Anti-CD154 antibodies and uses thereof

Abstract

The present disclosure relates to anti-human CD154 antibodies having a modified effector function. The present disclosure also relates to the use of these anti-human CD154 antibodies in the treatment of conditions associated with CD154 activation, the conditions associated with CD154 activation are, for example, transplant rejection, inflammatory conditions and diseases, dysfunctional immune responses associated with viral infections and diseases, autoimmune conditions and diseases, allergic conditions, atherosclerotic conditions, or neurodegenerative conditions and diseases. The present disclosure also relates to the use of these anti-human CD154 antibodies in inducing central tolerance and hematopoietic cell chimerism in transplanted patients.

Description

[0001] This application is a divisional application of the patent application with the application date of July 1, 2020, application number 202080059891.1, and invention title "Anti-CD154 Antibodies and Their Uses".

[0002] Cross - Reference to Related Applications

[0003] This application claims the priority of U.S. Provisional Application No. 62 / 869,489 filed on July 1, 2019 and U.S. Provisional Application No. 63 / 018,123 filed on April 30, 2020, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 1, 2020 is named 104545-0037-WO1_SL.txt and is 753,090 bytes in size.

[0006] Field of the Disclosure

[0007] The present disclosure relates to isolated antibodies that bind to CD154 and have modified (e.g., selectively reduced) effector functions. The present disclosure also relates to nucleic acid molecules encoding such antibodies, compositions comprising such antibodies, and methods of using such antibodies and compositions for inhibiting immune responses, e.g., in the treatment of transplant rejection, inflammatory responses, autoimmune responses, dysfunctional immune responses associated with viral infections and diseases, allergic, atherosclerotic or neurodegenerative conditions and diseases.

[0008] Background of the Disclosure

[0009] CD154 (also known as CD40 ligand, CD40L, gp39, TNF-related activation protein (TRAP), 5c8 antigen, T-BAM) is a protein expressed mainly on activated CD4+ T cells and is thought to be the molecular basis for T cell helper function (Lederman, S. et al., J. Exp. Med. 175:1091-1101 (1992)). CD154 is a member of the TNF molecular superfamily and is functionally expressed as a homotrimer. However, some of the CD154 units have truncated peptide chains, such that the CD154 trimer can 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), which results in a number of effects, depending on the target cell type. The main binding partner of CD154 is CD40, although other binding partners have been described, αMβ2 (Mac-1), α5β1 integrin, and αIIbβ3 (ElFakhry 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 functions as a co-stimulatory molecule for B cells and affects the function of CD4+ T follicular helper cells (TFH cells). On TFH cells, CD154 promotes B cell maturation and function by engaging CD40 on the B cell surface and thereby contributing to 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)). The absence of CD154 (e.g., 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 interaction is involved in normal T-B cell interaction, including increased co-stimulation, 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, A.M. et al., Journal of Immunol., 155:3329-3337 (1995); Muramatsu, M.K. et al., Cell 102:553 (2000); Xu Y and Song G, J. Biomed Sci. 11(4):426-38 (2004); Quezada S.A. et 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).

[0010] 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)), in other cell types, and in many cancers (Paulie, S et al., Cancer Immunol Immunother, 20, 23-8 (1985)). Supernatants from severe acute respiratory syndrome-related 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 inflammation, CD40 is expressed on endothelial cells, Müller glia (the major glia in the retina), 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 immune responses is generally tightly regulated over time in tissues. Dysfunctional immune responses can occur together with abnormal CD154 expression and play a role in certain tissues and at certain times, contributing to syndromes such as acute respiratory distress syndrome (ARDS), autoimmune diseases, vascular diseases, and cancer promotion.

[0011] The soluble form of CD154 (sCD154), generated by shedding of membrane-bound CD154, plays a role in the production of pro-inflammatory cytokines and has been associated with various 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, particularly soluble CD154, has been 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. 52(10):1435-41 (2003); and Charafeddine AH et al., Am J Transplant. 12(11):3143-51 (2012)).

[0012] The monoclonal antibody 5c8 is a murine anti-human CD154 that potently blocks CD154 function (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, A.M. et al., Journal of Immunol., 155:3329-3337 (1995)). A humanized anti-human CD154 IgG1 antibody (hu5c8, ruplizumab or ), and it has been tested in non-human primates and humans. The crystal structure of hu5c8 shows a unique binding of hu5c8 to the CD154 trimer and the antibody contacts the CD154 monomer (Karpusas M et al., Structure. 9(4):321-9(2001)).CD154 blockade has demonstrated efficacy in models of autoimmunity, humoral immunity, and allotransplantation (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 U S 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 stopped due to increased platelet activation and the incidence of thromboembolic events (Law and Grewal Adv Exp Med Biol. 647:8-36 (2009)). One mechanism regarding platelet activation may involve CD40 on platelets and activation via soluble CD154 (Inwald DP et al., Circ Res. 92(9):1041-8 (2003)). Several observations suggest that the mechanism regarding thrombosis in patients treated with anti-CD154 is mediated by FcγRIIa receptor (FcγRIIA, FCGR2A, CD32A)-dependent platelet activation by immune complexes (especially high-grade complexes) composed of anti-CD154 antibody and soluble CD154 (Robles-Carrillo L et al., J Immunol. 185(3):1577-83 (2010)). Eliminating the binding of Fc to Fc receptors by mutating the Fc region to prepare aglycosylated hu5c8 (IgG1 N297Q) has been shown to strongly 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 reduces the efficacy of the antibody in inhibiting or preventing transplant rejection in the rhesus monkey kidney and islet allograft models (Ferrant JL et al., International Immunol(11):1583 (2004)). Completely eliminating the Fc region (e.g., in the PEGylated Fab’ antibody fragment of anti-CD154) (dapirolizumab pegol; DZP, CDP7657, Biogen and UCB) also reduces the risk of thrombotic events, but fails 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, ) and a linker called "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 and also avoided platelet activation, thrombosis, or thromboembolism (Kim SC et al., Am J Transplant 17(5):1182-1192 (2017)).

[0013] U.S. Patent No. 9,765,150 also describes 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 the monkeys treated. The ADA were directed against the dAb (non-Fc) portion of the molecule, and these antibodies showed blocking of the binding of BMS-986003 to CD154, suggesting that the ADA might be neutralizing. Additionally, these ADA led to increased clearance of BMS-986003 in several monkeys. In contrast, the chimeric murine 5c8 human IgG1 chimeric antibody had an expected long plasma half-life (U.S. Patent No. 9,765,150).

[0014] Although BMS-986004 showed efficacy in an immune thrombocytopenic purpura (ITP) clinical trial, the pharmacokinetic property profile may not be optimal (NCT02273960; "Study to Evaluate Safety and Efficacy in Adult Subjects With ITP (ITP)"; results evaluated on July 1, 2019).

[0015] The mechanisms of anti-CD154 therapy for transplant rejection or autoimmunity are not fully understood. Anti-CD154 therapy appears to be more effective than anti-CD40 therapy, suggesting that the blockade of their interaction is not symmetric. The monoclonal antibody (mAb) hu5c8 appears to be more effective than the different humanized anti-CD154 mAb, IDEC-131 (O’Neill NA et al., Transplantation. 101(9):2038-2047 (2017)). Further, the increase in regulatory T cells (especially Foxp3+ T cells) associated with anti-CD154 therapy may be related to potency (Ferrer IR et al., Proc Natl Acad Sci U S A. 108(51):20701-6 (2011)). Another observation supporting the role of regulatory T cells is that dimeric soluble CD40, but not monomeric CD40, induces T regulatory cells (CD4+CD25+ T cells) and treats transplant rejection (Masunaga T et al., Transplantation. 80:1614-1622 (2005)). Those experiments also showed that the valence in CD154 ligation or blockade may be related to potency (Masunaga T et al., Transplantation. 80:1614-1622 (2005)). Anti-CD154 therapy has been shown to be particularly effective in facilitating allografts with mixed allogeneic chimerism (Kawai T et al., Am J Transplant. 4(9):1391-8 (2004)) and in xenotransplantation ( M et al., Nature. 564(7736):430-433 (2018)) is particularly effective.

[0016] Antibodies have a variety of Fc-mediated effector functions, including FcγR binding and complement C1q binding (the first component of complement activation), which are involved in effector functions and complement-dependent cytotoxicity. Engineering the complement can result in an increase or decrease in one or more effector functions. In extreme cases, the Fc can be removed, such as in an F(ab) construct, but for therapeutic efficacy, such constructs typically require other alterations to increase half-life, such as modification with polyethylene glycol or PEGylation. Complete elimination of the Fc region can affect the potency of anti-CD154 antibodies—the PEGylated Fab’ antibody fragment of anti-CD154 (Pegolizumab (DZP), CDP7657, Biogen and UCB) failed to treat SLE in a Phase IIb clinical trial (Waters J, Biocentury, October 26, (2018)). The “silent” N297Q IgG1 variant of hu5c8 (asialo or aglycosyl) lacks FcγR binding but is effective in inhibiting humoral immune responses (but not organ rejection) in non-human primates (Tao, M.H. and Morrison, S.L. J. Immunol. 143:2595-2601 (1989); Ferrant J.L. et al., International Immunol (11):1583 (2004)). Thus, one solution for balancing the potency and safety of anti-CD154 antibodies is to engineer anti-CD154 antibodies with a modified Fc with selectively reduced effector functions. Previous efforts to modify effector functions have focused on substitutions in the IgG hinge / CH2 region, which can affect FcγR and C1q binding. One consideration in antibody design is to maintain the CH2 / CH3 regions of the Fc domain, which are required for interaction with FcRn (the neonatal Fc receptor, which confers extended serum half-life).

[0017] Among IgG1 antibodies, substitution studies have identified several residues that affect the binding of Fc to C1q and to various FcγRs (FcγRI / CD64, FcγRIIa / CD32a, FcγRIIb / CD32b, and FcγRIIIa / CD16a and FcγRIIIb / CD16b). For example, Fc residues at positions 234 and 235 have been shown to have a modulating effect on Fc-CD64 binding (Canfield, S M and Morrison, S L J. Exp. Med. 173:1483-1491 (1991); Chappel, M S et al., Proc. Natl. Acad. Sci. USA, 88, 9036-9040 (1991); Alegre, M-L et al., J. Immunol. 148:3461-3468 (1992); Hezareh, M et al., J. Virol. 75, 12161-12168 (2001)), Fc-CD32A binding (Hezareh, M et al., J. Virol. 75, 12161-12168 (2001); Armour, K L et al., Mol. Immunol. 40, 585-593 (2003)), Fc-CD16 binding (Hezareh, M et al., J. Virol. 75, 12161-12168 (2001)), and Fc-C1q binding (Xu, D et al., Cell. Immunol. 200, 16-26 (2000); Hezareh, M et al., J. Virol. 75, 12161-12168 (2001)). In addition, CH2 position 331 has been shown to be involved in Fc-CD64 binding (Canfield, S M and Morrison, S L J. Exp. Med. 173:1483-1491 (1991)) and Fc-C1q binding (Tao, M H et al., J. Exp. Med. 178, 661-667 (1993); Hezareh, M et al., J. Virol. 75, 12161-12168 (2001); Idusogie, E E et al., J. Immunol. 164:4178-4184 (2000)). As an example, substitution of L234F / L235E / P331S in IgG1 has been shown to decrease the affinity for FcγRs (FcγI, FcγRIIa, and FcγRIIIa) and C1q (Oganesyan V et al., Acta Crystallogr D Biol Crystallogr. 64 (Pt 6):700-704 (2008)).As another example, teplizumab is a humanized OKT3 antibody with an IgG1 Fc having reduced effector function due to the substitutions (L234A, L235A) and is in development for the treatment of diabetes (Xu, D et al., Cell Immunol. 200, 16 - 26 (2000); Herold KC et al., N Engl J Med. 10.105 (2019)). Another modified (referred to as "silent") IgG1 Fc modification with substitutions C220S, C226S, C229S, and P238S is used to prepare a fusion protein expressing CTLA4 in CTL4 - Ig (abatacept, ). The cysteine substitutions remove the disulfide bonds (C226S and C229S) between the heavy chains (HC) in the dimer. Using this modified Fc, Kim et al. generated an Fc - silent humanized anti - human CD154 antibody, which is a mutant form of hu5c8 containing the same modified, non - Fc - binding IgG1 tail as anti - CD154 dAb BMS - 986004 ("hu5c8 - mod") (Kim SC et al., Am J Transplant 17(5):1182 - 1192 (2017)). U.S. Patent 9,765,150 also describes an anti - human CD154 "5c8" antibody generated by fusing a part or all of the variable domains of "5c8" with the Fc from abatacept and a linker of "CT Long" having the amino acid sequence AST - EPKSSDK (SEQ ID NO:326) or "CT Short" having the amino acid sequence AS (SEQ ID NO:214) (U.S. Patent No. 9,765,150).

[0018] Among IgG2 antibodies, the affinity for FcγR has been reduced by substitutions H268Q / 309L / A330S / P331S in a mutant form called "IgG2m4" (An, Z. et al., MAbs 1:572 - 579 (2009)). Another engineered Fc includes the substitutions V234A / G237A / P238S / H268A / V309L / A330S / P331S, which eliminates the affinity for FcgR and the C1q complement protein (Vafa, O. et al., Methods. 1, 65(1):114 - 26 (2014)).

[0019] Among IgG4 antibodies, the affinity for FcγR has been reduced by F234A and L235A substitutions. These substitutions have been combined with the S228P substitution in the core hinge (IgG1 and IgG2 have proline at position 228), which further stabilizes the IgG4 molecule to prevent Fab arm exchange (Silva, JP et al., J Biol Chem. 290(9):5462-5469 (2015)), in the "mutated" form of IgG4 called "IgG4 Pro-AlaAla" (Tao, MH and Morrison, SL J Immunol. 143:2595-2601 (1989); and Alegre, M-L et al., J Immunol. 148:3461-3468 (1992)). Dulaglutide is a fusion protein comprising a glucagon-like peptide-1 (GLP-1) agonist fused to an IgG4 Fc of the "IgG4 Pro-AlaAla" type, which is sold as Other modified IgG4 Fcs have substitutions F234A and L235A together with the S228P substitution (Xu, D et al., Cell Immunol. 200, 16-26 (2000)), or employ the substitution L235E together with the S228P substitution without modifying F234 (Reddy MP et al., J Immunol. 164:1925-1933 (2000); and Li, X et al., Int J Clin Exp Med. 8(3):3607-18 (2015)).

[0020] Antibody engineering is a complex and expensive technology with unpredictable in vivo outcomes (Saeed AF et al., Frontiers in Microbiology, Article 495, March (2017)). Antibody engineering strategies, including structural changes or other modifications optimized for an antibody, may implicate another antibody in vivo (Yan B et al., The Journal of Biol. Chem. 287(8):5891-97 (2012)). The effect of mutating a specific residue (e.g., N297Q) in a specific antibody (e.g., IgG1) can have very different clinical effects from another type of mutation in the same antibody (e.g., N297A in IgG1). In addition, the clinical effect of an intramolecular change within an antibody subtype (e.g., L235 mutation in IgG1) may be completely unpredictable when the same intramolecular change occurs within another antibody subtype (e.g., L235 mutation within IgG4). Intramolecular changes may also result in different clinical effects depending on how various mutations may pair within the antibody. Species-specific differences increase the unpredictability of the effect of Fc receptor mutations on the efficacy vs 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 many human patients experienced thromboembolic complications in clinical trials with the same antibodies (i.e., ruplizumab, tocilizumab, and ABI793) (Pinelli DF and Ford M.L. Immunotherapy 7(4):399-410 (2015)).

[0021] To date, there does not exist a fully human or humanized anti-CD154 antibody that meets the need for a product that can effectively prevent transplant rejection (including graft-versus-host disease), inflammatory conditions and diseases, autoimmune conditions and diseases, dysfunctional immune responses associated with viral infections and diseases, allergic conditions, atherosclerotic conditions, or neurodegenerative conditions and diseases in humans, with an acceptable level of side effects, such as thromboembolic side effects. This highlights the need for such antibodies and variants thereof that bind to CD154 with high affinity and inhibit downstream effects of CD154:CD40 binding without toxic side effects such as thrombosis. Summary of the Invention

[0023] The first aspect of the present disclosure provides an isolated anti-CD154 antibody that binds to mammalian CD154. In some embodiments, the anti-CD154 antibody comprises human or humanized variable domains, wherein the variable domains comprise a heavy chain variable region (VH) and a light chain variable region (VL), and wherein the VH is operably linked to a human Fc domain with modified effector functions. Optionally, one or more effector functions are reduced. Optionally, one or more effector functions are eliminated. In some embodiments, the antibody is bivalent. In other embodiments, the anti-CD154 antibody is functionally monovalent.

[0024] The second aspect of the present disclosure provides a nucleic acid molecule encoding the anti-CD154 antibody disclosed herein. In some embodiments, separate nucleic acid molecules encode the heavy and light chains of the anti-CD154 immunoglobulin. In other embodiments, the same nucleic acid molecule encodes both the heavy and light chains of the anti-CD154 immunoglobulin.

[0025] The third aspect of the present disclosure provides a vector comprising the disclosed nucleic acid molecule. In some embodiments, the vector comprises a nucleotide sequence encoding the heavy chain of the disclosed anti-CD154 antibody. Optionally, the vector comprises a nucleotide sequence encoding the light chain of the disclosed antibody. The vector may comprise nucleotide sequences encoding the heavy and light chains of the disclosed anti-CD154 antibody.

[0026] The fourth aspect of the present disclosure provides a transformed cell comprising the disclosed nucleic acid molecule or the disclosed vector.

[0027] The fifth aspect of the present disclosure provides a pharmaceutical composition comprising the anti-CD154 antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CD154 antibody disclosed herein.

[0028] The sixth aspect of the present disclosure provides a method for inhibiting CD154 activity. In some embodiments, CD154 activity is inhibited by contacting CD154 with the anti-CD154 antibody disclosed herein. Optionally, CD154 is inhibited by administering the anti-CD154 antibody disclosed herein to a subject in need thereof. In some embodiments, the anti-CD154 antibody is administered in one of the pharmaceutical compositions disclosed herein.

[0029] The seventh aspect of the present disclosure provides a method of suppressing an immune response in a subject. In some embodiments, the immune response is suppressed by administering to a subject in need thereof an anti-CD154 antibody disclosed herein. In some embodiments, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein. The immune response can be a humoral response, such as an antibody-mediated response. The immune response can be a cell-mediated response, such as 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 immune response can be a mixed humoral and cell-mediated response. The immune response can be a complement-mediated response. The immune response can be a primary response or a secondary response. The immune response can be a dysregulated immune response, such as an immune response to a virus that may result in cytokine release syndrome (CRS), cytokine storm, or acute respiratory distress syndrome (ARDS). The immune response can be a tissue-specific immune response, such as inflammation of the lung, heart, or kidney, such as lung inflammation.

[0030] The eighth aspect of the present disclosure provides a method of inducing hematopoietic cell chimerism in a transplant recipient. In some embodiments, the method comprises administering to the recipient an anti-CD154 antibody disclosed herein and transplanting hematopoietic stem cells into the recipient, thereby inducing hematopoietic cell chimerism in the recipient. In some embodiments, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein.

[0031] The ninth aspect of the present disclosure provides a method of inducing central tolerance in a transplant recipient. In some embodiments, the method comprises administering to the recipient an anti-CD154 antibody disclosed herein, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the transplanted hematopoietic stem cells generate immune cells that are tolerant to the donor tissue, thereby inducing central tolerance in the recipient. In some embodiments, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein.

[0032] The tenth aspect of the present disclosure provides a method of suppressing or preventing xenograft rejection in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an anti-CD154 antibody disclosed herein. In some embodiments, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein.

[0033] Specific embodiments of the present disclosure are set forth in the numbered paragraphs below.

[0034] 1. An isolated antibody that binds to CD154, comprising a human or humanized variable domain, wherein the variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein the VH is operably linked to a human Fc domain having modified effector functions.

[0035] 2. The antibody of paragraph 1, wherein one or more effector functions are reduced.

[0036] 3. The antibody of paragraph 1 or 2, wherein one or more effector functions are eliminated.

[0037] 4. The antibody of any one of paragraphs 1-3, wherein the VH is operably linked to a human Fc region, wherein the human Fc region comprises a human hinge sequence and the human Fc domain, and wherein the human hinge sequence is between the VH and the human Fc domain.

[0038] 5. The antibody of paragraph 4, wherein the hinge comprises the amino acid sequence of any one of SEQ ID NOs: 76-90.

[0039] 6. The antibody of any one of paragraphs 1-5, wherein the Fc domain is derived from an IgG4 Fc (or crystallizable fragment) region.

[0040] 7. The antibody of paragraph 6, wherein the Fc domain comprises one or more amino acid modifications that modify effector functions.

[0041] 8. The antibody of paragraph 7, wherein the antibody comprises an amino acid modification at any one of the positions selected from the group consisting of: S228, L235, G237, E318, and N297, or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman.

[0042] 9. The antibody of paragraph 8, wherein the antibody comprises an amino acid modification selected from the group consisting of: S228P, F234A, L235A, L235E, G237A, E318A, and N297Q, or a combination thereof.

[0043] 10. The antibody of any one of paragraphs 1-5, wherein the Fc domain is derived from an IgG1 Fc (or crystallizable fragment) region and comprises one or more amino acid modifications that modify effector functions.

[0044] 11. The antibody of paragraph 10, wherein the antibody comprises an amino acid modification at any one of the positions 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 thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman.

[0045] 12. The antibody of paragraph 11, wherein the antibody comprises an amino acid modification 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, Q347E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T366V, N390R, N390K, N390D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S400K, F405A, F405I, F405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T411N, T411R, T411Q, T411K, T411D, T411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR236 / 237, InR237 / 238, InV238 / 239, 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.

[0046] 13. The antibody of any one of paragraphs 1-5, wherein the Fc domain is derived from the IgG2 Fc (or fragment crystallizable) region.

[0047] 14. The antibody of paragraph 13, wherein the antibody comprises an amino acid modification at any one of the positions selected from the group consisting of: V234, G237, P238, H268, V309, A330 and P331 or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman.

[0048] 15. The antibody of paragraph 14, wherein 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.

[0049] 16. The antibody of any one of paragraphs 1-5, wherein the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3-9, 12-18, and 238-241.

[0050] 17. The antibody according to paragraph 4, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21-37, 40-56, and 243-251.

[0051] 18. The antibody of any one of paragraphs 1-17,

[0052] wherein the VH comprises

[0053] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 57,

[0054] (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and

[0055] (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 59; and

[0056] wherein the VL comprises

[0057] (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60,

[0058] (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61, and

[0059] (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 62.

[0060] 19. The antibody of any one of paragraphs 1-18, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 64, 252, and 253.

[0061] 20. The antibody of any one of paragraphs 1-19, wherein the VL comprises the amino acid sequence of SEQ ID NO: 65 or 66.

[0062] 21. An antibody according to any one of paragraphs 4-20, wherein the antibody further comprises a CH1 domain, and wherein the CH1 domain is operably linked to (a) the C-terminus of the VH, and (b) the N-terminus of the hinge.

[0063] 22. The antibody of paragraph 21, wherein the CH1 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 67, 70, and 73.

[0064] 23. An antibody according to any one of paragraphs 1-22, wherein the antibody comprises a linker between the VH and the Fc domain.

[0065] 24. An antibody according to any one of paragraphs 4-22, wherein the antibody comprises a linker between the VH and the hinge.

[0066] 25. The antibody of paragraph 21 or 22, wherein the antibody comprises a linker between the VH and the CH1 domain.

[0067] 26. An antibody according to any one of paragraphs 23-25, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 199-223 and 327-330.

[0068] 27. An antibody according to any one of paragraphs 1-26, wherein the VH is operably linked to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, and 238-241 and 243-251.

[0069] 28. An antibody according to any one of paragraphs 1-27, wherein the heavy chain comprises 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.

[0070] 29. An antibody according to any one of paragraphs 1-28, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 195 or 196.

[0071] 30. An antibody according to any one of paragraphs 1-29, wherein the antibody is monoclonal.

[0072] 31. An antibody according to any one of paragraphs 1-30, wherein the antibody is chimeric.

[0073] 32. An antibody according to any one of paragraphs 1-31, wherein the antibody is humanized.

[0074] 33. An antibody according to any one of paragraphs 1-17, 21-27, and 30, wherein the antibody is human.

[0075] 34. An antibody according to any one of paragraphs 1-33, wherein binding of the antibody to human CD154 inhibits the interaction between human CD154 and human CD40.

[0076] 35. An antibody according to any one of paragraphs 1-34, wherein the antibody blocks activation of one or more of B cells, macrophages, dendritic cells, or endothelial cells by inhibiting binding of CD154 to CD40.

[0077] 36. An antibody according to any one of paragraphs 1-35, wherein when administered to a subject, the antibody has one or more of the following effects:

[0078] (a) A reduced risk of thrombosis or thromboembolic events as compared to the hu5c8 antibody;

[0079] (b) Reduced activation of platelets expressing CD154;

[0080] (c) Inhibition of CD154 shedding; and

[0081] (d) Alteration of the expression or activity of downstream targets of CD154-CD40 signaling.

[0082] 37. An antibody according to any one of paragraphs 1-36, wherein the antibody has a K for CD154 of less than 50 pM, such as 5-25 pM or 9.5-23 pM D .

[0083] 38. An antibody according to any one of paragraphs 1-37, wherein the antibody does not comprise an amino acid sequence consisting of any one of SEQ ID NO: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278.

[0084] 39. An isolated nucleic acid molecule encoding the light and heavy chains of an anti-CD154 antibody according to any one of paragraphs 1-38.

[0085] 40. An isolated nucleic acid molecule encoding an anti-CD154 antibody comprising an amino acid sequence consisting of any one of SEQ ID NO: 121-132, 135-146, 149, 151-160, 163, 165-174, 195, 196, 266-277, and 279-288.

[0086] 41. A first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule encodes a heavy chain comprising an amino acid sequence selected from any one of SEQ ID NO: 121 - 132, 135 - 146, 149, 151 - 160, 163, 165 - 174, 266 - 277, and 279 - 288, and the second isolated nucleic acid molecule encodes a light chain comprising an amino acid sequence of SEQ ID NO: 195 or 196.

[0087] 42. A first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule and the second isolated nucleic acid molecule encode the heavy chain and the light chain of an anti - CD154 antibody of any one of paragraphs 1 - 38, respectively.

[0088] 43. A vector comprising the isolated nucleic acid molecule according to paragraph 39 or 40.

[0089] 44. A first vector and a second vector, wherein the first vector comprises the first isolated nucleic acid molecule according to paragraph 41 or 42, and the second vector comprises the second isolated nucleic acid molecule according to paragraph 41 or 42.

[0090] 45. A transformed cell comprising the isolated nucleic acid molecule according to paragraph 39 or 40, the first and second isolated nucleic acid molecules according to paragraph 41 or 42, the vector according to paragraph 43, or the first and second vectors according to paragraph 44.

[0091] 46. A pharmaceutical composition comprising an anti - CD154 antibody of any one of paragraphs 1 - 38 and a pharmaceutically acceptable carrier.

[0092] 47. A pharmaceutical composition comprising the isolated nucleic acid molecule according to paragraph 39 or 40, the first and second isolated nucleic acid molecules according to paragraph 41 or 42, the vector according to paragraph 43, or the first and second vectors according to paragraph 44, and a pharmaceutically acceptable excipient.

[0093] 48. A pharmaceutical composition comprising the transformed cell according to paragraph 45 and a pharmaceutically acceptable excipient.

[0094] 49. A method for inhibiting an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of any one of paragraphs 1 - 38 or a pharmaceutical composition of any one of paragraphs 46 - 48.

[0095] 50. The method of paragraph 49, wherein the immune response is a humoral response.

[0096] 51. The method of paragraph 50, wherein the immune response is an antibody - mediated response.

[0097] 52. The method of paragraph 49, wherein the immune response is a cell-mediated response.

[0098] 53. The method of paragraph 52, wherein the cell-mediated response is 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.

[0099] 54. The method of paragraph 49, wherein the immune response is a mixed humoral and cell-mediated response.

[0100] 55. The method of paragraph 54, wherein the mixed response is 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.

[0101] 56. The method of any one of paragraphs 49-55, wherein the subject is a human.

[0102] 57. The method of any one of paragraphs 49-55, wherein the subject is non-human.

[0103] 58. The method of paragraph 57, wherein the subject is a monkey.

[0104] 59. The method of any one of paragraphs 49-58, wherein the subject has received or will receive a cell, tissue, or organ transplant.

[0105] 60. The method of paragraph 59, wherein the transplant is an allograft, an autograft, or a xenograft.

[0106] 61. The method of paragraph 59 or 60, wherein the cell is a modified cell or an ex vivo expanded cell.

[0107] 62. The method of paragraph 61, wherein one or more genes in the cell are modified by using one or more techniques selected from the group consisting of transduction to express cDNA, the CRISPR / Cas9 system, RNAi technology, and retroviral technology.

[0108] 63. The method of paragraph 61 or 62, wherein the cell is modified to express a chimeric antigen receptor (CAR) on its surface.

[0109] 64. The method of any one of paragraphs 59-63, wherein the cell is selected from the group consisting of stem cells, regulatory T (Treg) cells, CAR-T cells, CAR-B cells, and tumor-infiltrating lymphocytes (TIL).

[0110] The method of any one of paragraphs 59-64, wherein the method comprises treating or preventing transplant rejection in the subject.

[0111] 66. The method of paragraph 65, wherein the transplant rejection is acute or chronic humoral rejection of the transplanted cells, tissues or organs.

[0112] 67. The method of paragraph 65 or 66, wherein the transplant rejection is acute or chronic graft rejection in a graft recipient of an allograft or xenograft.

[0113] 68. The method of paragraph 66 or 67, wherein the method promotes long-term graft survival of the transplanted cells, tissues or organs, wherein the long-term graft survival is selected from the group consisting of:

[0114] (a) at least 6 months after transplantation;

[0115] (b) at least 1 year after transplantation; and

[0116] (c) at least 5 years after transplantation.

[0117] 69. The method of any one of paragraphs 65-68, wherein the transplant rejection is associated with hematopoietic cell or bone marrow transplantation, allotransplantation of islet cells, graft-versus-host disease or solid organ transplantation, the solid organ transplantation being selected from the group consisting of: heart transplantation, kidney transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney-pancreas transplantation, heart-lung transplantation, kidney-heart transplantation, kidney-heart-pancreas transplantation, heart-liver transplantation, heart-liver-kidney transplantation, heart-lung-kidney transplantation, heart-lung-liver transplantation, lung-kidney transplantation, lung-liver transplantation, liver-intestine-pancreas transplantation, intestine-pancreas transplantation, liver-kidney-intestine-pancreas transplantation and kidney-intestine transplantation.

[0118] 70. The method of any one of paragraphs 56-58, wherein the subject has an immune-related disease, an atherosclerotic disorder or a neurodegenerative disorder.

[0119] 71. The method of any one of paragraphs 56-58 and 70, wherein the subject already has or is at risk of having a stroke, transient ischemic attack (TIA), aneurysm or dissecting aneurysm.

[0120] 72. The method of paragraph 70, wherein the immune-related disease is selected from the group consisting of: type I diabetes, juvenile diabetes, autoimmune diabetes, autoimmune hemolytic anemia, rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, inflammatory bowel disease, Addison's disease, Crohn's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, Guillain-Barré syndrome, ankylosing spondylitis, primary biliary cirrhosis, lupus nephritis, Goodpasture's disease, polymyositis, dermatomyositis, psoriasis, temporal arteritis, Churg-Strauss syndrome, transverse myelitis, thyroiditis, ulcerative colitis, sarcoidosis, hemolytic anemia, idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorder, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, Behçet's disease, diabetic retinopathy (DR), diabetic macular edema (DME), age-related macular degeneration (AMD), and macular edema following retinal vein occlusion (MEfRVO).

[0121] 73. The method of paragraph 70, wherein the immune-related disease is selected from the group consisting of: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), cytokine release syndrome (CRS), and cytokine storm syndrome.

[0122] 74. The method of paragraph 70, wherein the immune-related disease is selected from the group consisting of: acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, lung tissue inflammation, and chronic obstructive pulmonary disease (COPD).

[0123] 75. The method of paragraph 70, wherein the neurodegenerative disorder is selected from the group consisting of: Alzheimer's disease, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), amyotrophic lateral sclerosis (ALS), and Parkinson's disease.

[0124] 76. The method of paragraph 70, wherein the atherosclerotic disorder is selected from the group consisting of: angina pectoris, myocardial infarction, carotid artery stenosis, transient ischemic attack, and cerebrovascular accident (CVA).

[0125] 77. The method according to paragraph 70, wherein the immune-related disease is an allergic condition.

[0126] 78. The method according to paragraph 77, wherein the allergic condition is selected from the group consisting of: allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergy, drug allergy, and food allergy.

[0127] 79. The method of paragraph 49, wherein the immune response is a primary response or a secondary response.

[0128] 80. The method of any one of paragraphs 49-79, wherein the antibody is administered systemically.

[0129] 81. The method of paragraph 80, wherein the anti-CD154 antibody is administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally or rectally.

[0130] 82. The method of any one of paragraphs 49-79, wherein the antibody is administered locally.

[0131] 83. The method of any one of paragraphs 49-82, wherein the anti-CD154 antibody is administered in combination with one or more additional reagents selected from the group consisting of: antithrombotic drugs, antiplatelet drugs, non-steroidal anti-inflammatory drugs (NSAIDs) and anti-allergic drugs.

[0132] 84. The method of paragraph 83, wherein the anti-CD154 antibody is administered before, after or simultaneously with the one or more additional reagents.

[0133] 85. The method of paragraph 83 or 84, wherein the antithrombotic drug is selected from the group consisting of: glycoprotein IIb / IIIa receptor antagonists, direct or indirect factor Xa inhibitors and anticoagulants.

[0134] 86. The method of paragraph 85, wherein the anticoagulant is selected from the group consisting of: heparin, warfarin, rivaroxaban ximelagatran dabigatran apixaban edoxaban enoxaparin and fondaparinux

[0135] 87. The method of paragraph 85, wherein the glycoprotein IIb / IIIa receptor antagonist is selected from the group consisting of: abciximab rivaroxaban apixaban edoxaban idrabiotaparinux, tirofiban and eptifibatide

[0136] The method of paragraph 85, wherein the direct or indirect factor Xa inhibitor is selected from the group consisting of: apixaban idrabiotaparinux, fondaparinux and rivaroxaban

[0137] 89. The method of paragraph 83 or 84, wherein the antiplatelet drug is selected from the group consisting of: drugs that inhibit the TXA2 pathway, adenosine diphosphate (ADP) pathway inhibitors, thrombin inhibitors, protease-activated receptor-1 (PAR-1) inhibitors, and phosphodiesterase (PDE) inhibitors.

[0138] 90. The method of paragraph 89, wherein the ADP pathway inhibitor is selected from the group consisting of: clopidogrel ticlopidine prasugrel ticagrelor cangrelor and elinogrel.

[0139] 91. The method of paragraph 89, wherein the PDE inhibitor is selected from the group consisting of: dipyridamole and cilostazol

[0140] 92. The method of paragraph 83 or 84, wherein the NSAID is selected from the group consisting of: acetylsalicylic acid (aspirin), celecoxib diclofenac diflunisal etodolac ( SR, ), ibuprofen indomethacin ketoprofen ketorolac nabumetone naproxen oxaprozin piroxicam salsalate sulindac tolmetin and prasugrel

[0141] The method of any one of paragraphs 49-92, wherein the anti-CD154 antibody is administered in combination with one or more supplementary reagents selected from the group consisting of: immunosuppressive drugs, immunomodulatory drugs, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD28 antibodies, anti-CD52 antibodies, mTOR inhibitors, calcineurin inhibitors, and antiviral drugs.

[0142] The method of paragraph 93, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the one or more supplementary reagents.

[0143] The method of paragraph 93 or 94, wherein the immunosuppressive drug or immunomodulatory drug is selected from the group consisting of: cyclosporin A, tacrolimus (FK-506), doxorubicin azathioprine busulfan cyclophosphamide fludarabine, 5-fluorouracil, methotrexate mycophenolate mofetil imidazole riboside (BREDININ TM ), leflunomide, non-steroidal anti-inflammatory drugs, corticosteroids, rapamycin deoxyspergualin, FTY720, muromonab-CD3 (ORTHOCLONE ), alemtuzumab basiliximab daclizumab eculizumab rituximab bortezomib cilizumab, antithymocyte globulin leronlimab, siltuximbab sarilumab tocilizumab bevacizumab ranibizumab aflibercept and inhibitors of Bruton's tyrosine kinase (BTK), including zanubrutinib acalabrutinib and ibrutinib

[0144] 96. The method of paragraph 93 or 94, wherein the mTOR inhibitor is selected from the group consisting of: rapamycin everolimus temsirolimus ridaforolimus and deforolimus.

[0145] 97. The method of paragraph 93 or 94, wherein the calcineurin inhibitor is selected from the group consisting of: cyclosporine tacrolimus (FK506, ) and pimecrolimus

[0146] 98. The method of paragraph 93 or 94, wherein the antiviral agent is selected from the group consisting of: ribavirin, interferon (alfacon-1), chloroquine, hydroxychloroquine, EIDD-2801, EIDD-1931, GS-5734, GS-441524, ivermectin, favipiravir, indomethacin, chlorpromazine, penciclovir, nafamostat, nitazoxanide, and remdesivir.

[0147] 99. A method of inducing hematopoietic cell chimerism in a transplant recipient, the method comprising administering to the recipient one or more doses of an anti-CD154 antibody and transplanting hematopoietic stem cells into the recipient, thereby inducing hematopoietic cell chimerism in the recipient, wherein the anti-CD154 antibody is an anti-CD154 antibody according to any one of paragraphs 1-38.

[0148] 100. The method of paragraph 99, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

[0149] 101. The method of paragraph 99 or 100, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0150] 102. The method of any one of paragraphs 99-101, wherein the anti-CD154 antibody is administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally.

[0151] 103. The method of any one of paragraphs 99-102, wherein the method further comprises the step of conditioning the transplant recipient before transplantation with stem cells.

[0152] 104. The method of paragraph 103, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the conditioning step.

[0153] 105. The method of paragraph 103 or 104, wherein the conditioning step is selected from the group consisting of: total body irradiation, 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 cyclosporin A (CsA), administration of tacrolimus (FK-506), administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of an IL-15 receptor inhibitor, administration of rapamycin, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, administration of CD34+ hematopoietic stem cells and CD3+ T-cells from a renal donor (MDR-101 cell therapy), or a combination thereof.

[0154] 106. The method of paragraph 105, wherein the one or more T cell depleting antibodies are selected from the group consisting of: anti-CD4 antibody, anti-CD8 antibody, anti-CD45 antibody, anti-CTLA4 antibody, anti-CD20 antibody, and anti-CD33 antibody, or a combination thereof.

[0155] 107. The method of any one of paragraphs 99-106, wherein the transplant recipient has cancer.

[0156] 108. The method of any one of paragraphs 99-107, wherein the transplant is a bone marrow transplant.

[0157] 109. A method of inducing central tolerance in a transplant recipient, the method comprising administering to the recipient one or more doses of an anti-CD154 antibody, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the hematopoietic stem cells generate immune cells that are tolerant to the donor tissue, thereby inducing central tolerance in the recipient, and wherein the anti-CD154 antibody is the anti-CD154 antibody according to any one of paragraphs 1-38.

[0158] 110. The method of paragraph 109, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

[0159] 111. The method of paragraph 109 or 110, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0160] The method of any one of paragraphs 109-111, wherein the anti-CD154 antibody is administered subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally or rectally.

[0161] 113. The method of any one of paragraphs 109-112, wherein the method further comprises one or more treatments for conditioning a recipient for hematopoietic stem cell transplantation.

[0162] 114. The method of paragraph 113, wherein the one or more treatments for conditioning a recipient for hematopoietic stem cell transplantation are 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 cyclosporin A (CsA), administration of FK-506, administration of one or more interleukin-2 (IL-2) inhibitors, administration of rapamycin, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or a combination thereof.

[0163] 115. The method of paragraph 114, wherein the one or more T cell-depleting antibodies are selected from the group consisting of anti-CD4 antibody, anti-CD8 antibody, anti-CD45 antibody, anti-CTLA4 antibody, anti-CD20 antibody and anti-CD33 antibody, or a combination thereof.

[0164] 116. A method of inhibiting xenograft rejection in a subject, which comprises administering to the subject an effective amount of an anti-CD154 antibody, wherein the anti-CD154 antibody is the anti-CD154 antibody of any one of paragraphs 1-38.

[0165] 117. The method of paragraph 116, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0166] 118. The method of paragraph 116 or 117, wherein the xenograft is from a non-human donor selected from the group consisting of pigs, miniature pigs and non-human primates.

[0167] 119. The method of paragraph 118, wherein the non-human donor is a pig or a miniature pig that has been modified to reduce or eliminate the expression of one or more genes selected from the group consisting of porcine endogenous retrovirus (PERV), α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β1,4-N-acetylgalactosaminyltransferase (β4GalNT2) and class I MHC.

[0168] The method of paragraph 119, wherein the PERV is PERV A, PERV B, or PERV C.

[0169] The method of paragraph 119 or 120, wherein the expression of all PERV genes has been eliminated in the pig or miniature pig.

[0170] The method of any one of paragraphs 119-121, wherein the expression of the one or more genes is reduced or eliminated by using CRISPR / Cas9 gene editing.

[0171] The method of any one of paragraphs 118-122, wherein the non-human donor has been modified to express one or more human proteins selected from the group consisting of complement regulatory proteins, human α-galactosidase, thrombomodulin, human anti-inflammatory proteins, and human CTLA-4-Ig or a combination thereof.

[0172] The method of paragraph 123, wherein the one or more human proteins are expressed in all tissues of the non-human donor.

[0173] The method of paragraph 123, wherein the one or more human proteins are expressed in a tissue-specific manner in the non-human donor.

[0174] The method of any one of paragraphs 123-125, wherein the complement regulatory protein is selected from the group consisting of human decay-accelerating factor (CD55), membrane cofactor protein (CD46), and CD59.

[0175] The method of any one of paragraphs 123-125, wherein the thrombomodulin is selected from the group consisting of thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39, and CD73.

[0176] The method of any one of paragraphs 123-125, wherein the human anti-inflammatory protein is selected from the group consisting of heme oxygenase 1 (HO-1) and A20.

[0177] The method of any one of paragraphs 116-128, wherein the xenograft rejection is associated with a solid organ transplant selected from the group consisting of heart transplant, kidney transplant, liver transplant, lung transplant, pancreas transplant, kidney-pancreas transplant, heart-lung transplant, kidney-heart transplant, kidney-heart-pancreas transplant, heart-liver transplant, heart-liver-kidney transplant, heart-lung-kidney transplant, heart-lung-liver transplant, lung-kidney transplant, lung-liver transplant, liver-intestine-pancreas transplant, intestine-pancreas transplant, liver-kidney-intestine-pancreas transplant, and kidney-intestine transplant.

[0178] The method of paragraph 49, wherein the immune response is complement-mediated.

[0179] The method of any one of paragraphs 49-98, wherein the anti-CD154 antibody is administered in combination with one or more fusion peptides that bind to CD28 and block the function of CD28, the fusion peptides optionally selected from the group consisting of abatacept and belatacept Brief Description of the Drawings

[0180] Figure 1 Titers and viabilities (%) of a stable pool (TNX01-TNX05) of five CHO cells expressing anti-CD154 mAb in 100 mL fed-batch production are provided.

[0181] Figure 2 SDS-PAGE analysis of each of the anti-CD154 mAbs (TNX01-TNX05) obtained during the process of protein production and purification is provided. MW is the protein standard, which indicates the molecular weight of each band obtained on a gel stained with Sypro Ruby under non-reducing conditions. The TNX04 antibody appears as two separate bands on the non-reducing gel because it contains a Cys-Ser substitution that breaks the disulfide bonds linking the heavy and light chains and linking the two heavy chains.

[0182] Figure 3 (a)-3(e) respectively provide representative CD154 sensorgrams of TNX01-TNX05 binding to cognate antigen CD154. Affinity measurements of the purified anti-CD154 antibody are performed by surface plasmon resonance (SPR), wherein a T200 instrument and a sensor chip CM-5 are used. By using the Biacore T200 evaluation software, the change in refractive index is plotted in the sensorgram as a response in resonance units (RU) relative to time. For each sensorgram, a 1:1 binding model is used to fit the CD154 binding data. Figure 3 (f) summarizes the resulting Ka (1 / Ms), Kd (1 / s), and KD (M) values.

[0183] Figure 4 (a) and 4(b) provide based on fitting to Figure 3Kinetic results of the 1:1 binding model of the sensorgrams. For each of the TNX01 - TNX05 anti - CD154 antibodies, the mean value of the binding affinity KD (M) for sCD40L (i.e., sCD154) is shown together with the standard deviation (n = 3).

[0184] Figure 5 (a) - 5(e) respectively provide representative CD154 sensorgrams regarding the binding of TNX01 - TNX05 to FcγRIA (CD64). The affinity measurement of the purified anti - CD154 antibodies was performed by surface plasmon resonance (SPR), where the T200 instrument and sensor chip CM - 5 were used. By using the Biacore T200 evaluation software, the change in refractive index was plotted in the sensorgram as the response in resonance units (RU) relative to time. For each sensorgram, a 1:1 binding model was used to fit the CD154 binding data. Figure 5 (f) Summarizes the resulting Ka (1 / Ms), Kd (1 / s), and KD (M) values.

[0185] Figure 6 (a) and 6(b) provide the kinetic results of the 1:1 binding model based on the sensorgrams fitted to Figure 5 . The mean value of the binding affinity KD (M) for FcγRIA (CD64) is shown together with the standard deviation (n = 3). A lower KD value indicates a tighter binding between the antibody and FcγRIA, and thus TNX02 binds FcγRIA most tightly, followed by (in order) TNX04, TNX05, TNX01, and TNX03.

[0186] Figure 7 (a) and 7(b) provide the kinetic results of the 1:1 binding model based on the sensorgrams fitted to Figure 3 . The mean value of the binding affinity KD (M) for the binding of TNX02, TNX04, and TNX05 to the low - affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) is shown together with the standard deviation (n = 3). The KD values for TNX01 and TNX03 could not be determined because these variants showed negligible binding activity. A lower KD value indicates a tighter binding between the antibody and FcγR.

[0187] Figure 8(a) and 8(b) provide graphical representations of the mean fluorescence intensity (MFI) as a function of concentration (nM) in the binding of each antibody to the surface of (a) CD154-positive Jurkat D1.1 cells or (b) CD154-negative Jurkat cells, as obtained by flow cytometry. Figure 8 (c) provides each anti-CD154 antibody (TNX01-TNX05), a positive control antibody (mouse anti-human CD40L or CD140), and a negative control antibody for the binding characteristics (Bmax (maximum number of binding sites), h (Hill slope), and apparent Kd (ligand concentration that binds to half of the receptor sites at equilibrium)) of D1.1 cells (CD154-positive), as analyzed by using a high-throughput FACS cell binding assay performed with various antibodies.

[0188] Figure 9 (a), 9(b), and 9(c) provide various amino acid modifications of the Fc region for IgG1 ( Figure 9 (a)), IgG2 ( Figure 9 (b)), and IgG4 ( Figure 9 (c)) that can modify (e.g., reduce) FcR effector function.

[0189] Figure 10 SDS-PAGE analysis of each of the anti-CD154 mAbs (TNX06-TNX13) obtained during the process of protein production and purification is provided. STD is the protein standard, which indicates the molecular weight of each band obtained on a Sypro Ruby-stained gel under non-reducing (NR) or reducing (R) conditions. The lanes in the gel are as follows from left to right: (1) STD; (2) TNX06 (NR); (3) TNX06 (R); (4) STD; (5) TNX07 (NR); (6) TNX07 (R); (7) STD; (8) TNX08 (NR); (9) TNX08 (R); (10) STD; (11) TNX09 (NR); (12) TNX09 (R); (13) STD; (14) TNX10 (NR); (15) TNX10 (R); (16) STD; (17) TNX11 (NR); (18) TNX11 (R); (19) STD; (20) TNX12 (NR); (21) TNX12 (R); (22) STD; (23) TNX13 (NR); (24) TNX13 (R); (25) STD; (26) STD.

[0190] Figure 11Provided are surface plasmon resonance (SPR) comparability scores of sCD154 binding by TNX06-13 relative to TNX02. The scores are represented as a percentage of similarity by pairwise comparison.

[0191] Figure 12 (a) Summarizes data generated from surface plasmon resonance (SPR) analysis and calculated apparent KD values (affinity; ratio of kd to ka) regarding the binding of TNX06-13 to FcγRIA (CD64) and TNX02. Figure 12 (b) and 12(c) show the mean and standard deviation (n = 3) of the KD (M) for FcγRIA (CD64) binding affinity. Lower KD values indicate tighter binding between the antibody and FcγRIA, such that TNX02 binds FcγRIA most tightly, followed in order by TNX13, TNX07, TNX12, TNX06, TNX10, TNX08, and TNX09.

[0192] Figure 13(a) provides the mean KD values based on sensorgram data obtained regarding the binding of TNX02 and TNX06-13 to low-affinity FcγR. Figure 13(b) shows the mean and standard deviation (n = 3) of the KD (M) for the binding affinity of TNX02, TNX06-10, TNX12, and TNX13 to the low-affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF). KD values could not be determined for TNX11 and for TNX09, TNX10, and TNX12 (for some FcγR panel components) because these variants showed negligible binding activity. Lower KD values indicate tighter binding between the antibody and FcγR.

[0193] Figure 14 (a) Provides a graphical representation of the mean fluorescence intensity (MFI) as a function of concentration (nM) in the binding of each antibody to the surface of CD154-positive Jurkat D1.1 cells. Figure 14 (b) Provides the binding characteristics (Bmax (maximum number of binding sites), h (Hill slope), and apparent Kd (ligand concentration that binds to half of the receptor sites at equilibrium)) for each anti-CD154 antibody (TNX06-TNX13), positive control antibody (mouse anti-human CD40L or CD154), and negative control antibody for D1.1 cells (CD154-positive), as analyzed by a high-throughput FACS cell binding assay using the various antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0195] Definitions and General Techniques

[0196] Unless otherwise defined herein, scientific and technical terms used in this application will have the meanings commonly understood by one of ordinary skill in the art. In general, the nomenclature used in connection with, and the laboratory procedures and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Where a conflict exists, the present specification, including definitions, will control.

[0197] The methods and techniques of the present disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the skill level of the art, unless otherwise indicated. Such techniques are well explained in the literature, for example, Green MR & Sambrook J. Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th ed., Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., John Wiley & Sons, Inc., 2003); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), each of which is incorporated herein by reference.

[0198] Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The specialized terms used herein in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as laboratory procedures and techniques in these areas, are those well known and commonly used in the art.

[0199] Standard techniques are used for chemical synthesis and chemical analysis.

[0200] Throughout this specification and the embodiments, the word "comprising" or variations thereof will be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers. "Comprising" may be synonymous with "including" or "containing".

[0201] It should be understood that wherever an embodiment is described herein in the language "comprising", additional similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. As used herein, "consisting of" is a closed term that includes only the recited specific elements, while "consisting essentially of" includes the recited specific elements and may include additional unrecited non-essential elements.

[0202] The term "comprising" is used to mean "including but not limited to". "Comprising" and "including but not limited to" are used interchangeably.

[0203] Any examples following the terms "for example" or "such as" are not intended to be exhaustive or limiting.

[0204] Unless the context otherwise requires, singular terms will include the plural and plural terms will include the singular.

[0205] As used herein, the articles "a", "an", and "the" refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element. As used herein, the term "about", when modifying a quantity of a component, parameter, calculation, or measurement employed in the compositions of the present disclosure or in the methods of the present disclosure, refers to variations in such numerical quantities that can occur, for example, through typical measurement and liquid handling procedures used for preparing isolated polypeptides or pharmaceutical compositions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the components employed to prepare the compositions or to perform the methods, and the like, without substantial effect on the chemical or physical properties of the compositions or methods of the present disclosure. Such variations can be within an order of magnitude, typically within 10% of a given value or range, more typically within 5%. The term "about" also encompasses quantities that differ due to different equilibrium conditions of the compositions resulting from a particular initial mixture. Whether or not modified by the term "about", these paragraphs include equivalents of the recited quantities. References to "about" a value or parameter herein include (and describe) embodiments directed to that value or parameter per se. For example, a description of "about X" includes a description of "X". Numerical ranges include the numbers defining the range.

[0206] Although the broad numerical ranges and parameters setting forth 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. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, the recited range of "1 to 10" is to be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (and including the endpoints); that is, all subranges beginning with a minimum value of 1 or more (e.g., 1 to 6.1) and ending with a maximum value of 10 or less (e.g., 5.5 to 10).

[0207] 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.

[0208] Definitions

[0209] The following terms are to be understood to have the following meanings, unless otherwise indicated:

[0210] As used herein, the term "antibody" or "Ab" refers to an immunoglobulin molecule (e.g., a full antibody, an antibody fragment, or a modified antibody) that is capable of recognizing and binding a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody that specifically binds to a given antigen (e.g., CD154), including but not limited to monoclonal antibodies, polyclonal antibodies, human antibodies, engineered antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single-chain antibodies, affinity matured antibodies, CDR-grafted antibodies, etc.). Further, "antibody" and / or "immunoglobulin" (Ig) refers to a polypeptide that comprises at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa), which are optionally linked to each other by disulfide bonds. There are two types of light chains: λ and κ. In humans, the λ and κ light chains are similar, but only one type is present in each antibody. Heavy chains are classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Generally, see Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)) (which is incorporated by reference in its entirety). The antibodies disclosed herein can be "functionally bivalent" or "functionally monovalent." In other words, the antibodies disclosed herein can have one antigen-binding site (monovalent) or typically two antigen-binding sites (bivalent) linked by disulfide bonds. In some embodiments, the anti-CD154 antibody is an IgG1 antibody. The anti-CD154 antibody can be an IgG2 antibody. Optionally, the anti-CD154 antibody is an IgG4 antibody. In some embodiments, the anti-CD154 antibody does not contain the final lysine residue at its C-terminus to improve antibody stability. See, e.g., 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 referred to 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 thus is not present in the final chain or the mature antibody.

[0211] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody produced by the same group of immune cells, which are clones of a unique parental cell. Monoclonal antibodies have monovalent affinity (i.e., they bind to the same epitope).

[0212] As used herein, the term "chimeric" antibody refers to an antibody and antigen-binding fragment thereof that contains portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced with a mouse variable region of desired specificity spliced onto a human constant domain gene segment (e.g., U.S. Patent No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical applications. The term "chimeric" can refer to a non-natural sequence that has been manipulated to have one or more changes relative to the natural sequence. A chimeric antibody as used herein means an antibody that contains regions from two or more different antibodies.

[0213] 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 its similarity to an antibody 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 sequences of an antibody) that contains a minimal sequence derived from a non-human immunoglobulin. Optionally, the humanized antibody is derived from a human immunoglobulin (i.e., the recipient antibody), wherein residues of one or more complementarity-determining regions (CDRs) from the recipient antibody are replaced with residues of one or more CDRs from an antibody (the donor antibody) from a non-human species having the desired specificity, affinity, and capacity. In some embodiments, the non-human species is a mouse, rat, or rabbit. Humanized or CDR-grafted mAbs are particularly useful as therapeutic agents for humans because they are not cleared from the circulation as rapidly as mouse antibodies and typically do not elicit an adverse immune response. Generally, a humanized antibody has one or more amino acid residues introduced therein from a non-human source.

[0214] In some embodiments, the chimeric antibody is a humanized antibody, such as a humanized anti-CD154 antibody. The humanized anti-CD154 antibody can comprise the amino acid sequence of one or more human framework regions and / or at least a portion of the amino acid sequence from a human constant region, and further comprise 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 in the humanized antibody are human framework regions.

[0215] Humanized antibodies can be generated by replacing non-human sequences of the Fv variable regions that do not directly participate in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by the following references: Morrison, S.L., 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. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213 (each incorporated herein by reference). Those methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable regions from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those of skill in the art and can be obtained, for example, from hybridomas that produce antibodies against a pre-determined target (as described above), germline immunoglobulin genes, or synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into a suitable expression vector. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are replaced with residues from analogous sites in a rodent antibody. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205 (each incorporated herein by reference). See also U.S. Patent No. 6,180,370 and PCT International Publication No. WO 01 / 27160 (each incorporated herein by reference), which disclose humanized antibodies and techniques for generating humanized antibodies with improved affinity for a pre-determined antigen. Further, humanized and chimeric antibodies can be modified to contain residues not found in the recipient antibody or in the donor antibody in order to further improve antibody properties, such as affinity or effector function.

[0216] 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 include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences (i.e., humanized antibodies). The term encompasses antibodies having sequences derived from human genes but that have been altered (e.g., to reduce possible immunogenicity, increase affinity, eliminate cysteines that may cause unwanted folding, etc.). The term also encompasses such antibodies recombinantly produced in non-human cells, which may confer glycosylation atypical for human cells. For the generation 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; Brüggemann et al., Arch. Immunol. Ther. Exp., 2015, vol. 63:101-108 (the disclosures of which are incorporated herein by reference in their entireties).Human antibodies and methods for their preparation are further discussed in the following documents: 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. US2006-0015957 A1; and International Patent Publication Nos. WO 98 / 24893; and WO 2007 / 117410. The disclosures of each of the patents, applications, and references cited above are incorporated herein by reference in their entirety.

[0217] 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 ordinary skill of the art. Certain modifications of the Fc region (including amino acid deletions, substitutions, and additions) have been shown 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 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); US Patent Publication 20040132101; 20070111260; 20110287032; 20180194860; US Patent No. US 8409568; International Publication No. WO2017 / 177337) (which are incorporated by reference in their entirety). Amino acid deletions are denoted by "Δ", and insertions are denoted by "In". For example, a deletion of the amino acid sequence from E216 to E222 is denoted by ΔE216-E222. For example, an insertion of arginine (R) between amino acid residues 234 and 235 would be denoted by InR234 / 235.

[0218] 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 of the hinge region and the second and third constant domains from IgA, IgD, and IgG antibody isotypes, or the hinge region and the second, third, and fourth constant domains from IgM and IgE antibody isotypes. The Fc domain is the part of the antibody that binds to cell surface Fc receptors and certain proteins of the complement system. The term "Fc region" refers to the Fc domain in combination 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 can vary, the human IgG heavy chain Fc region as defined herein comprises residues E216 to the carboxyl terminus of its CH3 domain (or CH4 domain for IgM and IgE antibodies), where the numbering is in the EU format as in Edelman GM et al., (1969) Proc. Natl. Acad. USA, 63, 78-85. "The EU format as set forth in Edelman" refers to the residue numbering of the human IgG1 EU antibody described in Edelman GM et al. (supra). The residue numbering of human IgG2 and human IgG4 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 in isolation, or in the context of an antibody, antibody fragment, or Fc fusion protein. Fc variant proteins can be antibodies, Fc fusions, or any protein or protein domain that contains an Fc 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) can contain amino acid modifications. Any new amino acid residue that appears in the sequence of a variant Fc domain or variant Fc region due to an insertion or substitution can be referred to as a non-naturally occurring amino acid residue. Polymorphisms have been observed at many Fc domain positions, including but not limited to positions 270, 272, 312, 315, 356, and 358, and thus there can be minor differences between the sequences presented and those in the prior art.

[0219] As used herein, the term "linker" refers to a polypeptide sequence that connects two or more antibody domains. The characteristics of linkers and their suitability for particular purposes are known in the art. See, e.g., 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), which is incorporated herein by reference. Linkers can be flexible, rigid, or cleavable in vivo. 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 consisting predominantly of stretches of Gly and Ser residues ("GS" linkers). Optionally, the flexible linker contains a repeating sequence of 5 Gly and Ser residues. Non-limiting examples of flexible linkers include (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), where n can be any integer from 1 to 5. The length of the linker is optionally from 5 to 25 amino acid residues. Other suitable linkers can 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, flexible linkers should provide good flexibility and solubility and can act as passive linkers to maintain the distance between functional domains. The length of the flexible linker can be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein.

[0220] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd ed., E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0221] A "conservative amino acid substitution" is a substitution 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 substantially does not alter the functional properties of the 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 of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).

[0222] Groups of amino acids having similar biochemical properties that can be used in conservative substitutions include: 1) amino acid residues having aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) amino acid residues having aliphatic hydroxyl side chains: serine and threonine; 3) amino acid residues having amide-containing side chains: asparagine and glutamine; 4) amino acid residues having aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) amino acid residues having basic side chains: lysine, arginine, and histidine; 6) amino acid residues having acidic side chains: aspartic acid and glutamic acid; and 7) amino acid residues having sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and glutamine-asparagine.

[0223] Each heavy chain is composed of a variable heavy domain (VH) and multiple constant heavy domains (CH). For IgA, IgD, and IgG antibodies, the heavy chain typically contains three domains: CH1, CH2, and CH3. For 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 variable light domain (VL) and a constant light domain. The light chain typically contains one domain: CL. The variable light domain is encoded by two gene segments: a variable (V) gene segment that encodes the first 95 - 101 amino acids of the light chain; and a joining (J) gene segment that encodes approximately 12 or more amino acids. The variable heavy domain is encoded by three gene segments and includes a diversity (D) gene segment between the V and J gene segments that encodes approximately 3 or more amino acids. The VH and VL domains can be further subdivided into hypervariable regions, called "complementary determining regions (CDRs)", which are separated by more conserved "framework regions" (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0224] 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 multi-functional / multi-specific (e.g., bifunctional or bispecific) antibodies, these two binding sites are identical. The Fc region of the constant region of the antibody 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 of the classical complement system (Clq).

[0225] Residues in the variable domains are numbered according to Edelman (also known as the EU numbering system), which is a numbering system for the heavy-chain variable domain or the light-chain variable domain of antibody compilation. See Edelman Proc Natl Acad Sci U S A. May, 63(1):78-85(1969); and Kabat, E.A., Wu, T.T., 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 for a given antibody can be determined by aligning with a "standard" EU-numbered sequence at the homologous regions of the antibody's sequence. Variable region CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR H3) are identified according to contacts based on crystal structure (as defined in Karpusas et al., Structure. Apr 4, 9(4):321-9(2001)) and numbered according to Edelman.

[0226] As used herein, the term "operably linked" refers to a first structure that has been placed in a functional relationship with a second structure. In the context of an antibody, a targeting structure can be operably linked to a structure that confers effector function. For example, the antigen-binding sequence of an antibody (e.g., the variable region or the VH or VL domain) can be operably linked to the Fc region. In the context of a polynucleotide, a coding sequence can be operably linked to non-coding regulatory sequences (e.g., promoters, enhancers, signal sequences, ribosome-binding sequences, splice acceptor sequences, splice donor sequences, termination sequences, etc.). Two operably linked structures can be directly connected. Alternatively, two operably linked structures can be connected through one or more intermediate structures. For example, the antigen-binding portion of an antibody can be operably linked to the Fc region through the CH1 domain, hinge region, and / or linker sequence. Similarly, operably linked non-coding regulatory sequences include both sequences adjacent to the coding sequence and sequences that act in trans or at a distance to control the coding sequence.

[0227] As used herein, the term "effector function" refers to responses triggered through the interaction of antibodies and antibody-antigen complexes with cells of the immune system. These effector functions typically involve one of three main 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) or macrophages bind to the Fc region of an antibody bound to a target cell, resulting in the secretion of substances (e.g., lytic enzymes, perforin, granzyme, and tumor necrosis factor) that mediate the destruction of the target cell. In CDC, cell death is induced through 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 Fc receptors on the surface of phagocytic cells, thereby inducing phagocytosis. Such effector functions generally require the combination of the Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using standard assays known in the art (see, e.g., WO 05 / 018572, WO 05 / 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 Fcγ receptor family on the cell surface and to Clq of the complement system. Linking effector proteins through clustered antibodies triggers a variety of responses, including the release of inflammatory cytokines, regulation of antigen production, endocytosis, and cell killing. These responses can cause unwanted side effects such as inflammation and thrombosis. Accordingly, the present disclosure further relates to anti-CD154 antibodies having modified effector functions, including antibodies in which one or more effector functions are reduced or eliminated. Without being bound by theory, it is believed that the anti-CD154 antibodies disclosed herein do not cause platelet activation or aggregation because antibodies containing a mutated Fc region do not bind to FcγRIIa (also known as CD32a) on the platelet surface.

[0228] 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 Fc domain or Fc region. In some embodiments, one or more effector functions are reduced. Optionally, one or more effector functions are eliminated. The modified or reduced effector function can be the result of a lower binding affinity of the Fc region of an antibody disclosed herein for effector molecules (e.g., FcγR and / or C1q). For example, the anti-CD154 antibodies disclosed herein have reduced Fc receptor binding and complement activation compared to wild-type anti-CD154 antibodies. In some embodiments, the variant Fc region has reduced antibody-dependent cell-mediated cytotoxicity (ADCC). The effector functions of the anti-CD154 antibodies can be determined by using one of many known assays, including the CDC assay, the ADCC assay, and the phagocytosis assay (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 the anti-CD154 antibodies can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the effector functions of wild-type anti-CD154 antibodies.

[0229] As used herein, the terms "CD154," "CD40 ligand," and "CD40L" are used interchangeably and refer to a mammalian protein expressed primarily on the surface of activated T cells. The soluble form of CD154, which is cleaved and released by activated T cells, is a member of the TNF superfamily and binds to the CD40 protein on antigen-presenting cells. The term CD154 is intended to include recombinant CD154 and recombinant chimeric forms of CD154, which can be prepared by standard recombinant expression methods. In some embodiments, CD154 refers to human CD154.

[0230] As used herein, the term "inhibit" refers to the property of an antibody or other molecule to prevent the interaction of CD154 with CD40, or to inhibit the binding of CD154 to CD40, or to inhibit 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 potential of an anti-CD154 antibody can be determined, for example, by its ability to inhibit the upregulation of specific downstream target genes of CD40. For example, an anti-CD154 antibody can alter the expression, activity, or activation of kinases and genes in response to CD154-CD40 signaling. An anti-CD154 antibody can inhibit the upregulation of CD23 expression, inhibit the upregulation of CD69 expression, inhibit the upregulation and activity of activation-induced cytidine deaminase (AID), inhibit rescue from apoptosis, inhibit the upregulation of NF-κB activity, inhibit immunoglobulin isotype class switch, inhibit somatic hypermutation of immunoglobulin CDRs, 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, alter kinase activation, or inhibit the expression of other genes in response to CD154-CD40 signaling.See, e.g., 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, A.M., 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. 194: 4319-4327 (2015); Ishida TK et al., Proc Natl Acad Sci U S 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 U S 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): 6555-62 (2010); Rowland SL et al., Journalof Immunol. 179(7): 4645-53 (2007); Benson RJ et al., European Journal of Immunol. 6(9): 2535-43 (2006).

[0231] As used herein, the term "immune response" refers to the reaction of the body's immune system to the presence of substances that are not recognized as components of the body itself. An immune response can be a humoral immune response, a cell-mediated immune response, or a mixed humoral and cell-mediated immune response. A humoral response can be an antibody-mediated response. A cell-mediated response can 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. A mixed humoral and cell-mediated response can 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. An immune response can refer to an adaptive and / or innate immune response. For various types of immune responses, see David Chaplin JAllergy Clin Immunol February, 125(2 Suppl 2):S3-23(2010).

[0232] As used herein, the "affinity" of an antibody refers to the strength of the interaction between the antigen-binding site of the antibody and the epitope. Typically, the affinity of an antibody for an antigen is expressed as the binding affinity equilibrium dissociation constant (K D ) of a particular antibody-antigen interaction. When K D ≤1 mM, preferably ≤100 nM, it is said that the antibody specifically binds the antigen. High-affinity antibodies are generally considered to have a K -9 in the low nM (10 D ) range, and very high-affinity antibodies are generally considered to have a K -12 in the pM (10 D ) range. The K D binding affinity constant can be measured by surface plasmon resonance, for example using System (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), as 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 incorporated herein by reference. K D Measurements can also be made by using system (Sapidyne Instruments, Hanover, Germany and Boise, ID).

[0233] 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 when equilibrium concentrations between the antibody and antigen are reached. K D is the ratio of kd to ka.

[0234] As used herein, the term “avidity” refers to the overall strength of the antibody-antigen complex. Avidity involves three main parameters: the affinity of the antibody for the epitope; the valency of both the antibody and the antigen; and the structural arrangement of those parts that interact. As used herein, “avidity” describes the increased affinity that arises due to multiple antigen-binding sites on the immunoglobulin.

[0235] As used herein, the term “transplantation” refers to the process of surgically removing cells or tissue from a first organism (the donor) and placing it into a second organism (the recipient). The donor can be a human or non-human organism. In some embodiments, the donor is a primate. The donor can be a non-human primate. Optionally, the donor is human. In some embodiments, the donor is a pig or a minipig. The recipient can be a human or non-human organism. Preferably, the recipient is human. Optionally, the recipient is a non-human primate. The transferred cells, tissue, or organ is referred to as a “graft.” “Xenotransplantation” refers to the transfer of cells or tissue or an organ from a donor of one species (e.g., monkey or pig) into a recipient of a different species (e.g., human).

[0236] As used herein, the term "engineered cell" refers to a cell that has been modified from its natural state. Engineered cells can be modified by using one or several techniques such as transduction to express cDNA, the CRISPR / Cas9 system, RNAi technology, and retroviral technology. For example, the cells can be modified to express a chimeric antigen receptor (CAR) on their surface. Examples of cells that can 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 (TIL) (Zhang L et al., Clin Cancer Res. May 15, 21 (10): 2278-88. doi: 10.1158 / 1078-0432.CCR-14-2085 (2015)).

[0237] As used herein, the term "ex vivo expanded cell" refers to a cell that is generated by an ex vivo method to increase the yield of a cell (such as a hematopoietic stem cell (HSC)) to be used in a clinical application (such as transplantation). See, for example, Xie J and Zhang C Sci China Life Sci. Sep, 58 (9): 839-53 (2015), which reviews methods for expanding the number of HSCs, including culture systems such as stroma / HSC co-culture, continuous perfusion, and fed-batch culture, and those supplemented with external ligands, membrane-permeable transcription factors, complement components, protein modifying enzymes, metabolites, or small molecule chemicals. Desired cells to be transplanted can also be ex vivo expanded by applying an endogenous Notch-signaling activator (see, for example, Ex vivo expansion of human hematopoietic stem and progenitor cells, Dahlberg A et al., Blood 117: 6083-6090 (2011)).

[0238] As used herein, the term "transplant rejection" refers to the phenomenon that occurs when transplanted donor cells, tissues, or organs are rejected by the recipient's immune system. The recipient's immune system can initiate an adaptive immune response (cellular immunity) mediated by cytotoxic T cells, which induces 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)).

[0239] As used herein, the term "immune-related disease" refers to a condition in which the host immune system plays an indispensable role in the mediated disease and contributes to the progression of the disease. This term encompasses "autoimmune diseases", which are conditions that occur when a specific adaptive immune response is initiated against self-antigens, as a result of which the effector pathways of immunity cause chronic inflammatory damage to tissues. Autoimmune diseases include, but are not limited to, type I diabetes, juvenile diabetes, autoimmune diabetes, autoimmune hemolytic anemia, rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, inflammatory bowel disease, Addison's disease, Crohn's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, Guillain-Barré syndrome, ankylosing spondylitis, primary biliary cirrhosis, lupus nephritis, Goodpasture's disease, polymyositis, dermatomyositis, psoriasis, temporal arteritis, Churg-Strauss syndrome, transverse myelitis, thyroiditis, ulcerative colitis, sarcoidosis, hemolytic anemia, idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorder, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and Behçet's disease. Immune-related diseases can also refer to allergic diseases, which include, but are not limited to, allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergy, drug allergy, and food allergy.

[0240] Immune-related diseases can also refer to immune responses that cause or contribute to a dysfunctional inflammatory condition of an organ (including but not limited to the lung, heart, and kidney). For example, acute respiratory distress syndrome (ARDS) is a form of hypoxemic respiratory failure characterized by severe impairment in gas exchange and lung mechanics, with a high case fatality rate. ARDS occurs when small blood vessels in the lung leak fluid, which enters and may fill the alveoli. Multiple immunological processes involving neutrophils, macrophages, and dendritic cells are involved in mediating tissue damage in ARDS. See Han, S and Mallampalli RK. Journal of immunology 194, 3: 855-60 (2015). Other inflammatory conditions of the lung include but are not limited to, pneumonia, bronchitis, pulmonary tissue inflammation, chronic obstructive pulmonary disease (COPD). See Moldoveanu, B et al., Journal of inflammation research (2): 1-11 (2009).

[0241] Furthermore, immune-related diseases, such as a dysregulated immune response that causes or contributes to an inflammatory condition of an organ, can be due to a viral infection. For example, ARDS, pneumonia, bronchitis, lung tissue inflammation, and COPD can occur in the context of a viral infection, such as cytomegalovirus, Epstein-Barr virus, influenza virus, variola virus, orthopoxvirus, or a coronavirus, such as SARS coronavirus (SARS-CoV), SARS-CoV-2, and MERS-CoV. Such viral infections can be associated with an inflammatory condition or a dysregulated immune response, such as cytokine release syndrome (CRS) or "cytokine storm" (which is a severe immune reaction in which the host releases too many cytokines into the bloodstream too quickly). For example, the host can release high amounts of pro-inflammatory cytokines, such as IP-10, MCP-1, MIP-1A, IL-6, and TNF-α, into the bloodstream (see Prompetchara E. et al., Asian Pac J Allergy Immunol, 38(1):1-9 (2020)). Cytokine storm can occur due to an infection, an autoimmune condition, or other diseases. For example, cytokine storm can occur in the context of a viral infection, such as cytomegalovirus, Epstein-Barr virus, influenza virus, variola virus, SARS-CoV, SARS-CoV-2, and MERS-CoV. Immune-related diseases can refer to conditions associated with a dysregulated immune response, cytokine storm, or cytokine storm syndrome in a host. Examples of such virus-mediated immune-related diseases include, but are not limited to, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19). The transition from pneumonia to ARDS has also been described as a "cytokine storm". Many patients with severe COVID-19 lung disease present with typical ARDS, but others present with an atypical form of ARDS (with relatively high compliance and well-preserved lung gas volume), which may be related to loss of pulmonary perfusion regulation, hypoxic vasoconstriction (Gattinoni L et al., Am J. Respir Crit Care Med. (2020)), and / or microvascular thrombosis and potential disseminated intravascular coagulation (Phend C. MedPage Today, Infectious Disease. April 8, 2020).

[0242] As used herein, the term "primary response" refers to the body's immune response to an antigen that occurs upon first encounter with the antigen. The term "secondary response" refers to an immune response that involves both B cells and T cells and more rapidly leads to the activation of previously generated memory cells following a later encounter with the same antigen. The secondary response has some quantitative and qualitative differences from the primary response, as reviewed in Ademokun and Dunn-Walters, Immune Responses: Primary and Secondary, John Wiley & Sons (2010) and Kuby Immunology Macmillan, 8th Edition (2018). In a primary immune response, the responding cells are naive B-cells and T-cells, while in a secondary response, the responding cells are memory cells.

[0243] As used herein, the term "hematopoietic chimerism" refers to the coexistence of both host and donor hematopoietic cells that occurs as a result of the migration of donor pluripotent hematopoietic stem cells into the host. The host and donor cells may be mutually tolerant. The 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". The mechanisms of "central tolerance" in such chimeras may involve central, intrathymic clonal deletion, the 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, the hematopoietic stem cells are isolated or purified. Optionally, the hematopoietic stem cells are passenger cells transplanted together with an organ (e.g., a kidney or liver graft). The stem cells may be derived from the donor's bone marrow or adipocytes / adipose tissue.

[0244] As used herein, the term "conditioning" or "conditioned" refers to preparing a recipient for undergoing a stem cell transplantation, such as a hematopoietic stem cell transplantation. Gyurkocza B and Sandmaier BM Blood 124:344-353(2014) provide a review of high-dose, low-intensity, and non-myeloablative conditioning regimens and the most commonly used agents such as total body irradiation, fludarabine phosphate, cyclophosphamide, T-cell depleting antibodies, cyclosporine A (CsA). Monoclonal antibodies such as anti-CD20 Ab, anti-CD33 Ab, and anti-CD45 Ab can also be used, either alone or in combination with conventional therapies, as part of a conditioning regimen to prevent transplant rejection. See, e.g., Topcuoglu P et al., Progress in StemCell Transplantation, December(2015). Other agents that can be used in a 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)). Conditioning regimens can include chemotherapeutic agents including, but not limited to, alemtuzumab (CAMPATH TM ), busulfan, carboplatin, carmustine, cyclophosphamide, cytarabine (Ara-C), daunorubicin, etoposide (VP-16), fludarabine, melphalan, rituximab, and vincristine.

[0245] The term "isolated antibody" refers to an antibody that is at least partially free of other biomolecules that are present in the cells used to produce them. Other biomolecules that are not present in an isolated antibody include nucleic acid molecules, proteins, lipids, carbohydrates, cell debris, and culture medium. The term "isolated antibody" does not require but encompasses the complete absence of such other biomolecules. The term "isolated antibody" also does not refer to the complete absence of other molecules, such as water, buffers, salts, or components of a pharmaceutical formulation. Thus, a molecule that is chemically synthesized or synthesized in a cell-free system would be "isolated" from its naturally associated components. Molecules can also be "isolated" by using purification techniques well known in the art.

[0246] As used herein, the terms "nucleic acid", "nucleic acid molecule", and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides that is at least 10 bases in length. Polynucleotides can contain ribonucleotides, deoxyribonucleotides, modified forms of either type of nucleotide, or combinations thereof. Polynucleotides can be single-stranded or double-stranded.

[0247] As used herein, the term "isolated polynucleotide" or "isolated nucleic acid molecule" means a polynucleotide of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that (1) is not associated with all or a portion of the polynucleotide with which the "isolated nucleic acid" is associated in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur as part of a larger sequence in nature. An isolated polynucleotide that "comprises" a particular sequence may also include coding sequences for other proteins or immunoglobulin chains, expression control sequences, or vector sequences.

[0248] Molecular purity or homogeneity can be assayed by many means well known in the art. For example, the purity of an antibody sample can be assayed by using polyacrylamide gel electrophoresis and staining the gel with techniques well known in the art to visualize the antibody. For some 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 by spectrophotometric absorbance of the sample at 260 nm to 280 nm using techniques well known in the art. For some purposes, higher resolution can be provided by using means well known in the art for purification.

[0249] Examples of isolated antibodies include, but are not limited to, anti-CD154 antibodies that have been affinity purified using CD154, and anti-CD154 antibodies that have been synthesized in vitro by cell lines.

[0250] As used herein, the term "vector" refers to a construct that is capable of delivering and preferably expressing one or more polynucleotide sequences of interest in a host cell. Non-limiting examples of vectors include viral vectors, naked DNA or RNA expression vectors, bacterial vectors, mammalian vectors, plasmids, cosmids, phage vectors, DNA or RNA expression vectors associated with cationic condensing reagents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells). In some embodiments, the vectors replicate autonomously in the host cells into which they are introduced. Optionally, the vectors integrate into the genome of the host cell and replicate along with the host genome. Vectors can be capable of directing the expression of coding sequences to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors".

[0251] As used herein, the term "host cell" refers to a cell into which a vector or polynucleotide has been introduced. It should be understood that "host cell" includes not only a particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to mutations or environmental influences, such progeny may not in fact be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein, provided that they still contain the vector, polynucleotide, or a portion thereof, either in episomal form or integrated into the host cell genome.

[0252] In the context of polynucleotide (or polypeptide) sequences, the term "percent sequence identity" is defined as the percentage of nucleic acid (or amino acid) residues in a candidate sequence that are identical to the nucleic acid (or amino acid) residues in a reference nucleotide (or polypeptide) sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Such conservative substitutions are considered in the calculation of the "percent sequence similarity" of two sequences (in addition to identical residues). Residue positions that are not identical but are similar differ by conservative amino acid substitutions.

[0253] Alignment for the purposes of determining the percent amino acid sequence identity, sequence similarity, or sequence homology (e.g., between a wild-type protein and its mutant protein) can be achieved in various ways within the skill in the art, such as by using publicly available sequence analysis computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Gap, and other programs in Wisconsin Package version 10.0 or Genetics Computer Group (GCG), Madison, Wisconsin software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. FASTA can also be used with default or recommended parameters to compare polypeptide sequences. In the context of polypeptide sequences, FASTA grabs the query amino acid sequence and uses local sequence alignment to search a database to identify similar sequences within that 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 incorporated herein by reference). BLAST (especially blastp or tblastn) with default parameters can be used to compare a query sequence to a database containing sequences from different 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 incorporated herein by reference).

[0254] As used herein, reference to a nucleotide sequence encompasses its complementary sequence unless otherwise specified. Accordingly, reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand having its complementary sequence.

[0255] The terms "patient", "subject", and "individual" are used interchangeably herein and refer to a human or non-human animal in need of treatment. These terms include mammals such as humans and primates (e.g., monkeys). Optionally, the subject is a human. In some embodiments, the subject is in need of suppression or attenuation of an immune response.

[0256] The term "primate" refers to a mammal of the order Primates, which includes anthropoids and prosimians, and is characterized by the elaborate development of the hands and feet, a shortened muzzle, and a large brain. Mammals of the order Primates include humans, apes, monkeys, and prosimians, or lower primates.

[0257] As used herein, a "therapeutically effective amount" refers to the amount of a therapeutic agent administered that will, to some extent, alleviate one or more of the symptoms of the condition being treated. With respect to the treatment of transplant rejection, a therapeutically effective amount refers to an amount having at least one of the following effects: reducing, inhibiting, or preventing acute or chronic rejection of the transplanted cells, tissues, or organs, and one or more symptoms associated with rejection; prolonging graft survival; reducing thrombosis; reducing the risk of life-threatening infections, cancer, or other complications such as cardiovascular disease and renal failure. With respect to the mechanisms of cellular rejection in transplantation, see, e.g., Romano et al., Front Immunol. 10:43 (2019) and Ingulli E., Pediatr Nephrol. 25(1):61-74 (2010). With respect to the treatment of autoimmune diseases and antibody-mediated inflammatory diseases, a therapeutically effective amount refers to an amount having at least one of the following effects: alleviating one or more symptoms associated with the autoimmune disease, such as fatigue, myalgia, low-grade fever, inflammation, rash, etc.

[0258] The pharmaceutical composition may comprise a therapeutically effective amount or a prophylactically effective amount of an antibody disclosed herein. The therapeutically effective amount of the antibody may vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody are outweighed by therapeutically beneficial effects. It is conventional for those skilled in the art to determine the therapeutically effective amount of an antibody disclosed herein based on these factors. A "prophylactically effective amount" refers to an amount that is effective for achieving the desired prophylactic result at the required dosage and for the required period of time. Typically, a prophylactically effective amount may be lower than a therapeutically effective amount because a prophylactic dose is used in a subject prior to transplantation or in the early stages of transplant rejection.

[0259] As used herein, the term "treatment" refers to the administration of a therapeutic agent, such as a composition comprising any of the antibodies disclosed herein, internally or externally to a subject or patient having one or more disease symptoms or suspected of having a disease, wherein the agent has therapeutic activity for the disease. "Treatment" refers to both therapeutic treatment and / or prophylactic treatment. Therapeutic treatment includes, for example, methods of alleviating or reducing the severity of a condition or eliminating the condition, and includes alleviating or reducing the severity of one or more symptoms of the condition. If treatment is administered prior to the clinical manifestation of a condition, then the treatment is considered prophylactic. Alleviation or reduction of disease symptoms can be evaluated by any clinical measure typically used by a physician or other skilled person to assess the severity or progression of that symptom. The term further refers to the delay in the development of one or more disease symptoms and / or the reduction in the severity of one or more disease symptoms. The term further includes improving existing uncontrolled or unwanted disease symptoms, preventing additional disease symptoms, and improving or preventing the root cause of such disease symptoms. Thus, the term implies that a beneficial result has been conferred on the subject.

[0260] With respect to the treatment of transplant rejection, treatment can refer to alleviating, reducing, or delaying the rejection of a transplanted cell, tissue, or organ or one or more symptoms associated with rejection. Treatment can also result in prolonging graft survival, reducing thrombosis, and / or reducing the risk of life-threatening infections, cancer, and other complications such as cardiovascular disease and renal failure. With respect to the treatment of autoimmune diseases, treatment can refer to slowing the body's immune response and controlling the autoimmune reaction. With respect to the treatment of antibody-mediated inflammatory diseases, treatment can refer to alleviating one or more symptoms associated with the autoimmune disease, such as fatigue, myalgia, low-grade fever, inflammation, rash, etc. With respect to treatment with the antibodies disclosed herein, the term can simply mean that the life expectancy and quality of life of an individual who has received a graft or who suffers from an autoimmune or inflammatory disease will be increased, or that one or more of the symptoms associated with transplant rejection or an autoimmune or inflammatory disease will be reduced.

[0261] As used herein, the term "prevention" refers to the prevention or delay of the recurrence or onset of a condition in a subject, or the reduction of one or more symptoms of the condition, due to the administration of the anti-CD154 antibody of the present disclosure. For example, in the context of administering a therapy to a subject, "prevention" refers to inhibiting, reducing, or delaying the development or onset of rejection of that graft or associated thrombosis, or preventing or delaying the recurrence, onset, or development of one or more symptoms associated with transplantation (e.g., solid organ transplantation) or a combination of therapies (e.g., solid organ transplantation and a combination of immunosuppressive agents) in a subject.

[0262] As used herein, the term "administering" an antibody or composition of the disclosure to a subject refers to bringing the antibody or composition into contact with the subject or the subject's cells, tissues, organs, or biological fluids. Such administration can be carried out by one of a variety of methods known to those of skill in the art. For example, an antibody or composition of the disclosure can be administered systemically or locally. In some embodiments, the composition can be administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally, or rectally. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, the administration includes direct administration (including self-administration) and indirect administration, including the act of prescribing.

[0263] As used herein, the term "high stringency conditions" refers to DNA hybridization to a filter-bound DNA in 0.1x sodium chloride / sodium citrate (SSC) at 65°C, followed by one or more washes in 0.1x SSC, 0.1% SDS at 50 - 65°C (Ausubel et al., eds., 1989, Current Protocols in Molecular Biology, Volume I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., N.Y., at p. 2.10.3).

[0264] Anti - CD154 Antibody

[0265] A first aspect of the disclosure provides an isolated anti-CD154 antibody having modified effector function that binds 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, an antibody having one or more reduced effector functions exhibits reduced binding to Fc receptors relative to an antibody having a wild-type IgG1 heavy chain. Optionally, the reduced binding to Fc receptors is 10 to 3200 times weaker than the binding exhibited by a wild-type IgG heavy chain. In some embodiments, the K of the Fc domain of an antibody having reduced effector function with respect to FcγR1A binding D is 0.1 - 100 nM, such as 0.3 - 92 nM. Optionally, the K of the Fc domain of an antibody having one or more reduced effector functions with respect to CD16aF binding D is greater than 2 μM. In some embodiments, the K of the Fc domain of an antibody having one or more reduced effector functions with respect to CD16aV binding DGreater than 0.4 μM. Optionally, the K of the Fc domain of an antibody having one or more reduced effector functions with respect to CD32aH binding D Greater than 0.5 μM. In some embodiments, the K of the Fc domain of an antibody having one or more reduced effector functions with respect to CD32bF binding D Greater than 1 μM.

[0266] 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, a human anti-CD154 antibody is produced by immunizing a non-human transgenic animal whose genome contains human immunoglobulin genes, such as a rodent, such that the transgenic animal produces human antibodies.

[0267] In some embodiments, the anti-CD154 antibody comprises a human or humanized variable region, wherein the variable region comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and wherein the VH is operably linked to a human Fc domain having modified effector functions. 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, wherein the human Fc region comprises a human hinge sequence and the human Fc domain, and wherein the human hinge sequence is 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.

[0268] In some embodiments, the human Fc domain is derived from the IgG1 Fc (or crystallizable fragment) region. Optionally, the human Fc domain is derived from the IgG1 constant region. The human Fc domain may be derived from the IgG2 Fc (or crystallizable fragment) region. In some embodiments, the Fc domain is derived from the IgG2 constant region. The human Fc domain may be derived from the IgG4 Fc (or crystallizable fragment) region. In some embodiments, the Fc domain is derived from the IgG4 constant region. In some embodiments, the human Fc region is derived from the IgG1 Fc (or crystallizable fragment) region. Optionally, the human Fc region is derived from the IgG1 constant region. In some embodiments, the human Fc region is derived from the IgG2 Fc (or crystallizable fragment) region. In some embodiments, the Fc region is derived from the IgG2 constant region. Optionally, the human Fc region is derived from the IgG4 Fc (or crystallizable fragment) region. The human Fc region may be derived from the IgG4 constant region.

[0269] In some embodiments, the Fc domain of the antibodies disclosed herein contains one or more amino acid modifications that modify effector functions, including reducing or eliminating one or more effector functions. In some embodiments, the Fc domain contains 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 contains 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 can contain 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 contains 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 contains 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 contains the amino acid sequence of SEQ ID NO: 3. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 5. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 6. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 7. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 9. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 13. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 15. Optionally, the Fc domain contains the amino acid sequence of SEQ ID NO: 16. In some embodiments, the Fc domain contains the amino acid sequence of SEQ ID NO: 17. Optionally, the Fc domain contains 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 NO: 236 and 237.

[0270] In some embodiments, the Fc region of the antibodies disclosed herein comprises one or more amino acid modifications that modify effector functions, 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 can 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 can 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 NO: 236, 237, and 242.

[0271] In some embodiments, an antibody comprising an Fc domain or Fc region derived from IgG4 comprises an amino acid modification at any one of the positions selected from the group consisting of: S228, L235, L236, G237, E318, and N297 or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). In a preferred embodiment, an antibody comprising an Fc domain or Fc region derived from IgG4 comprises an amino acid modification selected from the group consisting of: S228P, L235A, L235E, L236E, G237A, E318A, and N297Q or a combination thereof. See, for example Figure 9 c.

[0272] In some embodiments, an antibody comprising an Fc domain or Fc region derived from IgG1 comprises an amino acid modification at any one of the positions 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 set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).In preferred embodiments, antibodies comprising an Fc domain or Fc region derived from IgG1 comprise an amino acid modification 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, Q347E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T366V, N390R, N390K, N390D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S400K, F405A, F405I, F405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T411N, T411R, T411Q, T411K, T411D, T411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR236 / 237, InR237 / 238, InV238 / 239, 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 combinations thereof. See, e.g., Figure 9 a.

[0273] In some embodiments, an antibody comprising an Fc domain or Fc region derived from IgG2 comprises an amino acid modification at any one of the positions selected from the group consisting of V234, G237, P238, H268, V309, A330, and P331 or a combination thereof, wherein the numbering of the amino acid residues is according to the EU numbering set forth in Edelman. In preferred embodiments, an antibody comprising an Fc domain or Fc region derived from IgG2 comprises an amino acid modification selected from the group consisting of V234A, G237A, P238S, H268Q, H268A, V309L, A330S, P331S, or a combination thereof. See, e.g., Figure 9 b.

[0274] 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 SEQID NO:61, and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO:62.

[0275] 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 comprises the amino acid sequence of SEQ ID NO:253. In some embodiments, the VH does not comprise the amino acid sequence of SEQ ID NO:233.

[0276] 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.

[0277] In some embodiments, the antibody further comprises a CH1 domain, wherein the CH1 domain is 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 any one of the amino acid sequences of SEQ ID NO: 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 any one of the amino acid sequences of SEQ ID NO: 67, 70 and 73. In some embodiments, the CH1 domain comprises any one of the amino acid sequences of SEQ ID NO: 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.

[0278] In some embodiments, the antibody comprises a linker between the VH and the Fc domain. 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.

[0279] 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 the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. The VH can be operably linked to the amino acid sequence of any one 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 the amino acid sequence of SEQ ID NO: 3. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 4. The VH can be operably linked to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 6. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 7. The VH can be operably linked to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 9. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 12. The VH can be operably linked to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 14. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 15. The VH can be operably linked to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH is operably linked to the 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 NO: 236 and 237. Optionally, the VH is not operably linked to the amino acid sequences of SEQ ID NO: 236, 237, and 242.

[0280] 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 any one of the amino acid sequences 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 any one of the amino acid sequences 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 any one of the amino acid sequences 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 any one of the amino acid sequences 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 any one of the amino acid sequences 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 any one of the amino acid sequences 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 NO: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 NO:234 and 278. In some embodiments, the heavy chain does not comprise the amino acid sequences of 234, 266, 272 and 278. Optionally, the heavy chain does not comprise the amino acid sequences of SEQ ID NO:150, 164, 234 and 278. In some embodiments, the heavy chain does not comprise the amino acid sequences of 150, 164, 234, 266, 272 and 278.

[0281] 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 either SEQ ID NO:195 or SEQ ID NO: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 either SEQ ID NO:195 or SEQ ID NO:196. The light chain may comprise the amino acid sequence of SEQ ID NO:195 or SEQ ID NO: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.

[0282] 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.

[0283] In some embodiments, the binding of the antibody to human CD154 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 reduced level of thrombus formation is observed upon administration of the antibody as compared to the level of thrombus formation after administration of the 5c8 or hu5c8 antibody. Optionally, the antibody does not cause thrombus formation when administered to a subject.

[0284] In some embodiments, when administered to a subject, the antibody has one or more of the following effects: (a) a reduced risk of thrombosis or thromboembolic events compared to the hu5c8 antibody; (b) reduced activation of platelets expressing CD154; (c) inhibition of CD154 shedding; and (d) alteration in the 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 subjects to whom 5c8 or hu5c8 antibody has been administered. In some embodiments, administration of the antibody results in 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 an alteration in the expression or activity of downstream targets of CD154-CD40 signaling.

[0285] In some embodiments, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of any one of SEQ ID NO: 1, 2, 10, 11, 231, and 236. Optionally, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 2. Optionally, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 11. Optionally, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 231. In some embodiments, the human Fc domain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 236. In some embodiments, the antibody does not comprise an amino acid sequence consisting of any one of SEQ ID NO: 1, 2, 10, 11, 231, and 236. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 2. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 11. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 231. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 236.

[0286] Optionally, the human Fc region with modified effector function does not contain an 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 modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 19. Optionally, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 20. Optionally, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 39. Optionally, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the human Fc region with modified effector function does not contain an amino acid sequence consisting of SEQ ID NO: 235. Optionally, the antibody does not contain an 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 contain an amino acid sequence consisting of SEQ ID NO: 19. Optionally, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 20. Optionally, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 39. Optionally, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the antibody does not contain an amino acid sequence consisting of SEQ ID NO: 235.

[0287] In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of any one of SEQ ID NO: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278. Optionally, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 119. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 120. Optionally, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 133. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 134. Optionally, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 147. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 148. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 150. Optionally, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 161. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 162. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 164. Optionally, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 230. In some embodiments, the heavy chain having modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 234. Optionally, the heavy chain having modified effector function does not comprise an 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 NO: 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 234, and 278. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 119. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 120. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 133. In some embodiments, the antibody does not comprise an 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 modified 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 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 NO: 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.

[0288] Characteristics of the Anti - CD154 Antibody

[0289] Binding affinity of the anti-CD154 antibody for CD154

[0290] Binding affinity (K D ) and dissociation rate (k off ) of the anti-CD154 antibody for CD154 can be determined by methods known in the art. The binding affinity can be measured by ELISA, RIA, flow cytometry, or surface plasmon resonance (SPR) (e.g., using system). The dissociation rate can be measured by SPR. Optionally, the binding affinity and dissociation rate are measured by SPR. In some embodiments, the binding affinity and dissociation rate are measured using . One skilled in the art can determine whether an antibody disclosed herein has a K D substantially the same as that of another anti-CD154 antibody by using methods known in the art. Such methods for determining K D and k off can be used during the initial screening phase and during subsequent optimization phases. In some embodiments, the antibody has a K D for CD154 of less than 50 pM. Optionally, the antibody has a K for CD154 of less than 25 pM.D In some embodiments, the antibody has a K for CD154 of 5 - 25 pM D Optionally, the antibody has a K for CD154 of 9.5 - 23 pM D 。

[0291] CD154 activity is inhibited by anti - CD154 antibody

[0292] Anti - CD154 antibodies that inhibit CD154 binding to CD40 can be identified by using any of a number of assays, such as competitive binding assays, FACS analysis, B - cell activation assays, B - cell proliferation assays, T - activation assays, T - proliferation assays. See, e.g., 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 the up - regulation of CD154 - specific downstream target genes (e.g., CD23, CD44H, CD54, TRAF - 3, and NFκB). In some embodiments, the anti - CD154 antibody has an IC of no greater than 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 1 nM 50 。

[0293] Effector function - platelet assay

[0294] The effector function of the anti-CD154 antibody can be identified by using any of a number of assays, such as in vitro platelet activation and / or aggregation assays. See, e.g., U.S. Patent No. 9,765,150; Langer et al., Thromb Haemost. Jun, 93(6):1137-46(2005); McKenzie, S.E. 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 platelet activation markers P-selectin (CD62P) and PAC-1 (activated GPIIb / IIIa). Platelet aggregation analysis can be performed by using a small cell impedance device and quantifying the area under the curve as a measure of the platelet aggregation impedance curve.

[0295] Nucleic Acids, Vectors, Host Cells, and Recombinant Methods for Preparing Antibodies

[0296] Nucleic acid

[0297] A second aspect of the present disclosure provides nucleic acid molecules encoding the anti-CD154 antibodies disclosed herein. In some embodiments, separate nucleic acid molecules encode the heavy and light chains of the anti-CD154 immunoglobulin. In other embodiments, the same nucleic acid molecule encodes both the heavy and light chains of the anti-CD154 immunoglobulin.

[0298] In some embodiments, the nucleic acid molecule comprises a sequence encoding the VL of the anti-CD154 antibody disclosed herein.

[0299] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VL comprising the amino acid sequence of any one of SEQ ID NO: 65 and 66. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VL comprising the amino acid sequence of any one of SEQ ID NO: 65 and 66, or a portion thereof. In some embodiments, the nucleic acid encodes the amino acid sequence of one, two, or all three light chain CDRs of the antibody. In some embodiments, the portion encodes the contiguous region of CDR1-CDR3 from the light chain of the anti-CD154 antibody.

[0300] In some embodiments, the nucleic acid molecule encodes a VL that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VL amino acid sequence of SEQ ID NO: 65 or 66. Optionally, the nucleic acid molecule encodes a VL that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the VL amino acid sequence of SEQ ID NO: 65 or 66. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of the VL region of SEQ ID NO: 65 or 66.

[0301] In some embodiments, the nucleic acid molecule comprises a sequence encoding a VH of an anti-CD154 antibody disclosed herein.

[0302] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VH comprising the amino acid sequence of any one of SEQ ID NOs: 63, 64, 252 and 253. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VH comprising the amino acid sequence of any one of SEQ ID NOs: 63, 64, 252 and 253, or a portion thereof. In some embodiments, the nucleic acid encodes the amino acid sequence of one, two or all three heavy chain CDRs of the antibody. In some embodiments, the portion encodes an adjacent region of CDR1-CDR3 from the heavy chain of an anti-CD154 antibody.

[0303] In some embodiments, the nucleic acid molecule encodes a VH that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VH amino acid sequence of SEQ ID NO: 63, 64, 252 or 253. Optionally, the nucleic acid molecule encodes a VH that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the VH amino acid sequence of SEQ ID NO: 63, 64, 252 or 253. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of the VH region of SEQ ID NO: 63, 64, 252 or 253.

[0304] In some embodiments, the nucleic acid molecule comprises a sequence encoding the Fc domain of an anti-CD154 antibody disclosed herein.

[0305] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an Fc domain comprising an amino acid sequence of one of SEQ ID NOs: 3-9, 12-18, and 236-241. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an Fc domain comprising an amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 236-241, or a portion thereof.

[0306] In some embodiments, the nucleic acid molecule encodes an Fc domain comprising an amino acid sequence that is at least 70%, 75%, 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, and 238-241. Optionally, the nucleic acid molecule encodes an Fc domain comprising an amino acid sequence that is at least 70%, 75%, 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, and 238-241. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding an amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 238-241 or to the complementary sequence of a nucleic acid of the Fc region portion of a heavy chain nucleotide sequence comprising any one of SEQ ID NOs: 177-179, 192-194, 289, 291-297, 316, and 318-324. In some embodiments, the nucleic acid molecule encodes an Fc domain comprising an amino acid sequence that is at least 70%, 75%, 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, and 236-241. Optionally, the nucleic acid molecule encodes an Fc domain comprising an amino acid sequence that is at least 70%, 75%, 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, and 236-241. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding an amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 236-241 or to the complementary sequence of a nucleic acid of the Fc region portion of a heavy chain nucleotide sequence comprising any one of SEQ ID NOs: 177-179, 192-194, 289-297, and 316-324. In some embodiments, the nucleic acid molecule encoding the Fc region comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the Fc region portion of any one of SEQ ID NOs: 177-179, 192-194, 289-297, and 316-324. Optionally, the nucleic acid molecule encoding the Fc domain comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the Fc region portion of any one of SEQ ID NOs: 177-179, 192-194, 289-297, and 316-324.In some embodiments, the nucleic acid molecule encoding the Fc domain comprises the nucleotide sequence of the Fc region portion of any one of SEQ ID NOs: 177-179, 192-194, 289-297, and 316-324.

[0307] In some embodiments, the nucleic acid molecule comprises a sequence encoding the Fc region of the anti-CD154 antibody disclosed herein.

[0308] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, and 236-251. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, and 236-251, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 243-251. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 243-251, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 242-251. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 242-251, or a portion thereof.

[0309] In some embodiments, the nucleic acid molecule encodes an Fc region that 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, 21-37, 40-56, 238-241, and 243-251. Optionally, the nucleic acid molecule encodes an Fc region that 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, 21-37, 40-56, 238-241, and 243-251. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251. In some embodiments, the nucleic acid molecule encodes an Fc region that 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, 21-37, 40-56, and 236-251. Optionally, the nucleic acid molecule encodes an Fc region that 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, 21-37, 40-56, and 236-251. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, and 236-251. In some embodiments, the nucleic acid molecule encodes an Fc region that 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 nucleic acid molecule encodes an Fc region that 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 243-251.In some embodiments, the nucleic acid molecule encodes an Fc region that 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 nucleic acid molecule encodes an Fc region that 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56, and 242-251.

[0310] In some embodiments, the nucleic acid molecule comprises a sequence encoding the light chain of an anti-CD154 antibody disclosed herein. Optionally, the nucleic acid molecule encoding the light chain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 197 and 198. In some embodiments, the nucleic acid molecule encoding the light chain comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 197 and 198. In some embodiments, the nucleic acid molecule encoding the light chain comprises a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 197 and 198.

[0311] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 195 and 196. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 195 and 196, or a portion thereof.

[0312] In some embodiments, the nucleic acid molecule encodes a light chain amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of either SEQ ID NO: 195 or 196. Optionally, the nucleic acid molecule encodes a light chain amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of either SEQ ID NO: 195 or 196. The nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the light chain amino acid sequence of either SEQ ID NO: 195 or 196 or to the complementary sequence of a nucleic acid comprising the light chain nucleotide sequence of either SEQ ID NO: 197 or 198.

[0313] In some embodiments, the nucleic acid molecule comprises a sequence encoding the heavy chain of the anti-CD154 antibody disclosed herein. Optionally, the nucleic acid molecule encoding the heavy chain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 177-179, 182-184, 187-189 and 192-194. In some embodiments, the nucleic acid molecule encoding the heavy chain comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 177-179, 182-184, 187-189 and 192-194. In some embodiments, the nucleic acid molecule encoding the heavy chain comprises a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO: 177-179, 182-184, 187-189 and 192-194.

[0314] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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 nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, and 266-288, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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 nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277, and 279-288, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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 nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, and 266-288, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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 nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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, or a portion thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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 nucleic acid molecule comprises a nucleotide sequence that encodes a heavy chain comprising an 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, or a portion thereof.

[0315] In some embodiments, the nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the heavy chain amino acid sequence of any one of SEQ ID NOs: 121 - 132, 135 - 146, 149 - 160, 163 - 174 and 266 - 288 or to the complementary sequence of a nucleic acid comprising the heavy chain nucleotide sequence of any one of SEQ ID NOs: 177 - 179, 182 - 184, 187 - 189, 192 - 194 and 289 - 324. In some embodiments, the nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the heavy chain amino acid sequence of any one of SEQ ID NOs: 121 - 132, 135 - 146, 149 - 160, 163 - 174, 266 - 277 and 279 - 288 or to the complementary sequence of a nucleic acid comprising the heavy chain nucleotide sequence of any one of SEQ ID NOs: 177 - 179, 182 - 184, 187 - 189, 192 - 194 and 289 - 324.In some embodiments, the nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the heavy chain 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 or to the complementary sequence of a nucleic acid comprising the heavy chain nucleotide sequence of any one of SEQ ID NOs: 177 - 179, 182 - 184, 187 - 189, 192 - 194, and 289 - 324. In some embodiments, the nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule encodes a heavy chain 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 nucleic acid molecule can comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the heavy chain 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 or to the complementary sequence of a nucleic acid comprising the heavy chain nucleotide sequence of any one of SEQ ID NOs: 177 - 179, 182 - 184, 187 - 189, 192 - 194, and 289 - 324.In some embodiments, the nucleic acid molecule encodes a heavy chain 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 and 273-288. Optionally, the nucleic acid molecule encodes a heavy chain 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 and 273-288. The nucleic acid molecule may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complementary sequence of a nucleic acid encoding the heavy chain amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 267-271 and 273-288 or to the complementary sequence of a nucleic acid comprising the heavy chain nucleotide sequence of any one of SEQ ID NOs: 177-179, 182-184, 187-189, 192-194 and 289-324. In some embodiments, the nucleic acid molecule does not encode a heavy chain comprising the amino acid sequence of SEQ ID NO: 164 or SEQ ID NO: 150. Optionally, the nucleic acid molecule does not comprise the nucleotide sequence of SEQ ID NO: 188 or SEQ ID NO: 183. In some embodiments, the nucleic acid molecule does not encode a heavy chain comprising the amino acid sequence of SEQ ID NO: 278 or SEQ ID NO: 234. Optionally, the nucleic acid molecule does not comprise the nucleotide sequence of SEQ ID NO: 299 or SEQ ID NO: 308. In some embodiments, the nucleic acid molecule encoding the heavy chain comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 177-179, 192-194, 289-297 and 316-324. Optionally, the nucleic acid molecule encoding the heavy chain comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to any one of SEQ ID NOs: 177-179, 192-194, 289-297 and 316-324. In some embodiments, the nucleic acid molecule encoding the heavy chain comprises the nucleotide sequence of any one of SEQ ID NOs: 177-179, 192-194, 289-297 and 316-324.

[0316] A nucleic acid molecule encoding a heavy or light chain of an anti-CD154 antibody can be isolated from any source that produces such an antibody. In various embodiments, the nucleic acid molecule is isolated from B cells expressing an anti-CD154 antibody isolated from an animal immunized with CD154 or from immortalized cells derived from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. See, e.g., Sambrook J. & Russell D., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000). In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal. The nucleic acid molecule isolated from a non-human, non-transgenic animal can be used, for example, for humanized antibodies that contain one or more amino acid sequences from the human anti-CD154 antibodies disclosed herein.

[0317] vector

[0318] A third aspect of the present disclosure provides a vector that contains the disclosed nucleic acid molecule. In some embodiments, the vector contains a nucleotide sequence encoding the heavy chain of the disclosed anti-CD154 antibody. Optionally, the vector contains a nucleotide sequence encoding the light chain of the disclosed antibody. The vector can contain nucleotide sequences encoding the heavy and light chains of the disclosed anti-CD154 antibody.

[0319] In some embodiments, the anti-CD154 antibody is expressed by inserting the DNA encoding the partial or full-length light chain and / or heavy chain obtained as described above into an expression vector, thereby operably linking the gene to expression control sequences such as transcriptional and translational control sequences. In some embodiments, the DNA encoding the partial light chain and / or heavy chain may comprise VH and / or VL sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The polynucleotide encoding the heavy chain and / or light chain can be ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the polynucleotide. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In some embodiments, both genes are inserted into the same expression vector. In some embodiments, the polynucleotide encoding the VH and / or VL sequences is inserted into an expression vector containing the CH and / or CL nucleotide sequences. Optionally, the polynucleotide encoding the VH sequence is inserted into an expression vector containing the Fc domain nucleotide sequence. In some embodiments, the polynucleotide encoding the VH sequence is inserted into an expression vector containing the Fc region nucleotide sequence. The polynucleotide encoding the heavy chain and / or light chain is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present).

[0320] A convenient vector can be a vector encoding a functionally complete CH or CL immunoglobulin sequence, which has suitable modified restriction sites such that any VH or VL nucleotide sequence can be readily inserted at the 5' of the CH or CL nucleotide sequence and expressed, as described above. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site in front of the C domain, and also at the splice regions present within the CH exons. Optionally, the vector encodes an Fc domain with modified effector functions, having suitable modified restriction sites such that the VH nucleotide sequence can be readily inserted at the 5' of the Fc domain nucleotide sequence and expressed. In some embodiments, the vector contains a sequence encoding a linker at the 5' of the sequence encoding the Fc domain, having suitable modified restriction sites such that the VH nucleotide sequence can be readily inserted at the 5' of the linker sequence and expressed. Optionally, the vector encodes an Fc region with modified effector functions, having suitable modified restriction sites such that the VH nucleotide sequence can be readily inserted at the 5' of the Fc region nucleotide sequence and expressed. In some embodiments, the vector contains a sequence encoding a linker at the 5' of the sequence encoding the Fc region, having suitable modified restriction sites such that the VH nucleotide sequence can be readily inserted at the 5' of the linker polynucleotide sequence and expressed. In some embodiments, the vector contains a sequence encoding the CH1 domain at the 5' of the sequence encoding the Fc region, having suitable modified restriction sites such that the VH nucleotide sequence can be readily inserted at the 5' of the CH1 domain polynucleotide sequence and expressed.

[0321] In some embodiments, the vector contains a polyadenylation and / or transcription termination sequence downstream of the coding region. The recombinant expression vector can also encode a signal peptide, which aids in the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is in-frame linked to the amino terminus of the immunoglobulin chain. Optionally, the signal peptide is an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0322] In addition to the antibody chain encoding sequence, the recombinant expression vector may optionally carry regulatory sequences that control the expression of the antibody chain encoding sequence in a host cell. Those skilled in the art will appreciate that the design of the expression vector (including the choice of regulatory sequences) can depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as those derived from retroviral long terminal repeats (LTRs), cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyomavirus promoters and / or enhancers, and strong mammalian promoters such as native immunoglobulin and actin promoters. For further descriptions of viral regulatory elements and their sequences, see, e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants (including descriptions of promoters and vectors, and transformation of plants) are known in the art. See, e.g., U.S. Patent 6,517,529, which is incorporated herein by reference. Methods for expressing polypeptides in bacterial cells or fungal cells (e.g., yeast cells) are also well known in the art.

[0323] In addition to the antibody chain encoding sequence and regulatory sequences, the recombinant expression vectors disclosed herein may also carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. Optionally, the vector contains a selectable marker gene that facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, which are incorporated herein by reference). For example, the selectable marker gene can confer resistance to a drug (e.g., geneticin (G418), hygromycin, or methotrexate) on the host cells into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-deficient host cells selected / amplified with methotrexate), the neomycin resistance (neo) gene (for G418 selection), and the glutamate synthase gene.

[0324] Host cells and methods for recombinant production of proteins

[0325] Any nucleic acid molecule encoding an anti-CD154 antibody and vectors containing such nucleic acid molecules disclosed herein can be used to transfect suitable mammalian, plant, or yeast host cells or to transform bacterial host cells. Methods for transfecting / transforming host cells with polynucleotides are well known in the art. For example, methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells via viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, each of which is incorporated herein by reference. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0326] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples include, in particular: Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. Plant host cells include, for example, tobacco, Arabidopsis, duckweed, corn, wheat, potato, and the like. Bacterial host cells include Escherichia coli (E. coli) and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris. Selection of a cell line based on specific characteristics (e.g., expression level and glycosylation pattern) is within the skill in the art. When a recombinant expression vector encoding an antibody chain is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or, more preferably, secretion of the antibody into the medium in which the host cell is growing. The antibody can be recovered from the medium or the host cell by using standard protein purification methods.

[0327] Further, expression of the disclosed antibodies from production cell lines can be enhanced by using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. See, e.g., European Patent Nos. 0216846, 0256055, 0323997, and 0338841, each of which is incorporated herein by reference.

[0328] Antibodies expressed by different cell lines or in transgenic animals can have different glycosylation patterns. The antibodies disclosed herein can be expressed by any suitable host cell line and can thus have any glycosylation pattern.

[0329] Pharmaceutical Compositions and Administration

[0330] The fourth aspect of the present disclosure provides a pharmaceutical composition comprising the anti-CD154 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CD154 antibody described herein.

[0331] The antibodies disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody disclosed herein and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are used interchangeably and refer to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Pharmaceutically acceptable carriers are well known in the art. See, for example, Remington's Pharmaceutical Sciences and the United States Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984), which are incorporated herein by reference. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it will be preferable to include an isotonic agent in the composition, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffering agents, which enhance the shelf life or effectiveness of the antibody.The pharmaceutical compositions can be prepared by mixing the antibodies disclosed herein with an acceptable carrier, excipient or stabilizer in the form of, for example, lyophilized powder, slurry, aqueous solution or suspension (see, e.g., 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: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY; each incorporated herein by reference).

[0332] The pharmaceutical composition can be in a variety of forms, such as liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or 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 compositions similar to those used for passive immunization in 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 can be administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection. Preparations for injection can be in unit dosage form, e.g., in ampoules, prefilled syringes or multi-dose containers, with or without added preservatives. The pharmaceutical composition can take the form of, for example, a suspension, solution or emulsion (in an oily or aqueous vehicle) and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be prepared in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0333] Typically, the therapeutic composition must be sterile and stable under the conditions of preparation and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the anti-CD154 antibody in the required amount into a suitable solvent (with one or a combination of the ingredients listed above, as required), followed by filtration sterilization. Dispersions can be prepared by incorporating the anti-CD154 antibody into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and freeze drying, which yield a powder of the anti-CD154 antibody and any additional desired ingredients from their pre-sterile filtered solutions. The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size (in the case of dispersions), and / or by surfactants. Prolonged absorption of the injectable composition can be brought about by including agents that delay absorption (e.g., monostearates and gelatin) in the composition.

[0334] The pharmaceutical composition can be administered by a variety of methods known in the art. In some embodiments, the preferred route / mode of administration is subcutaneous, intramuscular, or intravenous infusion. In some embodiments, the mode of administration is intravitreal. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome.

[0335] In some embodiments, the pharmaceutical composition can be formulated with a carrier that will protect the antibody from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are generally known to those skilled in the art. See, for example Sustained and Controlled Release Drug Delivery Systems J.R. Robinson, editor, Marcel Dekker, Inc., New York, 1978, which is incorporated herein by reference.

[0336] Additional active compounds can also be incorporated into the composition. In certain embodiments, the anti-CD154 antibodies disclosed herein are 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 drugs, antiplatelet drugs, non-steroidal anti-inflammatory drugs (NSAIDs), and anti-allergic drugs. Such combination therapies may require lower doses of the anti-CD154 antibody as well as the co-administered agents, thus avoiding possible toxicities or complications associated with various single therapies.

[0337] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. It can be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term "dosage unit form" refers to physically discrete units suitable as unit doses for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required quantity of the pharmaceutical carrier. The specification for dosage unit forms can be dictated by and directly depends 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 sensitivity in individuals.

[0338] Exemplary, non-limiting ranges for a therapeutically or prophylactically effective amount of the antibodies disclosed herein are from 5 to 50 mg / kg. A therapeutically or prophylactically effective amount of the antibodies disclosed herein can be from about 5 to about 50 mg / kg. In some embodiments, a therapeutically or prophylactically effective amount of the antibodies disclosed herein is from 5 to 30 mg / kg. A therapeutically or prophylactically effective amount of the antibodies disclosed herein can be from about 5 to about 30 mg / kg. Optionally, a therapeutically or prophylactically effective amount of the antibodies disclosed herein 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, a therapeutically or prophylactically effective amount of the antibodies disclosed herein 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. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosing regimen can be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges set forth herein are only exemplary and are not intended to limit the scope or practice of the segmented composition.

[0339] Methods of Therapeutic Use

[0340] The fifth aspect of the present disclosure provides a method for inhibiting CD154 activity. In some embodiments, CD154 activity is inhibited by contacting CD154 with an anti-CD154 antibody disclosed herein. Optionally, CD154 is inhibited by administering to a subject in need thereof an anti-CD154 antibody disclosed herein. In some embodiments, a therapeutically effective amount of the anti-CD154 antibody is administered. Optionally, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein.

[0341] The sixth aspect of the present disclosure provides a method of suppressing an immune response in a subject. In some embodiments, the immune response is suppressed by administering to a subject in need thereof an anti-CD154 antibody disclosed herein. Optionally, a therapeutically effective amount of the anti-CD154 antibody is administered. In some embodiments, the anti-CD154 antibody is administered in a pharmaceutical composition disclosed herein. The immune response can be a humoral response, such as an antibody-mediated response. The immune response can be a cell-mediated response, such as 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 immune response can be a mixed humoral and cell-mediated response. The immune response can be a primary response or a secondary response.

[0342] Any antibody disclosed herein can be used therapeutically. In some embodiments, the anti-CD154 antibody is a human, chimeric, or humanized antibody. Optionally, the subject is human. The subject can be non-human, such as a monkey. Optionally, the anti-CD154 antibody is a human antibody and the subject is human. In some embodiments, the anti-CD154 antibody is a humanized antibody and the subject is human. Alternatively, the subject can be a mammal (e.g., a monkey) that expresses CD154 (with which the anti-CD154 antibody cross-reacts). The antibody can be administered to a non-human mammal (e.g., a cynomolgus monkey) that expresses CD154 (with which the antibody cross-reacts) for veterinary purposes or as an animal model of human transplantation or disease. Such animal models can be useful for evaluating the therapeutic efficacy of the antibodies disclosed herein.

[0343] The antibody can be administered once. Optionally, the antibody can be administered multiple times. The antibody can be administered from three times a day to once every six months or longer. The administration can 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 can also be administered continuously by a micropump. The antibody can be administered by mucosal, buccal, intranasal, inhalable, intravenous, intravitreal, subcutaneous, intramuscular, parenteral, or intratumoral routes. In some embodiments, the anti-CD154 antibody is administered systemically, such as subcutaneously, intravenously, orally, via inhalation, transdermally, or rectally. Optionally, the anti-CD154 antibody is administered locally. In some embodiments, the anti-CD154 antibody is administered intravitreally. The antibody can be administered once, at least twice, or for at least a period of time until the condition is treated, alleviated, or cured. Generally, the antibody will be administered as long as the condition exists. Generally, the antibody will be administered as part of the pharmaceutical composition described above. The dose of the antibody will generally be in the range of 5 to 50 mg / kg. The dose of the antibody can be about 5 to about 50 mg / kg. In some embodiments, the dose of the antibody is 5 to 30 mg / kg. The dose of the antibody can be about 5 to about 30 mg / kg. Optionally, the dose 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 dose 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.

[0344] In some embodiments, an anti-CD154 antibody disclosed herein is administered to a subject expressing an inappropriately high level of CD154. Optionally, the subject has received or will receive a cell, tissue, or organ transplant. The transplant can be an allograft, autograft, or xenograft of a cell, tissue, or organ. The cell can be a modified cell or an ex vivo expanded cell. For example, the cell can be modified by using one or more techniques such as transduction to express cDNA, the CRISPR / Cas9 system, RNAi technology, and retroviral technology. Optionally, the cell is modified to express a chimeric antigen receptor (CAR) on its surface. Examples of cells that can be transplanted include, but are not limited to, stem cells, regulatory T (Treg) cells, CAR-T cells, CAR-B cells, and tumor-infiltrating lymphocytes (TIL).

[0345] In some embodiments, any anti-CD154 antibody disclosed herein can be administered to a subject to prevent transplant rejection in the subject. For example, the anti-CD154 antibody can be administered to prevent acute or chronic humoral rejection of the transplanted cell, tissue, or organ. The rejection can be acute or chronic graft rejection in a graft recipient of an allograft or xenograft. The methods disclosed herein can promote long-term graft survival of the transplanted cell, tissue, or organ. Optionally, the long-term graft survival is at least 6 months post-transplant, at least 1 year post-transplant, or at least 5 years post-transplant.

[0346] The transplant rejection can be associated with transplantation of hematopoietic cells or bone marrow, allotransplantation of islet cells, graft-versus-host disease, or solid organ transplantation selected from the group consisting of: heart transplant, kidney transplant, liver transplant, lung transplant, pancreas transplant, kidney-pancreas transplant, heart-lung transplant, kidney-heart transplant, kidney-heart-pancreas transplant, heart-liver transplant, heart-liver-kidney transplant, heart-lung-kidney transplant, heart-lung-liver transplant, lung-kidney transplant, lung-liver transplant, liver-intestine-pancreas transplant, intestine-pancreas transplant, liver-kidney-intestine-pancreas transplant, and kidney-intestine transplant.

[0347] In some embodiments, any anti-CD154 antibody disclosed herein can be administered to a subject having one or more of an immune-related disease, an atherosclerotic disorder, or a neurodegenerative disorder. The subject can also have or be at risk of having one or more of a stroke, transient ischemic attack (TIA), aneurysm, or dissecting aneurysm.

[0348] Examples of immune-related diseases that can be treated / prevented by the compositions / methods disclosed herein include, but are not limited to: type I diabetes, juvenile diabetes, autoimmune diabetes, autoimmune hemolytic anemia, rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, inflammatory bowel disease, Addison's disease, Crohn's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, Guillain-Barre syndrome, ankylosing spondylitis, primary biliary cirrhosis, lupus nephritis, Goodpasture's disease, polymyositis, dermatomyositis, psoriasis, temporal arteritis, Churg-Strauss syndrome, transverse myelitis, thyroiditis, ulcerative colitis, sarcoidosis, hemolytic anemia, idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorder, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, dysregulated immune responses associated with virus-mediated diseases, cytokine release syndrome (CRS), cytokine storm syndrome, Behcet's disease, diabetic retinopathy (DR), diabetic macular edema (DME), age-related macular degeneration (AMD), and macular edema following retinal vein occlusion (MEfRVO). Immune-related diseases can also include allergic conditions, which include, but are not limited to, allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergy, drug allergy, and food allergy. Optionally, the immune-related disease is an inflammatory condition of an organ. In some embodiments, the organ is selected from the group consisting of: lung, heart, and kidney. Optionally, the organ is the lung. In some embodiments, the immune-related disease is acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, lung tissue inflammation, and chronic obstructive pulmonary disease (COPD). The immune-related disease can be selected from the group consisting of: ARDS, pneumonia, and lung tissue inflammation. Optionally, the immune-related disease is ARDS. In some embodiments, the immune-related disease is a condition associated with cytokine storm in a host, cytokine release syndrome (CRS), or cytokine storm syndrome.

[0349] In some embodiments, the immune-related disease is a dysregulated immune response associated with viral infection. Optionally, the virus is selected from the group consisting of: cytomegalovirus, Epstein-Barr virus, influenza virus, variola virus, orthopoxvirus, coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). In some embodiments, the virus is selected from the group consisting of: SARS-CoV, SARS-CoV-2, and MERS-CoV. The virus can be SARS-CoV-2. In some embodiments, the immune-related disease is selected from the group consisting of: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), cytokine release syndrome (CRS), and cytokine storm syndrome. The immune-related disease can be selected from the group consisting of: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), and influenza. Optionally, the immune-related disease is COVID-19.

[0350] Examples of neurodegenerative disorders that can be treated / prevented by the compositions / methods disclosed herein include, but are not limited to: Alzheimer's disease, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and Parkinson's disease.

[0351] Examples of atherosclerotic disorders that can be treated / prevented by the compositions / methods disclosed herein include, but are not limited to: angina pectoris, myocardial infarction, carotid artery stenosis, transient ischemic attack, and cerebrovascular accident (CVA).

[0352] In some embodiments, the method comprises administering to the mammal a therapeutically effective amount of the anti-CD154 antibody disclosed herein in combination with one or more additional agents. Optionally, the one or more additional agents are selected from the group consisting of: antithrombotic drugs, antiplatelet drugs, and non-steroidal anti-inflammatory drugs (NSAIDs). The anti-CD154 antibody can be administered simultaneously with the one or more additional agents. In some embodiments, the anti-CD154 antibody and the one or more additional agents are administered sequentially. Optionally, the anti-CD154 antibody is administered before the one or more additional agents. The anti-CD154 antibody can be administered after 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.

[0353] Examples of antithrombotic agents 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 ximelagatran dabigatran apixaban edoxaban enoxaparin and fondaparinux Examples of antithrombotic agents include, but are not limited to, those disclosed 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; 5,726,173; 5,753,635; 5,846,970; 5,849,759; 5,889,005; 6,107,280; 6,140,351; 6,150,329; 6,180,627; 6,200,976; 6,242,432; 6,248,770; 6,271,215; 6,280,731; 6,287,794; 6,300,330; 6,300,342; 6,333,338; 6,395,731; 6,417,203; 6,432,955; 6,444,672; 6,451,832; 6,458,793; 6,486,129; 6,500,803; 6,583,173; 6,599,881; 6,723,723; 6,730,672; 6,753,331; 6,774,110; 6,797,710; and 6,924,296 (which are incorporated herein by reference in their entirety for all purposes). Examples of glycoprotein IIb / IIIa receptor antagonists include, but are not limited to: abciximab rivaroxaban apixaban edoxaban idrabiotaparinux, tirofiban and eptifibatide The direct or indirect factor Xa inhibitors include, but are not limited to: apixaban idrabiotaparinux, fondaparinux and rivaroxaban

[0354] Examples of antiplatelet agents 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 ticlopidine prasugrel ticagrelor cangrelor and elinogrel. Non-limiting examples of PDE inhibitors include dipyridamole and cilostazol

[0355] Examples of NSAIDs include, but are not limited to: acetylsalicylic acid, celecoxib diclofenac diflunisal etodolac (LODINE )、ibuprofen indomethacin ketoprofen ketorolac nabumetone naproxen oxaprozin piroxicam salsalate sulindac tolmetin prasugrel ticagrelor and cangrelor

[0356] In some embodiments, the anti-CD154 antibody can be administered in combination with one or more supplementary agents, including but not limited to, immunosuppressive drugs, immunomodulatory drugs, and monoclonal and / or polyclonal antibodies. The anti-CD154 antibody can be administered simultaneously with the one or more supplementary agents. In some embodiments, the anti-CD154 antibody and the one or more supplementary agents are administered sequentially. Optionally, the anti-CD154 antibody is administered before the one or more supplementary agents. The anti-CD154 antibody can be administered after the one or more supplementary agents. In some embodiments, the anti-CD154 antibody is administered in the same composition as the one or more supplementary agents. Optionally, the anti-CD154 antibody and the one or more supplementary agents are administered in separate compositions. Examples of such one or more supplementary agents include but are not limited to: anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD28 antibody, anti-CD52 antibody, anti-C5 antibody, mTOR inhibitors, calcineurin inhibitors, antiviral drugs, and fusion peptides that bind to CD28 and block the function of CD28. Non-limiting examples of fusion peptides that bind to CD28 and block the function of CD28 include abatacept and belatacept For example, the anti-CD52 antibody can be alemtuzumab Optionally, the anti-C5 antibody is eculizumab

[0357] Non-limiting examples of immunosuppressive drugs or immunomodulatory drugs include: cyclosporin A, tacrolimus (FK-506), doxorubicin azathioprine busulfan cyclophosphamide fludarabine, 5-fluorouracil, methotrexate( TREXALL TM )、mycophenolate mofetil imidazole riboside (BREDININ TM )、leflunomide, non-steroidal anti-inflammatory drugs, corticosteroids, rapamycin deoxyspergualin, FTY720, muromonab-CD3 (ORTHOCLONE )、alemtuzumab basiliximab daclizumab eculizumab rituximab bortezomib Cilizumab, antithymocyte globulin leronlimab, siltuximbab sarilumab Tocilizumab Bevacizumab Ranibizumab Aflibercept and inhibitors of Bruton's tyrosine kinase (BTK), including zanubrutinib acalabrutinib and ibrutinib

[0358] Examples of mTOR inhibitors include, but are not limited to: rapamycin everolimus temsirolimus ridaforolimus and deforolimus. Examples of calcium-dependent phosphatase inhibitors include, but are not limited to: cyclosporine tacrolimus (FK506, ) and pimecrolimus

[0359] Examples of anti-allergic drugs include, but are not limited to: antihistamines, decongestants, corticosteroids, mast cell stabilizers, leukotriene inhibitors, epinephrine injections, az astin eye drops az astin nasal sprays beclomethasone( and )、betamethasone brompheniramine budesonide( and PULMICORT )、carbinoxamine cetirizine ciclesonide cyproheptadine, chlorpheniramine clemastine disodium cromoglycate desloratadine desonide diphenhydramine emedastine eye drops epinastine eye drops fexofenadine fluorometholone fluticasone furoate (FLONASE Fluticasone Propionate (FLONASE ALLERGY )、Hydrocortisone Hydroxyzine Ketotifen Levocabastine Eye Drops Levocabastine Oral Lodoxamide Loratadine Loteprednol Methylprednisolone Mometasone (ASMANEX )、Naphazoline and Pheniramine Combination Eye Drops Nedocromil Olopatadine Eye Drops Oxymetazoline Pemirolast Prednisolone PRED )、Prednisone (PREDNISONE )、Tetrahydrozoline and Triamcinolone (NASACORT ALLERGY 24 )。

[0360] Examples of antiviral drugs include, but are not limited to: Ribavirin, Interferon (alfacon-1), Chloroquine, Hydroxychloroquine, EIDD-2801, EIDD-1931, GS-5734, GS-441524, Ivermectin, Favipiravir, Indomethacin, Chlorpromazine, Penciclovir, Nafamostat, Nitazoxanide, and Remdesivir.

[0361] Methods of Inducing Chimerism

[0362] The seventh aspect of the present disclosure provides a method for inducing hematopoietic cell chimerism in a transplant recipient. In some embodiments, the method comprises administering to the recipient an anti-CD154 antibody disclosed herein and transplanting hematopoietic stem cells into the recipient, thereby inducing hematopoietic cell chimerism in the recipient. Optionally, a therapeutically effective amount of the anti-CD154 antibody is administered. In some embodiments, the anti-CD154 antibody is administered among the pharmaceutical compositions disclosed herein. The anti-CD154 antibody may be administered simultaneously with the transplanted hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered sequentially with the transplanted hematopoietic stem cells. Optionally, the anti-CD154 antibody is administered prior to the transplantation of hematopoietic stem cells. The anti-CD154 antibody may be administered after the transplantation of hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered in a single dose. Optionally, the anti-CD154 antibody is administered in multiple doses.

[0363] In some embodiments, the anti-CD154 antibody is administered at a dose of 5-50 mg / kg. The anti-CD154 antibody may be administered at a dose of about 5 to about 50 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of 5-30 mg / kg. The anti-CD154 antibody may be administered at a dose of about 5 to about 30 mg / kg. Optionally, the anti-CD154 antibody is administered at a dose of 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 anti-CD154 antibody is administered at a dose of 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. In some embodiments, the anti-CD154 antibody is administered systemically. The anti-CD154 antibody may be administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally or rectally. Optionally, the anti-CD154 antibody is administered locally.

[0364] In some embodiments, the transplant recipient is conditioned prior to transplantation with stem cells. The anti-CD154 antibody can be administered concurrently with the conditioning step. Optionally, the anti-CD154 antibody is administered sequentially with the conditioning step. In some embodiments, the anti-CD154 antibody is administered prior to the conditioning step. Optionally, the anti-CD154 antibody is administered after the conditioning step.

[0365] Methods for conditioning a recipient include, but are not limited to: total body irradiation, 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 cyclosporine A (CsA), administration of FK-506, administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of an IL-15 receptor inhibitor, administration of rapamycin, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists (which block both IL2 and IL15 signaling), administration of CD34+ hematopoietic stem cells and CD3+ T-cells from a kidney donor (MDR-101 cell therapy), or a combination thereof. Non-limiting examples of one or more T cell depleting antibodies include: anti-CD4 antibody, anti-CD8 antibody, anti-CD45 antibody, anti-CTLA4 antibody, anti-CD20 antibody, and anti-CD33 antibody, or a combination thereof. In some embodiments, the transplant recipient has cancer. In some embodiments, the transplantation is a bone marrow transplantation.

[0366] Methods of Inducing Central Tolerance

[0367] The eighth aspect of the present disclosure provides a method of inducing central tolerance in a transplant recipient. In some embodiments, the method includes administering to the recipient the anti-CD154 antibody disclosed herein, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the transplanted hematopoietic stem cells generate immune cells that are tolerant to the donor tissue, thereby inducing central tolerance in the recipient. Optionally, a therapeutically effective amount of the anti-CD154 antibody is administered. In some embodiments, the anti-CD154 antibody is administered among the pharmaceutical compositions disclosed herein. The anti-CD154 antibody can be administered concurrently 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 after the transplantation of hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered in a single dose. Optionally, the anti-CD154 antibody is administered in multiple doses.

[0368] In some embodiments, the anti-CD154 antibody is administered at a dose of 5 - 50 mg / kg. The anti-CD154 antibody can be administered at a dose of about 5 to about 50 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of 5 - 30 mg / kg. The anti-CD154 antibody can be administered at a dose of about 5 to about 30 mg / kg. Optionally, the anti-CD154 antibody is administered at a dose of 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 anti-CD154 antibody is administered at a dose of 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. In some embodiments, the anti-CD154 antibody is administered systemically. The anti-CD154 antibody can be administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally or rectally. Optionally, the anti-CD154 antibody is administered locally.

[0369] In some embodiments, the transplant recipient is conditioned prior to transplantation with stem cells. The anti-CD154 antibody can be administered concurrently with the conditioning step. Optionally, the anti-CD154 antibody is administered sequentially with the conditioning step. In some embodiments, the anti-CD154 antibody is administered prior to the conditioning step. Optionally, the anti-CD154 antibody is administered after the conditioning step.

[0370] Methods for conditioning a recipient include, but are not limited to: total body irradiation, 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 cyclosporin A (CsA), administration of FK-506, administration of one or more interleukin-2 (IL-2) inhibitors, administration of rapamycin, administration of one or more anti-αβ T cell receptor antibodies, and administration of one or more CD122 antagonists, or combinations thereof. Non-limiting examples of one or more T cell depleting antibodies include: anti-CD4 antibody, anti-CD8 antibody, anti-CD45 antibody, anti-CTLA4 antibody, anti-CD20 antibody and anti-CD33 antibody, or combinations thereof.

[0371] Methods of Preventing Xenograft Rejection

[0372] The ninth aspect of the present disclosure provides a method for preventing xenograft rejection in a transplant recipient. In some embodiments, the method comprises administering to the subject an effective amount of an anti-CD154 antibody disclosed herein. Optionally, a therapeutically effective amount of the anti-CD154 antibody is administered. In some embodiments, the anti-CD154 antibody is administered among the pharmaceutical compositions disclosed herein. The anti-CD154 antibody may be administered concurrently with the xenograft. In some embodiments, the anti-CD154 antibody is administered sequentially with the xenograft. Optionally, the anti-CD154 antibody is administered prior to the xenograft. The anti-CD154 antibody may be administered after the xenograft. In some embodiments, the anti-CD154 antibody is administered in a single dose. Optionally, the anti-CD154 antibody is administered in multiple doses. Optionally, the anti-CD154 antibody is administered in four doses. Optionally, the anti-CD154 antibody is administered in four doses of 30 mg / kg. Optionally, the anti-CD154 antibody is administered in four doses within two weeks. Optionally, the first dose of the anti-CD154 antibody is administered in two administrations, wherein the first half dose is administered prior to graft revascularization, and the second half dose is administered once it is confirmed that the xenograft procedure has been successful.

[0373] The anti-CD154 antibody can be administered at a dose of 5-50 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of about 5 to about 50 mg / kg. The anti-CD154 antibody can be administered at a dose of 5-30 mg / kg. In some embodiments, the anti-CD154 antibody is administered at a dose of about 5 to about 30 mg / kg. Optionally, the anti-CD154 antibody is administered at a dose of 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 anti-CD154 antibody is administered at a dose of 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. In some embodiments, the anti-CD154 antibody is administered systemically. The anti-CD154 antibody can be administered subcutaneously, intravenously, intravitreally, orally, by inhalation, transdermally or rectally. Optionally, the anti-CD154 antibody is administered locally.

[0374] The transplant recipient can be human, and the xenograft can be from a non-human donor. In some embodiments, the non-human donor is selected from the group consisting of: pigs, minipigs and non-human primates. Optionally, the non-human donor is a pig or minipig that has been modified to reduce or eliminate the expression of one or more genes. In some embodiments, the one or more genes whose expression is reduced or eliminated include, but are not limited to: porcine endogenous retrovirus (PERV), α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β1,4-N-acetylgalactosaminyltransferase (β4GalNT2) and class I MHC. Optionally, the one or more genes whose expression is reduced or eliminated include PERV. The PERV can be PERV A, PERV B or PERV C. The expression of all PERV genes can be eliminated in the pig or minipig. The expression of the one or more genes can be reduced or eliminated by using CRISPR / Cas9 gene editing.

[0375] In some embodiments, the non-human donor can be engineered to express one or more human proteins. Examples of the one or more human proteins include, but are not limited to: complement regulatory proteins, human alpha-galactosidase, coagulation regulatory proteins, human anti-inflammatory proteins, and human CTLA-4-Ig or combinations thereof. The one or more human proteins can be expressed in all tissues of the non-human donor. In some embodiments, the one or more human proteins are expressed in a tissue-specific manner in the non-human donor. Examples of the complement regulatory proteins include, but are not limited to: human decay-accelerating factor (CD55), membrane cofactor protein (CD46), and CD59. Examples of the coagulation regulatory proteins include, but are not limited to: thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39, and CD73. Examples of the human anti-inflammatory proteins include, but are not limited to: heme oxygenase 1 (HO-1) and A20.

[0376] The xenograft rejection can be associated with solid organ transplantation selected from the group consisting of: heart transplantation, kidney transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney-pancreas transplantation, heart-lung transplantation, kidney-heart transplantation, kidney-heart-pancreas transplantation, heart-liver transplantation, heart-liver-kidney transplantation, heart-lung-kidney transplantation, heart-lung-liver transplantation, lung-kidney transplantation, lung-liver transplantation, liver-intestine-pancreas transplantation, intestine-pancreas transplantation, liver-kidney-intestine-pancreas transplantation, and kidney-intestine transplantation. Examples

[0377] The following examples are provided for illustrative purposes only and do not limit the scope of the present disclosure or the paragraphs in any way. Indeed, various modifications of the present disclosure will become apparent to those skilled in the art from the foregoing description and fall within the scope of the paragraphs. The following examples and preparations use the following abbreviations: "MSX" represents Methionine Sulphoximine, "RU" represents ratio unit; "FACS" represents fluorescence-activated cell sorting; "MW" represents molecular weight; "His-Tag" represents a C-terminal polyhistidine (6xHis) (SEQ ID NO: 331) tag, which is used for rapid purification with nickel chelating resin and detection with anti-His (C-terminal) antibody; "BSA" represents bovine serum albumin; "EDTA" represents ethylenediaminetetraacetic acid; "DMSO" represents dimethyl sulfoxide; "MOPS" represents 3-(N-morpholino)propanesulfonic acid; "MES" represents 2-(N-morpholino)ethanesulfonic acid; "PBS" represents phosphate-buffered saline; "dPBS" represents Dulbecco's phosphate-buffered saline; "HEMA" represents 2-hydroxyethyl methacrylate; "DMEM" represents Dulbecco's modified Eagle medium; "FBS" represents fetal bovine serum; "NEAA" represents non-essential amino acids; "HEPES" represents N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; and "DMF" represents dimethylformamide.

[0378] Example 1. Generation of a Stable Pool of CHO Cells Expressing Anti - CD154 Antibody

[0379] DNA molecules encoding sequences for TNX01 - TNX05, i.e., anti-CD154 antibodies (SEQ ID NOs: 175 - 178 and 194) with various Fc domains, were codon-optimized for CHO cells (wherein the endogenous EcoR1 and BamH1 were removed) and synthesized by one of two vendors: GeneScript (GS) and ATUM (AT). SEQ ID NO: 228 (5’-GAATTCGGCCGGCCACC) and SEQ ID NO: 229 (3’-TAATGAACGCGTGGATCC) were included as EcoRI and BamH1 restriction sites, respectively, in the final sequences. The DNA molecules were cloned into the pTT109 TM (National Research Council, Canada) plasmid using EcoR1 and BamH1 and stably transfected into CHO55E1 TMCells (National Research Council, Canada). For all five constructs, 100 μg of industrial-grade plasmid in ddH2O was prepared. 50 μM of MSX was used as a selection reagent to generate a stable library. Generation of the stable library with 50 μM MSX was performed in duplicate. The stable library was generated in 6 ultra-deep well plates and fed-batch production was carried out with Feed 4 (FUJIFILM IRVINE ). Then, the stable library was generated in shake flasks at a small scale of 100 ml volume with Feed 12.7 (FUJIFILM IRVINE ). Figure 1 Summarizes the titers and availabilities of stable libraries of five CHO cells expressing anti-CD154 mAb in 100 mL fed-batch production. Table 1 summarizes the production of anti-CD154 mAb (TNX01 - 05; light chain contains SEQ ID NO:196; heavy chains contain SEQ ID NOs:147 - 150 and 165 respectively) starting from the supernatant of stably transfected CHO cells as the starting material. A similar process was used to generate TNX06 - TNX13, i.e., anti-CD154 antibodies with various Fc domains (SEQ ID NO:316 - 319 and 293 - 296). Table 2 summarizes the production of anti-CD154 mAb (TNX06 - 13; light chain contains SEQ ID NO:196; heavy chains contain SEQ ID NO:166, 234, 284, 285, 159, 160, 281 and 282 respectively) starting from the supernatant of stably transfected CHO cells as the starting material.

[0380] Table 1. Antibody production starting from stable CHO cells (100 mL fed-batch production)

[0381]

[0382]

[0383] Table 2. Antibody production starting from stable CHO cells (100 mL fed-batch production)

[0384]

[0385] Example 2. Purification of Anti - CD154 Monoclonal Antibody

[0386] Purify the anti-CD154 monoclonal antibodies (TNX01-TNX13) from Example 1 by Protein A affinity purification using a 4x1 mL HiTrap MabSelect SuRe column. Perform the following steps according to the manufacturer's instructions: (1) Equilibrate / wash with DPBS; (2) Load 4x10 mL of sample at a linear flow rate for binding set at approximately 45 cm / hour (0.3 mL / minute) to obtain a residence time of approximately 3.3 minutes; (3) Elute using 0.1 M citrate at a pH of 3.0; (4) Neutralize using 10% (v / v) 1 M HEPES buffer. Change the buffer of the sample in DPBS using a 5 mL Zeba Spin desalting column (ThermoFisher Scientific, Waltham MA) and perform sterile filtration at 0.22 μm. No precipitation was observed during elution and neutralization. Table 3 below summarizes the antibody characteristics obtained after Protein A purification and desalting, and the yields of thirteen purified anti-CD154 monoclonal antibody samples obtained from the CHO55E1 stable library. Note: TNX04 and TNX05 require further purification using preparative size exclusion chromatography (prep-SEC) to remove aggregates.

[0387] Table 3. Antibody recovery after purification

[0388]

[0389] During each step of the purification process using a gel stained with Sypro Ruby under non-reducing conditions, analyze the samples by SDS PAGE. Once the final product is obtained, also analyze the samples. As shown in Figure 2 , under non-reducing conditions, the mAbs TNX01-TNX05 are estimated to have a molecular weight of 160-170 kDa, except for TNX04, which exists as free heavy chain (HC) and free light chain (LC). In TNX04, the heavy and light chains migrate separately in denaturing SDS-PAGE under non-reducing conditions, given that the cysteines (C220, C226, and C229) that form disulfide bonds are mutated. UPLC-SEC results suggest that non-covalent interactions form intact mAbs under physiological conditions. The lower molecular weight (LMW) bands may be attributed to artifactual fragmentation of the mAbs during SDS-PAGE sample preparation. As shown in Figure 10 , the mAbs TNX06-TNX13 are estimated to have a molecular weight of 160-170 kDa, except for TNX11, which behaves like a half-antibody and presumably forms a non-covalent 2HC-2LC structure.

[0390] The final product was also analyzed by size exclusion ultra performance liquid chromatography (UPLC-SEC) according to the manufacturer's instructions.

[0391] Results: These results demonstrated that a highly pure sample of the anti-CD154 antibody of the present disclosure was obtained.

[0392] Example 3. Use of Determination of the Binding of Anti - CD154 Monoclonal Antibody to CD40L by Surface Plasmon Resonance (SPR) Figure 3

[0393] According to the manufacturer's instructions, use the T200 instrument and the sensor chip CM-5 to measure the binding of the purified anti-CD154 antibody from Example 2 to CD40L by surface plasmon resonance.

[0394] Specifically, commercial CD40L (SINO Cat#LC12AP2301) was injected onto a library of anti-CD154 antibodies (TNX01-05) captured on the surface of Protein A (2,000 Rus; Cat#Z02201 Piscataway, NJ), where PBST pH 7.4 (PBS with 0.05% Tween 20) was used as the running buffer. Protein A (Cat#Z02201 Piscataway, NJ), 2000 Rus was used to prepare the SPR antibody capture surface. The ligand was 1.25 - 2.5 μg / mL mAb (TNX01 - TNX05), which was captured for 10 seconds at a flow rate of 10 μL / minute to generate an approximately 50 RU antibody surface. The analyte was CD40L (SINO 10239-H08E), which was resuspended according to the manufacturer's recommendations. Single cycle kinetics was performed at 4 concentrations (6.0 / 1.5 / 0.38 / nM 3-fold dilutions, flow rate 75 μL / minute, 1800 seconds association, 1800 seconds dissociation. Regeneration was performed with 1x60s 10 mM glycine pH 1.5 at a flow rate of 30 μL / minute.

[0395] Use the T200 evaluation software to process the raw data (n = 3). Figure 4 Representative CD40L sensorgrams for TNX01 - TNX05 were provided and showed a 1:1 binding model. Since CD40L is a trimer, the sensorgrams deviated slightly from ideal binding behavior due to the possibility of affinity binding (multiple binding sites) via the trimeric antigen. All fits should be considered "apparent" K DValues because the sensorgram is deviating from 1:1 binding behavior. CD40L binding is based on a calculated fit to the sensorgram.

[0396] Figure 11 Summarizes data generated from SPR analysis and calculated apparent K D values (affinity; ratio of kd to ka). TNX05 does not bind to the A protein surface as effectively as TNX01 - TNX04. This weaker binding may represent a smaller affinity component in these sensorgrams. The lower surface density indicates that TNX05 molecules are further apart, which may be equivalent to fewer sCD40L molecules engaging multiple antibody paratopes at any one time. TNX01 - TNX05 are comparable in K D terms and are close to the lower limit of affinity measurements for this instrument platform in terms of the measured on-rate.

[0397] For TNX06 - TNX13, the sensorgrams were generated using T200 evaluation software and compared using the sensorgram comparability method. This method is only a statistical comparison of sensorgram shape, does not rely on kinetic analysis, and was developed by GE Healthcare for the Biacore T200 platform. An average 3SD window of variation in the sensorgram scatter was generated for one batch (TNX02) as a reference and then pairwise compared to other variants. Similarity scores were evaluated for each pairwise comparison based on the number of points within the variation window and the distance of points outside the reference window. Using this method, all samples were compared to the TNX02 scatter window collected in triplicate. All tested variants (TNX06 - TNX13) showed approximately 80% similarity to the TNX02 sensorgram in terms of sCD40L binding, with no significant large deviations in terms of on-rate or off-rate, indicating that they all bind in a similar manner. See Figure 4 .

[0398] Results: These results demonstrate that each of the anti - CD154 antibodies of the present disclosure binds to CD40L. TNX01 - TNX04 (purified anti - CD154 antibodies) bind to CD40L with a binding affinity of approximately 10 pM (see Figure 4 ). TNX05 has slightly weaker binding, around 20 pM (see Figure 4 ). TNX06 - 13 are approximately 80% similar to TNX02 in the way they bind to CD40L, with no significant large deviations.

[0399] Example 4. Use of Determination of the Binding of Anti - CD154 Monoclonal Antibody to FcγRIA by Surface Plasmon Resonance (SPR) Figure 5

[0400] Use according to the manufacturer's instructions The T200 instrument and the sensor chip CM-5 were used to measure the binding of the purified anti-CD154 antibody from Example 2 to the high-affinity FcγRIA (CD64) by surface plasmon resonance (SPR).

[0401] Specifically, FcγRIA was injected onto a library of anti-CD154 antibodies immobilized on the surface of Protein A (2,000 Rus; Cat# Z02201 Piscataway, NJ), where PBST pH 7.4 (PBS with 0.05% Tween 20) was used as the running buffer. The ligand was 1.25 - 2.5 μg / mL mAb (TNX01 - TNX05), which was captured for 10 seconds at a flow rate of 10 μL / minute to generate an antibody surface of approximately 250 - 400 RU. The analyte FcγRIA (CD64; National Research Council, Canada) was resuspended in PBST containing 3.4 mM EDTA and 0.05% Tween 20 and used in a concentration range of 300 to 0.14 nM, with an association time of 90 seconds and a dissociation time of 1200 seconds at a flow rate of 100 μL / minute. Regeneration was performed with 1x60s 10 mM glycine pH 1.5 at a flow rate of 30 μL / minute.

[0402] Use The T200 evaluation software was used to process the raw data (n = 3). Figure 6 Representative FcγRIA sensorgrams for TNX01 - TNX05 were provided and showed a 1:1 binding model.

[0403] Figure 12 Summarized the data generated from the SPR analysis and calculated apparent K D values (affinity; the ratio of kd to ka) for TNX01 - 05. Due to the high affinity of the ligand CD64 (TNX02 K D ~30 pM), accurate K D values could be determined for all loss-of-function variants. TNX04 showed an 11-fold loss of activity, followed by TNX05, which had a 300-fold loss. TNX01 and TNX03 both had a 3000-fold loss of activity (fast binding and fast dissociation), which required steady-state endpoint analysis rather than kinetic analysis.

[0404] Example 5. Use ofa-12c summarizes the data generated from SPR analysis of the binding of TNX06-13 to FcγRIA and the calculated apparent K D values (affinity; ratio of kd to ka). For binding to FcgRIa, TNX06 to TNX13 were all successfully assayed. Due to the high affinity of the ligand CD64 (TNX02 K D ~40 pM), accurate K D values could be determined for all loss-of-function variants. TNX06, 07, 11, 12, and 13 showed 6- to 12-fold weaker binding than TNX02. TNX08, 09, and 10 had 2600-, 3800-, and 1800-fold weaker binding, respectively (fast binding and fast dissociation).

[0405] Results: These results demonstrate that, as desired, the anti-CD154 antibodies of the present disclosure exhibit reduced binding to FcγRIA.

[0406] Determination of the Binding of Anti - CD154 Monoclonal Antibody to Low - Affinity FcγR by Surface Plasmon Resonance (SPR) Figure 7 Figure 13a

[0407] Using the T200 instrument and sensor chip CM-5, surface plasmon resonance (SPR) was used to measure the binding of the purified anti-CD154 antibodies from Example 2 to the low-affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) according to the manufacturer's instructions. This low-affinity FcγR antigen panel represents the most common allotypes of different receptors that have been used to evaluate the effector functions of the antibody of interest. See, for example, Geuijen et al., FEBS Open Bio 7 (2017) 1557-1574 (2017); Hanson and Barb, Biochemistry, 54, 2931-2942 (2015); Smith and Clatworthy Nature Reviews Immunol (10): 328-344 (2010); Weiner et al., Nature Reviews Immunol (10): 317-327 (2010).

[0408] Specifically, each antigen in the FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) was injected onto the A protein surface (2,000 Rus; Cat# Z02201 on an anti-CD154 antibody library (TNX01-05) in Piscataway, NJ), where PBST pH 7.4 (PBS with 0.05% Tween 20) was used as the running buffer. The ligand was 1.25 - 2.5 μg / mL mAb (TNX01 - TNX05), which was captured for 10 seconds at a flow rate of 10 μL / minute to generate an antibody surface of approximately 200 - 500 RU (surface-dependent). The analyte was an antigen in the FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF; National Research Council, Canada), which was resuspended in PBST containing 3.4 mM EDTA and 0.05% Tween 20. Single-cycle kinetics were performed at concentrations ranging from 12 μM to 0.062 μM, a flow rate of 25 μL / minute, 15-second association (CD32aH and CD32bF) and 40-second association (CD16aF and CD16aV), and a dissociation time of 120 seconds. Regeneration was performed with 1x60s 10 mM glycine pH 1.5 at a flow rate of 30 μL / minute (SPR data not shown). Thus, TNX01 - TNX05 were assayed by measuring the steady-state affinity of binding to the low-affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) of the National Research Council Canada, Human Health Therapeutics Research Centre.

[0409] Example 6. Anti - CD154 Monoclonal Antibody Binding in Jurkat D1.1 and Jurkat Cells Summarizes data generated from SPR analysis and calculated apparent K D values (affinity; ratio of kd to ka) regarding TNX01 - 05. In particular, TNX02 exhibited expected wild-type binding, while the other variants showed significantly reduced binding. In particular, TNX01 and TNX03 showed little binding activity at the FcγR concentrations tested. TNX05 showed a ~5 - to 20-fold loss of binding to various receptors. TNX04 showed a 20 - to 60-fold loss of binding to CD16aV, CD32aH, CD32bF, and undetectable binding to CD16aF (FcγRIIIaF).

[0410] Figure 8 -13b summarizes data generated from SPR analysis and calculated apparent K DData generated from the values (affinity; ratio of kd to ka). In particular, TNX02 demonstrated expected wild-type binding, while the other variants showed significant loss of function. In particular, TNX11 showed little binding activity to any member of the low affinity FcγR panel at the FcγR concentrations tested. TNX06 and 07 showed a ~2- to 10-fold loss of activity against various low affinity receptors. TNX08 showed a 2- to 17-fold loss of activity against various low affinity receptors. TNX09 and 10 showed undetectable activity against all low affinity receptors except CD16aV (FcγRIIIaV), for which they had a ~12-fold loss. TNX12 showed undetectable activity against all low affinity receptors except CD32bF (FcγRIIbF), for which it had an 8-fold loss. TNX13 showed undetectable activity against both CD16aV (FcγRIIIaV) and CD16aF (FcγRIIIaF), and had 6- and 17-fold losses of binding activity against CD32bF (FcγRIIbF) and CD32aH (FcγRIIaH), respectively.

[0411] Results: These results demonstrate that the anti-CD154 antibodies of the present disclosure do not bind or show reduced binding to various FcγR low affinity receptors.

[0412] Figure 8

[0413] The binding of the purified anti-CD154 antibodies from Example 2 to Jurkat D1.1 cells (CD40L positive) and Jurkat cells (CD40L negative) was evaluated. Yellin, MJ et al., J. Immunol. 147:3389-3395 (1991); Lederman, S et al., J. Exp. Med. 175:1091-1101 (1992); U.S. Patent No. 6,331,433. High throughput FACS cell binding assays were performed using the test antibodies (TNX01-TNX05) and a control antibody (mouse anti-human CD40L from (R&D MAB617)). Using as a human IgG1 isotype negative control antibody. The detection reagents were AF488 F(ab’)2 donkey anti-human IgG, Fc γ fragment specific (Jackson ImmunoResearch, Westgrove, PA), and goat anti-mouse IgG, Fc γ fragment specific (Jackson ImmunoResearch, Westgrove, PA). Briefly, 105 One Jurkat or D1.1 cell was incubated with 0 - 200 nM primary antibody for 2 hours at 4°C, and with the detection secondary antibody for 1 hour at 4°C, followed by viability staining with propidium iodide (1% solution). Flow cytometry acquisition of the signals in the samples was performed on the same day on an LSR FORTESSA TM (BD Biosciences). The cells were washed, and AlexaFluor488 fluorescence was measured using a 488 nm laser as the excitation light source and a 530 / 30 nm band - pass filter. The median fluorescence intensity (MFI) was determined for 5000 live single cells / sample. The FACS cell - binding assay was performed similarly for the test antibodies TNX06 - TNX13.

[0414] As shown in Figure 8 (a), all anti - CD154 antibodies (TNX01 - TNX05) bound to D1.1 cells with high affinity, while the negative control antibodies showed no detectable binding to D1.1 cells. The K D values were calculated by fitting with a Hill slope using specific binding parameters. Contrary to Example 7. Anti - CD154 Monoclonal Antibody Platelet Activation Study (a), Example 8. Anti - CD154 Monoclonal Antibody for the Treatment of Heart Transplant Rejection (b) demonstrated that all anti - CD154 antibodies and the negative control antibodies failed to bind to Jurkat cells that do not express CD40L on their surface. As shown in 14(a), all anti - CD154 antibodies (TNX06 - TNX13) bound to D1.1 cells with high affinity, while the negative control antibodies showed no detectable binding to D1.1 cells. The K D values were calculated by fitting with a Hill slope using specific binding parameters.

[0415] Results: These results demonstrate that the purified anti - CD154 antibodies of the present disclosure bind to D1.1 cells expressing CD40L with high affinity.

[0416] ​

[0417] The ability of the purified anti-CD154 antibody from Example 2 to activate platelets was evaluated. CD40L (CD154) is a strong activator of nuclear factor кB (NF-кB) in platelets, which initiates and enhances platelet activation in response to thrombotic stimuli. Further, the binding of the Fc domain of anti-CD40L mAb (e.g., hu5c8) to the activating FcgRIIa (CD32a) receptor on platelets results in platelet activation and aggregation. Kojok K et al., J Am HeartAssoc. 7:e009636 (2018); Shock et al., Arthritis Res Ther. Sep 3, 17:234 (2015); Xie et al., The Journal of Immunology, 192:4083-4092 (2014).

[0418] Blood samples from healthy human blood donors (18 years or older) were collected in sterile vacuum blood collection tubes (yellow top BD Vacutainer B364606, Canada), gently mixed four times by inversion, and tested within 60 minutes. A portion of the blood was pretreated with anti-CD32a (FcgRIIa) antibody and incubated at room temperature (RT) for 30 minutes. A portion of the untreated blood from each donor was also tested (i.e., not incubated with anti-CD32a antibody). Test conditions included: (1) soluble recombinant human CD40L (sCD40L) alone; (2) sCD40L complexed with human IgG1 anti-CD40L; (3) the direct platelet agonist adenosine-5′-diphosphate sodium salt; along with detection antibodies directed against the platelet phenotypic analysis cell surface markers CD41a and CD61, and the platelet activation markers P-selectin (CD62P) and activated GPIIb / IIIa (PAC1). For platelet activation, IC ratios of HuIgG1 anti-CD40L:sCD40L of 1:1, 1:2, 1:3.5, 1:4, 1:5, and 1:6 were tested. To determine the absolute count of each cell type, 50 μL of 123counteBeads (Invitrogen, NJ) was added to the wells. All conditions were tested in triplicate. Platelets were analyzed for activation by flow cytometry.

[0419] Table 4 below summarizes the final concentrations of the molecules used in the platelet assay.

[0420]

[0421]

[0422] Once stained and fixed, samples were acquired on a Cytoflex flow cytometer using Cytexpert version 1.2 software (Beckman Coulter, MN) and analyzed using FlowJo version 10.1 software (FlowJo LLC, OR). Platelet cells were defined based on forward scatter (FSC; an indicator of size) and side scatter (SSC; an indicator of granularity) profiles. Further, the activated and non-activated states of platelets identified based on gating were recorded. Absolute counts using 123count eBeads were calculated using the following formula: (cells / μL) = (cell count / eBead count) × eBead concentration.

[0423] From the recorded data, all wells from the same sample were binned, averaged, and the standard deviation and % coefficient of variation (CV) were calculated between replicates using LibreOffice TM Calc software. A table including the percentage of each cell population was generated from the resulting Calc file.

[0424] Next, in platelet assays conducted at the optimal molar ratio of each test antibody:sCD40L, the fold induction relative to control was calculated for each treatment group. The fold induction relative to control is the ratio of the median fluorescence intensity (MFI) of each treatment group relative to the MFI of the control blood group for each parameter (CD62P or PAC-1). Anti-CD32a antibodies expected to selectively block 5c8-IgG1 / sCD40L IC-induced platelet activation are not expected to show an effect on platelet activation induced by ADP (a direct platelet agonist). 5c8-IgG1 (positive control) is expected to show platelet activation. The test antibodies (TNX01-TNX13) of the present disclosure are expected to show reduced or no activity in the platelet activation assay.

[0425] Results: These results are expected to demonstrate that, unlike the positive control hu5c8-IgG1, the purified anti-CD154 antibodies of the present disclosure have a reduced ability to activate or aggregate platelets.

[0426] ​

[0427] Heterotopic heart transplantation was performed in cynomolgus monkeys. The heart was transplanted into the abdomen and connected to the descending aorta and vena cava. The anti-CD154 antibody TNX05 (hu5c8-mutated -IgG4 (IgG4 S228P / L235A)) was administered to the monkeys intravenously. Four administrations were given to the monkeys at a dose of 30 mg / kg within the first two weeks. Half of the first dose of the anti-CD154 antibody TNX05 was given before graft revascularization, and the other half was given when the success of the procedure was confirmed. After the first two weeks, the monkeys were administered at a dose of 10 mg / kg once a week for four weeks. Then, the monkeys were administered 20 mg / kg once every four weeks. On the 84th day, the dose was reduced from 20 mg / kg to 15 mg / kg.

[0428] Blood was collected on days 70, 77, and 84 and a complete blood count was performed. All the tested parameters were within the normal range, as seen in Table 5 below for the monkeys at the 30 mg / kg dose. By palpation, the transplanted heart of the monkeys was beating strongly and also maintained contractility, as observed by ultrasound. No signs of rejection of the transplanted heart were detected throughout the 84th day. No signs of thrombosis or thromboembolic events were detected throughout the 84th day.

[0429] Table 5

[0430]

[0431]

[0432] *WBC: white blood cell; LYM: lymphocyte (%: percentage of total cells); MONO: monocyte (%: percentage of total cells); GRAN: granulocyte (%: percentage of total cells); HCT: hematocrit; MCV: mean corpuscular volume; RDW: red blood cell distribution width (a: absolute value and % percentage); HGB: hemoglobin; MCHC: mean corpuscular hemoglobin concentration; MCH: mean corpuscular hemoglobin; RBC: red blood cell count; PLT: platelet count; MPV: mean platelet volume.

[0433] A biopsy was performed on the 91st day. Two days after the biopsy, the telemetry device showed a declining cardiac graft function. Ultrasound confirmed a reduced graft contractility. Abdominal exploration confirmed that the graft was beating weakly. The graft was removed and the monkeys were euthanized.

[0434] The autopsy did not show any evidence of thromboembolism outside the heart graft. Fresh thrombi limited to the ventricular cavity were found in the heart graft, as would be expected after the graft stopped beating and the blood in the heart became static. The lungs had no infarcts, and the brain and intestines were completely normal. Those are the sites where thrombi were mainly observed after administration of prior art antibodies. The graft anastomoses were widely open and free of any clots.

[0435] Results: These results demonstrate that the antibodies of the present invention are capable of preventing transplant rejection while avoiding problems regarding thrombosis caused by prior art antibodies.

[0436] Table 6. Sequences of humanized anti-CD154 antibodies

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

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[0501] Some embodiments of the present invention are as follows:

[0502] 1. An isolated antibody that binds to CD154, comprising a human or humanized variable domain, wherein the variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein the VH is operably linked to a human Fc domain having modified effector functions.

[0503] 2. The antibody of embodiment 1, wherein one or more effector functions are reduced.

[0504] 3. The antibody of embodiment 1 or 2, wherein one or more effector functions are eliminated.

[0505] 4. The antibody of any one of embodiments 1-3, wherein the VH is operably linked to a human Fc region, wherein the human Fc region comprises a human hinge sequence and the human Fc domain, and wherein the human hinge sequence is between the VH and the human Fc domain.

[0506] 5. The antibody of embodiment 4, wherein the hinge comprises the amino acid sequence of any one of SEQ ID NOs: 76-90.

[0507] 6. The antibody of any one of embodiments 1-5, wherein the Fc domain is derived from the IgG4 Fc (or crystallizable fragment) region.

[0508] 7. The antibody of embodiment 6, wherein the Fc domain comprises one or more amino acid modifications that modify effector function.

[0509] 8. The antibody of embodiment 7, wherein the antibody comprises an amino acid modification at any one of the positions selected from the group consisting of: S228, L235, G237, E318, and N297, or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman.

[0510] 9. The antibody of embodiment 8, wherein the antibody comprises an amino acid modification selected from the group consisting of: S228P, F234A, L235A, L235E, G237A, E318A, and N297Q, or a combination thereof.

[0511] 10. The antibody of any one of embodiments 1-5, wherein the Fc domain is derived from the IgG1 Fc (or crystallizable fragment) region and comprises one or more amino acid modifications that modify effector function.

[0512] 11. The antibody of embodiment 10, wherein the antibody comprises an amino acid modification at any one of the positions 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 thereof, wherein the numbering of the amino acid residues is according to the EU index as set forth in Edelman.

[0513] 12. The antibody of embodiment 11, wherein the antibody comprises an amino acid modification 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, Q347E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T366V, N390R, N390K, N390D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S400K, F405A, F405I, F405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T411N, T411R, T411Q, T411K, T411D, T411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR236 / 237, InR237 / 238, InV238 / 239, 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.

[0514] 13. The antibody of any one of embodiments 1-5, wherein the Fc domain is derived from the IgG2 Fc (or crystallizable fragment) region.

[0515] 14. The antibody of embodiment 13, wherein the antibody comprises an amino acid modification at any one of the positions selected from the group consisting of: V234, G237, P238, H268, V309, A330 and P331 or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index as set forth in Edelman.

[0516] 15. The antibody of embodiment 14, wherein 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.

[0517] 16. The antibody of any one of embodiments 1-5, wherein the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3-9, 12-18, and 238-241.

[0518] 17. The antibody of embodiment 4, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21-37, 40-56, and 243-251.

[0519] 18. The antibody of any one of embodiments 1-17,

[0520] wherein the VH comprises

[0521] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 57,

[0522] (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and

[0523] (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 59; and

[0524] wherein the VL comprises

[0525] (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60,

[0526] (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61, and

[0527] (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 62.

[0528] 19. The antibody of any one of embodiments 1-18, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 64, 252, and 253.

[0529] 20. The antibody of any one of embodiments 1-19, wherein the VL comprises the amino acid sequence of SEQ ID NO: 65 or 66.

[0530] 21. The antibody of any one of embodiments 4-20, wherein the antibody further comprises a CH1 domain, and wherein the CH1 domain is operably linked to (a) the C-terminus of the VH, and (b) the N-terminus of the hinge.

[0531] 22. The antibody of embodiment 21, wherein the CH1 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 67, 70, and 73.

[0532] 23. The antibody of any one of embodiments 1-22, wherein the antibody comprises a linker between the VH and the Fc domain.

[0533] 24. The antibody of any one of embodiments 4-22, wherein the antibody comprises a linker between the VH and the hinge.

[0534] 25. The antibody of embodiment 21 or 22, wherein the antibody comprises a linker between the VH and the CH1 domain.

[0535] 26. The antibody of any one of embodiments 23-25, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 199-223 and 327-330.

[0536] 27. The antibody of any one of embodiments 1-26, wherein the VH is operably linked to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, 21-37, 40-56, 238-241, and 243-251.

[0537] 28. The antibody of any one of embodiments 1-27, wherein the heavy chain comprises 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.

[0538] 29. The antibody of any one of embodiments 1-28, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 195 or 196.

[0539] 30. The antibody of any one of embodiments 1-29, wherein the antibody is monoclonal.

[0540] 31. The antibody of any one of embodiments 1-30, wherein the antibody is chimeric.

[0541] 32. The antibody of any one of embodiments 1-31, wherein the antibody is humanized.

[0542] 33. An antibody according to any one of embodiments 1-17, 21-27 and 30, wherein the antibody is human.

[0543] 34. An antibody according to any one of embodiments 1-33, wherein binding of the antibody to human CD154 inhibits the interaction between human CD154 and human CD40.

[0544] 35. An antibody according to any one of embodiments 1-34, wherein 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.

[0545] 36. An antibody according to any one of embodiments 1-35, wherein when administered to a subject, the antibody has one or more of the following effects:

[0546] (a) A reduced risk of thrombosis or thromboembolic events compared to the hu5c8 antibody;

[0547] (b) Reduced activation of platelets expressing CD154;

[0548] (c) Inhibition of CD154 shedding; and

[0549] (d) Alteration of the expression or activity of downstream targets of CD154-CD40 signaling.

[0550] 37. An antibody according to any one of embodiments 1-36, wherein the antibody has a K for CD154 of less than 50 pM, such as 5-25 pM or 9.5-23 pM D .

[0551] 38. An antibody according to any one of embodiments 1-37, wherein 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.

[0552] 39. An isolated nucleic acid molecule encoding the light and heavy chains of an anti-CD154 antibody according to any one of embodiments 1-38.

[0553] 40. An isolated nucleic acid molecule encoding an anti-CD154 antibody comprising an amino acid sequence consisting of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 195, 196, 266-277 and 279-288.

[0554] 41. A first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule encodes a heavy chain comprising an amino acid sequence selected from any one of SEQ ID NO: 121 - 132, 135 - 146, 149, 151 - 160, 163, 165 - 174, 266 - 277, and 279 - 288, and the second isolated nucleic acid molecule encodes a light chain comprising an amino acid sequence of SEQ ID NO: 195 or 196.

[0555] 42. A first isolated nucleic acid molecule and a second isolated nucleic acid molecule, wherein the first isolated nucleic acid molecule and the second isolated nucleic acid molecule encode the heavy chain and the light chain of an anti - CD154 antibody according to any one of embodiments 1 - 38, respectively.

[0556] 43. A vector comprising the isolated nucleic acid molecule according to embodiment 39 or 40.

[0557] 44. A first vector and a second vector, wherein the first vector comprises the first isolated nucleic acid molecule according to embodiment 41 or 42, and the second vector comprises the second isolated nucleic acid molecule according to embodiment 41 or 42.

[0558] 45. A transformed cell comprising the isolated nucleic acid molecule according to embodiment 39 or 40, the first and second isolated nucleic acid molecules according to embodiment 41 or 42, the vector according to embodiment 43, or the first and second vectors according to embodiment 44.

[0559] 46. A pharmaceutical composition comprising an anti - CD154 antibody according to any one of embodiments 1 - 38 and a pharmaceutically acceptable carrier.

[0560] 47. A pharmaceutical composition comprising the isolated nucleic acid molecule according to embodiment 39 or 40, the first and second isolated nucleic acid molecules according to embodiment 41 or 42, the vector according to embodiment 43, or the first and second vectors according to embodiment 44, and a pharmaceutically acceptable excipient.

[0561] 48. A pharmaceutical composition comprising the transformed cell according to embodiment 45 and a pharmaceutically acceptable excipient.

[0562] 49. A method for inhibiting an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of embodiments 1 - 38 or a pharmaceutical composition according to any one of embodiments 46 - 48.

[0563] 50. The method of embodiment 49, wherein the immune response is a humoral response.

[0564] 51. The method of embodiment 50, wherein the immune response is an antibody-mediated response.

[0565] 52. The method of embodiment 49, wherein the immune response is a cell-mediated response.

[0566] 53. The method of embodiment 52, wherein the cell-mediated response is 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.

[0567] 54. The method of embodiment 49, wherein the immune response is a mixed humoral and cell-mediated response.

[0568] 55. The method of embodiment 54, wherein the mixed response is 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.

[0569] 56. The method of any one of embodiments 49-55, wherein the subject is a human.

[0570] 57. The method of any one of embodiments 49-55, wherein the subject is non-human.

[0571] 58. The method of embodiment 57, wherein the subject is a monkey.

[0572] 59. The method of any one of embodiments 49-58, wherein the subject has received or will receive a cell, tissue, or organ transplant.

[0573] 60. The method of embodiment 59, wherein the transplant is an allograft, an autograft, or a xenograft.

[0574] 61. The method of embodiment 59 or 60, wherein the cell is a modified cell or an ex vivo expanded cell.

[0575] 62. The method of embodiment 61, wherein one or more genes in the cell are modified by using one or more techniques selected from the group consisting of transduction to express cDNA, the CRISPR / Cas9 system, RNAi technology, and retroviral technology.

[0576] 63. The method of embodiment 61 or 62, wherein the cell is modified to express a chimeric antigen receptor (CAR) on its surface.

[0577] The method of any one of embodiments 59 - 63, wherein the cells are selected from the group consisting of: stem cells, regulatory T cells, CAR-T cells, CAR-B cells, tumor infiltrating lymphocytes (TIL).

[0578] 65. The method of any one of embodiments 59 - 64, wherein the method comprises treating or preventing transplant rejection in the subject.

[0579] 66. The method of embodiment 65, wherein the transplant rejection is acute or chronic humoral rejection of the transplanted cells, tissues or organs.

[0580] 67. The method of embodiment 65 or 66, wherein the transplant rejection is acute or chronic graft rejection in a graft recipient of an allograft or xenograft.

[0581] 68. The method of embodiment 66 or 67, wherein the method promotes long-term graft survival of the transplanted cells, tissues or organs, wherein the long-term graft survival is selected from the group consisting of:

[0582] (a) at least 6 months after transplantation;

[0583] (b) at least 1 year after transplantation; and

[0584] (c) at least 5 years after transplantation.

[0585] 69. The method of any one of embodiments 65 - 68, wherein the transplant rejection is associated with hematopoietic cell or bone marrow transplantation, allogeneic transplantation of pancreatic islet cells, graft-versus-host disease or solid organ transplantation, and the solid organ transplantation is selected from the group consisting of: heart transplantation, kidney transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney-pancreas transplantation, heart-lung transplantation, kidney-heart transplantation, kidney-heart-pancreas transplantation, heart-liver transplantation, heart-liver-kidney transplantation, heart-lung-kidney transplantation, heart-lung-liver transplantation, lung-kidney transplantation, lung-liver transplantation, liver-intestine-pancreas transplantation, intestine-pancreas transplantation, liver-kidney-intestine-pancreas transplantation and kidney-intestine transplantation.

[0586] 70. The method of any one of embodiments 56 - 58, wherein the subject has an immune-related disease, an atherosclerotic disorder or a neurodegenerative disorder.

[0587] 71. The method of any one of embodiments 56 - 58 and 70, wherein the subject has had or is at risk of having a stroke, transient ischemic attack (TIA), aneurysm or dissecting aneurysm.

[0588] 72. The method of embodiment 70, wherein the immune-related disease is selected from the group consisting of: type ...

Claims

1. An isolated antibody that binds to CD154, comprising a human or humanized variable domain, wherein the variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein the VH is operably linked to a human Fc domain having modified effector functions.

2. The antibody of claim 1, wherein one or more effector functions are reduced.

3. The antibody of claim 1 or 2, wherein one or more effector functions are eliminated.

4. The antibody of any one of claims 1-3, wherein the VH is operably linked to a human Fc region, wherein the human Fc region comprises a human hinge sequence and the human Fc domain, and wherein the human hinge sequence is between the VH and the human Fc domain.

5. The antibody of claim 4, wherein the hinge comprises the amino acid sequence of any one of SEQ ID NO: 76-90.

6. The antibody of any one of claims 1-5, wherein the Fc domain is derived from an IgG4 Fc (or crystallizable fragment) region.

7. The antibody of claim 6, wherein the Fc domain comprises one or more amino acid modifications that modify effector functions.

8. The antibody of claim 7, wherein the antibody comprises an amino acid modification at any one of the positions selected from the group consisting of: S228, L235, G237, E318, and N297, or a combination thereof, wherein the numbering of the amino acid residues is according to the EU index set forth in Edelman.

9. The antibody of claim 8, wherein the antibody comprises an amino acid modification selected from the group consisting of: S228P, F234A, L235A, L235E, G237A, E318A, and N297Q, or a combination thereof.

10. The antibody of any one of claims 1-5, wherein the Fc domain is derived from an IgG1 Fc (or crystallizable fragment) region and comprises one or more amino acid modifications that modify effector functions.

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