Anti-CD154 antibodies and their uses

Abstract

The present disclosure relates to anti-human CD154 antibodies with modified effector functions. The present disclosure also relates to the use of these anti-human CD154 antibodies in treating conditions associated with CD154 activation, such as 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 chimerism in transplant patients.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to 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 entireties.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically 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.

[0005] FIELD OF THE DISCLOSURE

[0006] The present disclosure relates to isolated antibodies with modified (e.g., selectively reduced) effector functions that bind to CD154. 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 suppressing immune responses, such as 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.

[0007] Background of the Disclosure

[0008] CD154 (also known as CD40 ligand, CD40L, gp39, TNF-associated activation protein (TRAP), 5c8 antigen, T-BAM) is a protein expressed primarily on activated CD4+ T cells and is considered 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 superfamily of molecules and is functionally expressed as a homotrimer. However, some of the CD154 units have shortened peptide chains, so that the CD154 trimer can be considered to be 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 is combined with the CD40 on antigen presenting cells (APC), which leads to many effects, depending on the target cell type. The main binding partner of CD154 is CD40, although other binding partners αMβ2 (Mac-1), α5β1 integrin and αIIbβ3 have been described (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 works as a co-stimulatory molecule on 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 on the CD40 on the B cell surface and thereby contributing to cell-cell communication in humoral immune response. CD40 triggering through 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) leads to defects in germinal center formation, class switch recombination, and antibody affinity maturation (Webster EA et al., Arthritis Rheum. 42(6):1291-6(1999)).CD40-CD154 interactions are involved in normal TB cell interactions, including increased 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, AM et al., Journal of Immunol., 155: 3329-3337 (1995); Muramatsu, MK et al., Cell 102: 553 (2000); Xu Y and Song G, J. Biomed Sci. 11(4): 426-38 (2004); Quezada ... SA et al., 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).

[0009] 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)). Supernatant of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infects human lung epithelial Calu-3 cells and induces 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 (large 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 usually tightly regulated in tissues over time. Dysfunctional immune responses can occur with abnormal CD154 expression and play a role in certain tissues and at certain times, thereby contributing to syndromes such as acute respiratory distress syndrome (ARDS), autoimmune diseases, vascular diseases, and promoting cancer.

[0010] The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a role in the production of proinflammatory cytokines and has been linked to 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 linked to 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)).

[0011] 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, AM et al., Journal of Immunol., 155: 3329-3337 (1995)). Humanized anti-human CD154 IgG1 antibodies (hu5c8, rulizumab or ), and was tested in nonhuman primates and humans. The crystal structure of hu5c8 showed that hu5c8 binds exclusively to the CD154 trimer and that 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 allogeneic transplantation (Pierson RN 3rd et al., Transplantation. 68(11):1800-5 (1999); Chang AC et al., Transplant Proc. 31(1-2):95 (1999); Kenyon NS et al., Proc Natl Acad Sci US A. 96(14):8132-7 (1999); Kenyon NS et al., Diabetes. 48(7):1473-81 (1999); Elster EA et al., Transplantation. 72(9):1473-8 (2001); Elster EA et al., Transplant Proc. 33(1-2):675-6 (2001); Cho CS et al., Transplantation. 72(4):587-97 (2001); Pierson RN 3rd et al., Immunol Res. 23(2-3):253-62 (2001); Pfeiffer S et al., J Heart Lung Transplant. 20(2):250 (2001); Xu H et al., Transplant Proc. 33(1-2):223-4 (2001); Xu H, Transplantation. 74(7):940-3 (2002); Crowe JE Jr et al., Am J Transplant. 3(6):680-8 (2003); Ferrant JL et al., International Immunol October 5, 11:1583 (2004); Kawai T et al., Am J Transplant. 4(9):1391-8 (2004); Preston EH et al., Am J Transplant. 5(5):1032-41 (2005); Xu H et al., J Immunol. 170(5):2776-82 (2003); Wu G et al., Xenotransplantation. 12(3):197-208 (2005); Smith RN, Am J Transplant. 6(8):1790-8 (2006); Zhang T et al., Transplantation. 102(3):e90-e100 (2018)).However, clinical trials of hu5c8 in systemic lupus erythematosus (SLE) (Huang W et al., Arthritis Rheum. 46(6):1554-62 (2002); Boumpas DT et al., Arthritis Rheum. 48:719-27 (2003); Grammer AC et al., J Clin Invest. 112:1506-20 (2003)) and transplantation (Kawai T et al., Nat Med. 2000, 6:114 (2000); Koyama I et al., Transplantation. 77(3):460-2 (2004)) were 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 of platelet activation may involve CD40 on platelets and activation by soluble CD154 (Inwald DP et al., Circ Res. 92(9):1041-8 (2003)). Several observations suggest that the mechanism of thrombosis in patients treated with anti-CD154 is mediated by FcγRIIa receptor (FcγRIIA, FCGR2A, CD32A)-dependent platelet activation through immune complexes (particularly high-level complexes) composed of anti-CD154 antibodies and soluble CD154 (Robles-Carrillo L et al., J Immunol. 185(3):1577-83 (2010)). Ablation of Fc binding to Fc receptors by mutating the Fc region to produce aglycosyl hu5c8 (IgG1 N297Q) has been shown to strongly reduce or eliminate such thromboemboli (Shock, A et al., Arthritis Res Ther. 17:234 (2015); and Xie et al., Journal of Immunol. 192(9):4083 (2014)), but also reduced the efficacy of the antibody in inhibiting or preventing transplant rejection in rhesus monkey kidney and pancreatic islet allograft models (Ferrant JL et al., International Immunol (11):1583 (2004)). Complete elimination of the Fc region (e.g., in the PEGylated Fab' antibody fragment of anti-CD154) (Dapirolizumab pegol (DZP), CDP7657, Biogen and UCB) also reduced the risk of thrombotic events but failed to treat systemic lupus erythematosus (SLE) in a Phase IIb clinical trial (Waters J, Biocentury, October 26, (2018)).Kim et al. synthesized the variable domain of an anti-human CD154 dAb by conjugating it to a dAb derived from CTLA4-Ig (Abatacept). ) was fused to a linker called "CT Long Fc" having the amino acid sequence EPKSSDK (SEQ ID NO: 325) to produce an Fc-silent anti-human CD154 single-domain antibody (dAb; BMS-986004; Letolizumab, BMS2h-572-633-CT-L2) (Kim SC et al., Am J Transplant 17(5): 1182-1192 (2017); and U.S. Patent No. 9,765,150). BMS-986004 effectively prevented renal 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)).

[0012] 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 the non-native glycine residue at its amino terminus. However, BMS-986003 induced anti-drug antibodies (ADA) in the treated monkeys. The ADA were directed against the dAb (non-Fc) portion of the molecule, and these antibodies were shown to block the binding of BMS-986003 to CD154, suggesting that the ADA may be neutralizing. In addition, these ADA led to increased clearance of BMS-986003 in several monkeys. In contrast, the chimeric murine 5c8 human IgG1 chimeric antibody has an expected long plasma half-life (U.S. Patent No. 9,765,150).

[0013] Although BMS-986004 showed efficacy in a clinical trial for immune thrombocytopenic purpura (ITP), the pharmacokinetic profile may be suboptimal (NCT02273960; "Study to Evaluate Safety and Efficacy in Adult Subjects With ITP (ITP)"; results reviewed July 1, 2019).

[0014] The mechanism of transplant rejection or autoimmune anti-CD154 treatment is not fully understood. Anti-CD154 therapy appears to be more effective than anti-CD40 therapy, which suggests that the blocking of their interaction is not symmetrical. Monoclonal antibody (mAb) hu5c8 appears to be more effective than different humanized anti-CD154 mAbs, i.e., IDEC-131 (O'Neill NA et al., Transplantation.101(9):2038-2047(2017)). Further, the increase in regulatory T cells (particularly Foxp3+ T cells) associated with anti-CD154 therapy may be related to efficacy (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 monovalent CD40, induced T regulatory cells (CD4+CD25+ T cells) and treated transplant rejection (Masunaga T et al., Transplantation. 80: 1614-1622 (2005)). Those experiments also showed that the titer in CD154 ligation or blockade may be related to efficacy (Masunaga T et al., Transplantation. 80: 1614-1622 (2005)). Anti-CD154 treatment has been shown to be effective in promoting allogeneic transplantation 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.

[0015] Antibodies have a variety of effector functions mediated by Fc, including FcγR binding and complement C1q binding (the first component of complement activation), which are involved in effector functions and complement-dependent cytotoxicity. Modifying complement can lead to an increase or decrease in one or more effector functions. In extreme cases, Fc can be removed, such as in F(ab) constructs, but for therapeutic effects, such constructs typically require other changes to increase half-life, such as modification with polyethylene glycol or PEGylation. Complete elimination of the Fc region can affect the efficacy of anti-CD154 antibodies—the PEGylated Fab' antibody fragment of anti-CD154 (Peg-dapirizumab (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 (sialic acid-free or aglycosyl) lacks FcγR binding but is effective in suppressing humoral immune responses (but not organ rejection) in non-human primates (Tao, MH and Morrison, SL J Immunol. 143: 2595-2601 (1989); Ferrant J.L. et al., International Immunol (11): 1583 (2004)). Therefore, one solution for balancing the efficacy and safety of anti-CD154 antibodies is to engineer anti-CD154 antibodies with modified Fcs that selectively reduce effector function. Previous efforts to modify effector function have focused on substitutions in the IgG hinge / CH2 region, which can affect FcγR and C1q binding. One consideration in designing antibodies is to maintain the CH2 / CH3 region of the Fc domain, which is required for interaction with FcRn (the neonatal Fc receptor, which confers extended serum half-life).

[0016] Among IgG1 antibodies, substitution studies have identified several residues that affect Fc binding to C1q and to various FcγRs (FcγRI / CD64, FcγRIIa / CD32a, FcγRIIb / CD32b, and FcγRIIIa / CD16a and FcγRIIIb / CD16b). For example, the Fc residues at positions 234 and 235 have been shown to be important for Fc-CD64 binding ( SM and Morrison, SL J. Exp. Med. 173: 1483-1491 (1991); Chappel, MS et al., Proc. Natl Acad. Sci. USA, 88, 9036-9040 (1991); Alegre, ML 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, 1 2161-12168 (2001); Armour, KL 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 was shown to be involved in Fc-CD64 binding ( SM and Morrison, SL J. Exp. Med. 173: 1483-1491 (1991)) and Fc-Clq binding (Tao, MH et al., J. Exp. Med. 178, 661-667 (1993); Hezareh, M et al., J. Virol. 75, 12161-12168 (2001); Idusogie, EE et al., J. Immunol. 164: 4178-4184 (2000)). As an example, the affinity for FcγR (FcγI, FcγRIIa, and FcγRIIIa) and C1q has been reduced by the substitution of L234F / L235E / P331S in IgG1 (Oganesyan V et al., Acta Crystallogr D Biol Crystallogr. 64(Pt6):700-704 (2008)). As another example, tepliczumab is a humanized OKT3 antibody with an IgG1 Fc with reduced effector function due to the substitutions (L234A, L235A), which is being developed 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 (termed "silent") IgG1 Fc modification with substitutions C220S, C226S, C229S, and P238S was used to prepare CTL4-Ig (Abatacept, ) in a fusion protein expressing CTLA4. The cysteine ​​replacement removes the disulfide bond (C226S and C229S) between the heavy chain (HC) in the dimer. Using this modified Fc, Kim et al. generated a humanized anti-human CD154 antibody with Fc silence, which is a mutant form ("hu5c8-mod") of hu5c8 containing the same modified, non-Fc binding IgG1 tail as anti-CD154 dAb BMS-986004 (Kim SC et al., Am J Transplant 17(5): 1182-1192(2017)). U.S. Patent No. 9,765,150 also describes an anti-human CD154 "5c8" antibody produced by fusing part or all of the variable domain of "5c8" to 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).

[0017] Among IgG2 antibodies, affinity for FcγRs has been reduced by the substitutions H268Q / 309L / A330S / P331S in a mutant form known as "IgG2m4" (An, Z. et al., MAbs 1:572-579 (2009)). Another engineered Fc includes the substitutions V234A / G237A / P238S / H268A / V309L / A330S / P331S, which abolished affinity for FcgRs and C1q complement proteins (Vafa, O. et al., Methods. 1, 65(1):114-26 (2014)).

[0018] Among IgG4 antibodies, affinity for FcγRs has been reduced by F234A and L235A substitutions. These substitutions have been combined with an S228P substitution in the core hinge (IgG1 and IgG2 have a proline at position 228), which further stabilizes the IgG4 molecule against Fab arm exchange (Silva, JP et al., J Biol Chem. 290(9):5462-5469 (2015)), in a "mutant" form of IgG4 called "IgG4 Pro-AlaAla" (Tao, MH and Morrison, SL J Immunol. 143:2595-2601 (1989); and Alegre, ML et al., J. Immunol. 148:3461-3468 (1992)). Dulaglutide is a fusion protein consisting of a glucagon-like peptide-1 (GLP-1) agonist fused to an IgG4 Fc of the "IgG4 Pro-AlaAla" type. Others have modified IgG4 Fc with substitutions F234A and L235A along with the S228P substitution (Xu, D et al., Cell. Immunol. 200, 16-26 (2000)), or with substitution L235E along 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)).

[0019] Antibody modification is a complex and expensive technology with unpredictable in vivo results (Saeed AF et al., Frontiers in Microbiology, Article 495, March (2017)). Antibody modification strategies (including structural changes or other modifications optimized for one antibody) may affect another antibody in vivo (Yan B et al., The Journal of Biol. Chem. 287 (8): 5891-97 (2012)). The effect of mutations for specific residues (e.g., N297Q) in a specific antibody (e.g., IgG1) may have very different clinical effects than mutations of another class in the same antibody (e.g., N297A in IgG1). In addition, the clinical effects of intramolecular changes within one antibody subtype (e.g., L235 mutations in IgG1) may be completely unpredictable when the same intramolecular changes occur within another antibody subtype (e.g., L235 mutations within IgG4). Intramolecular changes may also lead to different clinical effects, depending on how the various mutations may be paired within the antibody. Species-specific differences increase the unpredictability of the effects of Fc receptor mutations on the efficacy versus toxicity of anti-CD154 antibodies. In the past, anti-CD154 antibodies developed for use in transplantation studies did not cause thrombosis in preclinical rodents and some nonhuman primates, but many human patients experienced thromboembolic complications in clinical trials with the same antibodies (i.e., rulizumab, tocilizumab, and ABI793) (Pinelli DF and Ford ML Immunotherapy 7(4):399-410 (2015)).

[0020] To date, there are no fully human or humanized anti-CD154 antibodies that meet the need for products 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 acceptable levels of side effects, such as thromboembolic side effects. This highlights the need for antibodies and variants thereof that bind to CD154 with high affinity and inhibit the downstream effects of CD154:CD40 binding without toxic side effects such as thrombosis. SUMMARY OF THE INVENTION

[0022] A first aspect of the present disclosure provides an anti-CD154 antibody for separation combined with mammalian CD154. In some embodiments, the anti-CD154 antibody comprises 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 connected to a human Fc domain with a modified effector function. Optionally, one or more effector functions are reduced. Optionally, one or more effector functions are eliminated. In some embodiments, the antibody is divalent. In other embodiments, the anti-CD154 antibody is functionally monovalent.

[0023] Second aspect of the present disclosure provides the nucleic acid molecules of coding anti-CD154 antibody disclosed in this article.In some embodiments, the heavy chain and the light chain of the anti-CD154 immunoglobulin of nucleic acid molecule encoding separation.In other embodiments, the heavy chain and the light chain of the anti-CD154 immunoglobulin of same nucleic acid molecule encoding.

[0024] A third aspect of the present disclosure provides a vector comprising a disclosed nucleic acid molecule. In some embodiments, the vector comprises a nucleotide sequence encoding a heavy chain of an anti-CD154 antibody disclosed herein. Optionally, the vector comprises a nucleotide sequence encoding a light chain of an antibody disclosed herein. The vector may comprise a nucleotide sequence encoding a heavy chain of an anti-CD154 antibody disclosed herein and a light chain.

[0025] A fourth aspect of the present disclosure provides a transformed cell comprising the disclosed nucleic acid molecule or the disclosed vector.

[0026] A fifth aspect of the present disclosure provides a pharmaceutical composition comprising an anti-CD154 antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an anti-CD154 antibody disclosed herein.

[0027] A sixth aspect of the present disclosure provides methods 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 an anti-CD154 antibody disclosed herein to a subject in need thereof. In some embodiments, the anti-CD154 antibody is administered in the context of a pharmaceutical composition disclosed herein.

[0028] A seventh aspect of the present disclosure provides a method for suppressing an immune response in a subject. In some embodiments, the immune response is suppressed by administering an anti-CD154 antibody disclosed herein to a subject in need thereof. In some embodiments, the anti-CD154 antibody is administered in the context of a pharmaceutical composition disclosed herein. The immune response may be a humoral response, such as an antibody-mediated response. The immune response may 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 may be a mixed humoral and cell-mediated response. The immune response may be a complement-mediated response. The immune response may be a primary response or a secondary response. The immune response may be a dysfunctional immune response, such as an immune response to a virus that may cause cytokine release syndrome (CRS), cytokine storm, or acute respiratory distress syndrome (ARDS). The immune response may be a tissue-specific immune response, such as inflammation of the lungs, heart, or kidneys, such as lung inflammation.

[0029] An eighth aspect of the present disclosure provides a method for inducing hematopoietic cell chimerism in a transplant recipient. In some embodiments, the method comprises administering an anti-CD154 antibody disclosed herein to the recipient, 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 the context of a pharmaceutical composition disclosed herein.

[0030] A ninth aspect of the present disclosure provides a method for inducing central tolerance in a transplant recipient. In some embodiments, the method comprises administering an anti-CD154 antibody disclosed herein to the recipient, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the transplanted hematopoietic stem cells produce immune cells that tolerate the donor tissue, thereby inducing central tolerance in the recipient. In some embodiments, the anti-CD154 antibody is administered within a pharmaceutical composition disclosed herein.

[0031] The tenth aspect of the present disclosure provides a method for suppressing or preventing xenograft rejection in a subject. In some embodiments, the method comprises administering an effective amount of an anti-CD154 antibody disclosed herein to the subject. In some embodiments, the anti-CD154 antibody is administered in the context of a pharmaceutical composition disclosed herein.

[0032] Specific embodiments of the present disclosure are set forth in the following numbered paragraphs.

[0033] 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 with modified effector function.

[0034] 2. The antibody of paragraph 1, wherein one or more effector functions are reduced.

[0035] 3. The antibody of paragraph 1 or 2, wherein one or more effector functions are abolished.

[0036] 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, wherein the human hinge sequence is between the VH and the human Fc domain.

[0037] 5. The antibody of paragraph 4, wherein the hinge comprises the amino acid sequence of any one of SEQ ID NOs: 76-90.

[0038] 6. The antibody of any one of paragraphs 1-5, wherein the Fc domain is derived from an IgG4 Fc (or crystallizable fragment) region.

[0039] 7. The antibody of paragraph 6, wherein the Fc domain comprises one or more amino acid modifications that modify effector function.

[0040] 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 amino acid residues is according to the EU index as set forth in Edelman.

[0041] 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.

[0042] 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 function.

[0043] 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 amino acid residues is according to the EU index as set forth in Edelman.

[0044] 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, N390 R, N390K, N390D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S400R, S40 0K, F405A, F405I, F405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K409W, T 411N, T411R, T411Q, T411K, T411D, T411E, T411W, ΔE216-E222, K246R / L251E / T260R, InR234 / 235, InV235 / 236, InR 236 / 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.

[0045] 13. The antibody of any one of paragraphs 1-5, wherein the Fc domain is derived from an IgG2 Fc (or crystallizable fragment) region.

[0046] 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 amino acid residues is according to the EU index as set forth in Edelman.

[0047] 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.

[0048] 16. The antibody of any of paragraphs 1-5, wherein the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9, 12-18, and 238-241.

[0049] 17. The antibody according to paragraph 4, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56, and 243-251.

[0050] 18. The antibody of any one of paragraphs 1-17,

[0051] wherein the VH comprises

[0052] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 57,

[0053] (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and

[0054] (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 59; and

[0055] wherein the VL comprises

[0056] (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 60,

[0057] (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 61, and

[0058] (c) A light chain CDR3 having the amino acid sequence of SEQ ID NO: 62.

[0059] 19. The antibody of any of paragraphs 1-18, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 64, 252, and 253.

[0060] 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.

[0061] 21. The antibody of any one of paragraphs 4-20, wherein 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.

[0062] 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.

[0063] 23. The antibody of any one of paragraphs 1-22, wherein the antibody comprises a linker between the VH and Fc domains.

[0064] 24. The antibody of any one of paragraphs 4-22, wherein the antibody comprises a linker between the VH and the hinge.

[0065] 25. The antibody of paragraph 21 or 22, wherein the antibody comprises a linker between the VH and CH1 domains.

[0066] 26. The antibody of 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.

[0067] 27. The antibody of 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, 238-241, and 243-251.

[0068] 28. The antibody of 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.

[0069] 29. The antibody of any one of paragraphs 1-28, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 195 or 196.

[0070] 30. The antibody of any of paragraphs 1-29, wherein the antibody is monoclonal.

[0071] 31. The antibody of any of paragraphs 1-30, wherein the antibody is chimeric.

[0072] 32. The antibody of any of paragraphs 1-31, wherein the antibody is humanized.

[0073] 33. The antibody of any of paragraphs 1-17, 21-27 and 30, wherein the antibody is human.

[0074] 34. The antibody of any one of paragraphs 1-33, wherein binding of the antibody to human CD154 inhibits the interaction between human CD154 and human CD40.

[0075] 35. The antibody of 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.

[0076] 36. The antibody of any one of paragraphs 1-35, wherein when administered to a subject, the antibody has one or more of the following effects:

[0077] (a) reduced risk of thrombotic or thromboembolic events compared to hu5c8 antibody;

[0078] (b) reduced activation of CD154-expressing platelets;

[0079] (c) inhibition of CD154 shedding; and

[0080] (d) Alterations in the expression or activity of downstream targets of CD154-CD40 signaling.

[0081] 37. The antibody of any one of paragraphs 1-36, wherein the antibody has a K for CD154 of less than 50 pM, e.g., 5-25 pM or 9.5-23 pM. D .

[0082] 38. The antibody of any one of paragraphs 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.

[0083] 39. An isolated nucleic acid molecule encoding the light and heavy chains of the anti-CD 154 antibody of any of paragraphs 1-38.

[0084] 40. An isolated nucleic acid molecule encoding an anti-CD 154 antibody comprising the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 195, 196, 266-277, and 279-288.

[0085] 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 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, and the second isolated nucleic acid molecule encodes a light chain comprising the amino acid sequence of SEQ ID NO: 195 or 196.

[0086] 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 light chain, respectively, of the anti-CD 154 antibody of any of paragraphs 1-38.

[0087] 43. A vector comprising the isolated nucleic acid molecule according to paragraph 39 or 40.

[0088] 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.

[0089] 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.

[0090] 46. ​​A pharmaceutical composition comprising the anti-CD154 antibody of any one of paragraphs 1-38 and a pharmaceutically acceptable carrier.

[0091] 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 vector according to paragraph 44, and a pharmaceutically acceptable excipient.

[0092] 48. A pharmaceutical composition comprising the transformed cell according to paragraph 45 and a pharmaceutically acceptable excipient.

[0093] 49. A method of suppressing an immune response in a subject comprising administering to the subject a therapeutically effective amount of the antibody of any one of paragraphs 1-38 or the pharmaceutical composition of any one of paragraphs 46-48.

[0094] 50. The method of paragraph 49, wherein the immune response is a humoral response.

[0095] 51. The method of paragraph 50, wherein the immune response is an antibody-mediated response.

[0096] 52. The method of paragraph 49, wherein the immune response is a cell-mediated response.

[0097] 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.

[0098] 54. The method of paragraph 49, wherein the immune response is a mixed humoral and cell-mediated response.

[0099] 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.

[0100] 56. The method of any of paragraphs 49-55, wherein the subject is a human.

[0101] 57. The method of any of paragraphs 49-55, wherein the subject is non-human.

[0102] 58. The method of paragraph 57, wherein the subject is a monkey.

[0103] 59. The method of any of paragraphs 49-58, wherein the subject has received or will receive a cell, tissue or organ transplant.

[0104] 60. The method of paragraph 59, wherein the transplant is an allogeneic transplant, an autologous transplant, or a xenotransplant.

[0105] 61. The method of paragraph 59 or 60, wherein the cell is an engineered cell or an ex vivo expanded cell.

[0106] 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, CRISPR / Cas9 system, RNAi technology and retroviral technology.

[0107] 63. The method of paragraph 61 or 62, wherein the cell is modified to express a chimeric antigen receptor (CAR) on its surface.

[0108] 64. The method of any one of paragraphs 59-63, wherein the cells are selected from the group consisting of stem cells, regulatory T (Treg) cells, CAR-T cells, CAR-B cells, and tumor infiltrating lymphocytes (TILs).

[0109] 65. The method of any of paragraphs 59-64, wherein the method comprises treating or preventing transplant rejection in the subject.

[0110] 66. The method of paragraph 65, wherein the transplant rejection is acute or chronic humoral rejection of the transplanted cells, tissue or organ.

[0111] 67. The method of paragraph 65 or 66, wherein the transplant rejection is acute or chronic transplant rejection in an allogeneic or xenogeneic transplant recipient.

[0112] 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:

[0113] (a) At least 6 months after transplantation;

[0114] (b) at least 1 year after transplantation; and

[0115] (c) At least 5 years after transplantation.

[0116] 69. The method of any of paragraphs 65-68, wherein the transplant rejection is associated with a hematopoietic cell or bone marrow transplant, an allogeneic transplant of pancreatic islet cells, graft-versus-host disease, or a solid organ transplant selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a kidney-pancreas transplant, a heart-lung transplant, a kidney-heart transplant, a kidney-heart-pancreas transplant, a heart-liver transplant, a heart-liver-kidney transplant, a heart-lung-kidney transplant, a heart-lung-liver transplant, a lung-kidney transplant, a lung-liver transplant, a liver-intestine-pancreas transplant, an intestinal-pancreas transplant, a liver-kidney-intestine-pancreas transplant, and a kidney-intestine transplant.

[0117] 70. The method of any of paragraphs 56-58, wherein the subject has an immune-related disease, an atherosclerotic disorder, or a neurodegenerative disorder.

[0118] 71. The method of any of paragraphs 56-58 and 70, wherein the subject has had or is at risk of having a stroke, transient ischemic attack (TIA), aneurysm, or intramural aneurysm.

[0119] 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, The following conditions are listed as potential causes of cataract: inflammatory bowel disease, leukoderma, leukoderma, leukoderma, leukoderma lucidum, leukoderma spondylitis ...

[0120] 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.

[0121] 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, inflammation of lung tissue, and chronic obstructive pulmonary disease (COPD).

[0122] 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 encephalitis (CTE), amyotrophic lateral sclerosis (ALS), and Parkinson's disease.

[0123] 76. The method of paragraph 70, wherein the atherosclerotic condition is selected from the group consisting of angina pectoris, myocardial infarction, carotid artery stenosis, transient ischemic attack, and cerebrovascular accident (CVA).

[0124] 77. The method according to paragraph 70, wherein the immune related disease is an allergic condition.

[0125] 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.

[0126] 79. The method of paragraph 49, wherein the immune response is a primary response or a secondary response.

[0127] 80. The method of any of paragraphs 49-79, wherein the antibody is administered systemically.

[0128] 81. The method of paragraph 80, wherein the anti-CD 154 antibody is administered subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally, or rectally.

[0129] 82. The method of any of paragraphs 49-79, wherein the antibody is administered topically.

[0130] 83. The method of any of paragraphs 49-82, wherein the anti-CD 154 antibody is administered in combination with one or more additional agents selected from the group consisting of antithrombotic drugs, antiplatelet drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), and antiallergic drugs.

[0131] 84. The method of paragraph 83, wherein the anti-CD 154 antibody is administered before, after, or simultaneously with the one or more additional agents.

[0132] 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 coagulation factor Xa inhibitors, and anticoagulants.

[0133] 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

[0134] 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

[0135] 88. 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

[0136] 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.

[0137] 90. The method of paragraph 89, wherein the ADP pathway inhibitor is selected from the group consisting of: clopidogrel Ticlopidine Prasugrel Tigatran Cangrelo and elinogrel.

[0138] 91. The method of paragraph 89, wherein the PDE inhibitor is selected from the group consisting of: dipyridamole and cilostazol

[0139] 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

[0140] 93. The method of any of paragraphs 49-92, wherein the anti-CD154 antibody is administered in combination with one or more supplemental agents 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.

[0141] 94. The method of paragraph 93, wherein the anti-CD 154 antibody is administered before, after, or simultaneously with the one or more supplemental agents.

[0142] 95. The method of paragraph 93 or 94, wherein the immunosuppressive or immunomodulatory drug is selected from the group consisting of cyclosporine A, tacrolimus (FK-506), doxorubicin Azathioprine Busulfan Cyclophosphamide Fludarabine, 5-fluorouracil, methotrexate Mycophenolate mofetil Mizoribine (BREDININ TM ), leflunomide, nonsteroidal anti-inflammatory drugs, corticosteroids, rapamycin Deoxyspergualin, FTY720, Muromonab-CD3 (ORTHOCLONE ), alemtuzumab Basiliximab Daclizumab Eculizumab Rituximab Bortezomib Siliguzumab, antithymocyte globulin leronlimab, siltuximbab sarilumab Tocilizumab Bevacizumab Ranibizumab Aflibercept and Bruton's tyrosine kinase (BTK) inhibitors, including zanubrutinib acalabrutinib and ibrutinib

[0143] 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.

[0144] 97. The method of paragraph 93 or 94, wherein the calcineurin inhibitor is selected from the group consisting of: cyclosporine Tacrolimus (FK506, ) and pimecrolimus

[0145] 98. The method of paragraph 93 or 94, wherein the antiviral drug 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, nitazoxanil, and remdesivir.

[0146] 99. A method of inducing hematopoietic cell chimerism in a transplant recipient, the method comprising administering one or more doses of an anti-CD154 antibody to the recipient 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.

[0147] 100. The method of paragraph 99, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

[0148] 101. The method of paragraph 99 or 100, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0149] 102. The method of any of paragraphs 99-101, wherein the anti-CD 154 antibody is administered subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally, or rectally.

[0150] 103. The method of any of paragraphs 99-102, wherein the method further comprises the step of conditioning the transplant recipient prior to transplantation with stem cells.

[0151] 104. The method of paragraph 103, wherein the anti-CD 154 antibody is administered before, after, or simultaneously with the conditioning step.

[0152] 105. The method of paragraph 103 or 104, wherein the conditioning step is selected from the group consisting of: whole 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 tacrolimus (FK-506), administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of IL-15 receptor inhibitors, 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 derived from kidney donors (MDR-101 cell therapy), or a combination thereof.

[0153] 106. The method of paragraph 105, wherein the one or more T cell depleting antibodies are selected from the group consisting of: anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies and anti-CD33 antibodies, or a combination thereof.

[0154] 107. The method of any of paragraphs 99-106, wherein the transplant recipient has cancer.

[0155] 108. The method of any of paragraphs 99-107, wherein the transplant is a bone marrow transplant.

[0156] 109. A method of inducing central tolerance in a transplant recipient, the method comprising administering one or more doses of an anti-CD154 antibody to the recipient, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the hematopoietic stem cells produce immune cells that are tolerant to the donor tissue, thereby inducing central tolerance in the recipient, and wherein the anti-CD154 antibody is an anti-CD154 antibody according to any one of paragraphs 1-38.

[0157] 110. The method of paragraph 109, wherein the anti-CD154 antibody is administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

[0158] 111. The method of paragraph 109 or 110, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0159] 112. The method of any of paragraphs 109-111, wherein the anti-CD 154 antibody is administered subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally, or rectally.

[0160] 113. The method of any of paragraphs 109-112, wherein the method further comprises one or more treatments for conditioning the recipient for hematopoietic stem cell transplantation.

[0161] 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: whole 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.

[0162] 115. The method of paragraph 114, wherein the one or more T cell depleting antibodies are selected from the group consisting of: anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies and anti-CD33 antibodies, or a combination thereof.

[0163] 116. A method of inhibiting xenograft rejection in a subject, comprising administering to the subject an effective amount of an anti-CD154 antibody, wherein the anti-CD154 antibody is an anti-CD154 antibody according to any one of paragraphs 1-38.

[0164] 117. The method of paragraph 116, wherein the anti-CD154 antibody is administered at a dose of 5-50 mg / kg.

[0165] 118. The method of paragraph 116 or 117, wherein the xenograft is from a non-human donor selected from the group consisting of: pig, minipig, and non-human primate.

[0166] 119. The method of paragraph 118, wherein the non-human donor is a pig or minipig that has been engineered 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-acetylgalactosamine transferase (β4GalNT2), and class I MHC.

[0167] 120. The method of paragraph 119, wherein the PERV is PERV A, PERV B, or PERV C.

[0168] 121. The method of paragraph 119 or 120, wherein expression of all PERV genes has been eliminated in the pig or minipig.

[0169] 122. The method of any of paragraphs 119-121, wherein expression of the one or more genes is reduced or eliminated by using CRISPR / Cas9 gene editing.

[0170] 123. The method of any of paragraphs 118-122, wherein the non-human donor has been engineered 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.

[0171] 124. The method of paragraph 123, wherein the one or more human proteins are expressed in all tissues of the non-human donor.

[0172] 125. The method of paragraph 123, wherein the one or more human proteins are expressed in a tissue-specific manner in the non-human donor.

[0173] 126. The method of any 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.

[0174] 127. The method of any 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.

[0175] 128. The method of any of paragraphs 123-125, wherein the human anti-inflammatory protein is selected from the group consisting of heme oxygenase 1 (HO-1) and A20.

[0176] 129. The method of any 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, intestinal-pancreas transplant, liver-kidney-intestine-pancreas transplant, and kidney-intestine transplant.

[0177] 130. The method of paragraph 49, wherein the immune response is complement mediated.

[0178] 131. The method of any 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 Presented are the titers and viabilities (%) of five stable pools (TNX01-TNX05) of CHO cells expressing anti-CD154 mAb in 100 mL fed-batch production.

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

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

[0183] Figure 4 (a) and 4(b) provide the Figure 3Kinetic results for a 1:1 binding model of the sensorgrams of For each of the TNX01-TNX05 anti-CD154 antibodies, the mean values ​​of the binding affinity KD (M) for sCD40L (ie, sCD154) are shown together with the standard deviation (n=3).

[0184] Figure 5 (a)-5(e) provide representative CD154 sensorgrams for TNX01-TNX05 binding to FcγRIA (CD64), respectively. Affinity measurements of purified anti-CD154 antibodies were performed by surface plasmon resonance (SPR) using T200 instrument and sensor chip CM-5. By using Biacore T200 evaluation software, the change in refractive index was plotted in the sensorgram as a response expressed 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 obtained Ka (1 / Ms), Kd (1 / s) and KD (M) values.

[0185] Figure 6 (a) and 6(b) provide the Figure 5 Figure 1 shows the kinetic results of a 1:1 binding model for the sensorgrams of . The mean values ​​of the binding affinity KD (M) for FcγRIA (CD64) are shown together with the standard deviation (n=3). Lower KD values ​​indicate tighter binding between the antibody and FcγRIA, with TNX02 binding to FcγRIA the tightest, followed by (in order) TNX04, TNX05, TNX01, and TNX03.

[0186] Figure 7 (a) and 7(b) provide the Figure 3 Kinetic results for a 1:1 binding model based on sensorgrams of . Mean values ​​of KD (M) for the binding affinities of TNX02, TNX04, and TNX05 to a panel of low-affinity FcγRs (CD16aF, CD16aV, CD32aH, CD32bF) are shown along with standard deviations (n=3). KD values ​​for TNX01 and TNX03 could not be determined as these variants showed negligible binding activity. Lower KD values ​​indicate 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) Each anti-CD154 antibody (TNX01-TNX05), positive control antibody (mouse anti-human CD40L or CD140) and negative control antibody are provided Binding characteristics (Bmax (maximum number of binding sites), h (Hill slope), and apparent Kd (ligand concentration that binds half of the receptor sites at equilibrium)) for D1.1 cells (CD154 positive) as analyzed by using a high-throughput FACS cell binding assay with various antibodies.

[0188] Figure 9 (a), 9(b) and 9(c) provide methods for modifying (e.g., reducing) FcR effector function for IgG1 ( Figure 9 (a))、IgG2( Figure 9 (b)) and IgG4( Figure 9 (c)) A map of various amino acid modifications in the Fc region.

[0189] Figure 10 SDS-PAGE analysis of each of the anti-CD154 mAbs (TNX06-TNX13) obtained during the protein production and purification process is provided. STD is a protein standard that 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 D; (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 11Surface plasmon resonance (SPR) comparability scores for sCD154 binding by TNX06-13 compared to TNX02 are provided. The scores are expressed as percentage 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) for TNX06-13 binding to FcγRIA (CD64) and TNX02. Figure 12 (b) and 12 (c) show the mean and standard deviation (n=3) of the binding affinity KD (M) for FcγRIA (CD64). Lower KD values ​​indicate tighter binding between the antibody and FcγRIA, whereby TNX02 binds FcγRIA most tightly, followed by (in order) TNX13, TNX07, TNX12, TNX06, TNX10, TNX08, and TNX09.

[0192] Figure 13 (a) provides the average KD values ​​based on sensorgram data obtained for binding of TNX02 and TNX06-13 to low-affinity FcγRs. Figure 13 (b) shows the mean and standard deviation (n=3) of the binding affinity KD (M) for TNX02, TNX06-10, TNX12, and TNX13 to a panel of low-affinity FcγRs (CD16aF, CD16aV, CD32aH, CD32bF). KD values ​​for TNX11 and for TNX09, TNX10, and TNX12 (for some FcγR panel components) could not be determined 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) of each antibody binding to the surface of CD154-positive Jurkat D1.1 cells as a function of concentration (nM). Figure 14 (b) Each anti-CD154 antibody (TNX06-TNX13), positive control antibody (mouse anti-human CD40L or CD154) and negative control antibody are provided Binding characteristics (Bmax (maximum number of binding sites), h (Hill slope), and apparent Kd (ligand concentration that binds half of the receptor sites at equilibrium)) for D1.1 cells (CD154 positive) as analyzed by using a high-throughput FACS cell binding assay with various antibodies. Detailed Description of the Invention

[0195] Definition and general techniques

[0196] Unless defined otherwise in this article, the scientific and technical terms used in this application will have the implication that is commonly understood by those of ordinary skill in the art. Generally speaking, the technical terms used in this article and the technology of these aspects related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art. In the event of a conflict, this specification sheet (including definitions) will be used as the criterion.

[0197] The methods and techniques of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, for example, in Green MR & Sambrook J. Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th ed., Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, 5th ed., Wiley, John & Sons, Inc. (2003). Notebook (JECellis, editor, 1998) Academic Press; Animal CellCulture (RI Freshney, editor, 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell andTissue Culture: Laboratory Procedures (A. Doyle, JBG Griffins, and DG Newell, editors, 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., John Wiley & Sons, Inc., 2003); 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 performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0199] Standard techniques were used for chemical syntheses and chemical analyses.

[0200] Throughout this specification and embodiments, the word "comprise" or its variations will be understood to imply the inclusion of a recited integer or group of integers but not the exclusion of any other integer or group of integers. "Comprise" may be synonymous with "including" or "contains."

[0201] It should be understood that wherever an embodiment is described herein using the language "comprising," additional similar embodiments are also provided that are described as "consisting of" and / or "consisting essentially of. As used herein, "consisting of is a closed term that includes only the specified elements recited, while "consisting essentially of includes the specified elements recited and may include additional, non-essential elements that are not recited.

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

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

[0204] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0205] The articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. As an example, "element" means one element or more than one element. As used herein, the term "about," which modifies the amount of an ingredient, parameter, calculation, or measurement in a composition of the present disclosure or employed in a method of the present disclosure, refers to variations in such numerical quantities that may occur, for example, through typical measurement and liquid handling procedures used to prepare isolated polypeptides or pharmaceutical compositions in the real world; through unintentional errors in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to prepare the composition or perform the method, and so forth, without having a substantial effect on the chemical or physical properties of the composition or method of the present disclosure. Such variations may be within a certain order of magnitude, typically within 10%, more typically within 5%, of a given value or range. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition produced from a particular initial mixture. Whether or not modified by the term "about," these passages include equivalents to the stated quantities. Reference herein to a value or parameter that is "about" includes (and describes) embodiments directed to that value or parameter itself. For example, description of "about X" includes description of "X." Numerical ranges include the numbers defining the range.

[0206] Although the numerical ranges and parameters setting forth the broad scope of this disclosure are approximate, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily arising from the standard deviation found in its respective test measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges contained therein. For example, a recited range of "1 to 10" is to be considered to include any and all subranges between (and including) a minimum of 1 and a maximum of 10; that is, all subranges starting with a minimum of 1 or greater (e.g., 1 to 6.1) and ending with a maximum 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] definition

[0209] The following terms shall be understood to have the following meanings unless otherwise stated:

[0210] As used herein, the term "antibody" or "Ab" refers to an immunoglobulin molecule (e.g., a complete 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, modified antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single-chain antibodies, artificially selected antibodies, antibodies grafted with CDRs, etc.). Further, "antibody" and / or "immunoglobulin" (Ig) refers to a polypeptide comprising at least two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa), which are optionally linked to each other by disulfide bonds. There are two types of light chains: λ and κ. In humans, λ and κ light chains are similar, but only one type is present in each antibody. Heavy chain is classified as μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.Usually, referring to Fundamental Immunology, Chapter 7 (Paul, W., editor, 2nd edition, Raven Press, NY (1989)) (it is merged with its entirety by mentioning). Antibody disclosed in this article can be " functionally bivalent " or " functionally univalent ". In other words, antibody disclosed in this article can have an antigen binding site (univalent) or two antigen binding sites (bivalent) typically connected by a disulfide bond. In some embodiments, described anti-CD154 antibody is IgG1 antibody. Described anti-CD154 antibody can be IgG2 antibody. Optionally, described anti-CD154 antibody is IgG4 antibody. In some embodiments, described anti-CD154 antibody does not comprise last lysine residue at its C-terminal to improve antibody stability. See, for example, 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 to express each heavy and light chain containing a leader sequence (also known as a signal sequence) at its N-terminus, which is used to transport the newly synthesized chain into the endoplasmic reticulum. During post-translational processing, the leader sequence is removed and is therefore not present in the final chain or mature antibody.

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

[0212] As used in this article, the term "chimeric" antibody refers to an antibody and its antigen-binding fragment comprising a part from two or more different species (e.g., mouse and people). Chimeric antibodies can be produced with mouse variable regions with desired specificity spliced ​​onto human constant domain gene segments (e.g., U.S. Patent number 4,816,567). In this way, non-human antibodies can be modified so that they are more suitable for human clinical applications. The term "chimeric" can refer to non-natural sequences, which have been operated to have one or more variations relative to native sequences. Chimeric antibodies used in this article mean antibodies comprising 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 the antibody produced in humans. In some embodiments, a humanized antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody) comprising a minimal sequence derived from a non-human immunoglobulin. Optionally, a humanized antibody is derived from a human immunoglobulin (i.e., a recipient antibody), wherein the residues from one or more complementarity determining regions (CDRs) of the recipient antibody are replaced by residues from one or more CDRs of an antibody (donor antibody) from a non-human species with desired specificity, affinity, and ability. 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 people because they are not as quickly cleared from circulation as mouse antibodies and typically do not cause adverse immune responses. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.

[0214] In some embodiments, the chimeric antibody is a humanized antibody, for example, a humanized anti-CD154 antibody. Humanized anti-CD154 antibody can comprise the amino acid sequence of one or more human framework regions and / or the amino acid sequence of at least a portion from human constant region, and further comprises the sequence derived from non-human antibodies, for example, non-human (for example, mouse) CDR sequences. In some embodiments, the humanized antibody comprises a human constant region. Optionally, all 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 region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by the following literature: Morrison, SL, Science, 229: 1202-1207 (1985); Oi et al., BioTechniques, 4: 214 (1986); Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Staelens et al., 2006 Mol Immunol 43:1243-1257; and U.S. Patent No. 5,225,539; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 5,859,205; and U.S. Patent No. 6,407,213 (each of which is incorporated herein by reference). Those methods include isolating, manipulating and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable region from at least one of the heavy chain or light chain. The source of such nucleic acids is well known to those skilled in the art and can, for example, be obtained from hybridomas (as described above) that produce antibodies against a predetermined target, 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 rodent antibodies. See, e.g., U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205 (each of which is incorporated herein by reference). See also U.S. Patent No. 6,180,370 and PCT International Publication No. WO 01 / 27160 (each of which is incorporated herein by reference), which disclose humanized antibodies and techniques for producing humanized antibodies with improved affinity for a predetermined antigen. Further, humanized and chimeric antibodies can be modified to include 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 in this article, the term "human antibody" refers to an antibody with a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, by random or site-specific mutagenesis in vitro or by a mutation introduced by somatic mutation in vivo). However, the term "human antibody" does not include such antibodies, in which the CDR sequences of the germline derived from another mammalian species (for example, a mouse) are grafted onto human framework sequences (that is, humanized antibodies). The term encompasses antibodies with sequences derived from human genes but altered (for example, to reduce possible immunogenicity, increase affinity, eliminate the cysteine ​​that may cause undesirable folding, etc.). The term also encompasses such antibodies recombinantly produced in non-human cells, which can give atypical glycosylation 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. 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 entirety).Human antibodies and methods of making them are further discussed in the following: U.S. Patents 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. US 2006-0015957 A1; and International Patent Publication No. WO 98 / 24893; and WO 2007 / 117410. The disclosures of each of the above-cited patents, applications, and references 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 the wild-type amino acid sequence. Such modifications are within the ordinary skill of the skilled person. 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); U.S. Patent Publications 20040132101; 20070111260; 20110287032; 20180194860; U.S. Patent No. US 8409568; International Publication No. WO2017 / 177337) (which are incorporated by reference in their entirety). Amino acid deletions are indicated by "Δ," and insertions are indicated by "In." For example, a deletion of the amino acid sequence from E216 to E222 would be indicated as ΔE216-E222. For example, an insertion of arginine (R) between amino acid residues 234 and 235 would be indicated as InR234 / 235.

[0218] As used in this article, the term "Fc domain" refers to the crystallizable fragment of antibody after papain digestion. The Fc domain comprises two identical hinge regions and the second and third constant domains that are derived from IgA, IgD and IgG antibody isotypes or hinge regions and the second, third and fourth constant domains of IgM and IgE antibody isotypes. The Fc domain is that part of the antibody that is combined with certain proteins of cell surface Fc receptors and complement system. The term "Fc region" refers to the Fc domain combined with the hinge region. The hinge region is typically between the C-terminal end of the variable domains and the N-terminal end of the Fc domain. Although the boundaries of the Fc region may 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), wherein 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. (see above). Human IgG2 and human IgG4 residue numbering is also in 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" may refer to these sequences in isolation, or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein may be an antibody, an Fc fusion, or any protein or protein domain comprising 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) may comprise amino acid modifications. Any new amino acid residue that appears in the sequence of a variant Fc domain or variant Fc region due to 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 therefore slight differences may exist between the presented sequences and sequences in the prior art.

[0219] As used herein, the term "connector" refers to a polypeptide sequence that connects two or more antibody domains. The characteristics of the connector and its suitability for a particular purpose are known in the art. See, for example, Chen et al., Adv Drug Deliv Rev. October 15, 65(10): 1357-1369 (2013) (which discloses various types of connectors, their characteristics, and related connector design tools and databases), which are incorporated herein by reference. The connector can be flexible, rigid, or cleavable in vivo. Preferably, the connector is flexible. Flexible connectors typically contain small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The most commonly used flexible connector has a sequence consisting primarily of segments of Gly and Ser residues ("GS" connector). Optionally, the flexible connector contains a repeating sequence of 5 Gly and Ser residues. Non-limiting examples of flexible connectors 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), wherein n can be any integer from 1 to 5. The length of the connector is optionally 5 to 25 amino acid residues. Other suitable connectors can be selected from the group consisting of the following: 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, a flexible connector should provide good flexibility and solubility, and can serve as a passive connector to maintain the distance between the functional domains. The length of the flexible connector can be adjusted to allow correct folding, or to achieve the 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., ES Golub and DR 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 by a different amino acid residue with similar biochemical properties (e.g., charge, hydrophobicity, or size). Typically, a conservative amino acid substitution does not substantially change the functional properties of the protein. When comparing proteins with conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted to account for the conservative nature of the substitution. Such adjustments are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243: 307-31 (1994).

[0222] The group of amino acids with similar biochemical properties that can be used in conservative substitutions include: 1) amino acid residues with aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) amino acid residues with aliphatic hydroxyl side chains: serine and threonine;

[0223] 3) amino acid residues with amide side chains: asparagine and glutamine; 4) amino acid residues with aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) amino acid residues with basic side chains: lysine, arginine, and histidine; 6) amino acid residues with acidic side chains: aspartic acid and glutamic acid; and 7) amino acid residues with sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and glutamine-asparagine.

[0224] Each heavy chain consists of a heavy chain variable domain (VH) and multiple heavy chain constant domains (CH). For IgA, IgD, and IgG antibodies, the heavy chain typically comprises three domains: CH1, CH2, and CH3. For IgM and IgE antibodies, the heavy chain typically comprises four domains: CH1, CH2, CH3, and CH4. In some embodiments, the antibody comprises two domains: CH2 and CH3. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain. The light chain typically comprises one domain: CL. The light chain variable domain is encoded by two gene segments: the variable (V) gene segment, which encodes the first 95-101 amino acids of the light chain; and the joining (J) gene segment, which encodes approximately 12 or more amino acids. The heavy chain variable domain is encoded by three gene segments and includes a diversity (D) gene segment between the V and J gene segments, which encodes approximately 3 or more amino acids. The VH and VL domains can be further subdivided into hypervariable regions, termed "complementarity 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 from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0225] The pairing (VH and VL) of the variable domains of heavy chain and light chain forms an antibody binding site that interacts with antigen.Therefore, each antibody typically has two binding sites.Except for multifunctional / multispecific (e.g., bifunctional or bispecific) antibodies, these two binding sites are identical.The Fc district of the constant region of antibody typically mediates the combination of antibody with host tissue and factor (comprising various cells (e.g., effector cells) of immune system and the first component (C1q) of classical complement system).

[0226] The residues in the variable domain are numbered according to Edelman (also known as the EU numbering system), which is the numbering system for heavy chain variable domains or light chain variable domains compiled for antibodies. See Edelman Proc Natl Acad Sci US A. May, 63(1):78-85 (1969); and Kabat, EA, Wu, TT, Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, Bethesda, MD. The Eu numbering of residues can be determined for a given antibody by aligning the homology region of the antibody sequence with a "standard" EU numbered sequence. The variable region CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR H3) are identified according to crystal structure-based contacts as defined in Karpusas et al., Structure. Apr 4, 9(4):321-9 (2001) and numbered according to Edelman.

[0227] As used in this article, the term "operably connected" refers to the first structure that has been placed in a functional relationship with the second structure. In the context of an antibody, a targeting structure can be operably connected to a structure that imparts effector function. For example, the antigen-binding sequence (for example, variable region or VH or VL domain) of an antibody can be operably connected to the Fc district. In the context of a polynucleotide, a coding sequence can be operably connected to a non-coding regulatory sequence (for example, a promoter, enhancer, signal sequence, ribosome binding sequence, splice acceptor sequence, splice donor sequence, terminator sequence, etc.). Two operably connected structures can be directly connected. Alternatively, two operably connected structures can be connected by one or more intermediate structures. For example, the antigen-binding portion thereof of an antibody can be operably connected to the Fc district by CH1 domain, hinge region and / or connector sequence. Similarly, the non-coding regulatory sequence that is operably connected comprises a sequence adjacent to the coding sequence and works in trans or at a distance to control the sequence of the coding sequence.

[0228] As used herein, the term "effector function" refers to a response triggered by 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 combine with the Fc region of antibodies bound to target cells, resulting in the secretion of substances (e.g., lyases, perforins, granzymes, and tumor necrosis factors) that mediate the destruction of target cells. In CDC, cell death is induced by the activation of the complement cascade. See Daeron, Annu. Rev. Immunol., 15: 203-234 (1997); Ward and Ghetie, Therapeutic Immunol., 2: 77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). In opsonization and phagocytosis, the Fc region of the antibody bound to the pathogen binds to the Fc receptor on the surface of the phagocyte, thereby inducing phagocytosis. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using standard assays known in the art (see, for example, 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 the effector pathways of the immune system by combining with members of the cell surface Fcγ receptor family and with the C1q of the complement system. Various responses are triggered by the connection of the effector proteins of the clustered antibodies, including the release of inflammatory cytokines, the regulation of antigen production, endocytosis and cell killing. These responses can cause unwanted side effects such as inflammation and thrombosis. Therefore, the present disclosure further relates to anti-CD154 antibodies with 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 the antibodies comprising the mutated Fc region do not bind to FcγRIIa (also referred to as CD32a) on the platelet surface.

[0229] As used in this article, 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 the antibody disclosed herein to an effector molecule (e.g., FcγR and / or C1q). For example, the anti-CD154 antibody disclosed herein has reduced Fc receptor binding and complement activation compared to wild-type anti-CD154 antibody. In some embodiments, the variant Fc region has reduced antibody-dependent cell-mediated cytotoxicity (ADCC). The effector function of the anti-CD154 antibody can be determined by using one of many known assays, including CDC assays, ADCC assays, and phagocytosis assays (see Xu-Rong Jiang et al., Nature Reviews Drug Discovery 10: 101-111 (2011); and Liu et al. The Journal of Biological Chemistry 292: 1876-1883 (2017)). One or more of the effector functions of the anti-CD154 antibody can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% relative to the effector function of the wild-type anti-CD154 antibody.

[0230] 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, a soluble form of CD154 that is cleaved and released by activated T cells, 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 that can be prepared by standard recombinant expression methods. In some embodiments, CD154 refers to human CD154.

[0231] 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%, more preferably 80%, or more preferably 85%. Optionally, the antibody inhibits the cleavage or shedding of CD154 by at least about 20%, preferably 40%, more preferably 60%, more preferably 80%, or 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 that respond to CD154-CD40 signaling. Anti-CD154 antibodies 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 switching, inhibit immunoglobulin CDR somatic hypermutation, alter the expression or activity of molecules within the TNF-receptor-associated factor (TRAF) family, such as TRAF-2, TRAF3 (also known as CRAF1), TRAF-5, and TRAF-6, 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, AM, et al., Journal of Immunol., 155: 3329-3337 (1995); Cheng et al., Science. 267(5203): 1494-8 (1995); Bankert KC et al., Journal of Immunol. 194: 4319-4327 (2015); Ishida TK et al., Proc Natl Acad Sci US A.93(18):9437-42(1996); Muramatsu, MK et al., 2000. Cell 102:553(2000); Buchta CM and Bishop GA., Journal of Immunol. 192(1):145-50(2014); Arcipowski KM et al., International Immunology.26(3):149-58(2014); Mambetsariev N et al., Proc Natl Acad Sci US A.113(4):1032-7(2016); Arcipowski KM, Bishop GA., PLoS One.7(7)(2012); Bishop GA.Journal of Immunol.91(7):3483-5(2013); Peters AL and Bishop GA.Journal of Immunol. 185(11):6555-62 (2010); Rowland SL et al., Journal of Immunol. 179(7):4645-53 (2007); Benson RJ et al., European Journal of Immunol. 6(9):2535-43 (2006).

[0232] As used in this article, the term "immune response" refers to the reaction of the body's immune system to the presence of substances that are not recognized as the body's own components. 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 adaptive and / or innate immune responses. Regarding various types of immune responses, see David Chaplin J Allergy Clin Immunol February, 125(2 Suppl 2): ​​S3-23(2010).

[0233] As used herein, the term "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 equilibrium dissociation constant (K) for the binding affinity of a specific antibody-antigen interaction. D ). When K D An antibody is said to specifically bind to an antigen when its affinity is ≤1 mM, preferably ≤100 nM. High affinity antibodies are generally considered to have affinity in the low nM (10 -9 ) in the range of K D , and very high affinity antibodies are generally considered to have binding affinity in the pM (10 -12 ) in the range of K D . K D Binding affinity constants can be measured by surface plasmon resonance, e.g. using System (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), 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 of which is incorporated herein by reference. K D You can also use The measurements were performed using a Sigma-Aldrich system (Sapidyne Instruments, Hanover, Germany and Boise, ID).

[0234] 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. D It is the ratio of kd to ka.

[0235] As used herein, the term "affinity" refers to the overall strength of an antibody-antigen complex. Affinity involves three main parameters: the affinity of the antibody for the epitope; the valence of both the antibody and the antigen; and the structural arrangement of those interacting parts. As used herein, "affinity" describes the increased affinity that arises due to multiple antigen-binding sites on an immunoglobulin.

[0236] As used herein, the term "transplant" refers to the process of surgically removing cells or tissue from a first organism (the donor) and placing it in a second organism (the recipient). The donor can be a human or a non-human organism. In some embodiments, the donor is a primate. The donor can be a non-human primate. Optionally, the donor is a human. In some embodiments, the donor is a pig or a minipig. The recipient can be a human or a non-human organism. Preferably, the recipient is a human. Optionally, the recipient is a non-human primate. The transferred cells, tissues, or organs are called "grafts." "Xenotransplantation" refers to the transfer of cells, tissues, or organs from a donor of one species (e.g., monkey or pig) to a recipient of a different species (e.g., human).

[0237] As used herein, the term "modified cell" refers to a cell modified from its native state. Modified cells can be modified by using one or more techniques such as transduction to express cDNA, CRISPR / Cas9 systems, RNAi technology and retroviral technology. For example, the cell can be 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 (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)).

[0238] As used herein, the term "cells expanded in vitro" refers to cells that are produced in vitro to increase the yield of cells (e.g., hematopoietic stem cells (HSC)) to be used in clinical applications (e.g., transplantation). See, for example, Xie J and Zhang C Sci China Life Sci. Sep, 58 (9): 839-53 (2015), which review methods for expanding the number of HSCs, including culture systems such as matrix / HSC co-culture, continuous perfusion, and fed-batch culture, and supplemented with external ligands, membrane-transportable transcription factors, complement components, protein-modifying enzymes, metabolites, or small molecule chemicals. It is also possible to amplify the desired cells to be transplanted in vitro by applying endogenous Notch-signaling activators (see, for example, Ex vivo expansion of human hematopoietic stem and progenitor cells, Dahlberg A et al., Blood 117: 6083-6090 (2011)).

[0239] As used herein, the term "transplant rejection" refers to the phenomenon that occurs when transplanted cells, tissues or organs from a donor are rejected by the recipient's immune system. The recipient's immune system can initiate an adaptive immune response (cellular immunity) mediated by killer T cells, which induces apoptosis of donor cells; humoral immunity mediated by activated B cells that secrete antibodies; and / or innate immune responses 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)).

[0240] As used herein, the term "immune-related disease" refers to conditions in which the host immune system plays an integral role in the mediated disease and contributes to the progression of the disease. The term encompasses "autoimmune diseases," which are conditions that arise when a specific adaptive immune response is launched 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 onset 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 may 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.

[0241] Immune-related diseases can also refer to a dysfunctional immune response that causes or contributes to an inflammatory condition in 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 damage in gas exchange and lung mechanics, with a high case fatality rate. ARDS occurs when small blood vessels in the lungs leak fluid (which enters and may fill the alveoli). A variety of 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, lung tissue inflammation, chronic obstructive pulmonary disease (COPD). See Moldoveanu, B et al., Journal of inflammation research (2): 1-11 (2009).

[0242] Further, immune-related diseases, such as causing or contributing to the dysfunctional immune response of the inflammatory condition of an organ, can be due to viral infection. For example, ARDS, pneumonia, bronchitis, lung tissue inflammation and COPD can occur in viral infection, and the virus is, for example, cytomegalovirus, Epstein-Barr virus, influenza virus, smallpox virus, orthopoxvirus or coronavirus, such as SARS coronavirus (SARS-CoV), SARS-CoV-2 and MERS-CoV. Such viral infection can be associated with inflammatory conditions or dysfunctional immune responses such as cytokine release syndrome (CRS) or "cytokine storm" (which is a serious immune response in which the host releases too many cytokines into the bloodstream too quickly). For example, the host can release a high number of proinflammatory 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 infection, autoimmune conditions or other diseases.For example, cytokine storm can occur in viral infection, and the virus is, for example, cytomegalovirus, Epstein-Barr virus, influenza virus, smallpox virus, SARS-CoV, SARS-CoV-2 and MERS-CoV. Immune-related disease can refer to the situation associated with the dysfunctional immune response, cytokine storm or cytokine storm syndrome in the host. The example of such viral-mediated immune-related disease includes, but is 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 "cytokine storm". Many patients with severe COVID-19 lung disease present with classic ARDS, but others present with an atypical form of ARDS (with relatively high compliance and well-preserved lung gas volumes), which may be associated with loss of pulmonary perfusion regulation, hypoxic vasoconstriction (Gattinoni L et al., Am J. Respir Crit Care Med. (2020)) and / or microvascular thrombosis and potentially disseminated intravascular coagulation (Phend C. Med Page Today, Infectious Disease. April 8, 2020).

[0243] As used in this article, the term "primary response" refers to the immune response of the body to the antigen that occurs when encountering an antigen for the first time. The term "re-response" refers to such an immune response that involves both B cells and T cells after encountering the same antigen later and causes the activation of previously generated memory cells more quickly. Responding again 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 the primary immune response, responding cells are immature B- cells and T- cells, and in responding again, responding cells are memory cells.

[0244] As used herein, the term "hematopoietic cell chimerism" refers to the coexistence of host and donor hematopoietic cells due to the transplantation of donor multipotent hematopoietic stem cells into the host. The host and donor cells can be mutually tolerant. The mechanism of hematopoietic cell chimerism is 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 cell chimerism leads to "central tolerance". The mechanism of "central tolerance" in such chimeras can involve central, intrathymic clonal deletion, selection of regulatory T cells, and / or other related immune mechanisms. See, for example, 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 with an organ (e.g., a kidney or liver transplant). Stem cells can be derived from the donor's bone marrow or adipocytes / adipose tissue.

[0245] As used herein, the term "adaptation" or "adapted" refers to preparing the recipient for stem cell transplantation, such as hematopoietic stem cell transplantation. Gyurkocza B and Sandmaier BM Blood 124: 344-353 (2014) provide a review of high-dose, low-intensity and non-severe myelosuppressive conditioning regimes and the most commonly used agents such as whole-body irradiation, fludarabine phosphate, cyclophosphamide, T-cell depleting antibodies, cyclosporin A (CsA). Monoclonal antibodies such as anti-CD20 Ab, anti-CD33 Ab and anti-CD45 Ab can also be used alone or in combination with conventional therapy as part of the conditioning regime to prevent transplant rejection. See, for example, Topcuoglu P et al., Progress in Stem Cell Transplantation, December (2015). Other agents that can be used in the conditioning regime include, but are not limited to, BCL-2 inhibitors (Perini GF et al., Journal of Hematology & Oncology 11:65 (2018)) and anti-CTLA4 Ab (Pree I et al., Transplantation. Mar 15, 83(5):663-667 (2007)). The conditioning regime may 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.

[0246] The term "isolated antibody" refers to an antibody that is at least partially free of other biomolecules present in the cells used to produce it. Other biomolecules that an isolated antibody does not contain 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 pharmaceutical formulations. Thus, molecules synthesized chemically or in a cell-free system will be "isolated" from the components with which they are naturally associated. Molecules can also be "isolated" by using purification techniques well known in the art.

[0247] As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of at least 10 bases in length. A polynucleotide can comprise ribonucleotides, deoxynucleotides, modified forms of either type of nucleotide, or combinations thereof. A polynucleotide can be single-stranded or double-stranded.

[0248] 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, which (1) is not associated with all or part of a polynucleotide with which the "isolated nucleic acid" is associated in nature, (2) is operably linked to a polynucleotide with which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. An isolated polynucleotide "comprising" a particular sequence may also include coding sequences for other proteins or immunoglobulin chains, expression control sequences, or vector sequences.

[0249] Molecular purity or homogeneity can be tested by means well known in the art. For example, the purity of an antibody sample can be tested in the following manner: using polyacrylamide gel electrophoresis, and using technology well known in the art that the gel is dyed so that the antibody is visualized. 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 tested by using the spectrophotometric absorbance of a sample at 260nm to 280nm using technology well known in the art. For some purposes, higher resolution can be provided by using means well known in the art for purification.

[0250] 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.

[0251] As used in this article, 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. The non-limiting example of a vector includes a viral vector, a naked DNA or RNA expression vector, a bacterial vector, a mammalian vector, a plasmid, a clay, a phage vector, a DNA or RNA expression vector associated with a cationic coagulant, a DNA or RNA expression vector encapsulated in a liposome, and some eukaryotic cells (e.g., production cells). In some embodiments, the vector autonomously replicates in the host cell into which it is introduced. Optionally, the vector is integrated into the genome of the host cell and replicates along with the host genome. The vector can be capable of directing the expression of the coding sequence to which it is operably connected. Such vectors are referred to as "recombinant expression vectors" or "expression vectors" in this article.

[0252] As used in this article, the term "host cell" refers to the cell having wherein introduced a carrier or polynucleotide.Should be understood that "host cell" not only comprises specific test cell, but also comprises the offspring of this type of cell.Because some modification can appear in subsequent generations due to sudden change or environmental influence, so this type of offspring may in fact be different from the parental cell, but still be included within the scope of the term "host cell" as used in this article, condition is that they still comprise said carrier, polynucleotide or its part, no matter is with episomal form or be integrated in the host cell genome.

[0253] 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 with 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 not considering any conservative substitutions as part of the sequence identity. Such conservative substitutions (in addition to identical residues) are taken into account in calculating the "percent sequence similarity" of two sequences. Residue positions that are not identical but are similar differ by conservative amino acid substitutions.

[0254] Alignment for purposes of determining percent amino acid sequence identity, sequence similarity, or sequence homology (e.g., between a wild-type protein and a mutant thereof) can be achieved in various ways that are within the skill in the art, for instance, by using publicly available sequence analysis computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Gap, With other programs in Wisconsin Package version 10.0 or Genetics Computer Group (GCG), Madison, Wisconsin.software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for obtaining maximum alignment over the full length of the compared sequences. FASTA can also be used to compare polypeptide sequences using default or recommended parameters. In the context of polypeptide sequences, FASTA captures the query amino acid sequence and uses local sequence alignment to search the database to identify similar sequences within the database (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson Curr Protoc Bioinformatics. Mar 24, 53: 3.9.1-25 (2016); each of which is incorporated herein by reference). BLAST (especially blastp or tblastn) using default parameters can be used to compare a query sequence to a database containing sequences from different organisms. See, for example, Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997); Eser et al., PLoS One. 22, 9(12): e115445 (2014), each of which is incorporated herein by reference.

[0255] Reference to a nucleotide sequence herein encompasses its complementary sequence unless otherwise specified. Thus, reference to a nucleic acid having a specific sequence should be understood to encompass its complementary strand having its complementary sequence.

[0256] 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.

[0257] The term "primate" refers to mammals of the order Primates, which includes the great apes and prosimians, and is characterized by elaborate development of hands and feet, a shortened snout, and a large brain. Mammals of the order Primates include humans, apes, monkeys, and prosimians, or lower primates.

[0258] As used herein, a "therapeutically effective amount" refers to an amount of the administered therapeutic agent that will alleviate one or more of the symptoms of the treated condition to a certain extent. With regard to the treatment of transplant rejection, a therapeutically effective amount refers to an amount having at least one of the following effects: alleviating, inhibiting or preventing acute or chronic rejection of transplanted cells, tissues or organs and one or more symptoms associated with rejection; prolonging graft survival; alleviating thrombosis; reducing the risk of life-threatening infections, cancers or other complications such as cardiovascular disease and renal failure. With regard to the mechanism of cell rejection in transplantation, see, for example, Romano et al., Front Immunol. 10: 43 (2019) and Ingulli E. Pediatr Nephrol. 25 (1): 61-74 (2010). With regard 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 autoimmune diseases, such as fatigue, myalgia, low-grade fever, inflammation, rash, etc.

[0259] The pharmaceutical composition may include a therapeutically effective amount or a prophylactic effective amount of an antibody disclosed herein. The therapeutically effective amount of the antibody may vary according to factors such as individual disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody are surpassed 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 "prophylactic effective amount" refers to an amount effective for obtaining the desired prophylactic result at the necessary dosage and within the time period. Typically, due to the use of a prophylactic dose in a subject before transplantation or in the early stages of transplant rejection, the prophylactic effective amount may be lower than the therapeutically effective amount.

[0260] As used herein, the term "treating" refers to the internal or external administration of a therapeutic agent, such as a composition comprising any of the antibodies disclosed herein, to a subject or patient having one or more symptoms of a disease or suspected of having a disease, for which the agent is therapeutically active. "Treatment" refers to therapeutic treatment and / or prophylactic treatment. Therapeutic treatment includes, for example, methods that alleviate or lessen the severity of a condition or eliminate a condition, and includes alleviating or reducing the severity of one or more symptoms of the condition. If treatment is administered before the clinical manifestation of the condition, the treatment is considered prophylactic. Alleviation or reduction of disease symptoms can be assessed 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 amelioration of existing uncontrolled or unwanted disease symptoms, prevention of additional disease symptoms, and amelioration or prevention of the underlying cause of such disease symptoms. Thus, the term implies that a beneficial result has been achieved in the subject.

[0261] With regard to the treatment of transplant rejection, treatment can refer to mitigating, alleviating or delaying the rejection of the transplanted cells, tissues or organs or one or more symptoms associated with rejection. Treatment can also result in prolonged graft survival, alleviate thrombosis, and / or reduce the risk of life-threatening infections, cancers and other complications such as cardiovascular disease and renal failure. With regard to the treatment of autoimmune diseases, treatment can refer to slowing down the body's immune response and controlling the autoimmune reaction. With regard 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 regard to the treatment performed 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 transplant or an individual suffering from an autoimmune or inflammatory disease will increase, or one or more of the symptoms associated with transplant rejection or an autoimmune or inflammatory disease will be alleviated.

[0262] 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 alleviation of one or more symptoms of a condition, as a result of administering an anti-CD154 antibody of the present disclosure. For example, in the context of administering therapy to a subject, "prevention" refers to inhibiting, reducing or delaying the development or onset of rejection of that transplant 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 administering a combination of therapy (e.g., a combination of solid organ transplantation and an immunosuppressant) in a subject.

[0263] As used herein, the term "administering" an antibody or composition of the present disclosure to a subject refers to contacting the antibody or composition with the subject or the subject's cells, tissues, organs, or biological fluids. Such administration can be performed using one of the various methods known to those skilled in the art. For example, the antibody or composition of the present disclosure can be administered systemically or topically. In some embodiments, the composition can be administered subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally, or rectally. It can also be administered, for example, once, multiple times, and / or over one or more extended time periods. In some embodiments, the administration includes direct administration (including self-administration) and indirect administration, including the act of prescribing a drug.

[0264] As used herein, the term "high stringency conditions" refers to hybridization to 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, Vol. 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY, at p. 2.10.3).

[0265] Anti-CD154 antibody

[0266] A first aspect of the present disclosure provides an isolated anti-CD154 antibody with 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, the antibody with one or more reduced effector functions exhibits reduced binding to an Fc receptor relative to an antibody with a wild-type IgG1 heavy chain. Optionally, the reduced binding to the Fc receptor 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 the antibody with reduced effector function is about 10 to 3200 times weaker than the binding exhibited by the wild-type IgG heavy chain. D is 0.1-100 nM, such as 0.3-92 nM. Optionally, the K for CD16aF binding to the Fc domain of an antibody with one or more reduced effector functions is D Greater than 2 μM. In some embodiments, the K for CD16aV binding to the Fc domain of an antibody with one or more reduced effector functions is greater than 2 μM. DGreater than 0.4 μM. Optionally, the K for CD32aH binding to the Fc domain of an antibody with reduced effector function(s) is greater than 0.4 μM. D Greater than 0.5 μM. In some embodiments, the K for CD32bF binding to the Fc domain of an antibody with one or more reduced effector functions is greater than 0.5 μM. D Greater than 1μM.

[0267] In some embodiments, the antibody of the separation is a fully human monoclonal antibody. In some embodiments, the antibody of the separation is a chimeric antibody. In some embodiments, the antibody of the separation is a humanized antibody. In some embodiments, people's anti-CD154 antibodies are produced by the following manner: immunization of a non-human transgenic animal, such as a rodent, whose genome comprises human immunoglobulin genes, so that the transgenic animal produces human antibodies.

[0268] 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 connected to a human Fc domain with a modified effector function. In some embodiments, one or more effector functions of the human Fc domain are reduced. Optionally, one or more effector functions of the human Fc domain are eliminated. Optionally, the VH is operably connected to a human Fc region, wherein the human Fc region comprises a human hinge sequence and the human Fc domain, 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.

[0269] In some embodiments, the human Fc domain is derived from an IgG1 Fc (or crystallizable fragment) region. Optionally, the human Fc domain is derived from an IgG1 constant region. The human Fc domain may be derived from an IgG2 Fc (or crystallizable fragment) region. In some embodiments, the Fc domain is derived from an IgG2 constant region. The human Fc domain may be derived from an IgG4 Fc (or crystallizable fragment) region. In some embodiments, the Fc domain is derived from an IgG4 constant region. In some embodiments, the human Fc domain is derived from an IgG1 Fc (or crystallizable fragment) region. Optionally, the human Fc domain is derived from an IgG1 constant region. In some embodiments, the human Fc domain is derived from an IgG2 Fc (or crystallizable fragment) region. In some embodiments, the Fc domain is derived from an IgG2 constant region. Optionally, the human Fc domain is derived from an IgG4 Fc (or crystallizable fragment) region. The human Fc domain may be derived from an IgG4 constant region.

[0270] In some embodiments, the Fc domain 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 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 238-241. Optionally, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 238-241. The Fc domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9, 12-18 and 238-241. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 236-241. Optionally, the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 236-241. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 3. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 5. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 7. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 9. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 13. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 15. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 17. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 18.In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 238. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 240. Optionally, the Fc domain comprises the amino acid sequence of SEQ ID NO: 241. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain does not comprise the amino acid sequences of SEQ ID NOs: 236 and 237.

[0271] 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 an 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 an amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 243-251. The Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56 and 243-251. In some embodiments, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 242-251. Optionally, the Fc region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 242-251. The Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-37, 40-56 and 242-251. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 21. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 23. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 25. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 27. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 29. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 31. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 33.Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 35. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 40. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 42. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 44. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 46. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 48. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 50. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 52. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 54. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 242. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 243. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 244. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 246. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 247. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 248. Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 250.Optionally, the Fc region comprises the amino acid sequence of SEQ ID NO: 251. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 236. Optionally, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 237. In some embodiments, the Fc domain does not comprise the amino acid sequence of SEQ ID NO: 242. Optionally, the Fc domain does not comprise the amino acid sequences of SEQ ID NOs: 236, 237, and 242.

[0272] In some embodiments, the 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 as set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). In a preferred embodiment, the antibody comprising an Fc domain or 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, e.g. Figure 9 c.

[0273] In some embodiments, the 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, T252, , D350, L351, K360, T366, N390, K392, T394, D399, S400, F405, Y407, K409, T411, or a combination of such amino acid modifications, wherein the numbering of amino acid residues is according to the EU index as set forth in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).In a preferred embodiment, the antibody comprising an Fc domain or Fc region derived from IgG1 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, K 322A, P329G, P329A, A330S, P331S, Q347R, Q347E, Q347K, T350V, L351Y, K360D, K360E, T366A, T366I, T366L, T366M, T 366V, N390R, N390K, N390D, K392V, K392M, K392R, K392L, K392F, K392E, T394W, D399R, D399W, D399K, S400E, S400D, S4 00R, S400K, F405A, F405I, F405M, F405T, F405S, F405V, F405W, Y407A, Y407I, Y407L, Y407V, K409F, K409I, K409S, K4 09W, 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. See, for example, Figure 9 a.

[0274] In some embodiments, the 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 as set forth in Edelman. In a preferred embodiment, the 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.

[0275] In some embodiments, the VH comprises (a) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 57, (b) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 58, and (c) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 59; and the VL comprises (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO: 60, (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO: 61, and (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO: 62.

[0276] 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.

[0277] 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.

[0278] 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 the amino acid sequence of any one of SEQ ID NOs: 67, 70 and 73. The CH1 domain may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 67, 70 and 73. In some embodiments, the CH1 domain comprises the amino acid sequence of any one of SEQ ID NOs: 67, 70 and 73. The CH1 domain may comprise the amino acid sequence of SEQ ID NO: 67. Optionally, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO:73.

[0279] In some embodiments, the antibody comprises a connector between the VH and Fc domains. Optionally, the connector comprises the amino acid sequence of any one of SEQ ID NOs: 199-223 and 327-330. In some embodiments, the connector comprises the amino acid sequence of any one of SEQ ID NOs: 199-223. The connector may comprise the amino acid sequence of any one of SEQ ID NOs: 327-330. In some embodiments, the connector comprises the amino acid sequence of SEQ ID NO: 199. Optionally, the connector comprises the amino acid sequence of SEQ ID NO: 200. In some embodiments, the connector comprises the amino acid sequence of SEQ ID NO: 201. Optionally, the connector comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the connector comprises the amino acid sequence of SEQ ID NO: 203. Optionally, the connector comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the connector 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.

[0280] 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 may 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 may 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 may 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 may 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 may be operably linked to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 22. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 23. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 25. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 26.The VH may be operably linked to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 28. Optionally, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 29. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 31. Optionally, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 32. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 34. Optionally, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 35. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH may be operably linked to the amino acid sequence of SEQ ID NO: 37. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 40. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 42. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 43. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 45. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 46. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 48. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 49. The VH may be operably linked to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the VH is operably linked to the amino acid sequence of SEQ ID NO: 51. Optionally, the VH is operably linked to the amino acid sequence of SEQ ID NO: 52. The VH 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 NOs: 236 and 237. Optionally, the VH is not operably linked to the amino acid sequences of SEQ ID NOs: 236, 237, and 242.

[0281] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174 and 266-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174 and 266-288. The heavy chain can comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, and 266-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277, and 279-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277 and 279-288. The heavy chain may comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149-160, 163-174, 266-277 and 279-288. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174 and 266-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174 and 266-288. The heavy chain may comprise the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, and 266-288.In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277 and 279-288. Optionally, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 121-132, 135-146, 149, 151-160, 163, 165-174, 266-277 and 279-288. The heavy chain can 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 sequence 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 sequence of SEQ ID NO: 234 and 278. In some embodiments, the heavy chain does not comprise the amino acid sequence of 234, 266, 272, and 278. Optionally, the heavy chain does not comprise the amino acid sequence of SEQ ID NO: 150, 164, 234, and 278. In some embodiments, the heavy chain does not comprise the amino acid sequence of 150, 164, 234, 266, 272, and 278.

[0282] In some embodiments, the light chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 195 and 196. Optionally, the light chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similar to the amino acid sequence of any one of SEQ ID NOs: 195 and 196. The light chain may comprise the amino acid sequence of SEQ ID NOs: 195 or 196. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 195. Optionally, the light chain comprises the amino acid sequence of SEQ ID NO: 196.

[0283] In some embodiments, the antibody is monoclonal. Optionally, the antibody is a chimeric antibody. The antibody can be a humanized antibody. In some embodiments, the antibody is a human antibody.

[0284] 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, reduced levels of thrombosis are observed after administration of the antibody compared to the levels of thrombosis after administration of 5c8 or hu5c8 antibodies. Optionally, the antibody does not cause thrombosis when administered to a subject.

[0285] In some embodiments, when administered to a subject, the antibody has one or more of the following effects: (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) altered expression or activity of downstream targets of CD154-CD40 signaling. Optionally, administration of the antibody results in a reduced risk of thrombosis or thromboembolic events compared to a subject to which a 5c8 or hu5c8 antibody is 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 altered expression or activity of downstream targets of CD154-CD40 signaling.

[0286] In some embodiments, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of any one of SEQ ID NOs: 1, 2, 10, 11, 231, and 236. Optionally, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 1. In some embodiments, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 2. Optionally, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 10. In some embodiments, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 11. Optionally, the human Fc domain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 231. In some embodiments, the human Fc domain with 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 NOs: 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.

[0287] Optionally, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of any one of SEQ ID NOs: 19, 20, 38, 39, 232, and 235. In some embodiments, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 19. Optionally, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 20. Optionally, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 39. Optionally, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the human Fc region with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 235. Optionally, the antibody does not comprise an amino acid sequence consisting of any one of SEQ ID NOs: 19, 20, 38, 39, 232, and 235. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 19. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 20. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 38. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 39. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 232. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 235.

[0288] In some embodiments, the heavy chain with modified effector function 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. Optionally, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 119. In some embodiments, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 120. Optionally, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 133. In some embodiments, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 134. Optionally, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 147. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 148. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 150. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 161. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 162. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 164. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 230. In some embodiments, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 234. Optionally, the heavy chain with modified effector function does not comprise the amino acid sequence consisting of SEQ ID NO: 278. In some embodiments, the antibody does not comprise 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. 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 an amino acid sequence consisting of SEQ ID NO: 147. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 148. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 161. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 162. In some embodiments, the heavy chain with modified effector function does not comprise an amino acid sequence consisting of SEQ ID NO: 164. Optionally, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 230. In some embodiments, the antibody does not comprise an amino acid sequence consisting of SEQ ID NO: 234. Optionally, the heavy chain with 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 NOs: 1, 2, 10, 11, 19, 20, 38, 39, 119, 120, 133, 134, 147, 148, 150, 161, 162, 164, 230, 231, 232, 234, 235, 236, and 278.

[0289] Characteristics of the anti-CD154 antibody

[0290] Binding affinity of anti-CD154 antibodies to CD154

[0291] The binding affinity of anti-CD154 antibodies to CD154 (K D ) and dissociation rate (k off ) can be determined by methods known in the art. The binding affinity can be determined by ELISA, RIA, flow cytometry or surface plasmon resonance (SPR) (e.g., using 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 by using A skilled artisan can determine whether an antibody disclosed herein has substantially the same K as another anti-CD154 antibody by using methods known in the art. D Determination of K D and k off Such methods can be used during the initial screening phase and during subsequent optimization phases. In some embodiments, the antibody has a K for CD154 of less than 50 pM. D 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 .

[0292] CD154 activity is inhibited by anti-CD154 antibodies

[0293] Anti-CD154 antibodies that inhibit the binding of CD154 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, for example, Barr et al., Immunology, 102(1):39-43 (2001); and Blair et al., J. Exp. Med., 191(4):651-660 (2001). For example, neutralizing anti-CD154 antibodies can be identified by their inhibition of upregulation of specific downstream target genes of CD154 (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 .

[0294] Effector function – platelet assays

[0295] 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, for example, U.S. Patent No. 9,765,150; Langer et al., Thromb Haemost.Jun, 93(6): 1137-46(2005); McKenzie, SE et al., J Immunol 162(7)4311-4318(1999); and Scholthauer T et al., Protein Engineering, Design and Selection, 29(10): 457-466(2016). Blood from human donors or mice expressing FcγRIIA (CD32a) on platelets can be used to assay platelet function. Platelet activation can be detected by flow cytometry using antibodies against platelet activation markers P-selectin (CD62P) and PAC-1 (activated GPIIb / IIIa). Platelet aggregation analysis can be performed by using a minicell impedance device and quantifying the area under the curve as a measure of the platelet aggregation impedance curve.

[0296] Nucleic acid, vector, host cell and recombinant method for preparing antibodies

[0297] Nucleic Acids

[0298] Second aspect of the present disclosure provides the nucleic acid molecules of coding anti-CD154 antibody disclosed in this article.In some embodiments, the heavy chain and the light chain of the anti-CD154 immunoglobulin of nucleic acid molecule encoding separation.In other embodiments, the heavy chain and the light chain of the anti-CD154 immunoglobulin of same nucleic acid molecule encoding.

[0299] In some embodiments, the nucleic acid molecule comprises a sequence encoding the VL of an anti-CD 154 antibody disclosed herein.

[0300] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VL comprising an amino acid sequence of any one of SEQ ID NOs: 65 and 66. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VL comprising an amino acid sequence of any one of SEQ ID NOs: 65 and 66, or a portion thereof. In some embodiments, the nucleic acid encoding one, two, or all three light chain CDR amino acid sequences of the antibody. In some embodiments, the portion encoding is from the contiguous region of CDR1-CDR3 of the light chain of an anti-CD154 antibody.

[0301] In some embodiments, the nucleic acid molecule encodes a VL comprising 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 comprising 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding the amino acid sequence of the VL region of SEQ ID NO: 65 or 66.

[0302] In some embodiments, the nucleic acid molecule comprises a sequence encoding the VH of an anti-CD 154 antibody disclosed herein.

[0303] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding 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 encoding 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 the contiguous region of CDR1-CDR3 from the heavy chain of an anti-CD154 antibody.

[0304] In some embodiments, the nucleic acid molecule encodes a VH comprising 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 comprising 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding the amino acid sequence of the VH region of SEQ ID NO: 63, 64, 252 or 253.

[0305] In some embodiments, the nucleic acid molecule comprises a sequence encoding the Fc domain of an anti-CD 154 antibody disclosed herein.

[0306] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding 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 encoding 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.

[0307] 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 the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 238-241. The nucleic acid molecule may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18, and 238-241, or to the complement of a nucleic acid comprising the Fc region portion of the heavy chain nucleotide sequence of 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 the amino acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 236-241. The nucleic acid molecule may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 3-9, 12-18 and 236-241, or to the complement of a nucleic acid comprising the Fc region portion of the heavy chain nucleotide sequence 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 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.

[0308] In some embodiments, the nucleic acid molecule comprises a sequence encoding the Fc region of an anti-CD 154 antibody disclosed herein.

[0309] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an Fc region comprising an 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 an 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 an 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 an 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 an 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 an 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 an 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.

[0310] In some embodiments, the nucleic acid molecule encodes an Fc region comprising 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 comprising 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 may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 comprising 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 comprising 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 may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 comprising 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 comprising 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 may comprise a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 comprising 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 comprising 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding the amino acid sequence of any one of SEQ ID NOs: 21-37, 40-56 and 242-251.

[0311] In some embodiments, the nucleic acid molecule comprises a sequence encoding a light chain of an anti-CD154 antibody disclosed herein. Optionally, the nucleic acid molecule encoding a light chain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 197 and 198. In some embodiments, the nucleic acid molecule encoding a light chain comprises a nucleotide sequence 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: 197 and 198. In some embodiments, the nucleic acid molecule encoding a light chain is included in a nucleotide sequence hybridized under high stringency conditions with a complementary sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO: 197 and 198.

[0312] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding 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 encoding a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 195 and 196, or a portion thereof.

[0313] 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 any one of SEQ ID NOs: 195 and 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 any one of SEQ ID NOs: 195 and 196. The nucleic acid molecule can include a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding a light chain amino acid sequence of any one of SEQ ID NOs: 195 and 196, or to the complement of a nucleic acid comprising a light chain nucleotide sequence of any one of SEQ ID NOs: 197 and 198.

[0314] In some embodiments, the nucleic acid molecules include the sequence of the heavy chain of anti-CD154 antibody disclosed in this article. Optionally, the nucleic acid molecules of the heavy chain of the coding include the 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 molecules of the heavy chain of the coding include at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical nucleotide sequence of the 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 complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 177-179, 182-184, 187-189, and 192-194.

[0315] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding 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 encoding 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 encoding 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 encoding 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 encoding 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 encoding 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 encoding 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 encoding 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 encoding a heavy chain comprising 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 nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain comprising 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, or a portion thereof.

[0316] 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 include a nucleotide sequence that hybridizes under high stringency conditions to the complement of a nucleic acid encoding a 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 complement of a nucleic acid comprising a 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 complement 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 complement 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 may include a nucleotide sequence that hybridizes under high stringency conditions to the complement 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 complement 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 a 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 a 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 a 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.

[0317] The nucleic acid molecules of the heavy chain or light chain of coding anti-CD154 antibody can be separated from any source that produces this type of antibody.In various embodiments, described nucleic acid molecules are separated from the B cell of the expression anti-CD154 antibody separated from the animal that carries out immunization with CD154 or are separated from the immortalized cell that is derived from this type of B cell.The method for separating the nucleic acid of coding antibody is well known in the art.See, for example, Sambrook J.&Russell D., Molecular Cloning:A Laboratory Manual, the 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000).In another embodiment, described nucleic acid is separated from non-human non-transgenic animal.The nucleic acid molecules separated from non-human non-transgenic animal can be used for such as humanized antibody, and it comprises one or more amino acid sequences of the anti-CD154 antibody of people disclosed in this article.

[0318] carrier

[0319] A third aspect of the present disclosure provides a vector comprising a disclosed nucleic acid molecule. In some embodiments, the vector comprises a nucleotide sequence encoding a heavy chain of an anti-CD154 antibody disclosed herein. Optionally, the vector comprises a nucleotide sequence encoding a light chain of an antibody disclosed herein. The vector may comprise a nucleotide sequence encoding a heavy chain of an anti-CD154 antibody disclosed herein and a light chain.

[0320] In some embodiments, described anti-CD154 antibody is expressed by the following manner: the DNA of the coding portion or full-length light chain and / or heavy chain obtained as described above is inserted in the expression vector, thereby described gene and expression control sequence are for example transcribed and translation control sequence are operably connected.In some embodiments, the DNA of described coding portion light chain and / or heavy chain can comprise VH and / or VL sequence.Expression vector comprises plasmid, retrovirus, adenovirus, adeno-associated virus (AAV), plant virus is for example cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAC, EBV derived episome, etc.The polynucleotide of coding heavy chain and / or light chain can be connected in the carrier, thereby the transcription and translation control sequence in described carrier brings into play their desired function of transcribing and translating described polynucleotide of regulation and control.Expression vector and expression control sequence can be selected to be compatible with employed expression host cell.Antibody light chain gene and antibody heavy chain gene can be inserted in the carrier that separates.In some embodiments, these two genes are inserted in the same expression vector. In some embodiments, the polynucleotide encoding the VH and / or VL sequence is inserted into an expression vector comprising a CH and / or CL nucleotide sequence. Optionally, the polynucleotide encoding the VH sequence is inserted into an expression vector comprising an Fc domain nucleotide sequence. In some embodiments, the polynucleotide encoding the VH sequence is inserted into an expression vector comprising an 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).

[0321] A convenient vector may be one encoding a functionally complete CH or CL immunoglobulin sequence with appropriately engineered restriction sites such that any VH or VL nucleotide sequence can be readily inserted 5' to the CH or CL nucleotide sequence and expressed, as described above. In such vectors, splicing typically occurs between a splice donor site in the inserted J region and a splice acceptor site preceding the C domain, and also occurs at splice regions present within CH exons. Optionally, the vector encodes an Fc domain with modified effector function with appropriately engineered restriction sites such that a VH nucleotide sequence can be readily inserted 5' to the Fc domain nucleotide sequence and expressed. In some embodiments, the vector comprises a linker-encoding sequence 5' to the sequence encoding the Fc domain with appropriately engineered restriction sites such that a VH nucleotide sequence can be readily inserted 5' to the linker sequence and expressed. Optionally, the vector encodes an Fc region with modified effector function, with suitable engineered restriction sites such that the VH nucleotide sequence can be readily inserted 5' to the Fc region nucleotide sequence and expressed. In some embodiments, the vector comprises a linker-encoding sequence 5' to the sequence encoding the Fc region, with suitable engineered restriction sites such that the VH nucleotide sequence can be readily inserted 5' to the linker polynucleotide sequence and expressed. In some embodiments, the vector comprises a CH1 domain-encoding sequence 5' to the sequence encoding the Fc region, with suitable engineered restriction sites such that the VH nucleotide sequence can be readily inserted 5' to the CH1 domain polynucleotide sequence and expressed.

[0322] In some embodiments, the vector comprises a polyadenylation and / or transcription termination sequence downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector so that the signal peptide is linked to the amino terminus of the immunoglobulin chain in a frame-matched manner. Optionally, the signal peptide is an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0323] In addition to the antibody chain coding sequence, the recombinant expression vector can also optionally carry a regulatory sequence that controls the expression of the antibody chain coding sequence in the host cell. Those skilled in the art will appreciate that the design of the expression vector (including the selection of regulatory sequences) can depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct the high protein expression level in mammalian cells, such as those derived from retroviral long terminal repeats (LTR), 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 natural immunoglobulin and actin promoters. For further description of viral regulatory elements and sequences thereof, see, for example, 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, for example, U.S. Patent No. 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.

[0324] In addition to antibody chain coding sequences and regulatory sequences, the recombinant expression vector disclosed herein can also carry additional sequences, such as sequences (e.g., replication origins) and selective marker genes for regulating the replication of the vector in the host cell. Optionally, the vector includes a selective marker gene (see, e.g., U.S. Patent numbers 4,399,216, 4,634,665, and 5,179,017, which are incorporated herein by reference) that contributes to the selection of the host cell into which the vector has been introduced. For example, the selective marker gene can confer resistance to drugs (e.g., geneticin (G418), hygromycin, or methotrexate) to the host cell into which the vector has been introduced. For example, selective marker genes include dihydrofolate reductase (DHFR) genes (for use in DHFR-deficient host cells employing methotrexate selection / amplification), neomycin resistance (neo) genes (for G418 selection), and glutamate synthetase genes.

[0325] Host cells and methods for recombinant protein production

[0326] Any nucleic acid molecules of the coding anti-CD154 antibodies disclosed herein and the vectors comprising these nucleic acid molecules can be used for transfection of suitable mammals, plants or yeast host cells or transformed bacterial host cells. The method for transfecting / transforming host cells with polynucleotides is well known in the art. For example, the method for introducing heterologous polynucleotides into mammalian cells is well known in the art, and includes but is not limited to transfection mediated by dextran, calcium phosphate precipitation, transfection mediated by Polybrene, protoplast fusion, electroporation, polynucleotides are encapsulated in liposomes and DNA is directly microinjected into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. The method for transforming cells is 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, which are each incorporated herein by reference. The method for transforming plant cells is well known in the art, including, 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.

[0327] The 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-restrictive examples especially include: Chinese hamster ovary (CHO) cells, NSO 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 (for example, Hep G2), A549 cells, and many other cell lines. Other operable cell lines are insect cell lines, such as Sf9 or Sf21 cells. Plant host cells include, for example, tobacco, Arabidopsis thaliana, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia coli (E.coli) and Streptomyces (Streptomyces) species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris. It is within the skill of the art to select cell lines based on specific characteristics (e.g., expression levels and glycosylation patterns). 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, secreting the antibody into a culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium or the host cell using standard protein purification methods.

[0328] Furthermore, expression of the disclosed antibodies from production cell lines can be enhanced 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, for example, European Patent Nos. 0216846, 0256055, 0323997, and 0338841, each of which is incorporated herein by reference.

[0329] Antibodies expressed by different cell lines or in transgenic animals may have different glycosylation patterns.The antibodies disclosed herein may be expressed by any suitable host cell line and therefore may have any glycosylation pattern.

[0330] Pharmaceutical compositions and administration

[0331] A fourth aspect of the present disclosure provides a pharmaceutical composition comprising an anti-CD154 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an anti-CD154 antibody described herein.

[0332] The antibodies disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibodies 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 Pharmacopoeia: 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 is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.Pharmaceutical compositions can be prepared by mixing an antibody disclosed herein with an acceptable carrier, excipient, or stabilizer in the form of, for example, a lyophilized powder, a slurry, an aqueous solution, or a 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 of which is incorporated herein by reference).

[0333] 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 a composition similar to those used for passive immunization of humans. Optionally, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the mode of administration is intravitreal injection. The pharmaceutical composition can be administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection. Preparations for injection can exist in unit dosage form, such as in ampoules, prefilled syringes or multidose containers, with or without added preservatives. The pharmaceutical composition may take forms such as suspensions, solutions or emulsions (in oily or aqueous vehicles) and may contain formulation agents such as suspending agents, stabilizing agents and / or dispersing agents. Alternatively, the active ingredient may be prepared in powder form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

[0334] Typically, therapeutic compositions 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 structures suitable for high drug concentrations. Sterile injectable solutions can be prepared in the following manner: the anti-CD154 antibody is incorporated into a suitable solvent (having one of the ingredients listed above or its combination, as required) with the required amount, followed by filtration sterilization. Dispersion can be prepared in the following manner: the anti-CD154 antibody is incorporated into a sterile carrier comprising an alkaline dispersion medium and required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying, which produce the powder of the anti-CD154 antibody and any other desired composition from their solution previously aseptically filtered. The suitable fluidity of the solution can be, for example, by using a coating such as lecithin, by keeping required particle size (in the case of dispersion), and / or by maintaining a surfactant. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0335] The pharmaceutical composition can be administered by various 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.

[0336] In some embodiments, the pharmaceutical composition can be prepared with a carrier that will protect the antibody from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems JR Robinson, ed., Marcel Dekker, Inc., New York, 1978, which is incorporated herein by reference.

[0337] Alternatively, the active compound may be incorporated into the composition. In certain embodiments, the anti-CD154 antibody disclosed herein is formulated and / or administered in combination with one or more other therapeutic agents. These agents include, but are not limited to, antibodies, antithrombotic agents, antiplatelet agents, nonsteroidal anti-inflammatory drugs (NSAIDs) and antiallergic agents in conjunction with other targets. Such conjoint therapies may require lower dosages of the anti-CD154 antibody and the administered agents, thereby avoiding possible toxicity or complications associated with various monotherapies.

[0338] The dosing regime can be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and dosage consistency. As used herein, the term "dosage unit form" refers to physically dispersed units suitable as unit doses for mammalian subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in conjunction with the required amount of pharmaceutical carrier. Specifications for dosage unit form can be determined by and directly depend on the following aspects: (a) the unique characteristics of the anti-CD154 antibody and the specific therapeutic or prophylactic effect to be achieved; and (b) the inherent limitations of the technology for compounding such antibodies for treating sensitivity in individuals.

[0339] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the antibodies disclosed herein is 5 to 50 mg / kg. A therapeutically or prophylactically effective amount of the antibodies disclosed herein may be about 5 to about 50 mg / kg. In some embodiments, a therapeutically or prophylactically effective amount of the antibodies disclosed herein is 5 to 30 mg / kg. A therapeutically or prophylactically effective amount of the antibodies disclosed herein may be 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, the 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 may be adjusted over time according to individual needs and the professional judgment of the individual administering or supervising the administration of the composition, and the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the segmented compositions.

[0340] Therapeutic use

[0341] A fifth aspect of the present disclosure provides methods 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 an anti-CD154 antibody disclosed herein to a subject in need thereof. In some embodiments, a therapeutically effective amount of the anti-CD154 antibody is administered. Optionally, the anti-CD154 antibody is administered in the context of a pharmaceutical composition disclosed herein.

[0342] A sixth aspect of the present disclosure provides a method for suppressing an immune response in a subject. In some embodiments, the immune response is suppressed by administering an anti-CD154 antibody disclosed herein to a subject in need thereof. Optionally, a therapeutically effective amount of the anti-CD154 antibody is administered. In some embodiments, the anti-CD154 antibody is administered in the context of 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.

[0343] Any antibody disclosed in this article can be used therapeutically. In some embodiments, the anti-CD154 antibody is a human, chimeric or humanized antibody. Optionally, the subject is a human. The subject can be non-human, such as monkey. Optionally, the anti-CD154 antibody is a human antibody, and the subject is a human. In some embodiments, the anti-CD154 antibody is a humanized antibody, and the subject is a human. Alternatively, the subject can be a mammal (e.g., monkey) expressing CD154 (the anti-CD154 antibody cross-reacts with it). The antibody can be administered to a non-human mammal (e.g., cynomolgus macaque) expressing CD154 (the antibody cross-reacts with it), for veterinary purposes or as an animal model for human transplantation or disease. Such animal models can be useful for evaluating the therapeutic efficacy of the antibodies disclosed in this article.

[0344] The antibody can be used once. Optionally, the antibody can be used repeatedly. The antibody can be used three times a day to every six months or longer. The use can be on schedule, for example, 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 used continuously by a mini pump. The antibody can be used by mucosal, buccal, intranasal, inhalable, intravenous, intravitreal, subcutaneous, intramuscular, parenteral or tumor approach. In some embodiments, the anti-CD154 antibody is used systemically, for example, subcutaneously, intravenously, orally, via suction, transdermally or rectally. Optionally, the anti-CD154 antibody is used topically. In some embodiments, the anti-CD154 antibody is used in the intravitreal mode. 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. Typically, the antibody will be administered as long as the condition exists. Typically, the antibody will be administered as part of the pharmaceutical composition described above. The dosage of the antibody will typically be in the range of 5 to 50 mg / kg. The dosage of the antibody can be about 5 to about 50 mg / kg. In some embodiments, the dosage of the antibody is 5 to 30 mg / kg. The dosage of the antibody can be about 5 to about 30 mg / kg. Optionally, the dosage of the antibody is 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg. In some embodiments, the 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.

[0345] 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 allogeneic transplant, an autologous transplant, or a xenotransplant of a cell, tissue, or organ. The cell can be a modified cell or a cell amplified in vitro. For example, the cell can be modified by using one or more techniques such as transduction to express cDNA, 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, tumor infiltrating lymphocytes (TIL).

[0346] In some embodiments, any anti-CD154 antibody disclosed in this article can be used to prevent transplant rejection in the experimenter.For example, the anti-CD154 antibody can be used to prevent the acute or chronic humoral rejection of the transplanted cell, tissue or organ. The rejection can be the acute or chronic graft rejection in the transplant recipient of an allograft or xenotransplantation. The method disclosed in this article can promote the long-term graft survival of the transplanted cell, tissue or organ. Optionally, the long-term graft survival is at least 6 months after transplanting, at least 1 year after transplanting or at least 5 years after transplanting.

[0347] The transplant rejection may be associated with a transplant of hematopoietic cells or bone marrow, an allogeneic transplant of pancreatic islet cells, graft-versus-host disease, or a solid organ transplant selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a kidney-pancreas transplant, a heart-lung transplant, a kidney-heart transplant, a kidney-heart-pancreas transplant, a heart-liver transplant, a heart-liver-kidney transplant, a heart-lung-kidney transplant, a heart-lung-liver transplant, a lung-kidney transplant, a lung-liver transplant, a liver-intestine-pancreas transplant, an intestinal-pancreas transplant, a liver-kidney-intestine-pancreas transplant, and a kidney-intestine transplant.

[0348] 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, a transient ischemic attack (TIA), an aneurysm, or an intramural aneurysm.

[0349] 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, Sjögren'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 disorders, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, the dysfunctional immune response associated with viral-mediated diseases, cytokine release syndrome (CRS), cytokine storm syndrome, Behcet's disease, diabetic retinopathy (DR), diabetic macular edema (DME), age-related wet macular degeneration (AMD) and macular edema after retinal vein occlusion (MEfRVO). Immune-related diseases can also include allergic conditions, including but not limited to allergic rhinitis, asthma, atopic eczema, allergic reactions, insect venom allergic reactions, drug allergic reactions and food allergic reactions. 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 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 a cytokine storm in the host, cytokine release syndrome (CRS), or cytokine storm syndrome.

[0350] In some embodiments, the immune-related disease is a dysfunctional immune response associated with a viral infection. Optionally, the virus is selected from the group consisting of: cytomegalovirus, Epstein-Barr virus, influenza virus, smallpox virus, orthopoxvirus, coronavirus, such as SARS coronavirus (SARS-CoV), SARS-CoV-2 and 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.

[0351] Examples of neurodegenerative disorders that may be treated / prevented by the compositions / methods disclosed herein include, but are not limited to, Alzheimer's disease, traumatic brain injury (TBI), chronic traumatic encephalitis (CTE), and Parkinson's disease.

[0352] Examples of atherosclerotic conditions 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).

[0353] In some embodiments, described method comprises the anti-CD154 antibody disclosed in this article to described mammal administration therapeutic effective amount in combination with one or more other reagents.Optionally, described one or more other reagents are selected from the group consisting of the following: antithrombotic drug, antiplatelet drug and non-steroidal anti-inflammatory drug (NSAID).Described anti-CD154 antibody can be used simultaneously with described one or more other reagents.In some embodiments, described anti-CD154 antibody and described one or more other reagents are used sequentially.Optionally, described anti-CD154 antibody is used before described one or more other reagents.Described anti-CD154 antibody can be used after described one or more other reagents.In some embodiments, described anti-CD154 antibody is used in the composition identical with described one or more other reagents.Optionally, described anti-CD154 antibody and described one or more other reagents are used in the composition separated.

[0354] Examples of antithrombotic drugs include, but are not limited to, glycoprotein IIb / IIIa receptor antagonists, direct or indirect factor Xa inhibitors, and anticoagulants. Examples of anticoagulants include, but are not limited to, heparin, warfarin, rivaroxaban Ximelagatran Dabigatran Apixaban Edoxaban Enoxaparin and fondaparinux Examples of antithrombotic drugs are, but are not limited to, those described in U.S. Patent Nos. 4,782,069; 5,332,822; 5,492,895; 5,612,363; 5,691,364; 5,693,641; 5,721,214; 5,726,173; 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; 6,280,7 Examples of glycoprotein IIb / IIIa receptor antagonists include, but are not limited to, those disclosed in 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 in their entirety for all purposes). Rivaroxaban Apixaban Edoxaban Idrabiotaparinux, tirofiban and eptifibatide The direct or indirect coagulation factor Xa inhibitors include but are not limited to: apixaban Idrabiotaparinux, fondaparinux and rivaroxaban

[0355] Examples of antiplatelet drugs include, but are not limited to, TXA2 pathway inhibitors, adenosine diphosphate (ADP) pathway inhibitors, thrombin inhibitors, protease activated receptor-1 (PAR-1) inhibitors, and phosphodiesterase (PDE) inhibitors. Examples of ADP pathway inhibitors include, but are not limited to, clopidogrel. Ticlopidine Prasugrel Tigatran Cangrelo Non-limiting examples of PDE inhibitors include dipyridamole and cilostazol

[0356] 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 Tigatran and Cangrelo

[0357] In some embodiments, the anti-CD154 antibody can be used 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 used simultaneously with the one or more supplementary agents. In some embodiments, the anti-CD154 antibody and the one or more supplementary agents are used sequentially. Optionally, the anti-CD154 antibody is used before the one or more supplementary agents. The anti-CD154 antibody can be used after the one or more supplementary agents. In some embodiments, the anti-CD154 antibody is used in the composition identical with the one or more supplementary agents. Optionally, the anti-CD154 antibody and the one or more supplementary agents are used in a separate composition. The example of such one or more supplementary agents includes but is not limited to: anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD28 antibody, anti-CD52 antibody, anti-C5 antibody, mTOR inhibitor, calcineurin inhibitor, antiviral drug and a fusion peptide that is combined with CD28 and blocks 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

[0358] Non-limiting examples of immunosuppressive or immunomodulatory drugs include: cyclosporine A, tacrolimus (FK-506), doxorubicin Azathioprine Busulfan Cyclophosphamide Fludarabine, 5-fluorouracil, methotrexate ( TREXALL TM ), mycophenolate mofetil Mizoribine (BREDININ TM ), leflunomide, nonsteroidal anti-inflammatory drugs, corticosteroids, rapamycin Deoxyspergualin, FTY720, Muromonab-CD3 (ORTHOCLONE ), alemtuzumab Basiliximab Daclizumab Eculizumab Rituximab Bortezomib Siliguzumab, antithymocyte globulin leronlimab, siltuximbab sarilumab Tocilizumab Bevacizumab Ranibizumab Aflibercept and Bruton's tyrosine kinase (BTK) inhibitors, including zanubrutinib Acalabrutinib and ibrutinib

[0359] Examples of mTOR inhibitors include, but are not limited to, rapamycin Everolimus Temsirolimus ridaforolimus and deforolimus. Examples of calcineurin inhibitors include, but are not limited to, cyclosporine Tacrolimus (FK506, ) and pimecrolimus

[0360] Examples of antiallergic medications include, but are not limited to, antihistamines, decongestants, corticosteroids, mast cell stabilizers, leukotriene inhibitors, epinephrine injections, nitrous oxide, Sting Eye Drops nitrogen Sting nasal spray Beclomethasone ( and ), betamethasone Brompheniramine Budesonide ( and ), 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 medication Lodoxamide Loratadine Loteprednol Methylprednisolone Mometasone (ASMANEX ), naphazoline and pheniramine combination eye drops Nedocromil Olopatadine eye drops Oxymetazoline Pemirolast Prednisolone ( PRED ), prednisone ), tetrahydrozoline and Triamcinolone (NASACORT ALLERGY24 ).

[0361] 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, nitazoxanil, and remdesivir.

[0362] Methods for inducing chimerism

[0363] A 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 an anti-CD154 antibody disclosed herein to the recipient, 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 in the context of a pharmaceutical composition disclosed herein. The anti-CD154 antibody can be administered simultaneously with the transplantation of hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered sequentially with the transplantation of hematopoietic stem cells. Optionally, the anti-CD154 antibody is administered before 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.

[0364] In some embodiments, the anti-CD154 antibody is used with a dosage of 5-50mg / kg. The anti-CD154 antibody can be used with approximately 5 to approximately 50mg / kg. In some embodiments, the anti-CD154 antibody is used with a dosage of 5-30mg / kg. The anti-CD154 antibody can be used with approximately 5 to approximately 30mg / kg. Optionally, the anti-CD154 antibody is used with a dosage of 5mg / kg, 10mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg or 50mg / kg. In some embodiments, the anti-CD154 antibody is administered in an amount 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, via suction, transdermally or rectally. Optionally, the anti-CD154 antibody is administered topically.

[0365] In some embodiments, the transplant recipient is adapted before transplanting with stem cells. The anti-CD154 antibody can be administered simultaneously with the adaptation step. Optionally, the anti-CD154 antibody and the adaptation step are administered sequentially. In some embodiments, the anti-CD154 antibody is administered before the adaptation step. Optionally, the anti-CD154 antibody is administered after the adaptation step.

[0366] The method for adapting the recipient includes but is not limited to: whole 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 depletion antibodies, administration of cyclosporin A (CsA), administration of FK-506, administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of IL-15 receptor inhibitors, administration of rapamycin, administration of one or more anti-αβT 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 (MDR-101 cell therapy) derived from kidney donors, or a combination thereof. Non-limiting examples of one or more T cell depletion antibodies include: anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies and anti-CD33 antibodies or a combination thereof. In some embodiments, the transplant recipient has cancer. In some embodiments, the transplant is a bone marrow transplant.

[0367] Methods for inducing central tolerance

[0368] An eighth aspect of the present disclosure provides a method for inducing central tolerance in a transplant recipient. In some embodiments, the method comprises administering an anti-CD154 antibody disclosed herein to the recipient, transplanting hematopoietic stem cells into the recipient, and transplanting donor tissue into the recipient, wherein the transplanted hematopoietic stem cells produce immune cells that tolerate 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 in the context of a pharmaceutical composition disclosed herein. The anti-CD154 antibody can be administered simultaneously with the transplantation of hematopoietic stem cells. In some embodiments, the anti-CD154 antibody is administered sequentially with the transplantation of hematopoietic stem cells. Optionally, the anti-CD154 antibody is administered before 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-CD 154 antibody is administered in multiple doses.

[0369] In some embodiments, the anti-CD154 antibody is used with a dosage of 5-50mg / kg. The anti-CD154 antibody can be used with approximately 5 to approximately 50mg / kg. In some embodiments, the anti-CD154 antibody is used with a dosage of 5-30mg / kg. The anti-CD154 antibody can be used with approximately 5 to approximately 30mg / kg. Optionally, the anti-CD154 antibody is used with a dosage of 5mg / kg, 10mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg or 50mg / kg. In some embodiments, the anti-CD154 antibody is administered in an amount 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, via suction, transdermally or rectally. Optionally, the anti-CD154 antibody is administered topically.

[0370] In some embodiments, the transplant recipient is adapted before transplanting with stem cells. The anti-CD154 antibody can be administered simultaneously with the adaptation step. Optionally, the anti-CD154 antibody and the adaptation step are administered sequentially. In some embodiments, the anti-CD154 antibody is administered before the adaptation step. Optionally, the anti-CD154 antibody is administered after the adaptation step.

[0371] Methods for conditioning the recipient include, but are not limited to, whole 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 a combination thereof. Non-limiting examples of one or more T cell depleting antibodies include anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies, and anti-CD33 antibodies, or a combination thereof.

[0372] Methods for preventing xenotransplant rejection

[0373] A 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 within a pharmaceutical composition disclosed herein. The anti-CD154 antibody can be administered simultaneously with the xenograft. In some embodiments, the anti-CD154 antibody is administered sequentially with the xenograft. Optionally, the anti-CD154 antibody is administered before the xenograft. The anti-CD154 antibody can 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 30 mg / kg doses. Optionally, the anti-CD154 antibody is administered in four doses over two weeks. Optionally, the first dose of the anti-CD154 antibody is administered in two doses, wherein the first half dose is administered before the graft is revascularized, and the second half dose is administered once the xenograft procedure is confirmed to be successful.

[0374] In some embodiments, the anti-CD154 antibody is used at a dosage of 5 to 50 mg / kg. In some embodiments, the anti-CD154 antibody is used at a dosage of 5 to 30 mg / kg. In some embodiments, the anti-CD154 antibody is used at a dosage of 5 to 30 mg / kg. Optionally, the anti-CD154 antibody is used at a dosage 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 in an amount 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, via suction, transdermally or rectally. Optionally, the anti-CD154 antibody is administered topically.

[0375] The transplant recipient can be a 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, miniature pigs, and non-human primates. Optionally, the non-human donor is a pig or miniature pig that has been modified to reduce or eliminate the expression of one or more genes. In some embodiments, the one or more genes 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-acetylgalactosamine transferase (β4GalNT2) and class I MHC. Optionally, the one or more genes 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 miniature pig. The expression of the one or more genes can be reduced or eliminated by using CRISPR / Cas9 gene editing.

[0376] In some embodiments, the non-human donor can be modified 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 α-galactosidase, thrombomodulin, human anti-inflammatory proteins, and human CTLA-4-Ig, or a combination 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 thrombomodulin 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.

[0377] The xenograft rejection may be 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, intestinal-pancreas transplant, liver-kidney-intestine-pancreas transplant and kidney-intestine transplant. Example

[0378] The following examples are provided for illustrative purposes only and do not limit the scope of the present disclosure or paragraph in any way. Indeed, various modifications of the present disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the above description and fall within the scope of the paragraph. The following examples and preparations use the following abbreviations: "MSX" means Methionine Sulphoximine, "RU" means Ratio Units; "FACS" means Fluorescence Activated Cell Sorting; "MW" means Molecular Weight; "His-Tag" means C-terminal polyhistidine (6xHis) (SEQ ID NO:331) tag for rapid purification with nickel chelating resin and detection with anti-His (C-terminal) antibody; "BSA" denotes bovine serum albumin; "EDTA" denotes ethylenediaminetetraacetic acid; "DMSO" denotes dimethyl sulfoxide; "MOPS" denotes 3-(N-morpholino)propanesulfonic acid; "MES" denotes 2-(N-morpholino)ethanesulfonic acid; "PBS" denotes phosphate-buffered saline; "dPBS" denotes Dulbecco's phosphate-buffered saline; "HEMA" denotes 2-hydroxyethyl methacrylate; "DMEM" denotes Dulbecco's modified Eagle's medium; "FBS" denotes fetal bovine serum; "NEAA" denotes non-essential amino acids; "HEPES" denotes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; and "DMF" denotes dimethylformamide.

[0379] Example 1. Generation of a stable CHO library expressing anti-CD154 antibodies

[0380] DNA molecules encoding sequences for TNX01-TNX05, anti-CD154 antibodies with various Fc domains (SEQ ID NOs: 175-178 and 194), were codon-optimized for CHO cells (with the internal EcoRI and BamH1 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 in the final sequence as EcoRI and BamH1 restriction sites, respectively. The DNA molecules were cloned into pTT109 using EcoRI and BamH1. TM (National Research Council, Canada) plasmid and stably transfected into CHO55E1 TMCells (National Research Council, Canada). For all five constructs, 100 μg of technical plasmid was prepared in ddH2O. Stable pools were generated using 50 μM MSX as a selection agent. Stable pool generation was performed in duplicate using 50 μM MSX. Stable pools were generated in 6 ultra-deep well plates using Feed 4 (FUJIFILM IRVINE ) was used for fed-batch production. Then, Feed 12.7 (FUJIFILM IRVINE ) Stable pools were generated in shake flasks at a small scale of 100 ml. Figure 1 The titers and usability of five stable pools of CHO cells expressing anti-CD154 mAbs in a 100 mL fed-batch production are summarized. Table 1 summarizes the production of anti-CD154 mAbs (TNX01-05; light chain comprising SEQ ID NO: 196; heavy chain comprising SEQ ID NOs: 147-150 and 165, respectively) starting from the supernatant of stably transfected CHO cells as starting material. A similar process was used to generate TNX06-TNX13, anti-CD154 antibodies with various Fc domains (SEQ ID NOs: 316-319 and 293-296). Table 2 summarizes the production of anti-CD154 mAbs (TNX06-13; light chain comprising SEQ ID NO: 196; heavy chains comprising SEQ ID NOs: 166, 234, 284, 285, 159, 160, 281 and 282, respectively) starting from supernatants of stably transfected CHO cells as starting material.

[0381] Table 1. Antibody production starting from stable CHO cells (100 mL fed-batch production)

[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 antibodies

[0386] Anti-CD154 monoclonal antibodies (TNX01-TNX13) from Example 1 were purified by protein A affinity purification using 4x1 mL HiTrap MabSelect SuRe columns. The following steps were performed according to the manufacturer's instructions: (1) equilibration / washing with DPBS; (2) loading 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) elution with 0.1 M citrate at a pH of 3.0; (4) neutralization with 10% (v / v) 1 M HEPES buffer. The sample was buffer exchanged in DPBS using a 5 mL Zeba Spin desalting column (ThermoFisher Scientific, Waltham MA) and sterile filtered at 0.22 μm. No precipitation was observed during elution and neutralization. Table 3 below summarizes the antibody properties obtained after Protein A purification and desalting, and the yields of thirteen purified anti-CD154 monoclonal antibody samples obtained from the CHO55E1 stable pool. Note: TNX04 and TNX05 required 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 Sypro Ruby stained gels under non-reducing conditions, samples were analyzed by SDS PAGE. Once the final product was obtained, samples were also analyzed. Figure 2 As shown in , under non-reducing conditions, mAbs TNX01-TNX05 were estimated to have molecular weights of 160-170 kDa, with the exception of TNX04, which existed as free heavy chains (HC) and free light chains (LC). In TNX04, the heavy and light chains ran separately in denaturing SDS-PAGE under non-reducing conditions, given that the disulfide-forming cysteines (C220, C226, and C229) were mutated. UPLC-SEC results suggest that under physiological conditions, non-covalent interactions form intact mAbs. The lower molecular weight (LMW) bands may be due to artificial fragmentation of the mAbs during SDS-PAGE sample preparation. As shown in Figure 10 As shown in , mAbs TNX06-TNX13 were estimated to have molecular weights of 160-170 kDa, with the exception of TNX11, which behaved like a half-antibody and presumably formed a non-covalent 2HC-2LC structure.

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

[0391] Results: These results demonstrate that highly pure samples of the anti-CD 154 antibodies of the present disclosure were obtained.

[0392] Example 3. Use Anti-CD154 monoclonal antibody was detected by surface plasmon resonance (SPR). Binding of antibodies to CD40L

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

[0394] In particular, the commercial CD40L (SINO Cat#LC12AP2301) was injected onto the surface of protein A (2,000Rus; Cat#Z02201) The anti-CD154 antibody library (TNX01-05) was prepared on a plate in Piscataway, NJ, using PBST pH 7.4 (PBS with 0.05% Tween 20) as the run buffer. Protein A (Cat# Z02201) was used. The SPR antibody capture surface was prepared using 2000Rus of 10 μg / mL mAb (TNX01-TNX05) at a flow rate of 10 μL / min for 10 seconds. The analyte was CD40L (SINO 10239-H08E), which was resuspended according to the manufacturer's recommendations. Single cycle kinetics were performed with 4 concentrations (6.0 / 1.5 / 0.38 / nM 3-fold dilution, flow rate 75 μL / min, 10 seconds association, 1800 seconds dissociation. Regeneration was performed with 1 x 60s 10mM glycine pH 1.5, flow rate 30 μL / min.

[0395] use T200 evaluation software was used to process the raw data (n=3). Figure 3 Representative CD40L sensorgrams for TNX01-TNX05 are provided and a 1:1 binding model is shown. Since CD40L is a trimer, the sensorgrams deviate slightly from ideal binding behavior due to the possibility of affinity binding through this trimeric antigen (multiple binding sites). All fits should be considered "apparent" K DThe values ​​were not calculated because the sensorgrams were moving away from 1:1 binding behavior. CD40L binding was based on a calculated fit of the sensorgrams.

[0396] Figure 4 The apparent K values ​​calculated from SPR analysis are summarized. D The data were generated using the affinity values ​​(kd to ka ratio). TNX05 did not bind to the protein A surface as efficiently as TNX01-TNX04. This weaker binding likely represents a smaller affinity component in these sensorgrams. The lower surface density suggests that TNX05 molecules are further apart, which may equate to fewer sCD40L molecules engaging multiple antibody paratopes at any one time. TNX01-TNX05 has a lower Kd than TNX01-TNX04. D The binding affinity of the ligands was comparable to that of the ligands of interest and the measured on-rates were close to the lower limit of affinity measurements for this instrument platform.

[0397] For TNX06-TNX13, the sensor diagram uses The T200 evaluation software was used to generate and compare the sensorgram comparability method. This method is a statistical comparison of sensorgram shape only, independent of kinetic analysis, and was developed by GE Healthcare for the Biacore T200 platform. A mean 3SD window of the variation in sensorgram scatter plots was generated for one batch (TNX02) as a reference, which was then compared pairwise with the other variants. A similarity score was assessed for each pairwise comparison based on the number of points within the variation window and the distance to the points outside the reference window. Using this method, all samples were compared to a TNX02 scatter plot window collected in triplicate. All variants tested (TNX06-TNX13) showed approximately 80% similarity to the TNX02 sensorgram for sCD40L binding, with no significant deviations in on-rate or off-rate, indicating that they all bind in a similar manner. See Figure 11 .

[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 an affinity of approximately 10 pM (see Figure 4 TNX05 has slightly weaker binding, around 20 pM (see Figure 4 TNX06-13 were approximately 80% similar to TNX02 in the manner in which they bound to CD40L, with no significant deviations.

[0399] Example 4. Use Anti-CD154 monoclonal antibody was detected by surface plasmon resonance (SPR). Binding of antibodies to FcγRIA

[0400] According to the manufacturer's instructions, use Binding of the purified anti-CD154 antibody from Example 2 to high affinity FcγRIA (CD64) was measured by surface plasmon resonance (SPR) using a T200 instrument and sensor chip CM-5.

[0401] Specifically, FcγRIA was injected onto a protein A surface immobilized on a 2,000 Rus; Cat# Z02201 The anti-CD154 antibody collection library on Piscataway, NJ) was incubated with PBST pH 7.4 (PBS with 0.05% Tween 20) as a run-out 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 / min to generate approximately 250-400 RU antibody surfaces. The analyte FcγRIA (CD64; National Research Council, Canada) was resuspended in PBST containing 3.4 mM EDTA and 0.05% Tween 20 and used at a concentration range of 300 to 0.14 nM, at a flow rate of 100 μL / min, 90 seconds association time, and 1200 seconds dissociation time. Regeneration was performed with 1 x 60s 10 mM glycine pH 1.5 at a flow rate of 30 μL / min.

[0402] use T200 evaluation software was used to process the raw data (n=3). Figure 5 Representative FcγRIA sensorgrams for TNX01-TNX05 are provided, and a 1:1 binding model is shown.

[0403] Figure 6 The results of SPR analysis and calculated apparent K values ​​for TNX01-05 are summarized. D The data were generated based on the values ​​of affinity (ratio of Kd to Ka). Due to the high affinity of the ligand CD64 (TNX02 K D ~30pM), thus accurate K can be determined for all loss-of-function variants D 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 association and dissociation), which required steady-state endpoint analysis rather than kinetic analysis.

[0404] Figure 12a-12c summarizes the SPR analysis and calculated apparent K values ​​for TNX06-13 binding to FcγRIA. D The data were generated by analyzing the affinity values ​​(ratio of kd to ka). TNX06 to TNX13 were successfully tested for binding to FcgRIa. Due to the high affinity of the ligand CD64 (TNX02 K D ∼40 pM), thus allowing accurate K determination for all loss-of-function variants. D TNX06, 07, 11, 12, and 13 showed 6-12-fold weaker binding than TNX02. TNX08, 09, and 10 had 2600-, 3800-, and 1800-fold weaker binding, respectively (fast association and fast dissociation).

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

[0406] Example 5. Use Anti-CD154 monoclonal antibody was detected by surface plasmon resonance (SPR). Binding of antibodies to low-affinity FcγRs

[0407] According to the manufacturer's instructions, use T200 instrument and sensor chip CM-5, by surface plasmon resonance (SPR) to measure the binding of the purified anti-CD154 antibody from Example 2 to a low-affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF). The low-affinity FcγR antigen panel represents the most common allotypes of different receptors that have been used to evaluate the effector function 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, antigens in the FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) were injected onto the surface of protein A (2,000 Rus; Cat# Z02201 Piscataway, NJ) on an anti-CD154 antibody library (TNX01-05), using PBST pH 7.4 (PBS with 0.05% Tween 20) as a run-out 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 / min to generate approximately 200-500 RU antibody surface (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, at a flow rate of 25 μL / min, with 15-second association (CD32aH and CD32bF) and 40-second association (CD16aF and CD16aV), and a dissociation time of 120 seconds. Regeneration was performed using 1 x 60 s of 10 mM glycine pH 1.5 at a flow rate of 30 μL / min (SPR data not shown). Thus, TNX01-TNX05 was assayed for binding to the National Research Council Canada, Human Health Therapeutics Research Centre low-affinity FcγR panel (CD16aF, CD16aV, CD32aH, CD32bF) by measuring steady-state affinity.

[0409] Figure 7 The results of SPR analysis and calculated apparent K values ​​for TNX01-05 are summarized. D Data were generated using the values ​​of affinity (kd to ka ratio). In particular, TNX02 exhibited the expected wild-type binding, while the other variants showed significantly reduced binding. In particular, TNX01 and TNX03 showed almost no 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 13a -13b summarizes the SPR analysis and calculated apparent K values ​​for TNX06-13 binding to low-affinity FcγRs. DThe data generated by the analysis of the values ​​(affinity; ratio of kd to ka) were used. In particular, TNX02 exhibited the expected wild-type binding, while the other variants showed significant loss of function. In particular, TNX11 showed almost no binding activity for 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 for various low-affinity receptors. TNX08 showed a 2 to 17-fold loss of activity for various low-affinity receptors. TNX09 and 10 showed undetectable activity for all low-affinity receptors, with the exception of CD16aV (FcγRIIIaV), for which they all had a ~12-fold loss. TNX12 showed undetectable activity for all low-affinity receptors, with the exception of CD32bF (FcγRIIbF), for which there was an 8-fold loss. TNX13 showed undetectable activity against both CD16aV (FcγRIIIaV) and CD16aF (FcγRIIIaF), and had 6- and 17-fold loss 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 to or exhibit reduced binding to various FcγR low affinity receptors.

[0412] Example 6. Anti-CD154 Monoclonal Antibody Binding in Jurkat D1.1 and Jurkat Cells

[0413] The purified anti-CD154 antibody from Example 2 was evaluated for binding to Jurkat D1.1 cells (CD40L positive) and Jurkat cells (CD40L negative). 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. A high-throughput FACS cell binding assay was performed using the test antibodies (TNX01-TNX05) and a control antibody (mouse anti-human CD40L from (R&D MAB617)). As a negative control antibody of human IgG1 isotype, the detection reagents were AF488 F(ab')2 donkey anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch, Westgrove, PA) and goat anti-mouse IgG, Fcγ fragment specific (Jackson ImmunoResearch, Westgrove, PA). 5Jurkat or D1.1 cells were incubated with 0-200 nM primary antibody for 2 hours at 4°C and with detection secondary antibody for 1 hour at 4°C, followed by viability staining with propidium iodide (1% solution). Flow cytometry of the signals in the samples was acquired on the same day at LSR FORTESSA TM (BD Biosciences). Cells were washed and AlexaFluor 488 fluorescence was measured using a 488 nm laser as the excitation light source and a 530 / 30 nm bandpass filter. The median fluorescence intensity (MFI) was determined for 5,000 live single cells per sample. FACS cell binding assays were similarly performed for the test antibodies TNX06-TNX13.

[0414] As in Figure 8 As shown in (a), anti-CD154 antibodies (TNX01-TNX05) all bind to D1.1 cells with high affinity, while the negative control antibody No detectable binding to D1.1 cells. K was calculated by Hill slope fitting using specific binding parameters. D value. Figure 8 (a) On the contrary, Figure 8 (b) demonstrated that all anti-CD154 antibodies and negative control antibodies As shown in Figure 14(a), the anti-CD154 antibodies (TNX06-TNX13) all bind to D1.1 cells with high affinity, while the negative control antibody No detectable binding to D1.1 cells. K was calculated by Hill slope fitting using specific binding parameters. D value.

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

[0416] Example 7. Anti-CD154 monoclonal antibody platelet activation study

[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 triggers and enhances platelet activation in response to thrombosis stimulation. Further, the binding of the Fc domain of anti-CD40L mAb (e.g., hu5c8) to the activating FcgRIIa (CD32a) receptor on platelets leads to platelet activation and aggregation. Kojok K et al., J Am Heart Assoc. 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-capped 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 untreated blood from each donor (i.e., not incubated with anti-CD32a antibody) was also tested. Test conditions included: (1) soluble recombinant human CD40L (sCD40L) alone; (2) sCD40L complexed with human IgG1 anti-CD40L; (3) a direct platelet agonist, adenosine-5′-diphosphate sodium salt; along with detection antibodies for the platelet phenotyping 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 counts of each cell type, 50 μL of 123counteBeads (Invitrogen, NJ) were 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 molecules used in the platelet assay.

[0420]

[0421]

[0422] Once stained and fixed, samples were obtained 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 indication of size) and side scatter (SSC; an indication of granularity) characteristic spectra. Further, the activated and unactivated states of the 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 grouped, averaged, and analyzed using LibreOffice. TM The Calc software calculated the standard deviation and % coefficient of variation (CV) between replicates. From the generated Calc file, a table was generated that included the percentage of each cell population.

[0424] Next, the fold induction relative to the control was calculated for each treatment group in a platelet assay performed at the optimal molar ratio for each test antibody:sCD40L. The fold induction relative to the 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 that selectively block 5c8-IgG1 / sCD40L IC-induced platelet activation are expected to have no effect on platelet activation induced by ADP (a direct platelet agonist). 5c8-IgG1 (positive control) is expected to show platelet activation. The test antibodies of the present disclosure (TNX01-TNX13) 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 reduced ability to activate or aggregate platelets.

[0426] Example 8. Antibodies for the treatment of cardiac transplant rejection -CD154 Monoclonal antibodies

[0427] Heterotopic heart transplantation is carried out in cynomolgus monkeys. The heart is transplanted into the abdomen and connected to the descending aorta and vena cava. Anti-CD154 antibody TNX05 (hu5c8 mutated-IgG4 (IgG4 S228P / L235A)) is administered intravenously to the monkey. Four times in the first two weeks, 30mg / kg dosage is administered to the monkey. Half of the first dosage of anti-CD154 antibody TNX05 is administered before graft revascularization, and after confirming that the procedure is successful, the other half is administered. After the first two weeks, 10mg / kg dosage is administered to the monkey once a week for four weeks. Then, 20mg / kg is administered to the monkey once every four weeks. On the 84th day, the dosage is reduced from 20mg / kg to 15mg / kg.

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

[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 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 day 91. Two days after the biopsy, telemetry indicated declining cardiac graft function. Ultrasound confirmed decreased graft contractility. Abdominal exploration confirmed a weak beating graft. The graft was removed and the monkey was euthanized.

[0434] Autopsy revealed no evidence of thromboembolism outside the heart graft, where fresh thrombi confined to the ventricular cavity were found, as would be expected after the graft stops beating and the blood in the heart becomes stagnant. The lungs were free of infarction, and the brain and intestines were completely normal. These were the sites where thrombi were primarily observed after administration of prior art antibodies. The graft anastomosis was widely patent and free of any clots.

[0435] Results: These results demonstrate that the antibodies of the present invention are able to prevent transplant rejection while avoiding the problems associated with 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]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500] Sequence Listing <110> TONIX PHARMA HOLDINGS LIMITED <120> Anti-CD154 antibodies and their uses <130> 104545-0037-WO1 <140> <141> <150> 63 / 018,123 <151> 2020-04-30 <150> 62 / 869,489 <151> 2019-07-01 <160> 331 <170> PatentIn version 3.5 <210> 1 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 1 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 2 <211> 216 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 3 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 3 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 4 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 4 Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 5 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 5 Ala Pro Glu Phe Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly 210 215 <210> 6 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 6 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 7 <211> 216 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 7 Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 8 <211> 216 <212> PRT <213> Homo sapiens <400> 8 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly 210 215 <210> 9 <211> 215 <212> PRT <213> Homo sapiens <400> 9 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly 210 215 <210> 10 <211> 217 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 10 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 11 <211> 217 <212> PRT <213> Homo sapiens <400> 11 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 12 <211> 217 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic peptide" <400> 12 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 13 <211> 217 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic peptide" <400> 13 Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 14 <211> 217 <212> PRT <213> Artificial sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic peptide" <400> 14 Ala Pro Glu Phe Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 ...

Claims

1. An isolated antibody that binds to CD154, comprising a 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 region derived from IgG4 and comprising S228P and L235A amino acid modifications; wherein the VH comprises (a) a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, (b) a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and (c) a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:59; and wherein the VL comprises (a) a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, (b) a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and (c) a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

62.

2. The antibody of claim 1, wherein the antibody comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 196, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 151 or 165.

3. The antibody of claim 1, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 23 or 42.

4. The antibody of claim 1, wherein the heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 95 or 109.

5. The antibody of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:

64.

6. The antibody of claim 1, wherein the VL comprises the amino acid sequence of SEQ ID NO:

66.

7. The antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 or 165.

8. The antibody of claim 1, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

196.

9. An isolated nucleic acid molecule encoding the light chain and the heavy chain of the antibody of any one of claims 1-8.

10. 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 the antibody of any one of claims 1 to 8, respectively.

11. A vector comprising the isolated nucleic acid molecule of claim 9.

12. A first vector and a second vector, wherein the first vector comprises the first isolated nucleic acid molecule of claim 10, and the second vector comprises the second isolated nucleic acid molecule of claim 10.

13. A transformed cell comprising the isolated nucleic acid molecule of claim 9, the first and second isolated nucleic acid molecules of claim 10, the vector of claim 11, or the first and second vectors of claim 12.

14. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

15. Use of the antibody according to any one of claims 1 to 8 in the preparation of a medicament for (a) suppressing an immune response in a subject; (b) treating or preventing transplant rejection in a transplant recipient; or (c) Inhibition of xenograft rejection in transplant recipients.

16. The use of claim 15, wherein the immune response is a humoral response, an antibody-mediated response or a cell-mediated response.

17. The use of claim 16, 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.

18. The use of claim 15, wherein the immune response is a mixed humoral and cell-mediated response.

19. The use of claim 18, wherein the mixed humoral and cell-mediated 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.

20. The use of claim 15, wherein the subject is a human.

21. The use of claim 15, wherein the subject is non-human.

22. The use of claim 21, wherein the subject is a monkey.

23. The use of claim 15, wherein the subject has received or will receive a cell, tissue or organ transplant.

24. The use of claim 23, wherein the transplant is an allogeneic transplant, an autologous transplant or a xenotransplant.

25. The use according to claim 23, wherein the cell is a modified cell or a cell expanded ex vivo.

26. The use of claim 25, 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, CRISPR / Cas9 system, RNAi technology and retroviral technology.

27. The use of claim 26, wherein the cell is modified to express a chimeric antigen receptor (CAR) on its surface.

28. The use of claim 27, 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).

29. The use of claim 28, wherein the medicament is for treating or preventing transplant rejection in the subject.

30. The use of claim 29, wherein the transplant rejection is acute or chronic humoral rejection of the transplanted cells, tissues or organs.

31. The use of claim 29, wherein the transplant rejection is acute or chronic transplant rejection in an allogeneic or xenogeneic transplant recipient.

32. The use of claim 31, wherein the medicament promotes long-term graft survival of transplanted cells, tissues or organs, wherein the long-term graft survival is selected from the group consisting of: (a) At least 6 months after transplantation; (b) at least 1 year after transplantation; and (c) At least 5 years after transplantation.

33. The use of claim 32, wherein the transplant rejection is associated with a hematopoietic cell or bone marrow transplant, an allogeneic transplant of pancreatic islet cells, graft-versus-host disease, or a solid organ transplant selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a kidney-pancreas transplant, a heart-lung transplant, a kidney-heart transplant, a kidney-heart-pancreas transplant, a heart-liver transplant, a heart-liver-kidney transplant, a heart-lung-kidney transplant, a heart-lung-liver transplant, a lung-kidney transplant, a lung-liver transplant, a liver-intestine-pancreas transplant, an intestinal-pancreas transplant, a liver-kidney-intestine-pancreas transplant, and a kidney-intestine transplant.

34. The use of claim 20, wherein the subject has an immune-related disease, an atherosclerotic disorder, or a neurodegenerative disorder.

35. The use of claim 34, wherein the subject has had or is at risk of having a stroke, a transient ischemic attack (TIA), or an aneurysm.

36. The use of claim 34, wherein (a) 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, Sjögren'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, special Idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorder, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, Behcet's disease, diabetic retinopathy (DR), diabetic macular edema (DME), wet age-related macular degeneration (AMD), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), cytokine release syndrome (CRS), cytokine storm syndrome, acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, inflammation of lung tissue, chronic obstructive pulmonary disease (COPD), and macular edema after retinal vein occlusion (MEfRVO); (b) the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, traumatic brain injury (TBI), chronic traumatic encephalitis (CTE), amyotrophic lateral sclerosis (ALS) and Parkinson's disease; or (c) The atherosclerotic disorder is selected from the group consisting of angina pectoris, myocardial infarction, carotid artery stenosis, transient ischemic attack and cerebrovascular accident (CVA).

37. The use of claim 36, wherein the subject has an immune-related disease selected from the group consisting of systemic lupus erythematosus (SLE), multiple sclerosis, Sjögren's syndrome, and type I diabetes.

38. The use of claim 37, wherein the immune-related disease is SLE.

39. The use of claim 37, wherein the immune-related disease is multiple sclerosis.

40. The use of claim 37, wherein the immune-related disease is Sjögren's syndrome.

41. The use of claim 37, wherein the immune-related disease is type I diabetes.

42. The use according to claim 34, wherein the immune related disease is an allergic condition selected from the group consisting of allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergy, drug allergy and food allergy.

43. The use of claim 15, wherein the immune response is a primary response or a secondary response.

44. The use of claim 15, wherein the antibody is formulated for systemic administration.

45. The use of claim 44, wherein the antibody is formulated for administration subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally or rectally.

46. ​​The use of claim 15, wherein the antibody is formulated for topical administration.

47. The use of claim 15, wherein the antibody is to be administered in combination with one or more additional agents selected from the group consisting of antithrombotic drugs, antiplatelet drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), and antiallergic drugs.

48. The use of claim 47, wherein the antibody is to be administered before, after or simultaneously with the one or more additional agents.

49. The use of claim 15, wherein the antibody is to be administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

50. The use of claim 15, wherein the antibody is to be administered at a dose of 5-50 mg / kg.

51. The use of claim 15, wherein the transplant recipient is to be conditioned prior to transplantation with stem cells.

52. The use of claim 51, wherein the antibody is to be administered before the conditioning step, after the conditioning step or simultaneously with the conditioning step.

53. The use of claim 51 or 52, wherein the conditioning step is selected from the group consisting of: whole 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 tacrolimus (FK-506), administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of IL-15 receptor inhibitors, administration of rapamycin, administration of one or more anti-αβT cell receptor antibodies, and administration of one or more CD122 antagonists, administration of CD34+ hematopoietic stem cells and CD3+ T-cells derived from kidney donors, or a combination thereof.

54. The use of claim 53, wherein the one or more T cell depleting antibodies are selected from the group consisting of anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies and anti-CD33 antibodies or a combination thereof.

55. The use of claim 15, wherein the transplant recipient has cancer.

56. The use of claim 15, wherein the transplant is a bone marrow transplant.

57. The use of claim 15, wherein the xenograft is from a non-human donor selected from the group consisting of: pig and non-human primate.

58. The use of claim 57, wherein the non-human donor is a pig that has been engineered 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-acetylgalactosamine transferase (β4GalNT2) and class I MHC.

59. The use of claim 58, wherein the PERV is PERV A, PERV B or PERV C.

60. The use of claim 59, wherein the expression of all PERV genes has been eliminated in the pig.

61. The use of claim 60, wherein the expression of the one or more genes is reduced or eliminated by using CRISPR / Cas9 gene editing.

62. The use of claim 61, wherein the non-human donor has been engineered 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.

63. The use of claim 62, wherein the one or more human proteins are expressed in all tissues of the non-human donor.

64. The use of claim 62, wherein the one or more human proteins are expressed in a tissue-specific manner in the non-human donor.

65. The use of claim 62, wherein the complement regulatory protein is selected from the group consisting of human decay accelerating factor (CD55), membrane cofactor protein (CD46), and CD59.

66. The use of claim 62, wherein the thrombomodulin is selected from the group consisting of thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39 and CD73.

67. The use of claim 62, wherein the human anti-inflammatory protein is selected from the group consisting of heme oxygenase 1 (HO-1) and A20.

68. The use of claim 67, 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, intestinal-pancreas transplant, liver-kidney-intestine-pancreas transplant and kidney-intestine transplant.

69. Use of the pharmaceutical composition of claim 14 in the preparation of a medicament for (a) suppressing an immune response in a subject; (b) treating or preventing transplant rejection in a transplant recipient; or (c) Inhibition of xenograft rejection in transplant recipients.

70. The use of claim 69, wherein the immune response is a humoral response, an antibody-mediated response, or a cell-mediated response.

71. The use of claim 70, 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.

72. The use of claim 69, wherein the immune response is a mixed humoral and cell-mediated response.

73. The use of claim 72, wherein the mixed humoral and cell-mediated 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.

74. The use of claim 69, wherein the subject is a human.

75. The use of claim 69, wherein the subject is non-human.

76. The use of claim 75, wherein the subject is a monkey.

77. The use of claim 69, wherein the subject has received or will receive a cell, tissue or organ transplant.

78. The use of claim 77, wherein the transplant is an allogeneic transplant, an autologous transplant or a xenogeneic transplant.

79. The use of claim 77, wherein the cell is a modified cell or a cell expanded ex vivo.

80. The use of claim 79, 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, CRISPR / Cas9 system, RNAi technology and retroviral technology.

81. The use of claim 80, wherein the cell is modified to express a chimeric antigen receptor (CAR) on its surface.

82. The use of claim 81, 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).

83. The use of claim 82, wherein the medicament is for treating or preventing transplant rejection in the subject.

84. The use of claim 83, wherein the transplant rejection is acute or chronic humoral rejection of the transplanted cells, tissues or organs.

85. The use of claim 83, wherein the transplant rejection is acute or chronic transplant rejection in an allogeneic or xenotransplanted transplant recipient.

86. The use of claim 85, wherein the medicament promotes long-term graft survival of transplanted cells, tissues or organs, wherein the long-term graft survival is selected from the group consisting of: (a) At least 6 months after transplantation; (b) at least 1 year after transplantation; and (c) At least 5 years after transplantation.

87. The use of claim 86, wherein the transplant rejection is associated with a hematopoietic cell or bone marrow transplant, an allogeneic transplant of pancreatic islet cells, graft-versus-host disease, or a solid organ transplant selected from the group consisting of a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a pancreas transplant, a kidney-pancreas transplant, a heart-lung transplant, a kidney-heart transplant, a kidney-heart-pancreas transplant, a heart-liver transplant, a heart-liver-kidney transplant, a heart-lung-kidney transplant, a heart-lung-liver transplant, a lung-kidney transplant, a lung-liver transplant, a liver-intestine-pancreas transplant, an intestinal-pancreas transplant, a liver-kidney-intestine-pancreas transplant, and a kidney-intestine transplant.

88. The use of claim 74, wherein the subject has an immune-related disease, an atherosclerotic disorder, or a neurodegenerative disorder.

89. The use of claim 88, wherein the subject has had or is at risk of having a stroke, a transient ischemic attack (TIA), or an aneurysm.

90. The use of claim 88, wherein (a) 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, Sjögren'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, special Idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorder, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, Behcet's disease, diabetic retinopathy (DR), diabetic macular edema (DME), wet age-related macular degeneration (AMD), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), cytokine release syndrome (CRS), cytokine storm syndrome, acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, inflammation of lung tissue, chronic obstructive pulmonary disease (COPD), and macular edema after retinal vein occlusion (MEfRVO); (b) the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, traumatic brain injury (TBI), chronic traumatic encephalitis (CTE), amyotrophic lateral sclerosis (ALS) and Parkinson's disease; or (c) The atherosclerotic disorder is selected from the group consisting of angina pectoris, myocardial infarction, carotid artery stenosis, transient ischemic attack and cerebrovascular accident (CVA).

91. The use of claim 90, wherein the subject has an immune-related disease selected from the group consisting of systemic lupus erythematosus (SLE), multiple sclerosis, Sjögren's syndrome, and type I diabetes.

92. The use of claim 91, wherein the immune-related disease is SLE.

93. The use of claim 91, wherein the immune-related disease is multiple sclerosis.

94. The use of claim 91, wherein the immune-related disease is Sjögren's syndrome.

95. The use of claim 91, wherein the immune-related disease is type I diabetes.

96. Use according to claim 88, wherein the immune related disease is an allergic condition selected from the group consisting of allergic rhinitis, asthma, atopic eczema, anaphylaxis, insect venom allergy, drug allergy and food allergy.

97. The use of claim 69, wherein the immune response is a primary response or a secondary response.

98. The use of claim 69, wherein the antibody is formulated for systemic administration.

99. The use of claim 98, wherein the antibody is formulated for administration subcutaneously, intravenously, intravitreally, orally, via inhalation, transdermally or rectally.

100. The use of claim 69, wherein the antibody is formulated for topical administration.

101. The use of claim 69, wherein the antibody is to be administered in combination with one or more additional agents selected from the group consisting of: antithrombotic drugs, antiplatelet drugs, nonsteroidal anti-inflammatory drugs (NSAIDs) and antiallergic drugs.

102. The use of claim 101, wherein the antibody is to be administered before, after, or simultaneously with the one or more additional agents.

103. The use of claim 69, wherein the antibody is to be administered before, after, or simultaneously with the transplantation of hematopoietic stem cells.

104. The use of claim 69, wherein the antibody is to be administered at a dose of 5-50 mg / kg.

105. The use of claim 69, wherein the transplant recipient is to be conditioned prior to transplantation with stem cells.

106. The use of claim 105, wherein the antibody is to be administered before the conditioning step, after the conditioning step or simultaneously with the conditioning step.

107. The use of claim 105 or 106, wherein the conditioning step is selected from the group consisting of: whole 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 tacrolimus (FK-506), administration of one or more interleukin-2 (IL-2) receptor inhibitors, administration of IL-15 receptor inhibitors, administration of rapamycin, administration of one or more anti-αβT cell receptor antibodies, and administration of one or more CD122 antagonists, administration of CD34+ hematopoietic stem cells and CD3+ T-cells derived from kidney donors, or a combination thereof.

108. The use of claim 107, wherein the one or more T cell depleting antibodies are selected from the group consisting of anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45 antibodies, anti-CTLA4 antibodies, anti-CD20 antibodies and anti-CD33 antibodies, or a combination thereof.

109. The use of claim 69, wherein the transplant recipient has cancer.

110. The use of claim 69, wherein the transplant is a bone marrow transplant.

111. The use of claim 69, wherein the xenograft is from a non-human donor selected from the group consisting of: pig and non-human primate.

112. The use of claim 111, wherein the non-human donor is a pig that has been engineered 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-acetylgalactosamine transferase (β4GalNT2) and class I MHC.

113. The use of claim 112, wherein the PERV is PERV A, PERV B or PERV C.

114. The use of claim 113, wherein the expression of all PERV genes has been eliminated in the pig.

115. The use of claim 114, wherein the expression of the one or more genes is reduced or eliminated by using CRISPR / Cas9 gene editing.

116. The use of claim 115, wherein the non-human donor has been engineered 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.

117. The use of claim 116, wherein the one or more human proteins are expressed in all tissues of the non-human donor.

118. The use of claim 116, wherein the one or more human proteins are expressed in a tissue-specific manner in the non-human donor.

119. The use of claim 116, wherein the complement regulatory protein is selected from the group consisting of human decay accelerating factor (CD55), membrane cofactor protein (CD46) and CD59.

120. The use of claim 116, wherein the thrombomodulin is selected from the group consisting of thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39 and CD73.

121. The use of claim 116, wherein the human anti-inflammatory protein is selected from the group consisting of heme oxygenase 1 (HO-1) and A20.

122. The use of claim 121, 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, intestinal-pancreas transplant, liver-kidney-intestine-pancreas transplant and kidney-intestine transplant.

123. The use of any one of claims 57, 58, 60, 111, 112 and 114, wherein the pig is a miniature pig.

124. The use of claim 35 or 89, wherein the aneurysm is an intramural aneurysm.

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