Methods for inducing immune tolerance with engineered anti-CD154 antibodies

Abstract

The present disclosure relates to a method of inducing tolerance during transplantation by administering to a transplant recipient an anti-CD154 antibody with altered (e.g., selectively reduced) effector function and transplanting into the recipient hematopoietic stem cells that produce immune cells that are tolerant to donor organs, donor tissues, or donor cells. The tolerance can be central tolerance, peripheral tolerance, or organ-specific tolerance. A first aspect of the present disclosure provides a method of inducing immune tolerance in a transplant recipient.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 63 / 134,413, filed January 6, 2021, the contents of which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on January 6, 2022, is named 104545-0079-WO1_-_Sequence_Listing.txt, and is 753,073 bytes in size.

[0003] Field of the Disclosure The present disclosure relates to methods of inducing immune tolerance during transplantation by administering to the transplant recipient an anti-CD154 antibody with altered (eg, selectively reduced) effector function. [Background technology]

[0004] Background of the Disclosure CD154 (also known as CD40 ligand, CD40L, gp39, TNF-related activation protein (TRAP), 5c8 antigen, T-BAM) is a protein expressed primarily on activated CD4+ T cells and is recognized as the molecular basis for helper T cell function (Lederman, S., et al. J. Exp. Med. 175:1091-1101 (1992)). CD154 is a member of the TNF superfamily of molecules and is functionally expressed as a homotrimer. However, some of the CD154 units have truncated peptide chains, so the CD154 trimer may also be considered a heterotrimer of elements all encoded by the CD154 gene (Karpusas M, et al. Structure. 3(10):1031-9 (1995);Hsu YM, et al. J Biol Chem. 272(2):911-5 (1997)). CD154 binds to CD40 on antigen-presenting cells (APCs) and has many effects depending on the target cell type. The major binding partner for CD154 is CD40, but other binding partners αMβ2 (Mac-1), α5β1 integrin and αIIbβ3 have also been described (El Fakhry Y, et al. J Biol Chem. 287:18055 (2012);Wolf D, et al. Circ Res. 109:1269 (2011);Michel NA, et al Front Cardiovasc Med. 4:40 (2017)). CD154 acts as a costimulatory molecule for B cells and affects the function of CD4+ follicular helper T cells (TFH cells). In TFH cells, by engaging CD40 on the B cell surface, CD154 promotes B cell maturation and function, thereby facilitating cell-cell communication in the humoral immune response. CD40 triggering by CD154 stimulates adaptive immune system processes in B cells, including immunoglobulin class switch recombination and somatic hypermutation (Lederman S, et al. Curr Opin Hematol. 3(1):77-86 (1996)).For example, absence of CD154 in X-linked Hyper IgM syndrome results in defects in germinal center formation, class switch recombination and antibody affinity maturation. (Webster EA, et al. Arthritis Rheum. 42(6):1291-6 (1999)). CD40-CD154 interactions are involved in normal TB cell interactions, including increased costimulation, T cell priming, cytokine production, antibody class switching and affinity maturation, and antibody production (Lederman, S., et al. J. Exp. Med. 175:1091-1101 (1992);Lederman, S., et al., Journal of Immunol. 149:3817-3826 (1992);Lederman, S., et al., Journal of Immunol. 152:2163 (1994);Cleary, AM, et al., Journal of Immunol., 155:3329-3337 (1995);Muramatsu, MK et al. Cell 102: 553 (2000);Xu Y and Song G, J. Biomed Sci. 11(4):426-38 (2004);Quezada SA et al. al., Annu Rev Immunol. 22:307-28 (2004); and U.S. Patent Nos. 5,474,771; 5,933,816; 6,331,615; 6,340,459; 6,403,091; 6,451, 310; 6,455,044; 6,592,868; 6,610,294; 6,793,924; 7,070,777; and 9,765,150).

[0005] CD154 also interacts with CD40 on activated endothelial cells (Yellin MJ et al., J. Exp. Med 182:1857-1864 (1995)), on activated fibroblasts (Yellin, MJ et al.J Leukoc Biol. 58:209-216 (1995)), on other cell types and in many cancers (Paulie, S, et al. Cancer Immunol Immunother, 20, 23-8 (1985)). Supernatants of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infect human lung epithelial Calu-3 cells and induce CD40 on dendritic cells (Yoshikawa T, et al. J. Virol. 83(7): 3039-3048 (2009)). In the retina, during infection, CD40 is expressed on endothelial cells, Müller glia (retinal macroglia), microglia, ganglion cells, and retinal pigment epithelial cells (Subauste, CS, Front Immunol 10:2958 (2019);Portillo J-AC, et al. J Immunol. 181:8719-26 (2008);Portillo J-AC, et al. Diabetologia. 57:2222-31(2014);Portillo J-AC, et al., Mol Vis.15:1383-9 (2009)). The role of CD154 in the immune response is usually tightly regulated in tissues over time. Dysfunction of the immune response occurs with aberrant CD154 expression and function in certain tissues at certain times, which can lead to syndromes such as acute respiratory distress syndrome (ARDS), autoimmune diseases, vasculopathy, and cancer promotion.

[0006] The soluble form of CD154 (sCD154), generated by shedding of membrane-bound CD154, plays a role in the production of proinflammatory cytokines and is associated with a variety of autoimmune and vascular disorders (Yellin, MJ, et al., J. Immunol. 152:598 (1994);Yacoub D et al., J Biol Chem. 288(50):36083-93 (2013)). Activated platelets produce CD154, and platelet-derived CD154, especially soluble CD154, is associated with pathology. (Henn V, et al. Nature 391:591-594 (1998); Xu H, et al. Transplantation. 72(11):1858-61. (2001); Danese S, et al. Gut. 12(11):3143-51 (2012)).

[0007] Monoclonal antibody 5c8 is a murine anti-human CD154 that potently blocks the function of CD154. (Lederman, S., et al. J. Exp. Med. 175:1091-1101 (1992); Lederman, S., et al., Journal of Immunol. 149:3817-3826 (1992); Lederman, S., et al., Journal of Immunol. 152:2163 (1994); and Cleary, AM, et al., Journal of Immunol., 155:3329-3337 (1995)). Humanized anti-human CD154 IgG1 antibodies (hu5c8, ruplizumab or ANTOVA®) were generated and tested in non-human primates and humans. The crystal structure of hu5c8 demonstrated specific binding of hu5c8 to the CD154 trimer and antibody contacts with CD154 monomers (Karpusas M, et al. Structure. 9(4):321-9. (2001)). Blockade of CD154 has demonstrated efficacy in models of autoimmunity, humoral immunity and allogeneic transplantation (Pierson RN 3rd, et al. Transplantation. 68(11):1800-5 (1999);Chang AC, et al. Transplant Proc. 31(1-2):95 (1999);Kenyon NS, et al. Proc Natl Acad Sci US A. 96(14):8132-7 (1999);Kenyon NS, et al. Diabetes. 48(7):1473-81. (1999);Elster EA, et al. Transplantation. 72(9):1473-8. (2001);Elster EA, et al. Transplant Proc. 33(1-2):675-6 (2001);Cho CS, et al. Transplantation. 72(4):587-97 (2001);Pierson RN 3rd, et al. Immunol Res. 23(2-3):253-62 (2001);Pfeiffer S, et al.J Heart Lung Transplant. 20(2):250 (2001);Xu H, et al. Transplant Proc. 33(1-2):223-4 (2001);Xu H, Transplantation. 74(7):940-3 (2002);Crowe JE Jr, et al. Am J Transplant. 3(6):680-8 (2003);Ferrant JL, et al., International Immunol October 5;11:1583 (2004);Kawai T, et al., Am J Transplant. 4(9):1391-8 (2004);Preston EH, et al. Am J Transplant. 5(5):1032-41 (2005);Xu H, et al. J Immunol. 170(5):2776-82 (2003);Wu G, et al. Xenotransplantation. 12(3):197-208 (2005);Smith RN, Am J Transplant. 6(8):1790-8 (2006);Zhang T, et al. Transplantation. 102(3):e90-e100 (2018)). However, clinical trials of hu5c8 in systemic lupus erythematosus (SLE) (Huang W, et al. Arthritis Rheum. 46(6):1554-62 (2002);Boumpas DT, et al., Arthritis Rheum. 48:719-27. (2003);Grammer AC, et al. J Clin Invest. 112:1506-20. (2003)) and transplantation (Kawai T, et al. Nat Med. 2000;6:114. (2000);Koyama I, et al., Transplantation. 77(3):460-2. (2004)) were halted due to increased incidence of platelet activation and thromboembolic events (Law and Grewal Adv Exp Med Biol.647:8-36 (2009)). One mechanism of platelet activation may involve activation by CD40 and soluble CD154 on platelets (Inwald DP, et al, Circ Res. 92(9):1041-8 (2003)). Several observations suggest that the mechanism of thrombosis in patients treated with anti-CD154 is mediated by Fc gamma RIIa receptor (FcγRIIA, FCGR2A, CD32A)-dependent platelet activation by immune complexes, especially higher order complexes, composed of anti-CD154 antibodies and soluble CD154 (Robles-Carrillo L, et al., J Immunol.185(3):1577-83. (2010)). Eliminating Fc binding to Fc receptors by mutating the Fc region to create aglycosyl hu5c8 (IgG1 N297Q) has been shown to potently reduce or eliminate such thromboembolism (Shock, A, et al., Arthritis Res Ther. 17:234 (2015) and Xie et al., Journal of Immunol. 192(9):4083 (2014)), but also reduced the efficacy of the antibody in inhibiting or preventing transplant rejection in rhesus monkey kidney and islet allograft models (Ferrant JL et al., International Immunol (11):1583 (2004)). Complete elimination of the Fc region, such as in anti-CD154 pegylated Fab' antibody fragments (dapirorizumab pegol (DZP), CDP7657, Biogen and UCB), also reduced the risk of thrombotic events but failed to treat systemic lupus erythematosus (SLE) in a phase IIb clinical trial (Waters J, Biocentury; October 26, (2018)). Kim et al. generated an Fc-silent anti-human CD154 single domain antibody (dAb; BMS-986004; letolizumab, BMS2h-572-633-CT-L2) by fusing the variable domain of an anti-human CD154 dAb with the Fc from CTLA4-Ig (abatacept, ORENCIA®) ​​and a linker termed "CT Long Fc" having the amino acid sequence EPKSSDK (SEQ ID NO: 325) (Kim SC et al., Am J Transplant 17(5):1182-1192 (2017); and U.S. Patent No. 9,765,150). BMS-986004 effectively prevented kidney rejection in non-human primates without platelet activation, thrombosis or thromboembolism (Kim SC et al., Am J Transplant 17(5):1182-1192 (2017)). .

[0008] US Patent No. 9,765,150 also described BMS-986003 (BMS-2h572-633-CT), which shares the same amino acid sequence as BMS-986004, except for a non-native glycine residue at its amino terminus. However, BMS-986003 induced anti-drug antibodies (ADA) in treated monkeys. The ADAs were directed against the dAb (non-Fc) portion of the molecule, and these antibodies were shown to block the binding of BMS-986003 to CD154, suggesting that the ADAs may be neutralizing. Furthermore, these ADAs led to increased BMS-986003 clearance in some monkeys. In contrast, the chimeric mouse 5c8 human IgG1 chimeric antibody showed the expected prolonged plasma half-life. (US Patent No. 9,765,150).

[0009] BMS-986004 has demonstrated efficacy in immune thrombocytopenic purpura (ITP) clinical trials, although the pharmacokinetic profile may be suboptimal (NCT02273960; “Study to Evaluate Safety and Efficacy in Adult Subjects With ITP (ITP)”; results accessed July 1, 2019).

[0010] Antibodies have various effector functions mediated by Fc, including FcγR binding and complement C1q binding (the first component of complement activation) for effector function and complement-dependent cytotoxicity. Engineering of antibodies can result in an increase or decrease of one or more effector functions. In extreme cases, for example in F(ab) constructs, the Fc can be removed, but for therapeutic efficacy, such constructs typically require other changes to increase half-life, such as modification with polyethylene glycol or pegylation. Eliminating the Fc region altogether can affect the efficacy of anti-CD154 antibodies - anti-CD154 pegylated Fab' antibody fragments (dapirolizumab pegol (DZP), CDP7657, Biogen and UCB) failed to treat SLE in phase IIb clinical trials (Waters J, Biocentury; October 26, (2018)). The "silent" N297Q IgG1 variant of hu5c8 (asialo- or algyco-) lacked FcγR binding but was effective in inhibiting humoral immune responses other than organ rejection in non-human primates. (Tao, MH and Morrison, SLJ Immunol. 143:2595-2601. (1989); Ferrant JL et al., International Immunol (11):1583 (2004)). Thus, the solution to balancing efficacy and safety of anti-CD154 antibodies is to engineer anti-CD154 antibodies with altered Fc with selectively reduced effector function. Previous efforts to modify effector function have focused on substitutions in the IgG hinge / CH2 region that may affect FcγR and C1q binding. A consideration in designing the antibody is to maintain the CH2 / CH3 region of the Fc domain required for interaction with the neonatal Fc receptor, FcRn, which confers extended serum half-life.

[0011] Antibody engineering is a complex and costly technique with unpredictable outcomes in vivo (Saeed AF et al., Frontiers in Microbiology; Article 495; March (2017)). Antibody engineering strategies involving structural changes or other modifications that are optimized for one antibody may impair another antibody in vivo (Yan B, et al., The Journal of Biol. Chem. 287(8):5891-97 (2012)). The effect of a mutation (e.g., N297Q) to a particular residue in a particular antibody (e.g., IgG1) may have a very different clinical effect than another type of mutation (e.g., N297A in IgG1) of the same antibody. Furthermore, the clinical effect of an intramolecular change within one subtype of an antibody (e.g., L235 mutation in IgG1) may be quite unpredictable if the same intramolecular change occurs within another subtype of an antibody (e.g., L235 mutation in IgG4). Intramolecular changes may also result in different clinical effects depending on how the various mutations may pair within the antibody. Species-specific differences add to the unpredictability of the effect of Fc receptor mutations on the efficacy versus toxicity of anti-CD154 antibodies. In the past, anti-CD154 antibodies developed for use in transplantation studies did not cause thrombosis in preclinical rodents and certain non-human primates, but some human patients experienced thromboembolic complications in clinical trials using the same antibodies (i.e., ruplizumab, toralizumab, and ABI793) (Pinelli DF and Ford ML Immunotherapy 7(4): 399-410 (2015)).

[0012] To reduce graft rejection, immune tolerance to the donor graft is required. Immune tolerance is achieved when the immune system is non-responsive to the donor graft or organ. Immune tolerance is classified as central tolerance, where tolerance occurs in the thymus or bone marrow, or peripheral tolerance, where tolerance occurs in peripheral tissues and lymph nodes. Furthermore, different organs require different tolerance induction protocols to induce organ-specific tolerance (Madariaga, M, Kreisel, D, & Madsen, J, 20(4),392-399 (2015)). Tolerance can be established by acclimatizing the body to the graft and achieving mixed chimerism by transplanting donor hematopoietic stem cells. To achieve mixed chimerism, organ-specific protocols may be required. For example, a mixed chimerism protocol that achieves kidney transplant tolerance in monkeys failed to induce tolerance in heart transplant recipients (Kawai T, Cosimi AB, Wee SL, Houser S, Andrews D, Sogawa H, Phelan J, Boskovic S, Nadazdin O, Abrahamian G, Colvin RB, Sach DH, Madsen JC. Transplantation 73(11):1757-64 (2002)). The mechanism of anti-CD154 treatment of transplant rejection or autoimmunity is not fully understood. Anti-CD154 therapy appears to be more effective than anti-CD40 therapy, suggesting that blocking their interaction is not symmetric. Monoclonal antibody (mAb) hu5c8 appears to be more effective than IDEC-131, a different humanized anti-CD154 mAb (O'Neill NA, et al. Transplantation. 101(9):2038-2047 (2017)). Furthermore, the increase in regulatory T cells, especially Foxp3+ T cells associated with anti-CD154 therapy, may be related to efficacy (Ferrer IR, et al., Proc Natl Acad Sci US A. 108(51):20701-6 (2011)). Another observation supporting a role for regulatory T cells was that dimeric soluble CD40, but not monovalent CD40, induced T regulatory cells (CD4+CD25+ T cells) and treated transplant rejection (Masunaga T, et al. Transplantation. 80:1614-1622 (2005)). These experiments also showed that valency in ligating or blocking CD154 may be related to efficacy (Masunaga T, et al. Transplantation. 80:1614-1622 (2005)). Anti-CD154 treatment has been shown to be particularly effective in promoting allogeneic transplants with mixed chimerism (Kawai T, et al., Am J Transplant. 4(9):1391-8(2004)) and in xenotransplants (Laengin M, et al., Nature. 564(7736):430-433 (2018)). To date, there are no fully human or humanized anti-CD154 antibodies that satisfy the need for products that can effectively prevent inflammatory, autoimmune, viral infections and diseases, immune response dysfunction associated with allergic, atherosclerotic, or neurodegenerative conditions and diseases in human transplant rejection (including graft-versus-host disease), with acceptable levels of side effects, e.g., thromboembolic side effects. This highlights the need for antibodies and variants thereof that bind CD154 with high affinity and inhibit the downstream effects of CD154:CD40 binding in the absence of toxic side effects, such as thrombosis. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 9,765,150 [Non-patent literature]

[0014] [Non-Patent Document 1] O'Neill NA et al., Transplantation. (2017) 101(9): 2038-2047 [Non-Patent Document 2] Ferrer IR et al., Proc Natl Acad Sci US A. (2011)108(51):20701~6 [Non-Patent Document 3] Masunaga T et al. Transplantation. (2005) 80:1614~1622 Summary of the Invention [Means for solving the problem]

[0015] Summary of the Disclosure The present disclosure relates to the induction of immune tolerance in transplant recipients.The present disclosure also relates to inducing peripheral, central, or organ-specific tolerance by administering anti-CD154 and transplanting donor hematopoietic stem cells.The present disclosure also relates to the conditioning method used to prepare transplant recipients for hematopoietic stem cell transplantation and reduce graft rejection.The present disclosure further relates to the structure of anti-CD154 used to induce immune tolerance.

[0016] A first aspect of the present disclosure provides a method of inducing immune tolerance in a transplant recipient. In some embodiments, the method includes administering one or more doses of an isolated anti-CD154 antibody to the recipient, transplanting hematopoietic stem cells into the recipient, and transplanting a donor organ, donor tissue, or donor cells into the recipient, where the hematopoietic stem cells produce immune cells that are tolerant to the donor organ, donor tissue, or donor cells, thereby inducing immune tolerance in the recipient.

[0017] In some embodiments, the immune tolerance is central tolerance. Optionally, the method further comprises depleting T cells in the thymus. In some embodiments, the method further comprises depleting T cells in the bone marrow. Optionally, the method further comprises depleting T cells in both the thymus and the bone marrow.

[0018] In some embodiments, the immune tolerance is peripheral tolerance. Optionally, the method further comprises depleting T cells in lymph nodes. In some embodiments, the method further comprises depleting T cells in peripheral tissues. Optionally, the method further comprises depleting T cells in both lymph nodes and peripheral tissues.

[0019] In some embodiments, the method of inducing immune tolerance is organ-specific tolerance. In some embodiments, the method further comprises depleting T cells from the heart, kidney, both kidneys, liver, part of the liver, lung, both lungs, pancreas, intestine, islet cells, face, one hand, both hands, one arm, both arms, one foot, both feet, one leg, both legs, or skin, or a combination thereof.

[0020] In some embodiments, T cells are depleted by a method selected from the group consisting of total body irradiation, administration of abatacept, administration of one or more BCL-2 inhibitors, administration of busulfan, administration of fludarabine phosphate, administration of cyclophosphamide, administration of one or more immunosuppressive T cell depleting antibodies, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or a combination thereof.

[0021] In some embodiments, the one or more T cell depleting antibodies are selected from the group consisting of an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD45 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, and an anti-CD33 antibody, or a combination thereof.

[0022] In some embodiments, the method of inducing immune tolerance results in mixed chimerism.

[0023] In some embodiments, the anti-CD154 antibody is administered prior to hematopoietic stem cell transplantation. Optionally, the anti-CD154 antibody is administered following hematopoietic stem cell transplantation. In some embodiments, the anti-CD154 antibody is administered simultaneously with hematopoietic stem cell transplantation. In some embodiments, the hematopoietic stem cells are transplanted by bone marrow transplantation.

[0024] In some embodiments, the anti-CD154 antibody is administered at a dose of 5-50 mg / kg. Optionally, the anti-CD154 antibody is administered at a dose of 10-40 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of 20-30 mg / kg. Optionally, the anti-CD154 antibody is administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally.

[0025] In some embodiments, the anti-CD154 antibody comprises a human or humanized variable domain, the variable domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH being operably linked to a human Fc domain having an altered effector function. In some embodiments, the anti-CD154 antibody has one or more reduced effector functions. Optionally, one or more effector functions are eliminated. In some embodiments, the VH is operably linked to a human Fc region, the human Fc region comprising a human hinge sequence and a human Fc domain, the human hinge sequence being between the VH and the human Fc domain. In some embodiments, the hinge comprises the amino acid sequence of any one of SEQ ID NOs: 76-90.

[0026] In some embodiments, the Fc domain is derived from an IgG4 Fc (or crystallizable fragment) region and comprises one or more amino acid modifications that modify effector function. In some embodiments, the antibody comprises an amino acid modification at any one or combination of positions selected from the group consisting of S228, L235, G237, E318, and N297, where the numbering of amino acid residues is according to the EU index as set forth in Edelman. In some embodiments, the antibody comprises an amino acid modification or combination thereof selected from the group consisting of S228P, F234A, L235A, L235E, G237A, E318A, and N297Q.

[0027] In some embodiments, the Fc domain is derived from an IgG1 Fc (or crystallizable fragment) region and contains one or more amino acid modifications that modify effector function. In some embodiments, the antibody comprises one or more amino acid modifications, such as E216, R217, K218, C219, C220, C226, C229, P230, E233, L234, L235, G236, G237, P238, S239, V240, F241, K246, L251, T260, D265, V266, H268, W277, N297, E318, K322, P329, A330, E340, E341, E342, E343, E344, E345, E346, E347, E348, E349, E350, E351, E352, E353, E354, E355, E356, E357, E358, E359, E360, E361, E362, E363, E364, E365, E366, E367, E368, E369, E370, E372, E376, E377, E378, E379, E380, E381, E382, E383, E384, E385, E386, E387, E388, E389 ...90, E391, E392, E393, E , P331, Q347, N348, T350, L351, K360, T366, N390, K392, T394, D399, S400, F405, Y407, K409, T411, or any combination thereof, wherein the numbering of the amino acid residues is according to the EU index as set forth in Edelman.Optionally, the antibody is selected from the group consisting of C220S, C226S, C229S, P230S, E233P, L234A, L234F, L234V, L235A, L235E, L235V, G236E, G237A, P238S, D265S, D265A, H268Q, W277T, N297G, N297Q, N297D, N297A, E318A, K322A, P329G, P329A, A330S, P331S, Q347R, Q347 E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T366V, N390R, N390K, N390D, K392V, K392M, K39 2R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S400K, F405A, F405I, F405M, F405T, F4 05S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T411N, T411R, T411Q, T411K, T411D, T 411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR236 / 237, InR237 / 238, InV238 / 239, and InR237 / 238 / G236A.

[0028] In some embodiments, the Fc domain is derived from an IgG2 Fc (or crystallizable fragment) crystallizable fragment region and comprises one or more amino acid modifications that modify effector function. In some embodiments, the antibody comprises an amino acid modification at any one or combination of positions selected from the group consisting of V234, G237, P238, H268, V309, A330, and P331, where the numbering of the amino acid residues is according to the EU index as set forth in Edelman. In some embodiments, the antibody comprises an amino acid modification selected from the group consisting of V234A, G237A, P238S, H268Q, H268A, V309L, A330S, and P331S, or a combination thereof. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9, 12-18, and 238-241. Optionally, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56, and 243-251.

[0029] In some embodiments, the VH comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 57, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 59. Optionally, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 64, 252, and 253. Further, in some embodiments, the VL comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 62. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 65 or 66.

[0030] In some embodiments, the antibody further comprises a CH1 domain, the CH1 domain being operably linked to the C-terminus of the VH and the N-terminus of the hinge. In some embodiments, the CH1 domain comprises an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 67, 70, and 73. In some embodiments, the antibody comprises a linker between the VH and the Fc domain. In some embodiments, the antibody comprises a linker between the VH and the hinge. In some embodiments, the antibody comprises a linker between the VH and the CH1 domain.

[0031] In some embodiments, the linker comprises any one of the amino acid sequences of SEQ ID NOs: 199-223 and 327-330. In some embodiments, the VH is operably linked to any one of the amino acid sequences of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. In some embodiments, the heavy chain comprises any one of the amino acid sequences of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277, and 279-288. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 195 or 196.

[0032] In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is chimeric. Optionally, the antibody is humanized. In some embodiments, the antibody is human.

[0033] In some embodiments, the antibody binds to human CD154 and inhibits the interaction between human CD154 and human CD40. In some embodiments, the antibody blocks the activation of one or more of B cells, macrophages, dendritic cells, or endothelial cells by inhibiting the binding of CD154 to CD40. In some embodiments, the antibody has one or more of the following effects when administered to a subject: reduced risk of thrombosis or thromboembolic events compared to hu5c8 antibody, reduced activation of platelets expressing CD154, inhibited shedding of CD154, and altered expression or activity of downstream targets of CD154-CD40 signaling.

[0034] In some embodiments, the antibody has a K for CD154 of less than 50 pM, e.g., between 5 and 25 pM, or between 9.5 and 23 pM. D In some embodiments, the antibody does not comprise an amino acid sequence consisting of any one of SEQ ID NOs: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278.

[0035] In some embodiments, the transplant recipient is a human. Optionally, the transplant recipient is a non-human. In some embodiments, the transplant recipient is a non-human primate. In some embodiments, the transplant recipient is a monkey. In some embodiments, the transplant is an allograft, an autograft, or a xenograft.

[0036] In some embodiments, the donor's cells are engineered cells or ex-vivo expanded cells. In some embodiments, one or more genes in the donor's cells are modified using one or more techniques selected from the group consisting of transduction for expressing cDNA, CRISPR / Cas9 system, RNAi technology and retroviral technology. In some embodiments, the donor's cells are modified to express chimeric antigen receptor (CAR) on their surface. In some embodiments, the donor's cells are selected from the group consisting of stem cells, regulatory T cells, CAR-T cells, CAR-B cells, and tumor infiltrating lymphocytes (TIL).

[0037] In some embodiments, the methods of the disclosure promote long-term survival of a donor organ, donor tissue, or donor cells for at least six months after transplantation, at least one year after transplantation, and at least five years after transplantation.

[0038] In some embodiments, the anti-CD154 antibody is administered locally. Optionally, the anti-CD154 antibody is administered systemically. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 provides a T cell depletion regimen for inducing immune tolerance in non-human primate (NHP) transplant recipients. The recipients are treated with total body irradiation (TBI; 1.5 Gy x 2) on days -6, -5. The recipients also receive thymic irradiation (TI; 7 Gy) on day -1. Intravenous anti-T cell antibodies (50 mg / kg / day) are administered on days -2, -1, and 0. On day 0, the kidney and bone marrow (BM) are transplanted into the recipient. In addition, anti-CD154 mAb (20 mg / kg / day) is administered to the recipients on days 0, 3, 7, 14, 21, 28, 42, 56, 70, 84, and then monthly until the graft fails or 180 days after transplantation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Disclosure Definitions and General Techniques Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those skilled in the art.In general, the nomenclature used in connection with and in the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is that which is well known and commonly used in the art.In case of conflict, the present specification, including definitions, shall control.

[0041] The methods and techniques of the present disclosure employ, unless otherwise specified, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology that are within the skill of the art. Such techniques include, for example, those described in Green MR & Sambrook J. Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012);Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), each of which is incorporated herein by reference. thed., Wiley, John & Sons, Inc. (2002);Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); (2003);Oligonucleotide Synthesis (MJ Gait, ed., 1984);Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);Current Protocols in Molecular Biology (FM Ausubel et al., eds., John Wiley & Sons, Inc., 2003); eds., 1994).

[0042] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and medicinal chemistry described herein are those well known and commonly used in the art.

[0043] Standard techniques are used for chemical synthesis, and chemical analysis.

[0044] Throughout this specification and the embodiments, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integers or groups of integers. "Comprising" can be synonymous with "including" or "containing."

[0045] Whenever an embodiment is described herein with the word "comprising," it is understood that similar embodiments otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided. As used herein, "consisting of" is a closed term including only the specific elements recited, whereas "consisting essentially of" includes the specific elements recited and may include elements that do not consist of additional, unrecited, materials.

[0046] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0047] Any examples following the term "eg" or "for example" are not meant to be exhaustive or limiting.

[0048] Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0049] The articles "a", "an" and "the" are used herein to refer to one or more than one (i.e., at least one) of the object of the article. By way of example, "an element" means one or more than one element. As used herein, the term "about" to modify the amount of a component, parameter, calculation, or measurement of a composition of the present disclosure or used in a method of the present disclosure, refers to variations in quantity that may occur, for example, by typical measuring and liquid handling procedures used to make isolated polypeptides or pharmaceutical compositions in the real world; by inadvertent errors in these procedures; by differences in the manufacture, source, or purity of the components used to make the composition or carry out the method, and the like, without having a substantial effect on the chemical or physical contribution of the composition or method of the present disclosure. Such variations may be within an order of magnitude, typically within 10%, and even more typically within 5% of a given value or range. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition obtained from a particular initial mixture. Paragraphs include equivalent quantities, whether or not modified by the term "about." As used herein, reference to a value or parameter with "about" includes (and describes) embodiments directed to that value or parameter itself. For example, a description of "about X" includes a description of "X." Numeric ranges are inclusive of the numbers defining the range.

[0050] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein should be understood to encompass any and all subranges subsumed therein. For example, a range described as "1-10" should be considered to include any and all subranges between (and including) a minimum value of 1 and a maximum value of 10; i.e., all subranges beginning with a minimum value of 1 or greater, e.g., 1-6.1, and ending with a maximum value of 10 or less, e.g., 5.5-10.

[0051] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0052] definition The following terms, unless otherwise specified, shall be understood to have the following meanings:

[0053] As used herein, the term "antibody" or "Ab" refers to an immunoglobulin molecule (e.g., a complete antibody, an antibody fragment, or an engineered antibody) capable of recognizing and binding to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" may encompass any type of antibody, including, but not limited to, monoclonal, polyclonal, human, engineered (humanized, fully human, chimeric, single chain, artificially selected, CDR-granted, etc.) antibodies that specifically bind to a given antigen (e.g., CD154). Additionally, "antibody" and / or "immunoglobulin" (Ig) refers to a polypeptide comprising at least two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa), optionally interconnected by disulfide bonds. There are two types of light chains; λ and κ. In humans, the lambda and kappa light chains are similar, but only one type is present in each antibody. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), which is incorporated herein by reference in its entirety. The antibodies disclosed herein may be "functionally bivalent" or "functionally monovalent". In other words, the antibodies disclosed herein may have one antigen binding site (monovalent) or two antigen binding sites, typically linked by disulfide bonds (bivalent). In some embodiments, the anti-CD154 antibody is an IgG1 antibody. The anti-CD154 antibody may be an IgG2 antibody. Optionally, the anti-CD154 antibody is an IgG4 antibody. In some embodiments, the anti-CD154 antibody does not contain a final lysine residue at its C-terminus to improve the stability of the antibody.For example, see Jiang G et al., J Pharm Sci. Jul;105(7):2066-72 (2016); and Hintersteiner B. MAbs. 2016 Nov / Dec;8(8):1548-1560 (2016). It is known in the art that each heavy and light chain is expressed with a leader sequence (also known as a signal sequence) at its N-terminus, which is used to transport the newly synthesized chain into the endoplasmic reticulum. During post-translational processing, the leader sequence is removed and is therefore not present in the final chain or mature antibody.

[0054] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody produced by an identical set of immune cells, each of which is a clone of a unique parent cell. Monoclonal antibodies have a monovalent affinity (i.e., they bind to the same epitope).

[0055] As used herein, the term "chimeric" antibody refers to an antibody or antigen-binding fragment thereof that contains portions derived from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced with mouse variable regions of desired specificity spliced ​​onto human constant domain gene segments (e.g., U.S. Pat. No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical application. The term "chimeric" can refer to a non-native sequence that has been engineered to have one or more changes relative to the native sequence. Chimeric antibody, as used herein, refers to an antibody that contains regions derived from two or more different antibodies.

[0056] As used herein, the term "humanized" antibody refers to a chimeric antibody from a non-human species whose amino acid sequence has been modified to increase similarity to antibodies produced in humans. In some embodiments, a humanized antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. Optionally, a humanized antibody is derived from a human immunoglobulin (i.e., recipient antibody) in which residues from one or more complementarity determining regions (CDRs) of the recipient antibody have been replaced by residues from one or more CDRs of an antibody from a non-human species (donor antibody) with the desired specificity, affinity, and capacity. In some embodiments, the non-human species is mouse, rat, or rabbit. Humanized or CDR-grafted mAbs are particularly useful as therapeutic agents for humans because they are not cleared from circulation as rapidly as mouse antibodies and typically do not provoke adverse immune responses. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source.

[0057] In some embodiments, the chimeric antibody is a humanized antibody, such as a humanized anti-CD154 antibody. The humanized anti-CD154 antibody may comprise one or more amino acid sequences of human framework regions and / or amino acid sequences derived from at least a portion of a human constant region, and further comprises sequences derived from a non-human antibody, such as non-human (e.g., mouse) CDR sequences. In some embodiments, the humanized antibody comprises a human constant region. Optionally, all of the framework regions of the humanized antibody are human framework regions.

[0058] Humanized antibodies can be generated by replacing non-human sequences of Fv variable regions that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are described in Morrison, SL, Science, 229:1202-1207 (1985); Oi et al., BioTechniques, 4:214 (1986); Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), Staelens et al. 2006 Mol Immunol 43: 1243-1257, and U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of immunoglobulin Fv variable regions from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and may be obtained, for example, from hybridomas producing antibodies against a given target from germline immunoglobulin genes, or synthetic constructs, as described above. Recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are replaced by residues from analogous sites in rodent antibodies. See, e.g., U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; and 5,859,205, each of which is incorporated herein by reference.See also U.S. Patent No. 6,180,370 and PCT International Publication No. WO 01 / 27160, each of which is incorporated herein by reference, which disclose humanized antibodies and techniques for producing humanized antibodies with improved affinity for a given antigen. Furthermore, humanized and chimeric antibodies may be modified to include residues that are not found in the recipient antibody or in the donor antibody to further improve antibody properties, such as affinity or effector function.

[0059] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). The term encompasses antibodies derived from human genes but with sequences that have been altered, for example, to reduce potential immunogenicity, increase affinity, eliminate cysteines that may cause undesirable folding, etc. The term also encompasses such antibodies produced recombinantly in non-human cells, which may confer glycosylation that is not typical for human cells.For the production of human antibodies, see Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), Lonberg, Nature Biotechnology, Vol. 23(5): 1117-1125 (2005); Jackobovits, "Therapeutic Antibodies from XenoMouse Transgenic Mice," Chapter 7, Recombinant Antibodies for Immunotherapy, Cambridge University Press, New York, 2009, Murphy, "VelocImmune: Immunoglobulin Variable Region Humanized Mice," Chapter 8, Recombinant Antibodies for Immunotherapy, Cambridge University Press, New York, 2009, Murphy et al., PNAS, 2014, vol. 111(14): 5153-5158; Brueggemann et al., Arch. Immunol. Ther. Exp., 2015, vol. 63:101-108. Human antibodies and methods for making them are further discussed in U.S. Patent Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610; 6,657,103; 6,586,251; and U.S. Patent Application Publication No. 2006-0015957 A1; and International Patent Publication Nos. WO98 / 24893; and WO2007 / 117410. The disclosures of each of the above patents, applications, and references are hereby incorporated by reference in their entirety herein.

[0060] As used herein, the term "amino acid modification" refers to at least one amino acid substitution, insertion, deletion or mutation in an amino acid sequence compared to a wild-type amino acid sequence. Such modifications are within the skill of a person skilled in the art. Certain modifications, including amino acid deletions, substitutions, and additions of the Fc region, have been demonstrated to alter the binding of the Fc region to its ligands and / or receptors, resulting in concomitant changes in effector function (see, e.g., Shields et al., J Biol Chem 276:6591-6604 (2001); Presta et al., Biochem Soc Trans 30:487-490 (2002); Escobar-Cabrera E et al. Antibodies. 6: 7 (2017); Duncan AR et al. Nature. 1988; 332: 738-740 (1988); Duncan AR et al. Nature. 332: 563-564 (1988); Hezareh M et al. J Virol. 75: 12161-12168 (2001); Oganesyan V et al. Acta 2002: 123-124 (2002), all of which are incorporated by reference in their entireties. Crystallogr D Biol Crystallogr; 64: 700-704 (2008);Schlothauer T et al. Protein Eng Des Sel. Oct; 29(10):457-466 (2016);Tao MH et al. J. Immunol. 143: 2595-2601 (1989);Von Kreudenstein TS et al. MAbs. 5(5):646-654 (2013);Wang X et al. Protein Cell. 9(1):63-73 (2018); U.S. Patent Publication Nos. 20040132101; 20070111260; 20110287032; 20180194860; U.S. Patent No. 8409568; International Publication No. WO2017 / 177337). Amino acid deletions are indicated as "Δ" and insertions are indicated as "In". For example, the deletion of the amino acid sequence from E216 to E222 is indicated as ΔE216-E222.The insertion of an arginine (R) between amino acid residues 234 and 235 can be designated as InR234 / 235.

[0061] As used herein, the term "Fc domain" refers to the crystallizable fragment of an antibody after papain digestion. The Fc domain comprises two identical protein fragments derived from the hinge region and the second and third constant domains of IgA, IgD, and IgG antibody isotypes, or the hinge region and the second, third, and fourth constant domains of IgM and IgE antibody isotypes. The Fc domain is the part of an antibody that binds to cell surface Fc receptors and certain proteins of the complement system. The term "Fc region" refers to the Fc domain combined with the hinge region. The hinge region is typically between the C-terminus of the variable domain and the N-terminus of the Fc domain. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region as defined herein includes residue E216 at its carboxyl terminus of the CH3 domain (or the CH4 domain in IgM and IgE antibodies), numbering is in the EU format as in Edelman GM et al., (1969) Proc. Natl. Acad. USA, 63, 78-85. "EU format as shown in Edelman" refers to the residue numbering of the human IgG1 EU antibody described in Edelman GM et al., supra. Human IgG2 and human IgG4 residue numbering is also in the EU format (see Dillon TM, et al., J Biol Chem. Jun 6;283(23):16206-15 (2008); Aalberse RC and Schuurman J et al., Immunology 105:9-19 (2002); and Scholthauer T et al, Protein Engineering, Design and Selection, 29(10): 457-466, (2016)). The terms "Fc domain" and "Fc region" can refer to these sequences alone or in the context of an antibody, antibody fragment, or Fc fusion protein. The Fc variant protein can be an antibody, an Fc fusion, or any protein or protein domain that includes an F domain or Fc region.The amino acid sequence of a non-naturally occurring Fc domain or Fc region (also referred to herein as a "variant Fc domain" or "variant Fc region", respectively) may contain amino acid modifications. Any new amino acid residue that appears in the sequence of a variant Fc domain or variant Fc region as a result of insertion or substitution may be referred to as a non-naturally occurring amino acid residue. Polymorphisms have been observed at several Fc domain positions, including but not limited to positions 270, 272, 312, 315, 356, and 358, and thus slight differences may exist between the sequence(s) presented in the prior art.

[0062] As used herein, the term "linker" refers to a polypeptide sequence that joins two or more antibody domains. The characteristics of linkers and their suitability for specific purposes are known in the art. See, for example, Chen et al. Adv Drug Deliv Rev. October 15; 65(10): 1357-1369 (2013), which discloses various types of linkers, their properties, and related linker design tools and databases, incorporated herein by reference. Linkers may be flexible, rigid, or in vivo cleavable. Preferably, the linker is flexible. Flexible linkers typically contain small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The most commonly used flexible linkers have sequences that consist mainly of stretches of Gly and Ser residues ("GS" linkers). Optionally, the flexible linker contains five repeats of Gly and Ser residues. A non-limiting example of a flexible linker is (Gly-Gly-Gly-Gly-Ser). n (SEQ ID NO: 327), (Ser-Ser-Ser-Ser-Gly) n (SEQ ID NO: 328), (Gly-Ser-Ser-Gly-Gly) n (SEQ ID NO: 329), and (Gly-Gly-Ser-Gly-Gly) n(SEQ ID NO: 330) (wherein n can be any integer between 1 and 5). The linker is optionally between 5 and 25 amino acid residues in length. Other suitable linkers may be selected from the group consisting of AS (SEQ ID NO: 214), AST (SEQ ID NO: 215), TVAAPS (SEQ ID NO: 216), TVA (SEQ ID NO: 217), ASTSGPS (SEQ ID NO: 218), KESGSVSSEQLAQFRSLD (SEQ ID NO: 219), EGKSSGSGSESKST (SEQ ID NO: 220), (Gly)6 (SEQ ID NO: 221), (Gly)8 (SEQ ID NO: 222), and GSAGSAAGSGEF (SEQ ID NO: 223). In general, the flexible linker should provide good flexibility and solubility and may act as a passive linker to keep the distance between the functional domains. The length of the flexible linker may be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein.

[0063] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference.

[0064] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with a different amino acid residue having similar biochemical properties (e.g., charge, hydrophobicity, or size). Typically, a conservative amino acid substitution does not substantially change the functional properties of a protein. When comparing proteins with conservative substitutions, the percent sequence identity or degree of similarity can be adjusted to account for the conservative nature of the substitution. Such adjustments are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994).

[0065] The groups of amino acids with similar biochemical properties that can be used in conservative substitution include: 1) amino acid residues with aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) amino acid residues with aliphatic-hydroxyl side chains: serine and threonine; 3) amino acid residues with amide-containing side chains: asparagine and glutamine; 4) amino acid residues with aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) amino acid residues with basic side chains: lysine, arginine, and histidine; 6) amino acid residues with acidic side chains: aspartic acid and glutamic acid; and 7) amino acid residues with sulfur-containing side chains: cysteine ​​and methionine.Preferred conservative amino acid substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0066] Each heavy chain is composed of a heavy chain variable domain (VH) and multiple heavy chain constant domains (CH). In IgA, IgD, and IgG antibodies, the heavy chain typically contains three domains: CH1, CH2, and CH3. In IgM and IgE antibodies, the heavy chain typically contains four domains: CH1, CH2, CH3, and CH4. In some embodiments, the antibody contains two domains: CH2 and CH3. Each light chain contains a light chain variable domain (VL) and a light chain constant domain. The light chain typically contains one domain: CL. The light chain variable domain is encoded by two gene segments: a variable (V) gene segment that codes for the first 95-101 amino acids of the light chain, and a joining (J) gene segment that codes for about 12 or more amino acids. The heavy chain variable domain is encoded by three gene segments, with a diversity (D) gene segment between the V and J gene segments that codes for about three or more amino acids. The VH and VL domains can be further subdivided into regions of hypervariability called "complementarity determining regions" (CDRs) separated by more conserved "framework regions" (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0067] The pairing of the variable domains of the heavy and light chains (VH and VL) forms the antibody binding site that interacts with the antigen. Thus, each antibody typically has two binding sites. Except for multifunctional / multispecific (e.g., bifunctional or bispecific) antibodies, the two binding sites are identical. The Fc region of the antibody constant region typically mediates the binding of the antibody to host tissues and factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0068] Residues in variable domains are numbered according to Edelman, also known as the EU numbering system, which is the numbering system used for heavy or light chain variable domains of a collection of antibodies. See Edelman Proc Natl Acad Sci US A. May;63(1):78-85 (1969) and Kabat, EA, Wu, TT, Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD. The EU numbering of residues can be determined for a given antibody by alignment of the sequence of the antibody with the "standard" EU numbering sequence in the homology region. The variable region CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR H3) are identified by contacts based on the crystal structure defined in Karpusas et al. Structure. Apr 4;9(4):321-9 (2001) and numbered according to Edelman.

[0069] As used herein, the term "operably linked" refers to a first structure placed in a functional relationship with a second structure. In the context of an antibody, a targeting structure may be operably linked to a structure that confers an effector function. For example, an antigen-binding sequence of an antibody (e.g., a variable region or a VH or VL domain) may be operably linked to an Fc region. In the context of a polynucleotide, a coding sequence may be operably linked to a non-coding regulatory sequence, such as, for example, a promoter, an enhancer, a signal sequence, a ribosome binding sequence, a splice acceptor sequence, a splice donor sequence, a termination sequence, and the like. Two operably linked structures may be directly connected. Alternatively, two operably linked structures may be connected via one or more intermediate structures. For example, an antigen-binding portion of an antibody may be operably linked to an Fc region via a CH1 domain, a hinge region, and / or a linker sequence. Similarly, operably linked non-coding regulatory sequences include both sequences that are adjacent to the coding sequence and sequences that act in trans or at a distance to control the coding sequence.

[0070] As used herein, the term "effector function" refers to responses elicited by the interaction of antibodies and antibody-antigen complexes with cells of the immune system. These effector functions typically include one of three major mechanisms: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and opsonization and phagocytosis. In ADCC, Fc receptors on cytotoxic T cells, natural killer (NK) cells, or macrophages bind the Fc region of antibodies bound to target cells, resulting in the secretion of substances such as lytic enzymes, perforins, granzymes, and tumor necrosis factors that mediate the destruction of the target cells. In CDC, cell death is induced by the activation of the complement cascade. See Daeron, Annu. Rev. Immunol., 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol., 2:77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). In opsonization and phagocytosis, the Fc region of an antibody bound to a pathogen binds to an Fc receptor on the surface of a phagocyte, inducing phagocytosis. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using standard assays known in the art (see, e.g., WO05 / 018572, WO05 / 003175, and U.S. Patent No. 6,242,195). The Fc domain of an antibody mediates immune effector mechanisms. IgG antibodies activate effector pathways of the immune system by binding to members of the family of cell surface Fcγ receptors and Clq of the complement system. Ligation of effector proteins by clustered antibodies induces a variety of responses, including release of inflammatory cytokines, modulation of antigen production, endocytosis, and cell death. These responses can cause unwanted side effects, such as inflammation and thrombosis. Thus, the present disclosure further relates to anti-CD154 antibodies with altered effector functions, including antibodies with one or more effector functions reduced or eliminated.Without wishing to be bound by theory, it is believed that the anti-CD154 antibodies disclosed herein do not cause platelet activation or aggregation because antibodies containing mutated Fc regions do not bind to FcγRIIa (also known as CD32a) on the platelet surface.

[0071] As used herein, the term "modified effector function" refers to an Fc domain or Fc region whose effector function is different from that of a wild-type immunoglobulin. In some embodiments, one or more effector functions are reduced. Optionally, one or more effector functions are eliminated. The modified or reduced effector function may be the result of a reduced binding affinity of the Fc region of the antibody disclosed herein to an effector molecule (e.g., FcγR and / or C1q). For example, the anti-CD154 antibody disclosed herein has reduced Fc receptor binding and complement activation compared to that of the wild-type anti-CD154 antibody. In some embodiments, the variant Fc region has reduced antibody-dependent cell-mediated cytotoxicity (ADCC). The effector function of an anti-CD154 antibody can be determined using one of many known assays, including CDC assays, ADCC assays, and phagocytosis assays (see Xu-Rong Jiang et al., Nature Reviews Drug Discovery 10: 101-111 (2011) and Liu et al., The Journal of Biological Chemistry 292:1876-1883 (2017)). One or more of the effector functions of an anti-CD154 antibody can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the effector function of a wild-type anti-CD154 antibody.

[0072] As used herein, the terms "CD154", "CD40 ligand" and "CD40L" can be used interchangeably and refer to a mammalian protein that is primarily expressed on the surface of activated T cells, and the soluble form of CD154 that is cleaved and released by activated T cells is a member of the TNF superfamily and binds the CD40 protein on antigen-presenting cells. The term CD154 is intended to include recombinant CD154 and recombinant chimeric forms of CD154 that can be prepared by standard recombinant expression methods. In some embodiments, CD154 refers to human CD154.

[0073] As used herein, the term "inhibit" refers to the property of an antibody or other molecule that prevents the interaction of CD154 with CD40, or that inhibits the binding of CD154 to CD40, or that inhibits the cleavage or shedding of CD154. In some embodiments, the antibody inhibits the binding of CD154 to CD40 by at least about 20%, preferably 40%, more preferably 60%, even more preferably 80%, or even more preferably 85%. Optionally, the antibody inhibits the cleavage or shedding of CD154 by at least about 20%, preferably 40%, more preferably 60%, even more preferably 80%, or even more preferably 85%. The inhibitory ability of anti-CD154 antibodies can be determined, for example, by their ability to inhibit the upregulation of certain downstream target genes of CD40. For example, anti-CD154 antibodies can alter the expression, activity, or activation of kinases and genes that respond to CD154-CD40 signaling. Anti-CD154 antibodies can inhibit upregulation of CD23 expression, inhibit upregulation of CD69 expression, inhibit upregulation and activity of activation-induced cytidine deaminase (AID), inhibit rescue from apoptosis, inhibit upregulation of NF-κB activity, inhibit immunoglobulin isotype class switching, inhibit immunoglobulin CDR somatic hypermutation, alter the expression or activity of molecules within the TNF receptor-associated factor (TRAF) family, such as TRAF-2, TRAF3 (also known as CRAF1), TRAF-5 and TRAF-6, kinase activation, or inhibit the expression of other genes responsive to CD154-CD40 signaling.Furthermore, Lederman, S., et al. J. Exp. Med. 175:1091–1101. (1992);Lederman, S., et al., Journal of Immunol. 149:3817–3 (1992);Lederman, S., et al., Journal of Immunol. 152:2163. (1994);Cleary, AM, et al., Journal of Immunol., 155:3329-3337 (1995);Cheng et al., Science. 267(5203):1494-8 (1995);Bankert KC et al., Journal of Immunol. Proc Natl Acad Sci US A. 93(18):9437-42 (1996). Muramatsu, MK et al. 2000. Cell 102: 553 (2000);Buchta CM and Bishop GA., Journal of Immunol. 192(1):145-50. (2014) Arcipowski KM, et al. International Immunology. 26(3):149-58 (2014);Mambetsariev N, et al., Proc Natl Acad Sci US A. 113(4):1032-7 (2016);Arcipowski KM, Bishop GA, PLoS One. 7(7) (2012);Bishop GA. Journal of Immunol. 91(7):3483-5. (2013);Peters AL and Bishop GA. Journal of Immunol. 185(11):6555–6 (2010);Rowland SL, et al., Journal of Immunol. 179(7):4645–53. (2007)、Benson RJ, et al., European Journal of Immunol. 6(9):2535-43. (2006) is available.

[0074] As used herein, the term "immune response" refers to the reaction of the body's immune system to the presence of a substance that is not recognized as a component of the body itself. The immune response may be a humoral immune response, a cell-mediated immune response, or a mixed humoral and cell-mediated immune response. The humoral response may be an antibody-mediated response. The cell-mediated response may be one or more of a cytotoxic T cell-mediated immune response, a macrophage-mediated response, a natural killer (NK) cell-mediated immune response, or a cytokine-mediated response. The mixed humoral and cell-mediated response may be one or more of an antibody-mediated response, a cytotoxic T cell-mediated immune response, a macrophage-mediated response, a natural killer (NK) cell-mediated immune response, or a cytokine-mediated response. The immune response may refer to an adaptive and / or innate immune response. For various types of immune responses, see David Chaplin J Allergy Clin Immunol February; 125(2 Suppl 2): ​​S3-23 (2010).

[0075] As used herein, the term "affinity" of an antibody refers to the strength of interaction between the antigen-binding site of the antibody and the epitope. The affinity of an antibody for an antigen is typically measured by the binding affinity equilibrium dissociation constant (K D ) The antibody is expressed as K D High affinity antibodies are generally said to specifically bind an antigen when their affinity is less than 1 mM, and preferably less than 100 nM. -9 ) range of K D and very high affinity antibodies are generally at picomolar concentrations (10 -12 ) range of K D It is believed that K DThe binding affinity constant can be measured by surface plasmon resonance, for example, using the BIACORE® system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) discussed in Example 3. See also Jonsson et al., Ann. Biol. Clin. 51:19-26 (1993); Jonsson et al., Biotechniques 11:620-627 (1991); Jonsson et al., J. Mol. Recognit. 8:125-131 (1995); Johnsson et al., Anal. Biochem. 198:268-277 (1991); Hearty S et al., Methods Mol Biol. 907:411-42 (2012), each of which is incorporated herein by reference. D can also be measured using a KINEXA® system (Sapidyne Instruments, Hanover, Germany and Boise, ID).

[0076] As used herein, the terms "ka" or "affinity constant" and "kd" or "dissociation constant" refer to the amount of antibody-antigen complex present at the point where equilibrium concentrations are reached between the antibody and the antigen. D is the ratio of kd to ka.

[0077] As used herein, the term "avidity" refers to the overall strength of an antibody-antigen complex. Avidity relates to three main parameters: the affinity of the antibody to the epitope; the valency of both the antibody and the antigen; and the structural arrangement of the interacting moieties. As used herein, "avidity" describes the increased affinity that results from multiple antigen-binding sites on an immunoglobulin.

[0078] As used herein, the term "transplantation" refers to the process of surgically removing cells or tissues or organs from a first organism (donor) and placing them in a second organism (recipient). The donor can be a human or non-human organism. In some embodiments, the donor is a primate. The donor may be a non-human primate. Optionally, the donor is a human. In some embodiments, the donor is a pig or a mini-pig. The recipient can be a human or non-human organism. Preferably, the recipient is a human. Optionally, the recipient is a non-human primate. The transferred cells, tissues or organs are referred to as "transplants" or "grafts." "Xenotransplantation" refers to the transfer of cells or tissues or organs from a donor of one particular species (e.g., monkey or pig) to a recipient of a different species (e.g., human).

[0079] As used herein, the term "engineered cell" refers to a cell that is modified from its natural state.Engineered cells can be modified using one or more techniques, such as transduction to express cDNA, CRISPR / Cas9 system, RNAi technology and retroviral technology.For example, cells can be modified to express chimeric antigen receptor (CAR) on their surface. Examples of cells that may be transplanted include, but are not limited to, stem cells, regulatory T (Treg) cells, CAR-T cells (see Zhang, C et al. Biomarker Research (5)22. (2017)), CAR-B cells (Voss JE et al., Elife. Jan 17;8. (2019)), and tumor infiltrating lymphocytes (TILs) (Zhang L et al., Clin Cancer Res. May 15;21(10):2278-88. doi: 10.1158 / 1078-0432.CCR-14-2085 (2015)).

[0080] As used herein, the term "ex-vivo expanded cells" refers to cells produced in an ex-vivo method to enhance the yield of cells (e.g., hematopoietic stem cells (HSCs)) used in clinical applications such as transplantation. See, for example, Xie J, and Zhang C Sci China Life Sci. Sep;58(9):839-53 (2015), which discusses methods for expanding the number of HSCs, including stromal / HSC co-cultures, continuous perfusion and fed-batch cultures, and culture systems supplemented with exogenous ligands, membrane-transportable transcription factors, complement components, protein-modifying enzymes, metabolites, or small molecule chemicals. The desired cells to be transplanted can also be expanded ex-vivo by applying endogenous Notch signaling activators (see, for example, Ex vivo expansion of human hematopoietic stem and progenitor cells Dahlberg A, et al., Blood 117:6083-6090 (2011)).

[0081] As used herein, the term "transplant rejection" refers to a phenomenon that occurs when cells, tissues, or organs transplanted from a donor are rejected by the recipient's immune system. The recipient's immune system can mount an adaptive immune response (cellular immunity) mediated by killer T cells that induce apoptosis of donor cells, a humoral immune response mediated by activated B cells that secrete antibodies, and / or an innate immune response mediated by phagocytes and soluble immune proteins (see Ochanda J et al., Cell Mol Immunol. Apr;16(4):350-356 (2019); Koo J, and Wang HL. Surg Pathol Clin. Jun;11(2):431-452 (2018); Wang H, and Yang YG, Curr Opin Organ Transplant. Apr;17(2):162-7 (2012); and da Silva MB, World J Transplant. Feb 24;7(1):1-25 (2017)).

[0082] As used herein, the term "hematopoietic chimerism", "mixed chimerism", or "mixed allogeneic chimerism" refers to the coexistence of hematopoietic cells from both the host and the donor resulting from the engraftment of donor pluripotent hematopoietic stem cells into the host. The host and donor cells may be tolerant to each other. Mechanisms of hematopoietic chimerism are known in the art. See Pasquet L, et al. Front Immunol. 2:80 (2011) and Nikolic B, and Sykes M, Immunol Res. 16(3):217-28. (1997), which are incorporated herein by reference. In some embodiments, such hematopoietic chimerism results in "central tolerance". Mechanisms of such chimeric "central tolerance" may involve central, intrathymic clonal deletion, selection of regulatory T cells, and / or other related immune mechanisms. See, e.g., Nikolic B, and Sykes M, Immunol Res. 16(3):217-28. (1997) and Hogquist KA et al., Nature Reviews Immunology 5:772-782 (2005), which are incorporated herein by reference. In some embodiments, such hematopoietic chimerism results in "peripheral tolerance". The mechanism of such chimeric "peripheral tolerance" may involve peripheral clonal deletion, conversion to regulatory T cells, and / or other related immune mechanisms. See, e.g., Nikolic B, and Sykes M, Immunol Res. 16(3):217-28. (1997) and Mueller D, Nature Immunology. 11(1): 21-27 (2010). In some embodiments, such hematopoietic chimerism results in "organ-specific tolerance". The mechanism of such chimeric "organ-specific tolerance" may vary from organ to organ and require mixed chimerism procedures. See, for example, Madariaga, M, Kreisel, D, & Madsen, J, 20(4),392-399 (2015). In some embodiments, hematopoietic stem cells are isolated or purified. Optionally, hematopoietic stem cells are passenger cells that are transplanted with an organ, such as a kidney or liver transplant.The stem cells may be derived from the donor's bone marrow or adipocytes / adipose tissue.

[0083] As used herein, the term "conditioning" or "conditioning" refers to preparing a recipient for stem cell transplantation, e.g., hematopoietic cell transplantation. Gyurkocza B and Sandmaier BM Blood 124:344-353 (2014) provide a review of high-dose, reduced-intensity, and non-myeloablative conditioning regimens and the most commonly used agents, such as total body irradiation, fludarabine phosphate, cyclophosphamide, and T-cell depleting antibodies. Monoclonal antibodies, such as anti-CD20Ab, anti-CD33Ab, and anti-CD45Ab, may also be used alone or in combination with conventional therapy as part of conditioning regimens to prevent transplant rejection. See, for example, Topcuoglu P et al.; Progress in Stem Cell Transplantation; December (2015). Other agents that can be used in the conditioning regimen include, but are not limited to, BCL-2 inhibitors (Perini GF et al., Journal of Hematology & Oncology 11:65 (2018)) and anti-CTLA4 Ab (Pree I et al., Transplantation. Mar 15; 83(5): 663-667 (2007)). The conditioning regimen may include chemotherapeutic agents, including, but not limited to, alemtuzumab (CAMPATH™), busulfan, carboplatin, carmustine, cyclophosphamide, cytarabine (Ara-C), daunorubicin, etoposide (VP-16), fludarabine, melphalan, rituximab, and vincristine.

[0084] The term "isolated antibody" refers to an antibody that is at least partially free from other biological molecules present in the cells used to produce them. Other biological molecules that isolated antibodies do not include include nucleic acid molecules, proteins, lipids, carbohydrates, cell debris and culture medium. The term "isolated antibody" does not require, but includes, the complete absence of such other biological molecules. The term "isolated antibody" also does not refer to the complete absence of other molecules such as water, buffers, salts or components of pharmaceutical formulations. Thus, a molecule that is chemically synthesized or synthesized in a cell-free system will be "isolated" from its naturally associated components. A molecule may also be "isolated" using purification techniques well known in the art.

[0085] Molecular purity or homogeneity can be assayed by several means well known in the art. For example, the purity of an antibody sample can be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the antibody using techniques well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification. The purity of a nucleic acid sample can be assayed using the spectrophotometric absorbance of the sample at 260 nm to 280 nm using techniques well known in the art. For certain purposes, higher resolution can be provided by using means well known in the art for purification.

[0086] Examples of isolated antibodies include, but are not limited to, anti-CD154 antibodies that have been affinity purified using CD154 and anti-CD154 antibodies synthesized by cell lines in vitro.

[0087] The term "percent sequence identity" in the context of polypeptide sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such conservative substitutions are taken into account (in addition to identical residues) when calculating the "percent sequence similarity" of the two sequences. Positions that are not identical but similar differ by conservative amino acid substitutions.

[0088] Alignment, for example to determine percent amino acid sequence identity, sequence similarity or sequence homology between a wild-type protein and its mutein, can be accomplished in a variety of ways within the skill of the art, using publicly available sequence analysis computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Gap, BESTFIT®, and other programs in the Wisconsin Package Version 10.0 or Genetics Computer Group (GCG), Madison, Wisconsin programs. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters. In the context of polypeptide sequences, FASTA takes a query amino acid sequence and searches sequence databases using local sequence alignments to identify similar sequences within the database (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson Curr Protoc Bioinformatics. Mar 24;53:3.9.1-25 (2016), each of which is incorporated herein by reference). BLAST, particularly blastp or tblastn, using default parameters can be used to compare a query sequence to a database containing sequences from various organisms. See, e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); Eser et al., PLoS One. 22;9(12): e115445 (2014), each of which is incorporated herein by reference.

[0089] The terms "transplant recipient," "patient," "subject," and "individual" are used interchangeably herein and refer to either a human or a non-human animal in need of treatment. These terms include mammals, such as humans, and non-human primates (e.g., monkeys). Optionally, the transplant recipient is a human. The transplant recipient may also be a non-human primate, such as a monkey. In some embodiments, the subject is in need of an inhibited or reduced immune response.

[0090] The term "primate" refers to mammals of the order Primates, including anthropoids and prosimians, characterized by elaborate development of the hands and feet, short noses, and large brains. The mammalian order Primates includes humans, apes, monkeys, and prosimians, or lower primates.

[0091] As used herein, the term "therapeutically effective amount" refers to an amount of an administered therapeutic agent that relieves to some extent one or more of the symptoms of the condition being treated. In the context of the treatment of transplant rejection, a therapeutically effective amount refers to an amount that has at least one of the following effects: reducing, inhibiting or preventing acute or chronic rejection of transplanted cells, tissues or organs and one or more symptoms associated with the rejection, prolonging the survival of the graft, reducing thrombosis, and reducing the risk of life-threatening infections, cancer, and other complications such as cardiovascular disease and renal failure. See, for example, Romano et al. Front Immunol. 10:43 (2019) and Ingulli E. Pediatr Nephrol. 25(1):61-74 (2010) on the mechanisms of cell rejection in transplantation. With respect to the treatment of autoimmune diseases and antibody-mediated inflammatory diseases, a therapeutically effective amount refers to an amount that has at least one of the following effects: reducing one or more symptoms associated with an autoimmune disease, such as fatigue, muscle pain, low fever, inflammation, skin rash, etc.

[0092] The pharmaceutical composition may comprise a therapeutically effective amount or a prophylactically effective amount of the antibody disclosed herein. The therapeutically effective amount of the antibody may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to induce a desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody are outweighed by the therapeutically beneficial effects. It is routine in the art for a person skilled in the art to determine the therapeutically effective amount of the antibody disclosed herein based on these factors. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at the dosage and for the period required. Typically, a prophylactic dose is used in subjects before transplantation or at the early stage of transplant rejection, so that the prophylactically effective amount may be less than the therapeutically effective amount.

[0093] As used herein, the terms "treat", "treating" and "treatment" refer to the internal or external administration of a therapeutic agent, such as a composition containing any of the antibodies disclosed herein, to a subject or patient having or suspected of having one or more disease symptoms for which the agent has therapeutic activity. "Treat", "treating" and "treatment" refer to therapeutic and / or prophylactic treatment. Therapeutic treatment includes, for example, alleviating or reducing the severity of a condition or eliminating the condition, including methods of alleviating or reducing the severity of one or more symptoms of a condition. If the treatment is administered before the clinical manifestation of a condition, the treatment is considered to be prophylactic. Alleviation or reduction of a disease symptom can typically be assessed by any clinical measurement used by a physician or other skilled artisan to assess the severity or progression of the condition. The term further refers to a postponement of the onset of one or more disease symptoms and / or a reduction in the severity of one or more disease symptoms. The term further includes ameliorating existing uncontrolled or unwanted disease symptoms, preventing further disease symptoms, and ameliorating or preventing the underlying causes of such disease symptoms. Thus, the term indicates that a beneficial result has been imparted to the subject.

[0094] With respect to the treatment of transplant rejection, treatment can refer to the alleviation, reduction, or delay of the rejection of transplanted cells, tissues, or organs or one or more symptoms associated with the rejection. Treatment can also result in the prolongation of graft survival, reduction of thrombosis, and / or reduction of the risk of other complications such as life-threatening infections, cancer, and cardiovascular disease and renal failure. With respect to the treatment of autoimmune disease, treatment can refer to weakening the body's immune response and controlling the autoimmune reaction. With respect to the treatment of antibody-mediated inflammatory disease, treatment can refer to reducing one or more symptoms associated with autoimmune disease, such as fatigue, muscle pain, low fever, inflammation, skin rash, etc. With respect to treatment with the antibodies disclosed herein, these terms can simply mean that the life expectancy and quality of life of the individual who receives the transplant or the individual who suffers from the autoimmune or inflammatory disease is increased, or that one or more symptoms associated with transplant rejection or the autoimmune or inflammatory disease is reduced.

[0095] As used herein, the terms "prevent", "preventing" and "prevention" refer to preventing or delaying the recurrence or onset of, or reducing, one or more symptoms of a condition in a subject as a result of administration of an anti-CD154 antibody of the disclosure. For example, in the context of administration of a therapy to a subject, "prevent", "preventing" and "prevention" refer to inhibiting, reducing, or delaying the onset or onset of rejection of the transplant or associated thrombosis, or preventing or delaying the recurrence, onset, or onset of one or more symptoms associated with administration of a transplant (e.g., a solid organ transplant) or combination of therapies (e.g., a solid organ transplant and a combination of immunosuppressants) in a subject.

[0096] As used herein, the term "administering" an antibody or composition of the present disclosure to a subject or "administration of" an antibody or composition to a subject refers to contacting the antibody or composition with the subject or with the subject's cells, tissues, organs, or biological fluids. Such administration can be performed using one of a variety of methods known to those of skill in the art. For example, an antibody or composition of the present disclosure can be administered systemically or locally. In some embodiments, the composition can be administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally. Administering can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, administration includes both direct administration (including self-administration) and indirect administration, including the act of prescribing a drug.

[0097] Anti-CD154 antibody The disclosed methods utilize anti-CD154 antibodies with altered effector functions that bind to mammalian CD154, more preferably human CD154. In some embodiments, one or more effector functions are reduced. Optionally, one or more effector functions are eliminated. In some embodiments, the antibody with one or more reduced effector functions demonstrates reduced binding to an Fc receptor relative to an antibody with a wild-type IgG1 heavy chain. Optionally, the reduced binding to an Fc receptor is 10-3200 times weaker than the binding demonstrated by a wild-type IgG heavy chain. In some embodiments, the K for FcγR1A that binds the Fc domain of the antibody with reduced effector function. D is between 0.1 and 100 nM, for example, between 0.3 and 92 nM. Optionally, the K for CD16aF binding the Fc domain of an antibody with one or more reduced effector functions is D In some embodiments, the K for CD16aV that binds the Fc domain of an antibody with one or more reduced effector functions is greater than 2 μM. DOptionally, the K for CD32aH binding the Fc domain of an antibody having one or more reduced effector functions is greater than 0.4 μM. D In some embodiments, the K for CD32bF that binds the Fc domain of an antibody with one or more reduced effector functions is greater than 0.5 μM. D is greater than 1 μM.

[0098] In some embodiments, the isolated antibody is a fully human monoclonal antibody. In some embodiments, the isolated antibody is a chimeric antibody. In some embodiments, the isolated antibody is a humanized antibody. In some embodiments, the human anti-CD154 antibody is produced by immunizing a non-human transgenic animal, e.g., a rodent, whose genome contains human immunoglobulin genes such that the transgenic animal produces human antibodies.

[0099] In some embodiments, the anti-CD154 antibody comprises a human or humanized variable region, the variable region comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH being operably linked to a human Fc domain having an altered effector function. In some embodiments, one or more effector functions of the human Fc domain are reduced. Optionally, one or more effector functions of the human Fc domain are eliminated. Optionally, the VH is operably linked to a human Fc region, the human Fc region comprising a human hinge sequence and a human Fc domain, the human hinge sequence being between the VH and the human Fc domain. In some embodiments, one or more effector functions of the human Fc region are reduced. Optionally, one or more effector functions of the human Fc region are eliminated. The hinge may comprise the amino acid sequence of any one of SEQ ID NOs: 76-90. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 76. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 77. The hinge may comprise the amino acid sequence of SEQ ID NO: 78. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 79. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 80. The hinge may comprise the amino acid sequence of SEQ ID NO: 81. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 82. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 83. The hinge may comprise the amino acid sequence of SEQ ID NO: 84. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 85. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 86. The hinge may comprise the amino acid sequence of SEQ ID NO: 87. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 88. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO: 89. The hinge may comprise the amino acid sequence of SEQ ID NO: 90.

[0100] In some embodiments, the human Fc domain is derived from an IgG1 Fc (or crystalline fragment) region. Optionally, the human Fc domain is derived from an IgG1 constant region. The human Fc domain may be derived from an IgG2 Fc (or crystalline fragment) region. In some embodiments, the Fc domain is derived from an IgG2 constant region. The human Fc domain may be derived from an IgG4 Fc (or crystalline fragment) region. In some embodiments, the Fc domain is derived from an IgG4 constant region. In some embodiments, the human Fc region is derived from an IgG1 Fc (or crystalline fragment) region. Optionally, the human Fc region is derived from an IgG1 constant region. In some embodiments, the human Fc region is derived from an IgG2 Fc (or crystalline fragment) region. In some embodiments, the Fc region is derived from an IgG2 constant region. Optionally, the human Fc region is derived from an IgG4 Fc (or crystalline fragment) region. The human Fc region may be derived from an IgG4 constant region.

[0101] In some embodiments, the Fc domain of the antibody disclosed herein comprises one or more amino acid modifications that modify effector function, including reducing or eliminating one or more effector functions. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 238-241. Optionally, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 238-241. The Fc domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9, 12-18, and 238-241. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 236-241. Optionally, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 236-241. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 3. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 5. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 7. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 9. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 13. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 15. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 16.In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 17. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 238. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 240. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 241. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain does not comprise the amino acid sequences of SEQ ID NOs: 236 and 237.

[0102] In some embodiments, the Fc region of the antibody disclosed herein comprises one or more amino acid modifications that modify effector function, including reducing or eliminating one or more effector functions. In some embodiments, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 243-251. Optionally, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 243-251. The Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56 and 243-251. In some embodiments, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 242-251. Optionally, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 242-251. The Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56 and 242-251. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 21. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 23. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 25. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 27. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 29. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 30.In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:31. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:33. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:35. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:40. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:42. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:44. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:46. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 48. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 50. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 52. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 54. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 242. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 243. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 244. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 246. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 247. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:248.Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 250. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 251. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 242. Optionally, the Fc domain does not comprise the amino acid sequences of SEQ ID NOs: 236, 237 and 242.

[0103] In some embodiments, the antibody comprising an IgG4-derived Fc domain or Fc region comprises an amino acid modification at any one or combination of positions selected from the group consisting of S228, L235, L236, G237, E318, and N297, where the numbering of the amino acid residues is according to the EU index as set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). In a preferred embodiment, the antibody comprising an IgG4-derived Fc domain or Fc region comprises an amino acid modification selected from the group consisting of S228P, L235A, L235E, L236E, G237A, E318A, and N297Q, or a combination thereof.

[0104] In some embodiments, an antibody that comprises an Fc domain or region from IgG1 is selected from the group consisting of E216, R217, K218, C219, C220, C226, C229, P230, E233, L234, L235, G236, G237, P238, S239, V240, F241, K246, L251, T260, D265, V266, H268, W277, N297, E318, , K322, P329, A330, P331, Q347, N348, T350, L351, K360, T366, N390, K392, T394, D399, S400, F405, Y407, K409, T411, or a combination of such amino acid modifications, wherein the numbering of the amino acid residues is according to the EU index as set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).In a preferred embodiment, the antibody comprising an Fc domain or region from IgG1 is selected from the group consisting of C220S, C226S, C229S, P230S, E233P, L234A, L234F, L234V, L235A, L235E, L235V, G236E, G237A, P238S, D265S, D265A, H268Q, W277T, N297G, N297Q, N297D, N297A, E318A, K322A, P329G, P329A, A329G ... 30S, P331S, Q347R, Q347E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T366V, N390R, N390K, N390 D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S400K, F405A, F405I, F 405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T411N, T411R, T411Q, T41 1K, T411D, T411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR236 / 237, InR237 / 238, InV2 InN238 / 239, InL238 / 239, InE238 / 239, InG238 / 239, InS239 / 240, InG240 / 241, InE240 / 241, InG240 / 241, InL238 / 239 / P238Q, InE238 / 239 / N348A, InS239 / 240 / V266A, and InR237 / 238 / G236A or a combination thereof.

[0105] In some embodiments, an antibody comprising an IgG2-derived Fc domain or Fc region comprises an amino acid modification at any one or combination of positions selected from the group consisting of V234, G237, P238, H268, V309, A330, and P331, where the numbering of the amino acid residues is according to the EU numbering as set forth in Edelman. In a preferred embodiment, an antibody comprising an IgG2-derived Fc domain or Fc region comprises an amino acid modification selected from the group consisting of V234A, G237A, P238S, H268Q, H268A, V309L, A330S, P331S, or a combination thereof.

[0106] In some embodiments, the VH comprises (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 57, (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 59, and the VL comprises (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60, (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61, and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 62.

[0107] In some embodiments, the VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 63, 64, 252 or 253. Optionally, the VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of SEQ ID NO: 63, 64, 252 or 253. The VH may comprise the amino acid sequence of SEQ ID NO: 63, 64, 252 or 253. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 63. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 252. Optionally, the VH does not comprise the amino acid sequence of SEQ ID NO: 233.

[0108] In some embodiments, the VL comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 66. Optionally, the VL comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of SEQ ID NO: 65 or 66. The VL may comprise the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 65. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 66.

[0109] In some embodiments, the antibody further comprises a CH1 domain, the CH1 domain being operably linked to (a) the C-terminus of the VH and (b) the N-terminus of the hinge. Optionally, the CH1 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 67, 70, and 73. The CH1 domain may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 67, 70, and 73. In some embodiments, the CH1 domain comprises an amino acid sequence of any one of SEQ ID NOs: 67, 70, and 73. The CH1 domain may comprise the amino acid sequence of SEQ ID NO: 67. Optionally, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 73.

[0110] In some embodiments, the antibody comprises a linker between the VH and Fc domains. Optionally, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 199-223 and 327-330. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 199-223. The linker may comprise the amino acid sequence of any one of SEQ ID NOs: 327-330. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 199. Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 200. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 201. Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 203. Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 205. Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 206. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 207. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:208. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:209. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:210. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:211. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:212. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:213. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:214. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:215. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:216. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:217. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:218. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:219. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:220. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:221. Optionally, the linker comprises the amino acid sequence of SEQ ID NO:222. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:223.Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 327. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 328. Optionally, the linker comprises the amino acid sequence of SEQ ID NO: 329. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 330. The linker may be between the VH and the hinge. In some embodiments, the linker is between the VH and the CH1 domain.

[0111] In some embodiments, the VH is operably linked to an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. Optionally, the VH is operably linked to an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to any one of the amino acid sequences of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. The VH may be operably linked to any one of the amino acid sequences of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. In some embodiments, the VH is operably linked to an amino acid sequence of SEQ ID NO:3. Optionally, the VH is operably linked to an amino acid sequence of SEQ ID NO:4. The VH may be operably linked to an amino acid sequence of SEQ ID NO:5. In some embodiments, the VH is operably linked to an amino acid sequence of SEQ ID NO:6. Optionally, the VH is operably linked to an amino acid sequence of SEQ ID NO:7. The VH may be operably linked to an amino acid sequence of SEQ ID NO:8. In some embodiments, the VH is operably linked to an amino acid sequence of SEQ ID NO:9. Optionally, the VH is operably linked to an amino acid sequence of SEQ ID NO:12. The VH may be operably linked to an amino acid sequence of SEQ ID NO:13. In some embodiments, the VH is operably linked to an amino acid sequence of SEQ ID NO:14. Optionally, the VH is operably linked to an amino acid sequence of SEQ ID NO:15. The VH may be operably linked to an amino acid sequence of SEQ ID NO:16. In some embodiments, the VH is operably linked to an amino acid sequence of SEQ ID NO:17. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 18. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 22. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 23. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 24.In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:25. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:26. The VH may be operably linked to the amino acid sequence of SEQ ID NO:27. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:28. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:29. The VH may be operably linked to the amino acid sequence of SEQ ID NO:30. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:31. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:32. The VH may be operably linked to the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:34. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:35. The VH may be operably linked to the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:37. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 40. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 42. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 43. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 45. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 46. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 48. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 49. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 51. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 52. The VH may be operably linked to the amino acid sequence of SEQ ID NO:53.In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:54. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:55. The VH may be operably linked to the amino acid sequence of SEQ ID NO:56. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:236. The VH may be operably linked to the amino acid sequence of SEQ ID NO:237. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:238. The VH may be operably linked to the amino acid sequence of SEQ ID NO:239. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:240. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:241. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO:242. The VH may be operably linked to the amino acid sequence of SEQ ID NO:243. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO:244. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 245. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 246. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 247. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 248. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 249. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 250. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 251. Optionally, the VH is not operably linked to the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH is not operably linked to the amino acid sequence of SEQ ID NO: 237. Optionally, the VH is not operably linked to the amino acid sequence of SEQ ID NO: 242. In some embodiments, the VH is not operably linked to the amino acid sequences of SEQ ID NOs: 236 and 237. Optionally, the VH is not operably linked to the amino acid sequences of SEQ ID NOs: 236, 237 and 242.

[0112] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174 and 266-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174 and 266-288. The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174 and 266-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277, and 279-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277, and 279-288. The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277, and 279-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, and 266-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174 and 266-288.The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, and 266-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277, and 279-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277, and 279-288. The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277, and 279-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 267-271, 273-277, and 279-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 267-271, 273-277, and 279-288. The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 267-271, 273-277, and 279-288. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 121. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 122. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 123. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 124.Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 125. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 126. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 127. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 128. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 129. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 130. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 131. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 135. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 136. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 137. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 138. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 139. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 140. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 141. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 142. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 143. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 144. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 145. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 146. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 149. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 150. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 151. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 152. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 153. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 154. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 155. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 156. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 157. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 158. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 159. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 160. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 163. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164.The heavy chain may comprise the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 166. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 167. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 169. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 170. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 171. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 172. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 173. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 174. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 266. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 267. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 268. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 269. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 270. The heavy chain may comprise the amino acid sequence of SEQ ID NO:271. The heavy chain may comprise the amino acid sequence of SEQ ID NO:272. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:273. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO:274. The heavy chain may comprise the amino acid sequence of SEQ ID NO:275. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO:276. The heavy chain may comprise the amino acid sequence of SEQ ID NO:277. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:278. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:279. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO:280. The heavy chain may comprise the amino acid sequence of SEQ ID NO:281. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:282. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO:283. The heavy chain may comprise the amino acid sequence of SEQ ID NO:284. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:285. Optionally, the heavy chain comprises the amino acid sequence of SEQ ID NO: 286. The heavy chain may comprise the amino acid sequence of SEQ ID NO: 287. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 288. Optionally, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 150.In some embodiments, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 164. Optionally, the heavy chain does not comprise the amino acid sequences of SEQ ID NOs: 150 and 164. In some embodiments, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 234. Optionally, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 266. In some embodiments, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 278. Optionally, the heavy chain does not comprise the amino acid sequences of SEQ ID NOs: 234 and 278. In some embodiments, the heavy chain does not comprise the amino acid sequences of SEQ ID NOs: 234, 266, 272 and 278. Optionally, the heavy chain does not comprise the amino acid sequences of SEQ ID NOs: 150, 164, 234 and 278. In some embodiments, the heavy chain does not comprise the amino acid sequences of SEQ ID NOs: 150, 164, 234, 266, 272 and 278.

[0113] In some embodiments, the light chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 195 and 196. Optionally, the light chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 195 and 196. The light chain may comprise the amino acid sequence of SEQ ID NO: 195 or 196. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 195. Optionally, the light chain comprises the amino acid sequence of SEQ ID NO: 196.

[0114] In some embodiments, the antibody is monoclonal. Optionally, the antibody is a chimeric antibody. The antibody may be a humanized antibody. In some embodiments, the antibody is a human antibody.

[0115] In some embodiments, the antibody binds to human CD154 and inhibits the interaction between human CD154 and human CD40. Optionally, the antibody blocks the activation of one or more of B cells, macrophages, dendritic cells, or endothelial cells by inhibiting the binding of CD154 to CD40. In some embodiments, a reduction in the level of thrombosis is observed after administration of the antibody compared to the level of thrombosis after administration of 5c8 or hu5c8 antibody. Optionally, the antibody does not cause thrombosis when administered to a subject.

[0116] In some embodiments, the antibody has one or more of the following effects when administered to a subject: (a) a reduced risk of thrombosis or thromboembolic events compared to hu5c8 antibody; (b) a reduced activation of platelets expressing CD154; (c) inhibition of CD154 shedding; and (d) alteration of expression or activity of downstream targets of CD154-CD40 signaling. Optionally, administration of the antibody results in a reduced risk of thrombosis or thromboembolic events compared to a subject administered 5c8 or hu5c8 antibody. In some embodiments, administration of the antibody results in a reduced activation of platelets expressing CD154. Optionally, administration of the antibody results in inhibition of CD154 shedding. In some embodiments, administration of the antibody results in alteration of expression of activity of downstream targets of CD154-CD40 signaling.

[0117] In some embodiments, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of any one of SEQ ID NOs: 1, 2, 10, 11, 231, and 236. Optionally, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 2. Optionally, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 11. Optionally, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 231. In some embodiments, the human Fc domain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 236. In some embodiments, the antibody does not comprise the amino acid sequence consisting of any one of SEQ ID NOs: 1, 2, 10, 11, 231, and 236. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 2. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 11. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 231. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 236.

[0118] Optionally, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of any one of SEQ ID NO: 19, 20, 38, 39, 232, and 235. In some embodiments, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 19. Optionally, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 20. Optionally, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 39. Optionally, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the human Fc region with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 235. Optionally, the antibody does not comprise the amino acid sequence consisting of any one of SEQ ID NO: 19, 20, 38, 39, 232, and 235. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 19. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 20. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 39. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 235.

[0119] In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of any one of SEQ ID NOs: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 119. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 120. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 133. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 134. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 147. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 148. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 150. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 161. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 162. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 164. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 230. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 234. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 278. In some embodiments, the antibody does not comprise the amino acid sequence consisting of any one of SEQ ID NOs: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 119. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 120. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO:133.In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 134. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 147. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 148. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 161. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 162. In some embodiments, the heavy chain with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 164. Optionally, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 230. In some embodiments, the antibody does not comprise the amino acid sequence consisting of SEQ ID NO: 234. Optionally, the heavy chain with altered effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 278. In some embodiments, the antibody does not comprise an amino acid sequence consisting of any one of SEQ ID NOs: 1, 2, 10, 11, 19, 20, 38, 39, 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 231, 232, 234, 235, 236, and 278.

[0120] Anti-CD154 antibody properties Binding affinity of anti-CD154 antibodies to CD154 Binding affinity of anti-CD154 antibodies to CD154 (K D ) and dissociation rate (k off ) can be determined by methods known in the art. Binding affinity can be measured by ELISA, RIA, flow cytometry, or surface plasmon resonance (SPR), such as by a BIACORE® system. Dissociation rate can be measured by SPR. Optionally, binding affinity and dissociation rate are measured by SPR. In some embodiments, binding affinity and dissociation rate are measured using BIACORE®. One of skill in the art can determine whether an antibody disclosed herein has substantially the same K as another anti-CD154 antibody by using methods known in the art. D It can be determined whether or not the D and k offSuch methods of determining the K can be used during initial screening stages, and during subsequent optimization stages. In some embodiments, the antibody has a K of less than 50 pM for CD154. D Optionally, the antibody has a K for CD154 of less than 25 pM. D In some embodiments, the antibody has a K with respect to CD154 of 5 to 25 pM. D Optionally, the antibody has a K with respect to CD154 of between 9.5 and 23 pM. D has.

[0121] Inhibition of CD154 activity by anti-CD154 antibodies Anti-CD154 antibodies that inhibit the binding of CD154 to CD40 can be identified using any one of several assays, such as competitive binding assays, FACS analysis, B cell activation assays, B cell proliferation assays, T cell activation assays, and T cell proliferation assays. See, for example, Barr et al., Immunology, 102(1):39-43 (2001); and Blair et al., J. Exp. Med., 191(4):651-660 (2001). For example, neutralizing anti-CD154 antibodies can be identified by their inhibition of upregulation of specific downstream target genes of CD154, such as CD23, CD44H, CD54, TRAF-3, and NFκB. In some embodiments, anti-CD154 antibodies have an IC of 500nM, 300nM, 200nM, 150nM, 100nM, 50nM, 20nM, 10nM, or 1nM or less. 50 has.

[0122] Effector function-platelet assay The effector function of anti-CD154 can be determined using any one of several assays, such as in vitro platelet activation and / or aggregation assays. See, for example, U.S. Pat. No. 9,765,150; Langer F et al., Thromb Haemost. Jun; 93(6): 1137-46 (2005); McKenzie, SE et al., J Immunol 162 (7) 4311-4318 (1999); and Scholthauer T et al., Protein Engineering, Design and Selection, 29(10): 457-466, (2016). Blood from human donors or mice expressing FcγRIIA (CD32a) on platelets can be used to assay platelet function. Platelet activation can be detected by flow cytometry using antibodies against the platelet activation markers P-selectin (CD62P) and PAC-1 (activated GPIIb / IIIa). Platelet aggregation analysis can be analyzed using a minicell impedance device and quantifying the area under the curve as a measure of the platelet aggregation impedance curve.

[0123] Pharmaceutical Compositions and Administration The methods disclosed herein may also utilize a pharmaceutical composition comprising an anti-CD154 antibody described herein and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an anti-CD154 antibody described herein.

[0124] The antibody disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibody disclosed herein and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are used interchangeably and refer to any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutically acceptable carriers are well known in the art. See, for example, Remington's Pharmaceutical Sciences, and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984), which are incorporated herein by reference. Some examples of pharma- ceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharma- ceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.Pharmaceutical compositions can be prepared by mixing the antibodies disclosed herein with acceptable carriers, excipients, or stabilizers, for example in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Forms: Disperse Systems, Marcel Dekker, NY; see Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0125] The pharmaceutical composition may be in various forms, such as liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions, suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution, such as a composition similar to that used for passive immunization of humans. Optionally, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the mode of administration is intravitreal injection. The pharmaceutical composition may be administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection. The injectable formulation may be stored in unit dosage form, such as ampoules, prefilled syringes, or multi-dose containers, with or without added preservatives. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be prepared in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0126] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentration. Sterile injectable solutions may be prepared by incorporating the required amount of anti-CD154 antibody in a suitable solvent, with one or a combination of ingredients listed above as needed, followed by filtration sterilization. Dispersions may be prepared by incorporating anti-CD154 antibody into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods include vacuum drying and freeze-drying, which yield a powder of anti-CD154 antibody and any additional desired ingredients from the previously sterilized-filtered solution. The proper fluidity of the solution may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and / or by the use of surfactants. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0127] Pharmaceutical compositions can be administered by various methods known in the art.In some embodiments, preferred route / mode of administration is subcutaneous, intramuscular or intravenous injection.In some embodiments, mode of administration is intravitreal.As recognized by those skilled in the art, route and / or mode of administration vary according to desired results.

[0128] In some embodiments, pharmaceutical compositions can be prepared with carriers that protect antibody from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.The method of preparing such formulations is generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems JR Robinson, ed., Marcel Dekker, Inc., New York, 1978, which is incorporated herein by reference.

[0129] Additional active compounds can also be incorporated into the composition.In certain embodiments, the anti-CD154 antibody disclosed herein is co-formulated and / or co-administered with one or more additional therapeutic agents.These agents include, but are not limited to, antibodies that bind to other targets, antithrombotic agents, antiplatelet agents, nonsteroidal anti-inflammatory drugs (NSAIDs) and antiallergic agents.Such combination therapy requires lower dosages of anti-CD154 antibody and co-administered agents, thus avoiding possible toxicity or complications associated with various monotherapies.

[0130] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses may be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions into dosage unit forms. The term "dosage unit form" as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the dosage unit forms are subject to and directly depend on (a) the unique characteristics of the anti-CD154 antibody and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such antibodies for the treatment of susceptibility in individuals.

[0131] An exemplary non-limiting range for a therapeutically or prophylactically effective amount of an antibody disclosed herein is 5-50 mg / kg. A therapeutically or prophylactically effective amount of an antibody disclosed herein can be about 5-50 mg / kg. In some embodiments, a therapeutically or prophylactically effective amount of an antibody disclosed herein can be about 5-30 mg / kg. A therapeutically or prophylactically effective amount of an antibody disclosed herein can be about 5-30 mg / kg. Optionally, a therapeutically or prophylactically effective amount of an antibody disclosed herein can be 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg. In some embodiments, the therapeutically or prophylactically effective amount of the antibody disclosed herein is about 5mg / kg, about 10mg / kg, about 15mg / kg, about 20mg / kg, about 25mg / kg, about 30mg / kg, about 35mg / kg, about 40mg / kg, about 45mg / kg, or about 50mg / kg. Dosage values ​​may vary with the type and severity of the condition to be alleviated. For any particular subject, a particular dosage regimen may be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are merely examples and are not intended to limit the scope or practice of the compositions divided into paragraphs.

[0132] Methods for inducing tolerance A first aspect of the present disclosure provides a method of inducing tolerance in a transplant recipient. In some embodiments, the method includes administering an anti-CD154 antibody disclosed herein to the recipient, transplanting hematopoietic stem cells into the recipient, and transplanting a donor organ, donor tissue, or donor cells into the recipient, where the transplanted hematopoietic stem cells produce immune cells that are tolerant to the donor organ, donor tissue, or donor cells, thereby inducing tolerance in the recipient. Optionally, a therapeutically effective amount of an anti-CD154 antibody is administered. In some embodiments, the anti-CD154 is administered in a pharmaceutical composition disclosed herein. The anti-CD154 antibody can be administered simultaneously with the transplantation of hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered sequentially with the transplantation of hematopoietic stem cells. Optionally, the anti-CD154 antibody is administered prior to the transplantation of hematopoietic stem cells. The anti-CD154 antibody can be administered following the transplantation of hematopoietic stem cells. In some embodiments, the hematopoietic stem cells are transplanted by bone marrow transplantation. In some embodiments, anti-CD154 is administered in a single dose. Optionally, the anti-CD154 antibody is administered in multiple doses. In some embodiments, one of the multiple doses is administered simultaneously with the transplantation of hematopoietic stem cells, and the other doses are administered before and / or after the transplantation of hematopoietic stem cells. Optionally, one of the multiple doses is administered simultaneously with the transplantation of hematopoietic stem cells, and the other doses are administered before the transplantation of hematopoietic stem cells. In some embodiments, one of the multiple doses is administered simultaneously with the transplantation of hematopoietic stem cells, and the other doses are administered after the transplantation of hematopoietic stem cells. In some embodiments, one of the multiple doses is administered simultaneously with the transplantation of hematopoietic stem cells, and the other doses are administered before and after the transplantation of hematopoietic stem cells.

[0133] In some embodiments, the immune tolerance is central tolerance. In some embodiments, the method further comprises depleting T cells in the thymus. Optionally, the method of inducing immune tolerance further comprises depleting T cells in bone marrow. In some embodiments, the method of inducing immune tolerance further comprises depleting T cells in both the thymus and bone marrow.

[0134] In some embodiments, the immune tolerance is peripheral tolerance.In some embodiments, the method further comprises depleting T cells in lymph nodes.Optionally, the method further comprises depleting T cells in peripheral tissues.In some embodiments, the method further comprises depleting T cells in both lymph nodes and peripheral tissues.

[0135] In some embodiments, the method of inducing immune tolerance is organ-specific tolerance. In some embodiments, the method further comprises depleting T cells from the heart, kidney, both kidneys, liver, a portion of the liver, lung, both lungs, pancreas, intestine, islet cells, face, one hand, both hands, one arm, both arms, one foot, both feet, one leg, both legs, or skin or a combination thereof. In some embodiments, the method further comprises depleting T cells from the heart. In some embodiments, the method further comprises depleting T cells from the kidney. In some embodiments, the method further comprises depleting T cells from both kidneys. In some embodiments, the method further comprises depleting T cells from the liver. In some embodiments, the method further comprises depleting T cells from a portion of the liver. In some embodiments, the method further comprises depleting T cells from the lung. In some embodiments, the method further comprises depleting T cells from both lungs. In some embodiments, the method further comprises depleting T cells from the pancreas. In some embodiments, the method further comprises depleting T cells from the intestine. In some embodiments, the method further comprises depleting T cells from islet cells. In some embodiments, the method further comprises depleting T cells from the face. In some embodiments, the method further comprises depleting T cells from one hand. In some embodiments, the method further comprises depleting T cells from both hands. In some embodiments, the method further comprises depleting T cells from one arm. In some embodiments, the method further comprises depleting T cells from both arms. In some embodiments, the method further comprises depleting T cells from one foot. In some embodiments, the method further comprises depleting T cells from both feet. In some embodiments, the method further comprises depleting T cells from one leg. In some embodiments, the method further comprises depleting T cells from both legs. In some embodiments, the method further comprises depleting T cells from the skin.

[0136] In some embodiments, the method further comprises a treatment regimen for conditioning the recipient for hematopoietic stem cell transplantation. In some embodiments, the method further comprises more than one treatment regimen for conditioning the recipient for hematopoietic stem cell transplantation. In some embodiments, the method for conditioning the recipient for hematopoietic stem cell transplantation is selected from the group consisting of total body irradiation, administration of abatacept, administration of one or more BCL-2 inhibitors, administration of busulfan, administration of fludarabine phosphate, administration of cyclophosphamide, administration of one or more immunosuppressive T cell depleting antibodies, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or a combination thereof.

[0137] In some embodiments, T cells are depleted by a method selected from the group consisting of total body irradiation, administration of abatacept, administration of one or more BCL-2 inhibitors, administration of busulfan, administration of fludarabine phosphate, administration of cyclophosphamide, administration of one or more immunosuppressive T cell depleting antibodies, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or a combination thereof. In some embodiments, T cells are depleted by total body irradiation. In some embodiments, T cells are depleted by administration of abatacept. In some embodiments, T cells are depleted by administration of a BCL-2 inhibitor. In some embodiments, T cells are depleted by administration of more than one BCL-2 inhibitor. In some embodiments, T cells are depleted by administration of busulfan. In some embodiments, T cells are depleted by administration of fludarabine phosphate. In some embodiments, T cells are depleted by administration of cyclophosphamide. In some embodiments, T cells are depleted by administration of a T cell depleting antibody. In some embodiments, T cells are depleted by administration of more than one T cell depleting antibody. In some embodiments, T cells are depleted by administration of an anti-αβ T cell receptor antibody. In some embodiments, T cells are depleted by administration of more than one anti-αβ T cell receptor antibody. In some embodiments, T cells are depleted by administration of a CD122 antagonist. In some embodiments, T cells are depleted by administration of more than one CD122 antagonist.

[0138] In some embodiments, the one or more T cell depleting antibodies are selected from the group consisting of an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD45 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, and an anti-CD33 antibody, or a combination thereof.

[0139] In some embodiments, the method of inducing immune tolerance results in mixed allogeneic chimerism. Optionally, the method of inducing immune tolerance results in mixed chimerism.

[0140] In some embodiments, the anti-CD154 antibody is administered prior to hematopoietic stem cell transplantation. Optionally, the anti-CD154 antibody is administered following hematopoietic stem cell transplantation. In some embodiments, the anti-CD154 antibody is administered simultaneously with hematopoietic stem cell transplantation. In some embodiments, the hematopoietic stem cells are transplanted by bone marrow transplantation. In some embodiments, the anti-CD154 is administered in a single dose. Optionally, the anti-CD154 antibody is administered in multiple doses. In some embodiments, one of the multiple doses is concurrent with hematopoietic stem cell transplantation, and another dose is administered prior to and / or following hematopoietic stem cell transplantation. Optionally, one of the multiple doses is concurrent with hematopoietic stem cell transplantation, and another dose is administered prior to hematopoietic stem cell transplantation. In some embodiments, one of the multiple doses is concurrent with hematopoietic stem cell transplantation, and another dose is administered following hematopoietic stem cell transplantation. In some embodiments, one of the multiple doses is concurrent with hematopoietic stem cell transplantation, and other doses are administered prior to and following hematopoietic stem cell transplantation.

[0141] In some embodiments, the anti-CD154 antibody is administered at a dose of 5-50 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of 10-40 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of 20-30 mg / kg.

[0142] In some embodiments, the anti-CD154 antibody is administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally. In some embodiments, the anti-CD154 antibody is administered subcutaneously. In some embodiments, the anti-CD154 antibody is administered intravenously. In some embodiments, the anti-CD154 antibody is administered intravitreally. In some embodiments, the anti-CD154 antibody is administered orally. In some embodiments, the anti-CD154 antibody is administered by inhalation. In some embodiments, the anti-CD154 antibody is administered transdermally. In some embodiments, the anti-CD154 antibody is administered rectally.

[0143] Any of the antibodies disclosed herein can be used in the method of inducing tolerance. In some embodiments, the anti-CD154 antibody is a human, chimeric or humanized antibody. In some embodiments, the anti-CD154 antibody is a human antibody. In some embodiments, the anti-CD154 antibody is a chimeric antibody. In some embodiments, the anti-CD154 antibody is a humanized antibody.

[0144] Optionally, the transplant recipient is a human. In some embodiments, the transplant recipient is a non-human. The transplant recipient may be a non-human primate, such as a monkey. Optionally, the anti-CD154 antibody is a human antibody and the transplant recipient is a human. In some embodiments, the anti-CD154 antibody is a humanized antibody and the transplant recipient is a human. Alternatively, the transplant recipient may be a mammal (e.g., a monkey) that expresses a CD154 with which the anti-CD154 antibody cross-reacts. The antibody may be administered to a non-human mammal (e.g., a cynomolgus monkey) that expresses a CD154 with which the antibody cross-reacts for veterinary purposes or as an animal model of human transplantation or disease. Such animal models may be useful for evaluating the therapeutic efficacy of the antibodies disclosed herein.

[0145] The antibody may be administered once. If necessary, the antibody is administered multiple times. The antibody may be administered three times a day to once every six months or longer. Administration may be on a schedule such as three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, twice a week, three times a week, four times a week, twice every two weeks, three times every two weeks, and four times every two weeks. The antibody may also be administered continuously by a minipump. The antibody may be administered by mucosal, buccal, intranasal, inhalation, intravenous, intravitreal, subcutaneous, intramuscular, parenteral, or intratumoral routes. In some embodiments, the anti-CD154 antibody is administered systemically, e.g., subcutaneously, intravenously, orally, by inhalation, transdermally, or rectally. Optionally, the anti-CD154 antibody is administered topically. In some embodiments, the anti-CD154 antibody is administered intravitreally. The antibody may be administered once, at least twice, or for at least as long as the condition is treated, alleviated, or cured. The antibody is generally administered for as long as the condition exists. The antibody is generally administered as part of a pharmaceutical composition as described above. The dosage of the antibody is generally in the range of 5 to 50 mg / kg. The dosage of the antibody may be about 5 to about 50 mg / kg. In some embodiments, the dosage of the antibody is 5 to 30 mg / kg. The dosage of the antibody may be about 5 to about 30 mg / kg. Optionally, the dosage of the antibody is 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg. In some embodiments, the dosage of the antibody is about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg.

[0146] In some embodiments, the anti-CD154 antibody disclosed herein is administered to a subject expressing inappropriately high levels of CD154. The transplant can be an allograft, an autograft or a xenograft of donor cells, tissues or organs. In some embodiments, the transplant is an allograft. Optionally, the transplant is an autograft. In some embodiments, the transplant is a xenograft.

[0147] The donor's cells may be engineered cells or ex-vivo expanded cells. For example, the donor's cells may be modified using one or more techniques, such as transduction to express cDNA, CRISPR / Cas9 system, RNAi technology, and retroviral technology. Optionally, the donor's cells are modified to express a chimeric antigen receptor (CAR) on their surface. Examples of cells that may be transplanted include, but are not limited to, stem cells, regulatory T (Treg) cells, CAR-T cells, CAR-B cells, and tumor infiltrating lymphocytes (TILs). In some embodiments, the donor's cells are stem cells. In some embodiments, the donor's cells are regulatory T (Treg) cells. In some embodiments, the donor's cells are CAR-T cells. In some embodiments, the donor's cells are CAR-B cells. In some embodiments, the donor's cells are tumor infiltrating lymphocytes (TILs).

[0148] Induction of tolerance by any of the methods disclosed herein can prevent transplant rejection in a subject. For example, induction of tolerance can prevent acute or chronic humoral rejection of grafted cells, tissues, or organs. Rejection can be acute or chronic graft rejection in allograft or xenograft graft recipients. The methods disclosed herein can promote long-term graft survival of grafted cells, tissues, or organs. Optionally, long-term graft survival is at least 6 months after transplantation, at least 1 year after transplantation, or at least 5 years after transplantation. In some embodiments, long-term graft survival is at least 6 months after transplantation. In some embodiments, long-term graft survival is at least 1 year after transplantation. In some embodiments, long-term graft survival is at least 5 years after transplantation.

[0149] The transplant rejection may be associated with hematopoietic cell or bone marrow transplants, pancreatic islet cell allografts, graft-versus-host disease, or solid organ transplants selected from the group consisting of heart transplants, kidney transplants, liver transplants, lung transplants, pancreas transplants, kidney-pancreas transplants, heart-lung transplants, kidney-heart transplants, kidney-heart-pancreas transplants, heart-liver transplants, heart-liver-kidney transplants, heart-lung-kidney transplants, heart-lung-liver transplants, lung-kidney transplants, lung-liver transplants, liver-intestine-pancreas transplants, intestine-pancreas transplants, liver-kidney-intestine-pancreas transplants, and kidney-intestine transplants.

[0150] In some embodiments, the method comprises administering a therapeutically effective amount of an anti-CD154 antibody disclosed herein to said transplant recipient in combination with one or more additional agents. Optionally, the one or more additional agents are selected from the group consisting of antithrombotic agents, antiplatelet agents, and nonsteroidal anti-inflammatory drugs (NSAIDs). The anti-CD154 antibody may be administered simultaneously with the one or more additional agents. In some embodiments, the anti-CD154 antibody is administered sequentially with the one or more agents. Optionally, the anti-CD154 antibody is administered prior to the one or more additional agents. The anti-CD154 antibody may be administered subsequent to the one or more additional agents. In some embodiments, the anti-CD154 antibody is administered in the same composition as the one or more additional agents. Optionally, the anti-CD154 antibody and the one or more additional agents are administered in separate compositions.

[0151] Examples of antithrombotic drugs include, but are not limited to, glycoprotein IIb / IIIa receptor antagonists, direct or indirect factor Xa inhibitors, and anticoagulants. Examples of anticoagulants include, but are not limited to, heparin, warfarin, rivaroxaban (XARELTO®), ximelgatran (EXANTA®), dabigatran (PRADAXA®), apixaban (ELIQUIS®), edoxaban (SAVAYSA®), enoxaparin (LOVENOX®), and fondaparinux (ARIXTRA®). Examples of antithrombotic agents include, but are not limited to, those described in U.S. Patent Nos. 4,782,069; 5,332,822; 5,492,895; 5,612,363; 5,691,364; 5,693,641; 5,721,214; 5,726, No. 173; No. 5,753,635; No. 5,846,970; No. 5,849,759; No. 5,889,005; No. 6,107,280; No. 6,1 No. 40,351; No. 6,150,329; No. 6,180,627; No. 6,200,976; No. 6,242,432; No. 6,248,770; No. 6 ,271,215;No.6,280,731;No.6,287,794;No.6,300,330;No.6,300,342;No.6,333,338 ; No. 6,395,731; No. 6,417,203; No. 6,432,955; No. 6,444,672; No. 6,451,832; No. 6,458,79 Nos. 6,723,723; 6,730,672; 6,753,331; 6,774,110; 6,797,710; and 6,924,296.Examples of glycoprotein IIb / IIIa receptor antagonists include, but are not limited to, abciximab (REOPRO®), rivaroxaban (XARELTO®), apixaban (ELIQUIS®), edoxaban (SAVAYSA®), idrabiotaparinux, tirofiban (AGGRASTAT®), and eptifibatide (INTEGRILIN®). Direct or indirect factor Xa inhibitors include, but are not limited to, apixaban (ELIQUIS®), idrabiotaparinux, fondaparinux (ARIXTRA®), and rivaroxaban (XARELTO®).

[0152] Examples of antiplatelet drugs include, but are not limited to, TXA2 pathway inhibitors, adenosine diphosphate (ADP) pathway inhibitors, thrombin inhibitors, protease-activated receptor-1 (PAR-1) inhibitors, and phosphodiesterase (PDE) inhibitors. Examples of ADP pathway inhibitors include, but are not limited to, clopidogrel (PLAVIX®), ticlopidine (TICLID®), prasugrel (EFFIENT®), ticagrelor (BRILINTA®), cangrelor (KENGREAL®), and elinogrel. Non-limiting examples of PDE inhibitors include, but are not limited to, dipyridamole (PERSANTINE®) and cilostazol (PLETAL®).

[0153] Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, celecoxib (CELEBREX®), diclofenac (VOLTAREN®, PENNSAID®, SOLARAZE®, ZIPSOR®, CATAFLAM®, ZORVOLEX®), diflunisal (DOLOBID®), etodolac (LODINE®), SR®, ECCOXOLAC®), ibuprofen (BRUFEN®, ADVIL®, MOTRIN®), indomethacin (INDOCIN®), ketoprofen (ORUDIS®), ketorolac (TORADOL®, ACULAR®, SPRIX®), nabumetone (RELAFEN®), naproxen (AFLAXEN®, ALEVE®, ANAPROX®, NAPRELAN®), oxaprozin (DAYPRO®, DAYRUN®, DURAPROX®), piroxicam (FELDENE®), salsalate (MONO-GESIC®, SALFLEX®, DISALCID®, SALSITAB®), sulindac (CLINORIL®), tolmetin (TOLECTIN®), prasugrel (EFFIENT®), ticagrelor (BRILINTA®), and cangrelor (KENGREAL®).

[0154] In some embodiments, the anti-CD154 antibody may be administered in combination with one or more supplemental agents, including, but not limited to, immunosuppressants, immunomodulators, and monoclonal and / or polyclonal antibodies. The anti-CD154 antibody may be administered simultaneously with one or more supplemental agents. In some embodiments, the anti-CD154 antibody is administered sequentially with one or more supplemental agents. Optionally, the anti-CD154 antibody is administered before one or more supplemental agents. The anti-CD154 antibody may be administered next to one or more supplemental agents. In some embodiments, the anti-CD154 antibody is administered in the same composition as one or more supplemental agents. Optionally, the anti-CD154 antibody and one or more supplemental agents are administered in separate compositions. Examples of such one or more supplemental agents include, but are not limited to, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD28 antibodies, anti-CD52 antibodies, anti-C5 antibodies, mTOR inhibitors, calcineurin inhibitors, antiviral drugs, and fusion peptides that bind to and block the function of CD28. Non-limiting examples of fusion peptides that bind to and block the function of CD28 include abatacept and beratocept (NULOJIX®). For example, the anti-CD52 antibody can be alemtuzumab (CAMPATH®). Optionally, the anti-C5 antibody is eculizumab (SOLIRIS®).

[0155] Non-limiting examples of immunosuppressants or immunomodulators include cyclosporine A, tacrolimus (FK-506), doxorubicin (ADRIAMYCIN®), azathioprine (IMURAN®), busulfan (BUSULFEX®), cyclophosphamide (CYTOXAN®), fludarabine, 5-fluorouracil, methotrexate (OTREXUP®, RASUVO®, RHEUMATREX®, TREXALL™), mycophenolate mofetil (CELLCEPT®), mizoribine (BREDININ™), leflunomide, nonsteroidal anti-inflammatory drugs, corticosteroids, rapamycin (RAPAMUNE®), deoxyspergualin, FTY720, muromonab-CD3 (ORTHOCLONE®), cyclosporine A, cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine E, cyclosporine F ... OKT3®), alemtuzumab (CAMPATH®, MABCAMPATH®, CAMPATH-1H®, LEMTRADA®), basiliximab (SIMULECT®), daclizumab (ZINBRYTA®), eculizumab (SOLIRIS®), rituximab (RITUXAN®, MABTHERA®), bortezomib (VELCADE®, CHEMOBORT®, BORTECAD®), siplizumab, antithymocyte globulin (THYMOGLOBULIN®), ), ATGAM®), leronlimab, siltuximbab (SYLVANT®), sarilumab (KEVZARA®), tocilizumab (ACTEMRA®), bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (EYLEA®), and inhibitors of Bruton's tyrosine kinase (BTK), including zanubrutinib (BRUKINSA®), acalabrutinib (CALQUENCE®), and ibrutinib (IMBRUVICA®).

[0156] Examples of mTOR inhibitors include, but are not limited to, rapamycin (Rapamune®), everolimus (AFINITOR®), temsirolimus (TORISEL®), ridaforolimus, and deforolimus. Examples of calcineurin inhibitors include, but are not limited to, cyclosporine (NEORAL®, SANDIMMUNE®, GENGRAF®, RESTASIS®), tacrolimus (FK506, ENVARSUS®, HECORIA®, PROGRAF® PROTOPIC®, ASTRAGRAF®), and pimecrolimus (ELIDEL®). EXAMPLES

[0157] The following examples are provided for illustrative purposes only and do not limit the scope of the disclosure or the paragraphs in any way. Indeed, various modifications of the disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the paragraphs.

[0158] Example 1 Induction of tolerance To determine the efficacy of anti-CD154 in inducing tolerance, heterotopic kidney allograft experiments are performed in cynomolgus monkeys according to the regimen described in Figure 1. Briefly, transplant recipients are acclimatized to transplantation by two doses of 1.5 Gy total body irradiation (6 and 5 days before transplantation), 7 Gy thymus irradiation 1 day before transplantation, and three doses of 50 mg / kg / day of anti-T cell antibody [horse antithymocyte globulin (Atgam)] (2 days, 1 day before transplantation, and the day of transplantation). On day 0, transplant recipients receive kidney transplantation and bone marrow (containing hematopoietic stem cells) transplantation. Transplant recipients are given an initial dose of 20 mg / kg of anti-CD154 monoclonal antibody (mAb) (e.g., TNX02, TNX05, TNX06, etc.) within 24 hours of transplantation. The following doses of anti-CD154 antibody (20 mg / kg) are then given on post-operative days (POD) 3, 7, 14, 21, 28, 42, 56, 70, and 84 and monthly thereafter until POD 180 or graft rejection. On POD 180 or when grafts fail, a complete necropsy is performed to determine the level of thromboembolism in all animals.

[0159] Renal allograft survival is monitored regularly by urine output, blood chemistry measurements (twice weekly) and ultrasound examination of the kidney graft. Kidney biopsies are performed at regularly scheduled intervals after transplantation, e.g., on POD 42 and 84. Transplant recipients demonstrating an elevation in serum creatinine greater than 50% of baseline or alterations in renal blood flow are further biopsied to assess the health of the grafted tissue. Any animals with creatinine levels greater than 6 mg / dl are considered to have graft rejection and undergo explantation and necropsy of the graft. Histology is performed on the hearts of all animals at the time of graft rejection or POD 180 (whichever occurs first). Any incidence of thrombosis in the kidney allograft or other sites is measured and analyzed to determine the level of rejection and, conversely, the immune tolerance exhibited by the recipient.

[0160] The immune response of transplant recipients is measured and monitored serially. Baseline serum and peripheral blood lymphocytes are stored. Post-surgery serum and peripheral blood lymphocytes are then collected to determine T cell phenotype analyzed by fluorescence-activated cell sorting (FACS) for markers such as CD3, CD4, CD8, CD25, CD45, CD127, and FoxP3. Similarly, blood is drawn for serum and immune cells collected on POD7, 14, 21, 30, 60, 90, and monthly until POD180, which is the endpoint of transplant rejection or experiment. Table 1. Sequences of humanized anti-CD154 antibodies [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

Table 1-67

Claims

1. A pharmaceutical composition for use in hematopoietic stem cell transplantation and inducing immune tolerance in a transplant recipient who has received or will receive a donor organ, donor tissue or donor cell transplant, wherein said hematopoietic stem cells produce immune cells that are tolerant to said donor organ, donor tissue or donor cells, thereby inducing immune tolerance in said recipient; the pharmaceutical composition comprises an isolated anti-CD154 antibody comprising a humanized variable domain, the variable domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH being operably linked to a human Fc region derived from IgG4 and comprising S228P and L235A amino acid modifications; The VH is (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:57; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:58; and (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:59; wherein the VL is (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60; (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 62; Including, Pharmaceutical compositions.

2. The pharmaceutical composition for use according to claim 1 , wherein said tolerance is central tolerance.

3. A pharmaceutical composition for use as described in claim 2, characterized in that T cells in the thymus and / or bone marrow of the recipient have been or are to be depleted.

4. 2. The pharmaceutical composition for use according to claim 1, wherein said tolerance is (a) peripheral tolerance, or (b) organ-specific tolerance.

5. A pharmaceutical composition for use as described in claim 4, characterized in that (a) T cells in the lymph nodes and / or peripheral tissues of the recipient have been or will be depleted, or (b) T cells from the heart, kidneys, liver, lungs, pancreas, intestines, islet cells, face, hands, arms, feet, legs or skin or a combination thereof of the recipient have been or will be depleted.

6. 6. The pharmaceutical composition for use according to claim 3 or 5, characterized in that said T cells have been or are depleted by a method selected from the group consisting of total body irradiation, administration of abatacept, administration of one or more BCL-2 inhibitors, administration of busulfan, administration of fludarabine phosphate, administration of cyclophosphamide, administration of one or more immunosuppressive T cell depleting antibodies, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or a combination thereof.

7. 7. The pharmaceutical composition for use according to claim 6, wherein the one or more T cell depleting antibodies are selected from the group consisting of an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD45 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, and an anti-CD33 antibody or a combination thereof.

8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein the transplant recipient exhibits mixed chimerism.

9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the anti-CD154 antibody is formulated for administration prior to, following or simultaneously with the transplantation of the hematopoietic stem cells.

10. The pharmaceutical composition for use according to claims 1 to 9, wherein the anti-CD154 antibody is formulated for administration at a dose of 5 to 50 mg / kg.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the anti-CD154 antibody is formulated for administration subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally.

12. The pharmaceutical composition for use according to claim 11, wherein the anti-CD154 antibody is formulated for intravenous administration.

13. 13. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 23 or 42.

14. The pharmaceutical composition for use according to any one of claims 1 to 13, wherein the heavy chain comprises a constant region comprising an amino acid sequence of SEQ ID NO: 95 or 119.

15. 15. The pharmaceutical composition for use according to any one of claims 1 to 14, wherein the VH comprises the amino acid sequence of SEQ ID NO:

64.

16. 16. The pharmaceutical composition for use according to any one of claims 1 to 15, wherein the VL comprises the amino acid sequence of SEQ ID NO:

66.

17. 17. The pharmaceutical composition for use according to any one of claims 1 to 16, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 or 165.

18. 18. The pharmaceutical composition for use according to any one of claims 1 to 17, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

196.

19. 19. The pharmaceutical composition for use according to any one of claims 1 to 18, wherein the antibody is monoclonal.

20. The antibody has the following effects when administered to a subject: (a) a reduced risk of thrombosis or thromboembolic events compared to the hu5c8 antibody; (b) decreased activation of CD154-expressing platelets; (c) inhibition of CD154 shedding; and (d) altering the expression or activity of downstream targets of CD154-CD40 signaling 20. A pharmaceutical composition for use according to any one of claims 1 to 19, comprising one or more of:

21. 21. The pharmaceutical composition for use according to any one of claims 1 to 20, wherein the transplant recipient is a human.

22. 21. The pharmaceutical composition for use according to any one of claims 1 to 20, wherein the transplant recipient is non-human.

23. 23. The pharmaceutical composition for use according to claim 22, wherein the transplant recipient is a monkey.

24. 24. The pharmaceutical composition for use according to any one of claims 1 to 23, wherein the transplant is an allograft, an autograft or a xenograft.

25. 24. The pharmaceutical composition for use according to any one of claims 1 to 23, wherein said donor cells are engineered cells or ex-vivo expanded cells.

26. 26. The pharmaceutical composition for use according to claim 25, wherein one or more genes in the cells of the donor have been modified using one or more techniques selected from the group consisting of transduction to express cDNA, CRISPR / Cas9 system, RNAi technology and retroviral technology.

27. 27. The pharmaceutical composition for use according to claim 25 or 26, wherein the cells of the donor have been modified to express a chimeric antigen receptor (CAR) on their surface.

28. The pharmaceutical composition for use according to any one of claims 24 to 27, wherein the donor's cells are selected from the group consisting of stem cells, regulatory T cells, CAR-T cells, CAR-B cells, and tumor infiltrating lymphocytes (TILs).

29. wherein the pharmaceutical composition promotes long-term survival of the donor organ, donor tissue, or donor cells, and the long-term graft survival is (a) at least 6 months after transplantation; (b) at least 1 year after transplantation, and (c) At least 5 years after transplantation 29. A pharmaceutical composition for use according to any one of claims 1 to 28, selected from the group consisting of:

30. 30. The pharmaceutical composition for use according to any one of claims 1 to 29, characterized in that the anti-CD154 antibody is administered locally.

31. 30. The pharmaceutical composition for use according to any one of claims 1 to 29, characterized in that the anti-CD154 antibody is administered systemically.