodies, particularly those that bind human CD 154 may be synthesized by engineering similar mutations in the antibody constant region that eliminate FcR binding and optionally complement binding, which eliminate or reduce thrombosis or thrombocytopenia, while maintaining the antibody's ability to induce tolerance.

[0063] As disclosed in the working examples, the first step in engineering the hamster amurine CD 154 into a human IgGl is to clone and sequence the and γ heavy chains. This has been accomplished and the sequences are in Figure 2.

[0064] The generation and characterization of a series of Fc and C variants of the hlgGl form of MR1 is then performed. Mutagenesis of residue 322 from K -> A (K322A) of IgGl has been shown to abrogate complement activation. It has been shown that this variant binds human complement Clq with greatly lowered affinity and to inefficiently activate human C'(Hessell, A. J., Hangartner, L., Hunter, M., Havenith, C. E., Beurskens, F. J., Bakker, J. M., Lanigan, C. M., Landucci, G., Forthal, D. N., Parren, P. W., Marx, P. A. and Burton, D. R., Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007. 449: 101-104).

[0065] In addition the antibody was engineered in an effort to eliminate or reduce FcR binding in a manner that eliminates thrombotic or clotting toxic reactions in vivo while not impacting its desired effects on immunity such as tolerance. In this regard, specific residues in the Fc region, if mutated, have been reported to eliminate or reduce FcR binding. However, the effects of such modifications on anti-CD 154 antibody functionality (ability to induce antigen-specific tolerance) and toxicity (thrombosis) of anti-CD 154 antibodies were uncertain.

[0066] Examples of such sites are reported in patent and non-patent literature.

For example, Shields RL, Namenuk AK, Hong K, et al. (High resolution mapping of the binding site on Human IgGl for Fc for FcyRI, Fc for FcyRII, Fc for FcyRIII, and FcRn) report the design of IgGl variants with impaired binding to the Fc for FcyR. J BiolChem 2001; 276: 6591-604) In addition, some patents (US20070237767 and US20100104564) describe Fc mutagenesis.

[0067] Mutations reported to significantly reduce FcR binding are summarized below. Reported activities are conveyed as relative folds comparing to the wild type Fc.

• Table 2 Shields' 2001 paper

• Table 3 US20100104564

• Table 4 US20070237767

[0068] General Description of Inventive Methods

[0069] Preparation of MRl variants.

[0070] DNA encoding VH and VL of hamster otmurine CD 154 were cloned and fused to the human γ 1 CHI, ¾2, CH3 region or to described variants. The nucleotide sequences was verified using Megabace™ sequence analyzer. A plasmid expression vector, pEE12 containing both heavy and light chains of each of the MRl variants will be transfected into NSO cells and products purified by Protein A chromatography.

[0071] Binding to CD154.

[0072] Comparison of the binding activity of CD 154 antibody variants was determined by their binding to CHO cells transfected with mouse CD 154. CD 154- expressing CHO cells will be incubated with biotin-labeled aCD154 in the presence of unlabeled aCD154 heavy chain variants or isotype-matched antibodies for 1 hr at 4°C. Binding of biotinylated MRl will be detected using a streptavidin conjugated fluorochrome and flow cytometry will be performed. The percent of inhibition by variants will be deduced by recording reductions in the mean fluorescence intensity of MRl stained cells.

[0073] Antibody half-life using ELISA

[0074] An ahuman IgGl ELISA will be used to determine the half-life of all the IgGl variants. Serum concentrations of hlgGl will be determined over 1 month post- administration.

[0075] Binding of variants to FcRs.

[0076] Binding of each of the variant MRl IgGl mAbs to FcRs is determined by a solid phase assay. Briefly, Maxisorb ELISA plates will be coated with mouse or human FcyRI, FcyRIIA, FcyRIIB, or FcyRIIIA (R & D Systems). We will prepare biotinylated versions of the MRl variants γΐ (WT), yl^'c (K322A), yr^FcR (E233P), yL^{c " FcR} (K322A, E233P). Binding is determined by colorimetric detection using enzyme- coupled avidin. Reduction in binding is determined for each of the variants compared to the WT γΐ molecule.

[0077] Binding of aCD154 mAbs to human Clq

[0078] Purified human Clq will be titrated into wells in which the IgGl variants of MRl have been absorbed onto Maxisorb ELISA plates. Bound Cl will be detected with HRP-chicken anti-Clq. All variants will be compared to the binding of Clq to the WT IgGl MRl, as described (Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M._; Garber, E. A., Hess, D. M.₅ Shapiro, R. L, Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly, L. C. and Taylor, F. R., The contribution of Fc effector mechanisms in the efficacy of anti-CD 154 immunotherapy depends on the nature of the immune challenge. Int Immunol 2004. 16: 1583-1594.; and Taylor, P. A., Lees, C. J., Wilson, J. M., Ehrhardt, M. J., Campbell, M. T., Noelle, R. J. and Blazar, B. R., Combined effects of calcineurin inhibitors or sirolimus with anti-CD40L mAb on

alloengi'aftment under nonmyeloablative conditions. Blood 2002. 100: 3400-3407.

Induction of tolerance with mutant aCD154 mAbs. The hamster anti-murine CD 154 that was produced in our laboratory 40 MR1 routinely induces long-lived graft tolerance, as we have shown (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; Quezada, S. A., Bennett, K., Blazar, B. R., Rudensky, A. Y., 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, A. A., Parker, D. C, Phillips, N. E., Durie, F. H., Noelle, R. I, Mordes, J. P. and Greiner, D. L., Induction of immunological tolerance to islet allografts. Cell Transplant 1996. 5: 49-52). Tolerance is induced by the co-administration of alloantigen (in the form of donor spleen cells) and CD154. It has been shown that a humanized IgGl form of MR1 also induces graft tolerance 24, and therefore the WT al variant will serve as a positive control for tolerance

Table 5. Experimental groups to induction. The four hlgGl versions of MR1 (γ,, γ,^'0, y ^cR, y ^CIFcR) determine the tolerogenic activity of

□nti-CD154 variants will be tested for their ability to induce graft tolerance (see Table

5). Skin grafting is performed as a modification of the technique used by Markees et al.12. Briefly, age-matched male CB6F1 mice will be used as donors for both spleen cells (DST) and skin grafts. Recipient C57BL/6 mice will injected with or without 5 x 107 DST cells in 500 μΙ_, Hanks balanced salt solution by tail vein injection (intravenously) and 500 μg of CD154 (MR1 or yi, yi^~c, yi^"FcR, yi^"c/FcR)) or control immunoglobulin, hamster or human, (HIgGI) in phosphate-buffered saline (PBS) intraperitoneally on days -3, -5 and -7. Mice will treated with the appropriate antibody (250 ag/injection) 3 times per week, thereafter for the duration of the experiment. On day 0, recipient mice will be anesthetized with 50 μg per gram body weight of each of ketamine and xylazine injected intraperitoneally (15 mg/mL in PBS), and CB6F1 skin grafts will be prepared using established methods. Rejection will be defined as the day on which less than 20% of the skin graft remains. Animals will be evaluated for skin graft rejection for 100 days. In addition, for each of the tolerant groups, skin grafts will be taken at day 100 and evaluated by histochemistry for leukocyte infiltrates and scored based on the number of cells/area measured. Finally, third party transplants (H-2Kskin) will be transplanted on tolerized mice (in selected groups) to assure that the tolerance induced is antigen specific, as has been published previously in this system( Markees, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P., Greiner, D. L. and Rossini, A. A., Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand. Transplantation 1997. 64: 329-335, Markees, T., Phillips, N., Gordon, E., Noelle, R. J., Mordes, J. P., Greiner, D. L. and Rossini, A. A., Improved skin allograft tolerance induced by treatment with donor splenocytes and an extended course of anti-CD154 monoclonal antibody. Transplant Proc 1998. 30: 2444- 2446; Markees, T. G., Appel, M. C, Noelle, R. J., Mordes, J. P., Greiner, D. L. and Rossini, A. A., Tolerance to islet xenografts induced by dual manipulation of antigen presentation and co-stimulation. Transplantation Proceedings 1996. 28: 814-815) of humoral immunity with mutant aCD154 mAbs.

[0079] In addition to measuring the impact of yi , yi^'c, yf^FcR, y₁ ^{'c FcR} on tolerance, we also will measure the impact of antibody treatment on the development of primary and secondary humoral immune responses, as we have previously described 45- 47. Briefly, mice (4/group) will be immunized with chicken ovalbumin in CFA (200 ag/mouse) and treated with the MR1 variants (200 ag/mouse x 3 times/ week). On days 7, 14 and 21, IgM and IgG anti-OVA will be measured by a standardized anti-OVA ELISA and serum concentrations of anti-OVA will be quantified. It is anticipated that all of the variants will be effective at inhibiting humoral immunity.

[0080] Toxicity studies with mutant aCD154 mAbs.

[0081] The thrombogenic activity of CDl 54 has been demonstrated in a murine model using mice that express human FcyRllA. This model parallels toxicity findings in NHP using both intact and aglycosylated forms of anti-human CD 154.

Briefly, mice will be injected with preformed immune complexes (IC) of sCD154 (R & D Systems) and each variant of CD154 (138 g mAb and 5C^g Ag, approximating 500nM IC at a 1:3 (mAb/Ag) stoichiometric ratio). Following injection, if the mixture is thrombolytic, mice will exhibit prolonged disorientation, shallow breathing, and impaired mobility. Those exhibiting this activity are expected to have marked reductions in platelet counts. After 60 minutes, lungs will be harvested, fixed in formalin, sectioned and H&E- stained. Mouse lung sections will be evaluated for evidence of thrombosis (as measured by intravascular thrombi) and the number of thrombi/section will be counted. For each mouse, 10 sections will be counted and the total number of thrombi compared across all groups treated with the various variants of IgGl MR1. hi addition, total platelet counts (harvested by cardiac puncture at the time of euthanasia), will be evaluated by flow cytometry, and are expected to drop by 80% using those antibodies that are

thrombogenic. These findings will determine which of the MR1 variants are

thrombogenic and if alteration of the FcR binding alters this activity.

[0082] Blocking the development ofa T cell mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE).

[0083] It has not been reported that C activation is critical for anti- CD154 induced protection in EAE. Our data show that short-term intervention leads to long term remission, which suggests that it induces tolerance. It has been reported that MR1^aB,ys inhibits EAE49, however, this specific mAb only had a 50% reduction in Clq. . As we have extensive experience in anti-CD 154 in treatment of EAE ( we will evaluate each of the variants in this disease model) to address the potential of each in blocking cell- mediated immunity.

[0084] Female C57BL/6 mice 5-8 weeks old will be immunized

subcutaneously with 200 μg of MOG35-55 peptide emulsified in CFA supplemented with 5 mg/ml of Mycobacterium tuberculosis. The mice will receive intraperitoneal injections with 250 ng pertussis toxin at the time of immunization and 48 hours later. After 7 days, the mice will receive an identical booster immunization with MOG/CFA without pertussis toxin. Clinical disease usually commences between day 16 and day 20 after immunization. Mice will be administered each of the MRl variants, human IgG (as control for the variants), hamster Ig (as control for MRl) or hamster MRl (200 μg/mouse 3x/week) for the duration of the experiment (50 days).

[0085] Clinical evaluation. Mice will be scored four times per week as follows:

0, no detectable signs of EAE; 0.5, limp distal tail; 1, complete 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, total paralysis of both forelimbs and hind limbs; 5, death. Mice scoring greater than 4 but less than 5 will be euthanized.

[0086] Determination of toxicity

[0087] This will be assessed in a rodent engineered to express human FcRs. A desired antibody according to the invention will have greatly reduced or no toxicity in the disclosed thrombotic animal model.

[0088] Determination of efficacy

[0089] Efficacy (induction of tolerance) will be assessed in the disclosed skin graft model of tolerance. [0090] The following examples illustrate the efficacy of the invention in developing safe and improved, functionally active anti-CD 154 antibodies for use in immune therapies.

[0091] Example 1: . Design of Anti-CD 154 Antibodies with Impaired FcyR

Binding Activities And Functional Properties

Assessment of the capacity of MR1 and MRl-derived monoclonal anti-mouse CD154 antibodies to activate platelets in mice transgenic for human FcyRUA

[0092] As discussed herein, in early clinical trials, it was reported that mAbs targeting CD 154, which is important in autoimmune and other diseases, displayed an unexpected association with thrombosis (induced blood clots which may cause death or stroke). The mechanisms by which such mAbs are apparently associated with thi mbosis were unknown, in part because the disease conditions in which they were used are independently associated with thrombosis. Additionally, there is no known molecular mechanism by which antibodies directly activate coagulation (i.e., the blood clotting system that drives thrombosis); hence, one or more components intermediary between therapeutic mAbs and coagulation per se must be involved. In the case of heparin- induced thrombocytopenia (HIT), a single intermediary component has been identified: the platelet IgG receptor, FcyRIIa.

[0093] HIT is a drug-induced thrombotic autoimmune syndrome in which IgG antibodies can induce a thrombotic state in patients— not by directly activating coagulation, but rather by forming immune complexes (ICs) with a platelet antigen target, PF4 (bound to the drug, heparin), and subsequently activating platelet FcyRIIa, which leads to multiple platelet-dependent prothrombotic processes, including coagulation activation and thrombosis. Attempts to replicate HIT's thrombotic processes in a mouse model were hindered by the fact that mice lack the equivalent of the human FcyRILA gene. McKenzie and colleagues thus made mice transgenic for human FcyRIIa (FCGR2A mice) and went on to demonstrate that the HIT thrombotic phenotype could be fully replicated in FCGR2A mice, but not in mice lacking this IgG receptor McKenzie SE, Taylor SM, Malladi P, Yuhan H, Cassel DL, Chien P, Schwartz E, Schreiber AD, Surrey S, Reilly MP. The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: a transgenic mouse model. J Immunol. 1999;162:4311-8).

[0094] It was later shown that anti-CD 154 mAbs, when combined with CD 154

(human or mouse), rapidly induced thrombocytopenia and thrombosis in FCGR2A, but not wild type (WT) mice. (Robles-Carrillo L, Meyer T, Hatfield M, Desai H, Davila M, Langer F, Amaya M, Garber E, Francis JL, Hsu YM, Amirkhosravi A. Anti-CD 154 immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol. 2010;185: 1577-83). These studies suggested that any therapeutic mAb associated with thrombosis may depend, at least in part, on the activation of the platelet IgG receptor. It will thus be informative to evaluate the platelet- activating capacity of anti-CD 154 mAbs being developed for therapeutic uses by treating FCGR2A mice with such mAbs, and subsequently identifying how this affects, if at all, the onset of thrombocytopenia or thrombosis. Such testing will be particularly useful for anti-CD 154 mAbs that have been engineered to have reduced capacity for triggering FcyRIIa-dependent platelet activation.

[0095] It has been reported that a humanized form of M 1, when combined with its antigen target, mouse CD 154, rapidly induced severe thrombocytopenia (loss of circulating platelets) and pulmonary thrombosis in FCGR2A mice. (Robles-Carrillo L, Meyer T, Hatfield M, Desai H, Davila M, Langer F, Amaya M, Garber E, Francis JL, Hsu YM, Amirkhosravi A. Anti-CD 154 immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol. 2010;185:1577-83) In this same study, an aglycosylated humanized anti-mouse CD 154 mAb, MR1, which is presumed to have greatly reduced capacity to activate FcyRIIa, did not induce thrombocytopenia or thrombosis.

[0096] In the experiments herein, we tested variants of monoclonal anti-mouse

CD154, MR1, and derivatives thereof, in the above-described FCGR2A mouse model of thrombosis. The specific aim of this study was to inject FCGR2A mice with refomed ICs consisting of mouse CD154 plus MRl or various MRl derivatives and to identify: (1) any possible evidence of thrombocytopenia, (2) any possible evidence of pulmonary thrombosis, and (3) any possible behavioral signs of thrombotic stress subsequent to IC- induced platelet activation.

[0097] Materials and Methods

[0098] Materials:

[0099] Four anti-mouse CD154 antibodies were tested in FCGR2A mice:

[00100] PBS (baseline controls used for comparison with test mAbs, below)

[00101] MR1-WT (a humanized MRl anti-mouse CD154 mAb)

[00102] N325L (a variant of MR1-WT)

[00103] K326V (a variant of MR1-WT)

[00104] E269R (a variant of MRl -WT)

[00105] Murine soluble CD154 (or "sCD154") was purchased from Peprotech,

Inc. (Rocky Hill, NJ).

[00106] Methods:

[00107] Preparation and delivery of immune complexes (IC): Mouse sCD154

(60 μ ) was combined with anti-CD154 mAb (175 μg) in PBS to prepare 250 μΐ volume of mCD154+anti-CD154 IC solution, 200 μΐ, of which was injected intravenously into each FCGR2A mouse within 5 minutes of IC preparation.

[00108] Experimental animals:

[00109] Twenty four FCGR2A mice (8-12 week old, male or female) mice were divided into five groups (one per test mAb, and one PBS negative control) of six animals per group. The genotype of all FCGR2A animals used in the study were verified by PCR as per Tax Labs protocol. [00110] Intravenous injection of IC:

[00111] Unanesthetized mice were restrained in a standard mouse restrainer.

The lateral tail vein was dilated by warming with a heat lamp. IC solutions were then injected slowly (~10 seconds), and mice were transferred immediately to an empty cage for observation.

[00112] Observation of symptoms:

[00113] Following IC injection, each mouse was continuously monitored in isolation for ten minutes. During this period, observers assessed and recorded the mice's locomotion, gait, breathing, and monitored the mice for signs of thrombotic stress (such as disorientation and partial or temporary paralysis). Four categories were used to summarize the complex of symptoms observed in test animals: (1) None - no

abnormalities in locomotion, gait, breathing, and no sign of disorientation or paralysis; (2) Mild - no sign of disorientation or paralysis, normal locomotion, but signs of lethargy and rapid breathing; (3) Moderate - lethargy, rapid breathing, disruption of locomotion except following contact by observer; (4) Severe— disorientation, signs of paralysis or complete immobility.

[00114] Blood collection and platelet counting:

[00115] Ten minutes after IC injection, mice were anesthetized by isoflurane and approximately 500 μΐ of blood was collected into citrate anticoagulant by cardiac puncture using a 25 gauge needle. Platelet counts were determined electronically using an Coulter Act diff Counter within 2 minutes of blood collection. Platelet counts were adjusted for the volume of citrate in the collection tube and recorded for each animal.

[00116] Assessment of thrombosis in the pulmonary vasculature:

[00117] Immediately after blood draw, entire lungs were dissected, rinsed in

PBS buffer, and placed in buffered formalin. Twenty four hours later, paraffin blocks were prepared and 3 Dm slide sections were cut and stained with hematoxylin and eosin (H&E) for histological evaluation for the presence of thrombi. Five slides were prepared from the mid-organ region of each lung with spacing between cut section of

approximately 50-100 μιτι. Each slide was assessed by two independent observers blinded to the identity of the animal groups from which the slides were prepared. Five randomly chosen fields were assessed per slide. In cases where greater than 9 thrombi were observed per field, no attempt was made to determine the precise number of thrombi, and the value of 10 (ten) was entered as the nominal observation.

[00118] Statistical Analysis:

[00119] Data were analyzed by SigmaPlot .. Platelet counts and number of clots/field between groups were analyzed using the Kruskal-Wallis One Way Analysis of Variance on Ranks.

[00120] Results

[00121] The first group of animals were injected with PBS (200 iL delivered) in order to obtain baseline platelet counts and normal lungs for histological analysis. These values are compared below to test animal groups. Following PBS injection, all animals exhibited normal locomotion, gait, breathing, and showed no signs of thrombotic stress (such as disorientation and partial or temporary paralysis). Animals injected with MR1- WT mAb showed signs of moderate to severe signs of thrombotic stress (Figure 6), which correlated with loss of circulating platelets (Figure 7), and histologic observation of the prevalence of pulmonary thrombi (Figure 8). The injection of animals with N325L and K326V mAbs gave similar results (did not prevent thrombosis) . In many cases, histologic evidence of thrombosis greatly exceeded 10 clots per field. All animals injected with E269R mAb exhibited normal locomotion, gait, breathing, and showed no signs of thrombotic stress. The lung vasculature of all E269R-injected mice were free of thrombi. (See histologic data also in Figures 9-13) [00122] It should be noted that two of six mice injected with N325L did not experience thrombotic thrombocytopenia. The causes of these anomalies are unknown; however, in our experience, such occasional outliers can occur in experiments of this type. On the other hand, because the platelet counts correlated with the relative absence of pulmonary thrombi from these two mice, the data were included in the statistical analysis comparing the experimental groups.

[00123] Conclusions

[00124] In this mouse model of antibody-induced thrombocytopenia and thrombosis, MRl-WT, N325L, and K326V demonstrated potent activity, whereas E269R lacked activity and was comparable by all measures with the PBS negative control group.

[00125] Example 2: . Design of Anti-CD154 Antibodies with Impaired CDC

Activities And Functional Properties

[00126] Cloning and Synthesis of Chimeric Anti-CD154 Antibody (MR1) with Human IgG Constant Regions

[00127] It was initially theorized by the present inventor, in part based on prior literature, that anti-CD 154 antibodies lose their ability to induce tolerance when the CI q binding site is mutated Based thereon, we assumed that a model anti-CD 154 antibody, i.e., the murine anti-CD 154 (MR1) having the variable heavy and light sequences in Figure 2 would lose its ability to induce tolerance when the Clq binding site is mutated.

[00128] To this end, MR1 was converted into a human IgGl . It has previously reported that a human IgGl version of MR1 can induce tolerance (Daley, S.R., Cobbold, S.P. & Waldmann, H. Fc-disabled anti-mouse CD 154 antibodies retain efficacy in promoting transplantation tolerance. Am J Transplants, 2265-2271 (2008)). As described below a chimeric hlgGl form of MR1 was produced and then engineered to introduce mutations in the hlgGl Fc region that disrupt Clq binding. [00129] The first step in engineering the hamster anti-murine CD 154 into a human IgGl (MR1 hlgGl) is to clone and sequence the light and heavy chains of MR1, DNA encoding VH and VL of hamster anti-CD 154 MR1 have been cloned and fused to the human γ 1 CHI, CH2, CH3 region or to valiants described below. The nucleotide sequences have been verified using Megabace™ sequence analyzer and are shown in Figure 2. A plasmid expression vector, pEE12 containing both light and heavy chains of each of the MR1 variants was transfected into NSO cells and products purified by Protein A chromatography.

[00130] The generation and characterization of a series of C variants of the hlgGl form of MR1 was then effected.

[00131] Designing Fc variants with impaired CDC.

[00132] No single or combinations of Fc mutations have been reported to ablate the CDC activity while maintaining near wild type ADCC activity. However, CDC assay conditions may effect this analysis. For example, CDC activities can differ significantly depending on target cells, dilution factors of the complement, and species sources of the complement which could be from human, guinea pig, or rabbit as well as other factors. Given our analysis we believe that the best single and double mutation candidates for impaired CDC activity without significant effects on ADCC are: K322A, P331 G, and P331/K322A.

[00133] Mutagenesis of K322A and P331 G of IgGl have been shown to abrogate complement activation. It has been shown that this variant binds human complement Clq with greatly lowered affinity and inefficiently activates human C. (Hessell, A. J., et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature449, 101-104 (2007)).

[00134] Measurement of loss of Clq binding by MR1 hlgGl mutants. [00135] The binding of Clq to each of the MR1 hlgGl mutants was evaluated. For measuring Clq binding to MR1, purified MR1 variant antibody (Aragen Bioscience, Morgan Hill, CA), was diluted to 100, 10, 1 and 0.1 μ^ιηΐ in phosphate-buffered saline (PBS) to coat a 96-well enzyme-linked immunosorbent assay (ELISA)-grade plate (ThermoScientific, Florence, KY) overnight at 4°C. The plate was then washed three times with PBS-0,05% Tween 20 (Tw20) and blocked for 1 h with 1% bovine serum albumin (BSA)-Tw20-PBS at room temperature. Complement component Clq from human serum (Sigma, St. Louis, MO), was diluted to 1 μ^πύ in 1% BSA-Tw20-PBS then plated and allowed to incubate for 1 h at room temperature. The plate was washed three times with PBS-Tw20, and horseradish peroxidase-labeled sheep anti-human Clq (GenWay Biotech, San Diego, CA) was added. After a 1 h, room temperature incubation, the plate was washed three times with PBS~Tw20, then TMB (3,3',5,5'- tetramethylbenzidine) (ThermoScientific) provided a colorimetric change which was then quantitated at 450 nm by an ELISA reader (BioTek, Winooski, VT).

[00136] The results of these experiments are in Figure 4. As shown therein, all of the mutants had reduced Clq binding.

[00137] Functional studies with mutant anti-CD154 mabs.

[00138] The hamster anti-murine CD154 that was produced in our laboratory 3MR1 routinely induces long-lived graft tolerance, as we have shown (Noelle, R.J., et al.A novel ligand on activated T helper cells binds CD40 and transduces the signal for the cognate activation of B cells.Proc. Natl. Acad. Sci. USA89, 6550-6554 (1992)).

[00139] However, previous reports have suggested that complement deficient anti- Cdl54 antibodies do not elicit tolerance. ( Quezada, S.A., et al Analysis of the underlying cellular mechanisms of anti-CD154-induced graft tolerance: the interplay of clonal anergy and immune regulation. Jlmmunol 175, 771-779 (2005) ; Quezada, S.A., et al. Mechanisms of donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation. Bloodl02, 1920-1926 (2003).; Quezada, SA._} Jarvinen, L.Z., Lind, E.F. & Noelle, RJ. CD40/CD154 Interactions at the Interface of Tolerance and Immunity. Annu Rev Immunol!!, 307-328 (2004); Rossini, A. A., et al Induction of immunological tolerance to islet allografts. Cell Transplants, 49-52 (1996).) Therefore, we assessed whether our mutants were able to elicit tolerance. In these experiments, the four HIgGI versions of MR1 (MR1 WT, K322A, P331G, and

P331/K322A) were tested for their ability to induce graft tolerance and the results of these experiments are in Figure 5.

[00140] Said skin grafting was effected using a modification of the technique developed by Markees et al. (Markees, T.G., et at Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand. Transplantation^, 329-335 (1997)). Briefly, age-matched male CB6F1 mice were used as donors of both spleen cells (DST) and skin grafts. More specifically, Tail skin (μΐ cm2) from CB6F1 (Fl) female donors was transplanted onto the dorsal area of age- matched C57BL/6 females. To induce T cell tolerance, recipients received T-depleted spleen cells (DST) by IV tail injection from Fl donors on day -7 before skin graft (day 0) and 200 μg of MR-1 variants DP on days -7, -5, and -3. Grafts were observed 3 times per week starting on day 8. Grafts were considered rejected when 80% of the original graft disappeared or became necrotic.

[00141] Recipient mice were injected with or without 5 x 10 DST cells in 500 μΐ, Hanks balanced salt solution by tail vein injection (intravenously) and 500 μg of anti- CD 154 or control hamster immunoglobulin (H-Ig) in phosphate-buffered saline (PBS) intraperitoneally. Mice were injected with the MR1 variants or H-Ig 3 times per week for the duration of the experiment. On day 0, recipient mice were anesthetized with 50 μg per gram body weight of each of ketamine and xylazine injected intraperitoneally (15 mg/mL in PBS), and CB6Fi or C57BL/6 skin grafts were prepared using established methods. Rejection was defined as the day on which less than 20% of the skin graft remained.

[00142] Afterward the results were analyzed. Unexpectedly, treatment with control human IgGl and DST did not prolong rejection, as was anticipated. As can be seen in Figure 6, like WT H IgGl MR1, all of the mutant MR1 antibodies induced long lived graft acceptance. Hence, Clq binding and complement activation by anti-CD 154 antibodies IS NOT essential to induce graft tolerance. This is in contrast to what was observed in the complement deficient mice, and suggests that the complement deficient mice likely have some other anomalies that preclude the induction of graft tolerance.

[00143] REFERENCES CITED IN APPLICATION

[00144] The following references are cited. The contents of all are incorporated by reference herein.

1 Noelle, R. J., Mackey, M., Foy, T., Buhlmann, J. and Burns, C, CD40 and its ligand in autoimmunity. Ann N Y Acad Sci 1997. 815: 384-391.

2 Mackey, M. F., Barth, R. J., Jr. and Noelle, R. J., The role of CD40/CD 154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells. J Leukoc Biol 1998. 63: 418-428.

3 Noelle, R. J., CD40 and its ligand in cell-mediated immunity. Agents Actions Suppl 1998. 49: 17-22.

4 Quezada, S. A., Jarvinen, L. Z., Lind, E. F. and Noelle, R. J., CD40/CD154 Interactions at the Interface of Tolerance and Immunity. Annu Rev Immunol 2004. 22: 307-328.

5 Kenyon, N. S., Chatzipetrou, M., Masetti, M., Ranuncoli, A., Oliveira, M.₃ Wagner, J. L., Kirk, A. D., Harlan, D. M., Burkly, L. C. and Ricordi, C, Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A 1999. 96: 8132-8137.

6 Kirk, A. D., Burkly, L. C, Batty, D. S., Baumgartner, R. E., Berning, J. D., Buchanan, K., Fechner, J. H., Jr., Germond, R. L., Kampen, R. L., Patterson, N. B., Swanson, S. J._} Tadaki, D. K., TenHoor, C. N., White, L., Knechtle, S. J. and Harlan, D. M., Treatment with humanized monoclonal antibody against CD 154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 1999. 5: 686-693.

7 Sidiropoulos, P. I. and Boumpas, D. T., Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus 2004. 13: 391-397.

8 Sidiropoulos, P. I. and Boumpas, D. T., Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus 2004. 13: 391-397.

9 Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D. M., Shapiro, R. L, Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly, L.

C. and Taylor, F. R., The contribution of Fc effector mechanisms in the efficacy of anti- CD 154 immunotherapy depends on the nature of the immune challenge. Int Immunol 2004. 16: 1583-1594.

10 U.S. Patent No. 6,444,018

11 Gordon, E. J., Markees, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P., Rossini, A. A. and Greiner, D. L._; Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD 154 monoclonal antibody. Diabetes 1998. 47: 1199-1206.

12 Markees, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P., Greiner,

D. L. and Rossini, A. A., Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 Iigand. Transplantation 1997. 64: 329-335.

13 Jarvinen, L. Z._} Blazar, B. R., Adeyi, O. A., Strom, T. B. and Noelle, R. J., CD 154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance. Transplantation 2003. 76: 1375-1379.

14 Quezada, S. A., Fuller, B._} Jarvinen, L. Z., Gonzalez, M., Blazar, B. R.₃

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.

15 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.

16 Quezada, S., Eckert, M., Schned, A., Noelle, R. J. and Burns, C, Distinct mechanisms of action of anti-CD 154 in early versus late treatment of murine lupus nephritis. Arth Rheum. 2003. in press.

17 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 CD1 4-specific monoclonal antibody, hu5C8, prevents acute rejection of primary skin allografts in nonhuman primates. Transplantation 2001. 72: 1473-1478.

18 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.

19 Wekerle, T. and Sykes, M., Mixed chimerism and transplantation tolerance. Annual review of medicine 2001. 52: 353-370.

20 Camirand, G., Caron, N. J., Turgeon, N. A., Rossini, A. A. and Tremblay, J. P., Treatment with anti-CD 154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation. Transplantation 2002. 73: 453-461.

21 Tung, T. H., Mackinnon, S. E. and Mohanakumar, T., Long-term limb allograft survival using anti-CD40L antibody in a murine model. Transplantation 2003. 75: 644- 650. 22 Koyama, I., Kawai, T., Andrews, D., Boskovic, S., Nadazdin, 0., Wee, S. L., Sogawa, H., Wu, D. L., Smith, R. N., Colvin, R. B., Sachs, D. H. and Cosimi, A. B., Thrombophilia associated with anti-CD 154 monoclonal antibody treatment and its prophylaxis in nonhuman primates. Transplantation 2004. 77: 460-462.

23 Kawai, T., Andrews, D., Colvin, R. B., Sachs, D. H. and Cosimi, A. B.,

Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 2000. 6: 114.

24 Daley, S. R., Cobbold, S. P. and Waldmann, FL, Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance. Am J Transplant 2008. 8: 2265-2271.

25 Sanchez-Fueyo, A., Domenig, C, Strom, T. B. and Zheng, X. X._} The

complement dependent cytotoxicity (CDC) immune effector mechanism contributes to anti-CD 154 induced immunosuppression. Transplantation 2002. 74: 898-900.

26 Monk, N. J., Hargreaves, R. E., Marsh, J. E., Farrar, C. A., Sacks, S. EL, Millrain, M., Simpson, E., Dyson, J. and Jurcevic, S., Fc-dependent depletion of activated T cells occurs through CD40L-specific antibody rather than costimulation blockade. Nat Med 2003. 9: 1275-1280.

27 Tmscott, S. M., Abate, G., Price, J. D., Kemper, C, Atkinson, J. P. and Hoft, D. F., CD46 engagement on human CD4+ T cells produces T regulatory type 1 -like regulation of antimycobacterial T cell responses. Infection and immunity 2010. 78: 5295- 5306.

28 Cardone, J., Le Friec, G., Vantourout, P., Roberts, A., Fuchs, A., Jackson, I., Suddason, T., Lord, G., Atkinson, J. P., Cope, A., Hayday, A. and Kemper, C,

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells. Nature immunology 2010. 11 : 862-871. 29 Fuchs, A., Atkinson, J. P., Fremeaux-Bacchi, V. and Kemper, C, CD46-induced human Treg enhance B-cell responses. European journal of immunology 2009. 39: 3097- 3109.

30 Alford, S. K., Longmore, G. D., Stenson, W. F. and Kemper, C, CD46-induced immunomodulatory CD4+ T cells express the adhesion molecule and chemokine receptor pattern of intestinal T cells. Journal of immunology 2008. 181: 2544-2555.

31 Barchet, W., Price, J. D._; Cella, M._; Colonna, M.₅ MacMillan, S. K. , Cobb, J. P., Thompson, P. A., Murphy, K. M., Atkinson, J. P. and Kemper, C, Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation. Blood 2006. 107: 1497-1504.

32 Liszewski, M. K., Kemper, C, Price, J. D. and Atkinson, J. P., Emerging roles and new functions of CD46. Springer seminars in immunopafhology 2005. 27: 345-358.

33 Kawai, T., Andrews, D., Colvin, R. B., Sachs, D. H. and Cosimi, A. B._;

Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand [In Process Citation]. Nat Med 2000. 6: 114.

34 Langer, F., Ingersoll, S. B., Amirkhosravi, A., Meyer, T., Siddiqui, F. A., Ahmad, S., Walker, J. M., Amaya, M., Desai, H. and Francis, J. L., The role of CD40 in CD40L- and antibody-mediated platelet activation. Thrombosis and haemostasis 2005. 93: 1137- 1146.

35 Robles-Carrillo, L., Meyer, T., Hatfield, M., Desai, H., Davila, M._s Langer, F., Amaya, M., Garber, E., Francis, J. L., Hsu, Y. M. and Amirkhosravi, A., Anti-CD40L immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol 2010. 185: 1577-1583.

36 Couzin, J., Drug discovery. Magnificent obsession. Science 2005. 307: 1712- 1715. 37 Hessell, A. J., Hangartner, L., Hunter, M., Havenith, C. E., Beurskens, F. J., Bakker, J. M., Lanigan, C. M., Landucci, G., Forthal, D. N., Parren, P. W., Marx, P. A. and Burton, D. R,, Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007. 449: 101-104.

38 Armour, K. L., Clark, M. R., Hadley, A. G. and Williamson, L. M., Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol 1999. 29: 2613-2624.

39 Taylor, P. A., Lees, C. J., Wilson, J. M., Ehrhardt, M. J., Campbell, M. T., Noelle, R. J. and Blazai-, B. R., Combined effects of calcineurin inhibitors or sirolimus with anti- CD40L mAb on alloengraftment under nonmyeloablative conditions. Blood 2002. 100: 3400-3407.

40 Noelle, R. J., Roy, M., Shepherd, D. M., Stamenkovic, L, Ledbetter, J. A. and Aruffo, A., A novel ligand on activated T helper cells binds CD40 and transduces the signal for the cognate activation of B cells. Proc. Natl. Acad. Sci. USA 1992. 89: 6550- 6554.

41 Quezada, S. A., Bennett, K., Blazar, B. R., Rudensky, A. Y., Sakaguchi, S. and Noelle, R. J., Analysis of the underlying cellular mechanisms of anti-CD 154-induced graft tolerance: the inteiplay of clonal anergy and immune regulation. J Immunol 2005. 175: 771-779.

42 Rossini, A. A., Parker, D, C, Phillips, N. E., Durie, F. H., Noelle, R. J., Mordes, J. P. and Greiner, D. L., Induction of immunological tolerance to islet allografts. Cell Transplant 1996. 5: 49-52.

43 Markees, T., Phillips, N._} Gordon, E., Noelle, R. J,, Mordes, J. P., Greiner, D. L. and Rossini, A. A., Improved skin allograft tolerance induced by treatment with donor splenocytes and an extended course of anti-CD 154 monoclonal antibody. Transplant Proc 1998. 30: 2444-2446. 44 Markees, T. G., Appel, M. C, Noelle, R. J., Mordes, J. P., Greiner, D. L. and Rossini, A. A., Tolerance to islet xenografts induced by dual manipulation of antigen presentation and co-stimulation. Transplantation Proceedings 1996. 28: 814-815.

45 van den Eertwegh, A. J., Van Meurs, M., Foy, T. ._} Noelle, R. J., Boersma, W. J. and Claassen, E., In vivo gp39-CD40 interactions occur in the non-follicular compartments of the spleen and are essential for thymus dependent antibody responses and germinal center formation. Adv Exp Med Biol 1994. 355: 75-80.

46 van, den, Eertwegh, Aj, Van, M. M, Foy, T. M., Noelle, R. J., Boersma, W. J. and Claassen, E.₃ B vivo gp39-CD40 interactions occur in the non-follicular

compartments of the spleen and are essential for thymus dependent antibody responses and germinal center formation. Advances in experimental medicine and biology 1994. 355: 75-80.

47 Foy, T. M., Laman, J. D., Ledbetter, J. A., Aruffo, A., Claassen, E. and Noelle, R. J., gp39-CD40 interactions are essential for germinal center formation and the

development of B cell memory. J. Exp. Med. 1994. 180: 157-164.

48 Gerritse, K., Laman, J. D., Noelle, R. J., Aruffo, A., Ledbetter, J. A., Boersma, W. J. and Claassen, E._} CD40-CD40 ligand interactions in experimental allergic

encephalomyelitis and multiple sclerosis. National Academy of Sciences, Washington, D.c, Proceedings of the National Academy of Sciences 1996. 93: 2499-2504.

49 Nagelkerken, L., Haspels, I, van Rijs, W., Blauw, B., Ferrant, J. 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-CD 154 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., Vanderhigt, 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.

Claims

1. A human, chimeric or humanized antibody of the human IgGl, IgG2, IgG3 or IgG4 isotype specific to human CD 154 wherein the Fc region of the antibody is mutated to eliminate or substantially reduce FcR binding in a manner that renders it substantially non-thrombogenic in a rodent thrombosis model which is engineered to express human FcR, but which does not substantially impact the ability of the antibody to induce tolerance.

2. The antibody of Claim 1, which is of the IgGl isotype.

3. The antibody of Claim 1, which is of the IgG3 isotype.

4. The human, chimeric or humanized antibody of claim 1, wherein the rodent thrombosis model is a transgenic mouse that expresses human FcyRIIa (FCGR2A mouse).

5. The human, chimeric or humanized antibody of claim 1 , wherein the Fc region of the antibody is mutated to introduce a E269R mutation.

6. The human, chimeric or humanized antibody of claim 5, wherein the Fc region of the antibody is further mutated to introduce one or more other mutations.

7. The human, chimeric or humanized antibody of claim 1, 2, 3, 4, 5 or 6, wherein the antibody is further mutated at one or more sites in the Fc region to eliminate or substantially reduce Clq activity which mutation or mutations also does not substantially impact the ability of the antibody to induce tolerance.

8. The antibody of any one of claims 1-7 which contains the CDRs of IDEC-131 as shown in Figures 17 and 18.

9. The antibody of claim 8, which contains the humanized VL region in Figure 14 or 15 and the humanized VH region in Figure 16.

10. A method of inducing tolerance without eliciting thrombotic events in a patient in need thereof comprising administering to a patient an effective amount of an antibody according to any one of claims 1-9.

11. The method of claim 10 which further includes administration of an antigen.

12. A method of treating autoimmunity, allergy, transplant, GVHD, or inflammation by administering an antibody according to any one of claims 1-10.

13. The method of claim 12, wherein the disease is a T- cell mediated autoimmune disorder.

14. The method of claim 12, wherein the disease is a B- cell mediated autoimmune disorder.

15. The method of claim 12, wherein the disease is an autoimmune disease selected from rheumatoid arthritis, Myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetes mellitus, psoriasis, Addison's disease, and drug-induced autoimmune diseases, e.g., drug-induced lupus.

16. The method of claim 10 or 12 which includes administration of an antigen.

17. The method of claim 10 or 12 or 16 which is used to treat or prevent GVHD, bone marrow transplant (BMT), multiple sclerosis, lupus, ITP, rheumatoid arthritis, asthma., IBD or another inflammatory bowel disorder.