Antibodies specifically recognizing cd40 and uses thereof

Abstract

Provided are antibodies or antigen-binding fragments that specifically recognize CD40, methods of making and uses thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202110793032.1, filed on July 14, 2021, entitled "Antibody that specifically recognizes CD40 and its application", the entire contents of which are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic sequence list (text name: CN_202106295612_SEQLIST.xml, record date: 2022.07.12, size: 141KB) are incorporated herein by reference in their entirety. Technical Field

[0005] This application provides antibodies or antigen-binding fragments that specifically recognize CD40, their preparation methods, and their uses. Background Technology

[0006] CD40 is a co-stimulatory member of the tumor necrosis factor receptor (TNFR) superfamily (Croft, Michael et al. Nature reviews. Drug discovery vol. 12, 2(2013): 147-68). The function of CD40 was first identified in human B lymphocytes, where it is constitutively expressed, and CD40 signaling promotes B cell activation and proliferation (Clark, E.A., and J.A. Ledbetter. Proceedings of the National Academy of Sciences of the United States of America vol. 83, 12(1986): 4494-8). CD40 is known to be expressed on other hematopoietic cells (e.g., monocytes and dendritic cells (DCs)) to promote cell survival and cytokine production (Stout, RD, and J. Suttles. Immunology today vol. 17, 10(1996): 487-92), and can also signal to induce CD40L expression in CD4+ cells. +T cell activation, proliferation, and cytokine production (Grewal, IS, and R A Flavell. Immunology Today vol. 17, 9 (1996): 410-4). CD40 signaling can also affect the cellular responses of non-hematopoietic cells, including endothelial cells, epithelial cells, fibroblasts, and neurons (Hollenbaugh, D et al. The Journal of Experimental Medicine vol. 182, 1 (1995): 33-40; Karmann, K et al. Proceedings of the National Academy of Sciences of the United States of America vol. 92, 10 (1995): 4342-6; and Tan, Jun et al. The EMBO Journal vol. 21, 4 (2002): 643-52).

[0007] CD40L was first developed from activated CD4. + CD40 has been identified on the surface of T cells (Hollenbaugh, D et al. The EMBO journal vol. 11, 12 (1992): 4313-21), and the CD40-CD40L interaction between B and T cells has shown to be crucial in germinal center (GC) responses and in response to T-dependent antigen allotype switching (Kawabe, T et al. Immunity vol. 1, 3 (1994): 167-78). Furthermore, CD40L has been shown to be expressed in a variety of cell types, including mast cells, basophils, B cells, natural killer (NK) cells, macrophages, megakaryocytes, and platelets, highlighting the broad role of the CD40 pathway in cell biology. U, and P Libby. Cellular and molecular life sciences: CMLS vol. 58, 1 (2001): 4-43; and van Kooten, C, and J Banchereau. Journal of leukocyte biology vol. 67, 1 (2000): 2-17).

[0008] In numerous animal models, the CD40L / CD40 interaction has been demonstrated to be associated with many diseases, including systemic lupus erythematosus (Davidson, Anne et al. Annals of the New York Academy of Sciences vol. 987 (2003): 188-98), experimental allergic encephalomyelitis (Toubi, Elias, and Yehuda Shoeenfeld. Autoimmunity vol. 37, 6-7 (2004): 457-64), collagen-induced arthritis (Durie, FH et al. Science (New York, NY) vol. 261, 5126 (1993): 1328-30), immune rejection (Goldwater, R et al. American journal of transplantation vol. 13, 4 (2013): 1040-1046), and atherosclerosis-mediated cardiovascular disease (Jansen, Matthijs F et al. Basic research in...). Cardiology vol. 111, 4(2016): 38), Sjögren's syndrome (Okimura, K et al. American journal of transplantation vol. 14, 6(2014): 1290-9), systemic sclerosis (Boleto, (E.G. Frontiers in immunology vol.9 2998.18 Dec.2018), immune thrombocytopenic purpura (Kuwana, Masataka et al. Blood vol.103,4(2004):1229-36), inflammatory bowel disease (Senhaji, Nezha et al. Frontiers in immunology vol.6 529.16 Oct.2015), diabetes (Balasa, B et al. Journal of immunology (Baltimore, Md.:1950) vol.159,9(1997):4620-7), RA (Gotoh, Haruhiko et al. The Journal of rheumatology vol.31,8(2004):1506-12), and cancer (Li, Rui et al. Molecular biology reports) vol. 39, 9 (2012): 8741-7; and Li, Rui et al. Immunology letters vol. 131, 2 (2010): 120-5).

[0009] Therefore, neutralizing the binding of CD40 and CD40L could be a method for treating diseases and symptoms mediated by the CD40 / CD40L pathway. Patent application WO200563981A1 discloses the anti-human CD40 antibody ASKP1240 (used as a control antibody in the examples section of this application); WO201265950A1 discloses the anti-human CD40 antibody CFZ533 (used as a control antibody in the examples section of this application).

[0010] All publications, patents, patent applications, and disclosed information in published patent applications mentioned herein are incorporated herein by reference in their entirety.

[0011] Application Overview

[0012] In some embodiments, any of the isolated anti-CD40 antibodies described above, the isolated anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V HIncludes: a heavy chain complementarity determination region (HC-CDR) 1, which contains GYX1VN (SEQ ID NO: 81), wherein X1 is L, T, or V; HC-CDR 2, which contains X1IX2PX3X4GX5TX6YNQKFKG (SEQ ID NO: 82), wherein X1 is L or M, X2 is A, N, or V, X3 is E, K, L, M, R, S, T, or Y, X4 is H, N, S, or T, X5 is G or R, and X6 is A, S, T, or W; and HC-CDR 3, which contains SX1X2X3X4PYAX5DY (SEQ ID NO: 81). NO:83), where X1 is G, I, L, M, P, R, S or Y, X2 is W or Y, X3 is S or V, X4 is D, K, L, N, R, T or V, X5 is A, G, M, N, Q or S; and a light chain variable structural domain (V L ), the V L Includes: Light chain complementarity determination region (LC-CDR)1, which contains KASQNV H TX1VA (SEQ ID NO:84), wherein X1 is A, H, N or R; LC-CDR2, which includes LX1SDRRT (SEQ ID NO:85), wherein X1 is A or R; and LC-CDR3, which includes LQX1WX2X3PLT (SEQ ID NO:86), wherein X1 is A or H, X2 is N, S or T, and X3 is A, Q, R, S or Y.

[0013] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-3 or variants thereof, the variants comprising substitutions of up to about 3 amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs:9-26 or variants thereof, the variants comprising substitutions of up to about 3 amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs:32-52 or variants thereof, the variants comprising substitutions of up to about 3 amino acids; and V L The V L It comprises: LC-CDR1, which comprises any of the amino acid sequences shown in SEQ ID NOs:58-61 or variants thereof, the variants comprising substitutions of up to about 3 amino acids; LC-CDR2, which comprises any of the amino acid sequences shown in SEQ ID NOs:65-66 or variants thereof, the variants comprising substitutions of up to about 3 amino acids; and LC-CDR3, which comprises any of the amino acid sequences shown in SEQ ID NOs:70-77 or variants thereof, the variants comprising substitutions of up to about 3 amino acids.

[0014] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H V containing any of the amino acid sequences shown in SEQ ID NOs:87-119 H Includes HC-CDR1, HC-CDR2, and HC-CDR3, as well as V L The V L V containing any of the amino acid sequences shown in SEQ ID NOs:126-138 L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0015] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: (i) V H It contains V as shown in the amino acid sequence SEQ ID NO:87. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:126. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (ii)V H It contains V as shown in the amino acid sequence SEQ ID NO:88. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:127. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (iii)V H It contains V as shown in the amino acid sequence SEQ ID NO:89. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (iv)V H It contains V as shown in the amino acid sequence SEQ ID NO:94. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (v)V HIt contains V as shown in the amino acid sequence SEQ ID NO:96. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:132. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (vi)V H It contains V as shown in the amino acid sequence SEQ ID NO:101. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (vii)V H It contains V as shown in the amino acid sequence SEQ ID NO:110. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L Includes LC-CDR1, LC-CDR2 and LC-CDR3; (viii)V H It contains V as shown in the amino acid sequence SEQ ID NO:114. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L Includes LC-CDR1, LC-CDR2, and LC-CDR3; (ix)V H It contains V as shown in the amino acid sequence SEQ ID NO:115. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:129. L Includes LC-CDR1, LC-CDR2, and LC-CDR3; or (x)V H It contains V as shown in the amino acid sequence SEQ ID NO:117. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:129. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0016] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: (i) V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:9; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:32, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:58; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L (ii) V variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:10; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:32, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L (iii) V variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:13; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:34, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L (iv) V variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:37, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:73, or the V L The variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; (v)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:20; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:42, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L The variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; (vi)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:14; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:43, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V LThe V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L The variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; (vii)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:51, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70, or the V L The variants, whose LC-CDRs contain substitutions of up to about 5 amino acids; (viii)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:11; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:48, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:71, or the V L Variants whose LC-CDRs contain substitutions of up to about 5 amino acids; or (ix)V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:18; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:47, or the V HVariants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:71, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0017] In some embodiments, any of the isolated anti-CD40 antibodies described above, the isolated anti-CD40 antibody comprises: V H It comprises any of the amino acid sequences shown in SEQ ID NOs:87-119 or variants thereof, said variants having at least about 90% sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-119; and V L It comprises any of the amino acid sequences shown in SEQ ID NOs:126-138 or variants thereof, wherein the variants have at least about 90% sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-138.

[0018] In some embodiments, an isolated anti-CD40 antibody comprises: (i) V H It comprises the amino acid sequence SEQ ID NO:87 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:87; and V L It contains the amino acid sequence SEQ ID NO:126 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:126; (ii) V H It comprises the amino acid sequence SEQ ID NO:88 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:88; and V L It contains the amino acid sequence SEQ ID NO:127 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:127; (iii) V H It comprises the amino acid sequence SEQ ID NO:89 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:89; and V L It contains the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128; (iv)VH It comprises the amino acid sequence SEQ ID NO:94 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:94; and V L It contains the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128; (v)V H It comprises the amino acid sequence SEQ ID NO:96 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:96; and V L It contains the amino acid sequence SEQ ID NO:132 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:132; (vi)V H It comprises the amino acid sequence SEQ ID NO:101 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:101; and V L It contains the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128; (vii)V H It comprises the amino acid sequence SEQ ID NO:110 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:110; and V L It contains the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128; (viii)V H It comprises the amino acid sequence SEQ ID NO:114 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:114; and V L It contains the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128; (ix)V H It comprises the amino acid sequence SEQ ID NO:115 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:115; and V L It contains the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129; or (x)V HIt comprises the amino acid sequence SEQ ID NO:117 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:117; and V L It contains the amino acid sequence SEQ ID NO:129 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129.

[0019] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:120. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:139. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0020] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:53, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:78, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0021] In some embodiments, any of the isolated anti-CD40 antibodies described above, the isolated anti-CD40 antibody comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:120 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:120; and V L It contains the amino acid sequence shown in SEQ ID NO:139 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:139.

[0022] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:121. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:140. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0023] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:5; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:54, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:78, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0024] In some embodiments, any isolated anti-CD40 antibody as described above comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:121 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:121; and V L It contains the amino acid sequence shown in SEQ ID NO:140 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:140.

[0025] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:122. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:141.L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0026] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:6; HC-CDR2, which contains the amino acid sequence SEQ ID NO:28; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:55, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:63; LC-CDR2, which contains the amino acid sequence SEQ ID NO:68; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:79, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0027] In some embodiments, any isolated anti-CD40 antibody as described above comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:122 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:122; and V L It contains the amino acid sequence shown in SEQ ID NO:141 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:141.

[0028] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:123. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:142. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0029] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V HContains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:7; HC-CDR2, which contains the amino acid sequence SEQ ID NO:29; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:56, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:64; LC-CDR2, which contains the amino acid sequence SEQ ID NO:69; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:80, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0030] In some embodiments, any isolated anti-CD40 antibody as described above comprises: V H It comprises the amino acid sequence shown in SEQ ID NO: 123 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 123; and V L It contains the amino acid sequence shown in SEQ ID NO:142 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:142.

[0031] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:124. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:143. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0032] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:8; HC-CDR2, which contains the amino acid sequence SEQ ID NO:30; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:57, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:80, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0033] In some embodiments, any isolated anti-CD40 antibody as described above comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:124 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:124; and V L It contains the amino acid sequence shown in SEQ ID NO:143 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:143.

[0034] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H It contains V as shown in the amino acid sequence SEQ ID NO:125. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:144. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0035] In some embodiments, an isolated anti-CD40 antibody is provided, comprising: V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:31; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:53, or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:68; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:79, or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0036] In some embodiments, any isolated anti-CD40 antibody as described above comprises: V H It comprises the amino acid sequence shown in SEQ ID NO:125 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:125; and V L It contains the amino acid sequence shown in SEQ ID NO:144 or a variant thereof, the variant having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:144.

[0037] In some embodiments, the Kd value of the isolated anti-CD40 antibody binding to human CD40 is 0.1 pM to 1 nM.

[0038] In some embodiments, an isolated anti-CD40 antibody is provided that competes with any of the isolated anti-CD40 antibodies described above for specific binding to CD40. In some embodiments, an isolated anti-CD40 antibody is provided that specifically binds to the same epitope as any of the isolated anti-CD40 antibodies described above.

[0039] In some embodiments, the isolated anti-CD40 antibody, as described above, comprises an Fc fragment. In some embodiments, the isolated anti-CD40 antibody is a full-length IgG antibody. In some embodiments, the isolated anti-CD40 antibody is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated anti-CD40 antibody is a chimeric, fully human, or humanized antibody. In some embodiments, the isolated anti-CD40 antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, diabody, and linear antibody.

[0040] In some embodiments, an isolated nucleic acid molecule is provided, the nucleic acid molecule encoding any of the anti-CD40 antibodies described above. In some embodiments, a vector is provided, the vector comprising any of the nucleic acid molecules described above. In some embodiments, a host cell is provided, the host cell comprising any of the anti-CD40 antibodies described above, any of the nucleic acid molecules described above, or any of the vectors described above. In some embodiments, a method for preparing an anti-CD40 antibody is provided, comprising: a) culturing any of the above-described host cells under conditions that enable efficient expression of the anti-CD40 antibody; and b) obtaining the expressed anti-CD40 antibody from the host cell.

[0041] In some embodiments, a method of treating a desired individual disease or condition is provided, comprising administering an effective amount of any of the anti-CD40 antibodies described above to the individual. In some embodiments, the use of any of the anti-CD40 antibodies described above in the preparation of a pharmaceutical composition for treating a desired individual disease or condition is provided. In some embodiments, the use of any of the anti-CD40 antibodies described above or a pharmaceutical composition comprising an anti-CD40 antibody in the preparation of a medicament for treating a disease or condition is provided. In some embodiments, the disease or condition is related to the CD40 signaling pathway, including autoimmune diseases and / or inflammatory conditions and / or cancer and / or transplant-related diseases or conditions. In some embodiments, the disease or condition is selected from, for example, systemic lupus erythematosus (SLE), discoid lupus erythematosus, lupus nephritis, inflammatory arthritis: including but not limited to juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis and gouty arthritis, organ or tissue transplant rejection: hyperacute, acute or chronic rejection and / or graft-versus-host disease, multiple sclerosis, hyperIgE syndrome, polyinflammatory nodules, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, celiac disease (gluten-sensitive bowel disease), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, scleroderma, myasthenia gravis, idiopathic thrombocytopenic purpura, autoimmune thyroiditis, Grave's disease, Hashimoto's thyroiditis. Thyroiditis, immune complex diseases, chronic fatigue immune dysfunction syndrome (CFIDS), polymyositis, dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis, type I and type II diabetes, delayed-type hypersensitivity reactions of types 1, 2, 3 and 4, allergic or anaphylactic diseases, unwanted / accidental immune responses to therapeutic proteins (e.g., factor VII in hemophilia), asthma, Rich-Strauss syndrome (allergic granulomatous disease), atopic dermatitis, allergic and irritant contact dermatitis, urticaria, IgE-mediated allergy, atherosclerosis, vasculitis, idiopathic inflammatory myopathy, hemolytic diseases, Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, Sjögren's syndrome Diseases including psoriasis, hidradenitis suppurativa, purulent urethritis, sarcoidosis, ulcerative colitis, and tumors including but not limited to non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma, high / intermediate / low-grade B-cell lymphoma, acute B-cell lymphoblastic leukemia, granulocytic leukemia, and Hodgkin's disease; nephritis, acute respiratory distress syndrome, epididymitis, glomerulonephritis, and hyperthyroidism.

[0042] It also provides pharmaceutical compositions, kits, and manufactured products containing any of the anti-CD40 antibodies described above. Attached Figure Description

[0043] Figure 1A The results show that, compared with the control antibody ASKP1240, anti-CD40 antibodies D1, D6, or D7 inhibited human CD40L-induced CD95 expression in Ramos cells. Figure 1B The results show that after cross-linking with anti-immunoglobulin antibodies, anti-CD40 antibodies D1 and D7 and control antibody ASKP1240 have almost no side effects of activating the CD40 pathway, but control antibody CFZ533 shows some agonistic activity.

[0044] Figure 2 The results show that, compared with the control antibody ASKP1240, anti-CD40 antibodies D1, D6, or D7 inhibited CD40L-induced CD23 expression in PBMCs.

[0045] Figures 3A-3B The results show that, compared with the control antibody ASKP1240, anti-CD40 antibodies D1, D6, or D7 inhibited the secretion of CD40L-induced cytokines in iDCs. Figure 3A The results show that anti-CD40 antibodies D1, D6, or D7 inhibit CD40L-induced IL12p40 secretion in iDCs. Figure 3B The results show that anti-CD40 antibodies D1, D6, or D7 inhibit CD40L-induced TNF-α secretion in iDCs.

[0046] Figure 4 The results show that, compared with the control antibody ASKP1240, the optimized anti-CD40 antibodies D21, D26, D33, D42, D47, and D49 inhibited the activation of the human CD40L-induced NF-κB signaling pathway.

[0047] Figures 5A-5B The results shown demonstrate the binding affinity of the optimized anti-CD40 antibody to CD40 from different species compared to the control antibody ASKP1240. Figure 5A The figure shows the binding curves of the optimized anti-CD40 antibodies D21, D26, D33, D42, D47, and D49 to human CD40. Figure 5B The figure shows the binding curves of the optimized anti-CD40 antibodies D21, D26, D33, D42, D47, and D49 to CD40 in rhesus monkeys.

[0048] Figure 6The results show that, compared with the control antibody ASKP1240, the optimized anti-CD40 antibodies D21, D26, D33, D42, D47, and D49 inhibited human CD40L-induced CD95 expression in Ramos cells.

[0049] Figure 7 The results show that, compared with the control antibody ASKP1240, the optimized anti-CD40 antibodies D33, D42, and D47 inhibited CD40L-induced CD23 expression in PBMCs.

[0050] Figures 8A-8D The results shown demonstrate the blocking effect of anti-CD40 antibody on T cell-dependent antibody responses compared to the control antibody ASKP1240. Figure 8A The results show that the optimized anti-CD40 antibodies D33, D42, or D47 almost completely blocked the production of IgG specific to the MASP2 antigen at a dose of 3 mpk. Figure 8B The results show that the optimized anti-CD40 antibodies D33, D42, or D47 almost completely blocked the production of IgM specific to the MASP2 antigen at a dose of 3 mpk. Figure 8C The results show that the optimized anti-CD40 antibodies D33, D42, or D47 almost completely blocked the production of IgG specific to the KLH antigen at a dose of 1 mpk. Figure 8D The results show that the optimized anti-CD40 antibodies D33, D42, or D47 almost completely blocked the production of IgM specific to the KLH antigen at a dose of 1 mpk.

[0051] Figure 9 The results shown are the serum concentration curves of the optimized anti-CD40 antibodies D33, D42, or D47 in rats compared with the control antibody ASKP1240.

[0052] Detailed description of this application

[0053] This application provides an anti-CD40 antibody molecule. Through a combination of hybridoma technology, scFv phage library screening, humanization technology, affinity maturation, and appropriately designed biochemical and biological experiments, a highly efficient antibody molecule capable of binding to human CD40 and inhibiting the interaction between human CD40 and its receptor has been identified. The results presented herein demonstrate that, compared to the known anti-CD40 antibody ASKP1240, the antibody of this application binds to human and / or monkey CD40 with high affinity, and surprisingly, various biological experiments have shown that the antibody of this application is even more effective than ASKP1240.

[0054] The anti-CD40 antibodies provided in this application include, for example, full-length anti-CD40 antibodies, anti-CD40 single-chain antibodies (scFvs), anti-CD40Fc fusion proteins, multispecific (e.g., bispecific) anti-CD40 antibodies, anti-CD40 immunoconjugates, and the like.

[0055] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Includes: a heavy chain complementarity determination region (HC-CDR) 1, which contains GYX1VN (SEQ ID NO: 81), wherein X1 is L, T, or V; HC-CDR 2, which contains X1IX2PX3X4GX5TX6YNQKFKG (SEQ ID NO: 82), wherein X1 is L or M, X2 is A, N, or V, X3 is E, K, L, M, R, S, T, or Y, X4 is H, N, S, or T, X5 is G or R, and X6 is A, S, T, or W; and HC-CDR 3, which contains SX1X2X3X4PYAX5DY (SEQ ID NO: 81). NO:83), where X1 is G, I, L, M, P, R, S or Y, X2 is W or Y, X3 is S or V, X4 is D, K, L, N, R, T or V, X5 is A, G, M, N, Q or S; and a light chain variable structural domain (V L ), the V L Includes: Light chain complementarity determination region (LC-CDR)1, which contains KASQNV H TX1VA (SEQ ID NO:84), wherein X1 is A, H, N or R; LC-CDR2, which includes LX1SDRRT (SEQ ID NO:85), wherein X1 is A or R; and LC-CDR3, which includes LQX1WX2X3PLT (SEQ ID NO:86), wherein X1 is A or H, X2 is N, S or T, and X3 is A, Q, R, S or Y.

[0056] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Includes: heavy chain complementarity determination region (HC-CDR)1, which contains DAYMH (SEQ ID NO:4); HC-CDR2, which contains RIDPANGNTKYDPKFQG (SEQ ID NO:27); and HC-CDR3, which contains NYYGSRYPFAY (SEQ ID NO:53); and light chain variable domain (V L ), the V LIt includes: LC-CDR1, which contains SASSVSYMH (SEQ ID NO:62); LC-CDR2, which contains STSNLAS (SEQ ID NO:67); and LC-CDR3, which contains QQGSSYPLT (SEQ ID NO:78).

[0057] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H Heavy chain complementarity determination region (HC-CDR) 1, the V H Contains: DSYMH (SEQ ID NO:5); HC-CDR2, which contains RIDPANGNTKYDPKFQG (SEQ ID NO:27); and HC-CDR3, which contains NYYGAAYPFAY (SEQ ID NO:54); and a light chain variable structural domain (V L ), the V L It includes: LC-CDR1, which contains SASSVSYMH (SEQ ID NO:62); LC-CDR2, which contains STSNLAS (SEQ ID NO:67); and LC-CDR3, which contains QQGSSYPLT (SEQ ID NO:78).

[0058] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Contains: heavy chain complementarity-determining region (HC-CDR)1, which contains DSYIH (SEQ ID NO:6); HC-CDR2, which contains RIDPTNGNTNFDPKFRG (SEQ ID NO:28); and HC-CDR3, which contains NYYGSRYPFSY (SEQ ID NO:55); and light chain variable domain (V L ), the V L It includes: LC-CDR1, which contains SGSSSVSYMH (SEQ ID NO:63); LC-CDR2, which contains FTSNLAS (SEQ ID NO:68); and LC-CDR3, which contains QQGSTYPLT (SEQ ID NO:79).

[0059] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V HIncludes: heavy chain complementarity-determining region (HC-CDR)1, which contains DHGMH (SEQ ID NO:7); HC-CDR2, which contains VISTSYGVATYNQKFKG (SEQ ID NO:29); and HC-CDR3, which contains RDSKSPFDY (SEQ ID NO:56); and light chain variable domain (V L ), the V L It includes: LC-CDR1, which contains SASSSISSNYLH (SEQ ID NO:64); LC-CDR2, which contains RTSNLAS (SEQ ID NO:69); and LC-CDR3, which contains HQYHRSPLT (SEQ ID NO:80).

[0060] On the one hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Includes: heavy chain complementarity-determining region (HC-CDR)1, which contains DHAIH (SEQ ID NO:8); HC-CDR2, which contains AISTNYGAATYNQKFKG (SEQ ID NO:30); and HC-CDR3, which contains RGSHGDWFAY (SEQ ID NO:57); and light chain variable domain (V L ), the V L It includes: LC-CDR1, which contains SASSVSYMH (SEQ ID NO:62); LC-CDR2, which contains STSNLAS (SEQ ID NO:67); and LC-CDR3, which contains HQYHRSPLT (SEQ ID NO:80).

[0061] On the other hand, this application provides an anti-CD40 antibody, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Includes: heavy chain complementarity determination region (HC-CDR)1, which contains DAYMH (SEQ ID NO:4); HC-CDR2, which contains RIDPANGYTKSDPKFQG (SEQ ID NO:31); and HC-CDR3, which contains NYYGSRYPFAY (SEQ ID NO:53); and light chain variable structural domain (V L ), the V LIt includes: LC-CDR1, which contains SASSVSYMH (SEQ ID NO:62); LC-CDR2, which contains FTSNLAS (SEQ ID NO:68); and LC-CDR3, which contains QQGSTYPLT (SEQ ID NO:79).

[0062] It also provides nucleic acids encoding anti-CD40 antibodies, compositions containing anti-CD40 antibodies, and methods for preparing and using anti-CD40 antibodies.

[0063] definition

[0064] As described herein, “treatment” or “treating” is a method of obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, the beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction of disease severity, stabilization of the disease (e.g., prevention or delay of disease exacerbation), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, relief of the disease (partial or complete), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, improvement or enhancement of quality of life, weight gain, and / or prolonged survival. Simultaneously, “treatment” also includes a reduction in disease pathological outcomes (e.g., tumor volume in the case of cancer). The methods of this application consider any one or more aspects of these treatments.

[0065] The term "antibody" includes both full-length antibodies and their antigen-binding fragments. Full-length antibodies consist of two heavy chains and two light chains. Variable regions in both the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include three hypervariable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by having α, δ, ε, γ, and μ-type heavy chains, respectively. Several major antibody classes are subclassed, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0066] As described herein, the term "antigen-binding fragment" includes antibody fragments, such as diabody, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds-diabody), single-chain Fv (scFv), scFv dimers (bivalent diabody), multispecific antibodies, single-domain antibodies, nanobodies, domain antibodies, divalent domain antibodies, or any other antibody fragment capable of binding to an antigen but not containing a complete antibody structure. The antigen-binding fragment is capable of binding the same antigen to a parent antibody or a parent antibody fragment (such as a parental scFv). The antigen-binding fragment also includes fusion proteins comprising the antibody fragments described above. In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody that have been grafted into a frame region from one or more different human antibodies.

[0067] As described herein, the term "epitope" refers to a specific set of atoms or amino acids on an antigen that an antibody or antibody portion binds to. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen.

[0068] As used herein, when the first antibody inhibits the binding of the second antibody to the CD40 target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at an equimolar concentration, the first antibody “competitively” binds to the CD40 target with the second antibody, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody “epitope classification” method based on cross-competition.

[0069] As described herein, the terms "specifically bind," "specifically recognize," or "specific to" refer to measurable and reproducible interactions, such as antibody binding to a target that confirms the presence of that target in a heterogeneous group of molecules, including biomolecules. For example, an antibody's ability to specifically recognize a target (which may be an epitope) means that the antibody binds to that target with higher affinity, stronger binding, and / or more persistent binding compared to binding to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity at least 10 times greater than its binding affinity to other targets.

[0070] As described herein, an “isolated” anti-CD40 antibody is an anti-CD40 antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins of the same origin, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0071] As described herein, the term “isolated nucleic acid” refers to a nucleic acid or combination thereof of genomic, cDNA, or synthetic origin. Depending on its origin, “isolated nucleic acid” means that it is (1) unrelated to all or part of the polynucleotides in “isolated nucleic acids” found in nature, (2) operablely ligable to polynucleotides not naturally associated with it, or (3) not present in nature as part of a longer sequence.

[0072] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable domains of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions have been described in al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlaps or subsets of amino acid residues when compared with each other. However, any definition used to indicate a CDR of an antibody or transplanted antibody or its variants is included within the scope of the terminology defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the aforementioned references for comparison. Algorithms and interfaces for CDR prediction are known in the art, including, for example, those described in Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The entire contents of the references cited in this paragraph are incorporated herein by reference for the purposes of this application and one or more claims that may be included herein.

[0073] Table 1: CDR Definition

[0074] <![CDATA[V H CDR1]]> 31-35 26-32 30-35 27-38 25-40 <![CDATA[V H CDR2]]> 50-65 53-55 47-58 56-65 58-77 <![CDATA[V H CDR3]]> 95-102 96-101 93-101 105-117 109-137 <![CDATA[V L CDR1]]> 24-34 26-32 30-36 27-38 25-40 <![CDATA[V L CDR2]]> 50-56 50-52 46-55 56-65 58-77 <![CDATA[V L CDR3]]> 89-97 91-96 89-96 105-117 109-137

[0075] 1 The amino acid residue numbering follows the nomenclature method described in Kabat et al. above.

[0076] 2 The amino acid residue numbering follows the nomenclature method described in Chothia et al. above.

[0077] 3 The amino acid residue numbering follows the nomenclature method described in MacCallum et al. above.

[0078] 4 The amino acid residue numbering follows the nomenclature method described in Lefranc et al. above.

[0079] 5 The amino acid residue numbering follows the nomenclature method described in Honegger and Plückthun.

[0080] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody species or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody species or subclass, as well as fragments of such antibodies, provided they possess the biological activity described in this application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0081] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly and non-covalently linked. The folding of these two domains derives six hypervariable rings (three in the light chain and three in the heavy chain), which provide the antibody with amino acid residues for binding to the antigen and confer the antibody's specificity for antigen binding. However, even a single variable domain (or half of the Fv fragment, containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity is lower than that of the complete binding site.

[0082] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a type of Fv containing V molecules linked together to form a single polypeptide chain. H and V L Antibody fragments of antibody domains. In some embodiments, the scFv polypeptide further includes V H and V LThe linker polypeptide between the domains allows scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0083] The term "diabodies" is used in V H and V L Small antibody fragments are prepared by constructing scFv fragments (see above) using short linkers (e.g., 5–10 residues) between domains. This allows the variable domains to pair between chains rather than within chains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific double-chain antibodies are heterodimers of two “crossed” scFv fragments, where the V0 of the two antibodies... H and V L The domains are located on different polypeptide chains. Double-chain antibodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0084] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody, which includes a minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where the hypervariable region (HVR) residues of the receptor antibody are replaced by hypervariable region residues from non-human species such as mice, rats, rabbits, or non-human mammals that possess desirable antibody specificity, affinity, and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced by corresponding non-human residues. Additionally, humanized antibodies may include residues not present in either the receptor or donor antibody. These modifications can further improve antibody performance. Typically, humanized antibodies contain substantially all, at least one, and usually two variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. Humanized antibodies may also optionally include at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For details, please refer to Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0085] The "percentage of amino acid sequence identity (%)" or "homology" of the peptide and antibody sequences identified in this paper is defined as the percentage of identical amino acid residues in a candidate sequence compared to a peptide sequence, assuming that conserved substitutions are part of sequence identity. The percentage of amino acid sequence identity can be determined using various alignment methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to maximize alignment across the full length of the compared sequences. However, for the purposes of this paper, the percentage of amino acid sequence identity values ​​were generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0086] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of antibodies. In some embodiments, the FcR described in this application is an FcR that binds to IgG antibodies (a type of γ receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) (see M.in.). Annu.Rev.Immunol.15:203-234 (1997)). The term also includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med.126:330-41 (1995). In this application, the term FcR covers other types of FcRs, including those to be identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0087] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC), consisting of an α-chain non-covalently bound to β2-microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulin IgGs from the mother to the newborn and in regulating serum IgG levels. As a rescue receptor, FcRn can bind and transport endocytotic IgG in its intact form both intracellularly and intercellularly, protecting it from the default degradation pathway.

[0088] The "CH1 domain" of the human IgG Fc region typically extends from amino acid 118 to amino acid 215 (EU numbering system).

[0089] The "hinge region" is typically defined as extending from Glu at position 216 to Pro at position 230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). By placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond at the same positions as IgG1, it is possible to align the hinge regions of other IgG isotypes with the IgG1 sequence.

[0090] The "CH2 domain" of the human IgG Fc region typically extends from amino acid 231 to amino acid 340. The unique feature of the CH2 domain is that it does not pair tightly with another region; instead, two N-terminal branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the sugars may act as a substitute for domain-to-domain pairing, helping to maintain the stability of the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985)).

[0091] The “CH3” domain includes the extension from the C-terminal residue to the CH2 domain within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, typically amino acid 446 or 447 of IgG).

[0092] "Functional Fc fragments" possess the "effective functions" inherent in the natural Fc region sequence. Exemplary "effective functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (such as B cell receptors; BCR). These effector functions typically require the Fc region to bind to a binding domain (such as the antibody variable region) and can be evaluated using a variety of experimental methods known in the art.

[0093] Antibodies with IgG Fc variants exhibiting "altered" FcR binding affinity or ADCC activity show enhanced or weakened FcR binding and / or ADCC activity compared to the parent peptide or peptides containing the native Fc sequence. Fc variants exhibiting "enhanced binding" to FcRs have higher binding affinity (e.g., lower apparent Kd or IC50) to at least one FcR compared to the parent peptide or peptides containing the native IgG Fc sequence. 50(Value). In some embodiments, the binding affinity is enhanced by 3 times compared to the parent peptide, for example, 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500 times, or the binding affinity is increased by 25% to 1000%. Fc variants exhibit “reduced binding” to FcRs, having a lower affinity for at least one FcR compared to the parent peptide (e.g., higher apparent Kd or IC). 50 (Value). Compared with the parent peptide, its binding ability is reduced by 40% or more.

[0094] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages). This allows these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are essential for this killing. Among the main cell types mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). The ADCC activity of target molecules can be assessed by in vitro ADCC experiments, described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK cells). Alternatively, or additionally, the ADCC activity of the target molecule can also be assessed in vivo, as described, for example, in animal models disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0095] Peptides containing Fc region variants exhibit "enhanced ADCC activity" or are more effective at mediating ADCC effects in the presence of human effector cells compared to peptides containing wild-type IgG Fc or parental peptides. These peptides, when experimentally present in substantially the same quantity as those containing wild-type IgG Fc (or parental peptides), mediate ADCC more effectively both in vitro and in vivo. Such variants are typically identified using any in vitro ADCC assay known in the art, such as experiments or methods for identifying ADCC activity, for example in animal models. In some embodiments, such variants mediate ADCC with a 5 to 100-fold, for example, 25 to 50-fold, efficiency improvement compared to wild-type Fc (or parental peptide).

[0096] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (a subclass with a suitable structure) that binds to a homologous antigen. To assess complement activation, CDC assays can be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Peptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0097] Unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins or RNA may also include introns; for example, nucleotide sequences encoding proteins may contain introns in certain forms.

[0098] The term "operationally ligated" refers to a functional link between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling the latter to be expressed. For example, the first and second nucleotide sequences are operably ligated when they are in a functional relationship. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter and coding sequence are operably ligated. Typically, operably ligated DNA sequences are contiguous, and, if necessary, two protein-coding regions can be ligated within the same reading frame.

[0099] "Homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. If the same position in two compared sequences is the same base or amino acid monomer subunit, for example, if both DNA molecules have adenine at the same position, then the two DNA molecules are homologous at that position. The percentage of homology between two sequences is a function calculated by multiplying the number of matching or homologous positions in the two sequences to the total number of positions, and then multiplying by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, when comparing two sequences, the goal is to obtain the maximum homology.

[0100] The “effective amount” of the anti-CD40 antibody or composition disclosed herein refers to an amount sufficient to achieve a specific purpose. The “effective amount” can be determined empirically and by known methods associated with the stated purpose.

[0101] The term "therapeutic effective amount" refers to the amount of the anti-CD40 antibody or its composition disclosed herein that is effective in treating an individual's disease or symptoms. This includes an amount sufficient to reduce or improve the severity and / or duration of the disease or one or more of its symptoms; prevent disease progression; induce symptom resolution; prevent recurrence, development, onset, or progression of one or more disease-related symptoms; detect the disease; or enhance / improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent).

[0102] As used herein, "pharmaceuticalally acceptable" or "pharmacologically compatible" means a material that has no biological activity or other undesirable properties, such as the ability to be incorporated into a pharmaceutical composition given to a patient without causing significant adverse biological reactions, or that does not interact harmfully with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological or manufacturing testing and / or are included in the inactive ingredient guidelines prepared by the U.S. Food and Drug Administration.

[0103] The embodiments of this application described herein should be understood to include embodiments that are “composed of” and / or “substantially composed of”.

[0104] In this article, “about” is used to refer to a numerical value or parameter, including (and descriptions) variations for that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0105] As used in this article, mentioning "not" a value or parameter usually means and describes something other than a certain value or parameter. For example, "This method cannot be used to treat type X cancer" means that this method is generally used to treat other types of cancer besides type X cancer.

[0106] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the claims include plural objects.

[0107] Anti-CD40 antibody

[0108] On one hand, this application provides anti-CD40 antibodies that specifically bind to CD40. These anti-CD40 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs as described herein. On the other hand, this application provides isolated antibodies that bind to CD40. Anticipated anti-CD40 antibodies include, for example, full-length anti-CD40 antibodies (such as full-length IgG1 or IgG4), anti-CD40 single-chain antibodies, anti-CD40 Fc fusion proteins, multispecific (such as bispecific) anti-CD40 antibodies, anti-CD40 immunoconjugates, and the like. In some embodiments, the anti-CD40 antibody is a full-length antibody (such as full-length IgG1 or IgG4) or its antigen-binding fragment that specifically binds to CD40. In some embodiments, the anti-CD40 antibody is Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, or linear antibody. In some embodiments, an antibody that specifically binds to CD40 means that the antibody's affinity for binding to CD40 is at least 10 times greater than its affinity for binding to non-target antibodies (including, for example, 10, 10). 2 10 3 10 4 10 5 10 6 , or 10 7 (Multiple times). In some embodiments, non-target refers to an antigen that is not CD40. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). Kd value can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interference (BLI) technology.

[0109] While this document discusses anti-CD40 antibodies containing human sequences (e.g., human heavy and light chain variable domains containing human CDR sequences), non-human anti-CD40 antibodies are also considered. In some embodiments, a non-human anti-CD40 antibody comprises the human CDR sequence and non-human frame region sequence of the anti-CD40 antibody described herein. In some embodiments, the non-human frame region sequence comprises any sequence used to generate heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., apes, macaques), etc. In some embodiments, a non-human anti-CD40 antibody comprises an anti-CD40 antibody generated by transplanting one or more human CDR sequences as described herein into a non-human frame region (e.g., a mouse or chicken frame region sequence).

[0110] The complete amino acid sequence of the exemplary human CD40 contains or consists of the amino acid sequence shown in SEQ ID NO: 149.

[0111] In some embodiments, the anti-CD40 antibody described herein specifically recognizes an epitope in human CD40. In some embodiments, the anti-CD40 antibody cross-reacts with CD40 from species other than humans. In some embodiments, the anti-CD40 antibody is completely specific to human CD40 and does not cross-react with other non-human species.

[0112] In some embodiments, the anti-CD40 antibody cross-reacts with at least one allelic variant of the CD40 protein (or a fragment thereof). In some embodiments, the allelic variant has up to 30 amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) compared to the naturally occurring CD40 protein (or a fragment thereof) (e.g., conserved substitutions). In some embodiments, the anti-CD40 antibody does not cross-react with any allelic variant of the CD40 protein (or a fragment thereof).

[0113] In some embodiments, the anti-CD40 antibody cross-reacts with at least one interspecies variant of the CD40 protein. In some embodiments, for example, the CD40 protein (or a fragment thereof) is human CD40, and the interspecies variant of the CD40 protein (or a fragment thereof) is a variant found in cynomolgus monkeys. In some embodiments, the anti-CD40 antibody does not cross-react with any interspecies variant of the CD40 protein.

[0114] In some embodiments, any anti-CD40 antibody as described herein includes an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-CD40 antibody includes an IgG1 type heavy chain constant region. In some embodiments, the anti-CD40 antibody includes an IgG2 type heavy chain constant region. In some embodiments, the anti-CD40 antibody includes an IgG3 type heavy chain constant region. In some embodiments, the anti-CD40 antibody includes an IgG4 type heavy chain constant region. In some embodiments, the heavy chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:146. In some embodiments, the anti-CD40 antibody includes a κ light chain constant region. In some embodiments, the light chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:147. In some embodiments, the anti-CD40 antibody includes a λ light chain constant region. In some embodiments, the light chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:148. In some embodiments, the anti-CD40 antibody includes an antibody heavy chain variable domain and an antibody light chain variable domain.

[0115] In some embodiments, the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Includes: a heavy chain complementarity determination region (HC-CDR)1, which contains GYX1VN (SEQ ID NO:81), wherein X1 is L, T, or V; HC-CDR2, which contains X1IX2PX3X4GX5TX6YNQKFKG (SEQ ID NO:82), wherein X1 is L or M, X2 is A, N, or V, X3 is E, K, L, M, R, S, T, or Y, X4 is H, N, S, or T, X5 is G or R, and X6 is A, S, T, or W; and HC-CDR3, which contains SX1X2X3X4PYAX5DY (SEQ ID NO:81). IDNO:83), where X1 is G, I, L, M, P, R, S or Y, X2 is W or Y, X3 is S or V, X4 is D, K, L, N, R, T or V, X5 is A, G, M, N, Q or S; and a light chain variable structural domain (V L ), the V L Includes: LC-CDR1, which contains KASQNV HTX1VA (SEQ ID NO:84), wherein X1 is A, H, N or R; LC-CDR2, which includes LX1SDRRT (SEQ ID NO:85), wherein X1 is A or R; and LC-CDR3, which includes LQX1WX2X3PLT (SEQ ID NO:86), wherein X1 is A or H, X2 is N, S or T, and X3 is A, Q, R, S or Y.

[0116] In some embodiments, the anti-CD40 antibody comprises V H The V H It comprises: HC-CDR1, which comprises any of the amino acid sequences shown in SEQ ID NOs:1-3 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; HC-CDR2, which comprises any of the amino acid sequences shown in SEQ ID NOs:9-26 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; and HC-CDR3, which comprises any of the amino acid sequences shown in SEQ ID NOs:32-52 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0117] In some embodiments, the anti-CD40 antibody comprises V H The V H It comprises: HC-CDR1, which contains any of the amino acid sequences shown in SEQ ID NOs:1-3; HC-CDR2, which contains any of the amino acid sequences shown in SEQ ID NOs:9-26; and HC-CDR3, which contains any of the amino acid sequences shown in SEQ ID NOs:32-52.

[0118] In some embodiments, the anti-CD40 antibody comprises V L The V L It comprises: LC-CDR1, which comprises any of the amino acid sequences shown in SEQ ID NOs:58-61 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises any of the amino acid sequences shown in SEQ ID NOs:65-66 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises any of the amino acid sequences shown in SEQ ID NOs:70-77 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0119] In some embodiments, the anti-CD40 antibody comprises V L The V LIt includes: LC-CDR1, which contains any of the amino acid sequences shown in SEQ ID NOs:58-61; LC-CDR2, which contains any of the amino acid sequences shown in SEQ ID NOs:65-66; and LC-CDR3, which contains any of the amino acid sequences shown in SEQ ID NOs:70-77.

[0120] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-3 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs:9-26 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs:32-52 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and V L The V L It comprises: LC-CDR1, which comprises any of the amino acid sequences shown in SEQ ID NOs:58-61 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises any of the amino acid sequences shown in SEQ ID NOs:65-66 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises any of the amino acid sequences shown in SEQ ID NOs:70-77 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0121] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-3; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs:9-26; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs:32-52; and V L The V LIt comprises: LC-CDR1, which contains any of the amino acid sequences shown in SEQ ID NOs:58-61; LC-CDR2, which contains any of the amino acid sequences shown in SEQ ID NOs:65-66; and LC-CDR3, which contains any of the amino acid sequences shown in SEQ ID NOs:70-77.

[0122] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:9; HC-CDR3, which contains the amino acid sequence SEQ ID NO:32; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:58; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0123] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:9; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:32; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:58, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0124] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:10; HC-CDR3, which contains the amino acid sequence SEQ ID NO:32; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and VL The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0125] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:10; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:32; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0126] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:13; HC-CDR3, which contains the amino acid sequence SEQ ID NO:34; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0127] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:13; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:34; and VL The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0128] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; HC-CDR3, which contains the amino acid sequence SEQ ID NO:37; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:73; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0129] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:37; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:73.

[0130] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:20; HC-CDR3, which contains the amino acid sequence SEQ ID NO:42; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and VL The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0131] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:20; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:42; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0132] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:14; HC-CDR3, which contains the amino acid sequence SEQ ID NO:43; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0133] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:14; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:43; and VL The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0134] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; HC-CDR3, which contains the amino acid sequence SEQ ID NO:51; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:70; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0135] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:15; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:51; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:70.

[0136] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:11; HC-CDR3, which contains the amino acid sequence SEQ ID NO:48; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and VL The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:71; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0137] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:11; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:48; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:71.

[0138] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:18; HC-CDR3, which contains the amino acid sequence SEQ ID NO:47; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59; LC-CDR2, which contains the amino acid sequence SEQ ID NO:65; LC-CDR3, which contains the amino acid sequence SEQ ID NO:71; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0139] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:18; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:47; and VL The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:59, LC-CDR2, which contains the amino acid sequence SEQ ID NO:65, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:71.

[0140] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains any amino acid sequence from SEQ ID NOs:1-3,9-26,32-52 or a variant thereof, said variant containing substitutions of up to about 5 amino acids; and V L The V L Contains any amino acid sequence or a variant thereof from SEQ ID NOs:58-61,65-66,70-77, wherein the variant contains substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody contains V H The V H Contains any amino acid sequence from SEQ ID NOs:1-3,9-26,32-52; and V L The V L It contains any of the amino acid sequences in SEQ ID NOs:58-61,65-66,70-77.

[0141] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains amino acid sequences SEQ ID NOs:1, 9, and 32, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:58, 65, and 70, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 9, and 32; and V L The V L It contains amino acid sequences SEQ ID NOs:58, 65 and 70.

[0142] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 10, and 32, or the V HVariants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 70, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 10, and 32; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 70.

[0143] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 13, and 34, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 70, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 13, and 34; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 70.

[0144] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 15, and 37, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 73, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 15, and 37; and V L The V LIt contains amino acid sequences SEQ ID NOs:59, 65 and 73.

[0145] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 20, and 42, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 70, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 20, and 42; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 70.

[0146] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 14, and 43, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 70, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 14, and 43; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 70.

[0147] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 15, and 51, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 70, or the V LVariants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 15, and 51; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 70.

[0148] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 11, and 48, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 71, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 11, and 48; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 71.

[0149] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 18, and 47, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:59, 65, and 71, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 18, and 47; and V L The V L It contains amino acid sequences SEQ ID NOs:59, 65 and 71.

[0150] In some embodiments, the anti-CD40 antibody comprises V H The V HV containing any of the amino acid sequences shown in SEQ ID NOs:87-119 H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L The V L V containing any of the amino acid sequences shown in SEQ ID NOs:126-138 L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0151] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:87. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:88. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:89. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:94. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:96. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:101. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:110. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:114. In some embodiments, the anti-CD40 antibody contains V H The V HContains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:115. In some embodiments, the anti-CD40 antibody contains V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:117.

[0152] In some embodiments, the anti-CD40 antibody comprises V L The V L Contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:126. In some embodiments, the anti-CD40 antibody contains V L The V L Contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:127. In some embodiments, the anti-CD40 antibody contains V L The V L Contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody contains V L The V L Contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:129. In some embodiments, the anti-CD40 antibody contains V L The V L It contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:132.

[0153] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:87. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:126. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0154] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:88. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:127. LIt includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0155] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:89. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0156] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:94. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0157] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:96. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:132. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0158] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:101. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0159] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:110. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V LIt contains V as shown in the amino acid sequence SEQ ID NO:128. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0160] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:114. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:128. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0161] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:115. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:129. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0162] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:117. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:129. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0163] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-119 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-119; and V L The V LThe antibody comprises any of the amino acid sequences shown in SEQ ID NOs:126-138 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-138. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-119, and V L The V L It contains any of the amino acid sequences shown in SEQ ID NOs:126-138.

[0164] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:87 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:87; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:126 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:126. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:87, and V L The V L It contains the amino acid sequence SEQ ID NO:126.

[0165] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:88 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:88; and V L The V LThe antibody comprises the amino acid sequence SEQ ID NO:127 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:127. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:88, and V L The V L It contains the amino acid sequence SEQ ID NO:127.

[0166] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:89 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:89; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:89, and V L The V L It contains the amino acid sequence SEQ ID NO:128.

[0167] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:94 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:94; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody comprises VH The V H Contains the amino acid sequence SEQ ID NO:94, and V L The V L It contains the amino acid sequence SEQ ID NO:128.

[0168] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:96 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:96; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:132 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:132. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:96, and V L The V L It contains the amino acid sequence SEQ ID NO:132.

[0169] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:101 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:101; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:101, and V L The V L It contains the amino acid sequence SEQ ID NO:128.

[0170] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:110 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:110; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:110, and V L The V L It contains the amino acid sequence SEQ ID NO:128.

[0171] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:114 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:114; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:114, and V L The V L It contains the amino acid sequence SEQ ID NO:128.

[0172] In some embodiments, the anti-CD40 antibody comprises: V H The V HContains the amino acid sequence SEQ ID NO:115 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:115; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:115, and V L The V L It contains the amino acid sequence SEQ ID NO:129.

[0173] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:117 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:117; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:117, and V L The V L It contains the amino acid sequence SEQ ID NO:129.

[0174] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; HC-CDR3, which contains the amino acid sequence SEQ ID NO:53; or the V HVariants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; LC-CDR3, which contains the amino acid sequence SEQ ID NO:78; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0175] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:53; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62, LC-CDR2, which contains the amino acid sequence SEQ ID NO:67, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:78.

[0176] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:4, 27, and 53, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:62, 67, and 78, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:4, 27, and 53; and V L The V L It contains amino acid sequences SEQ ID NOs:62, 67 and 78.

[0177] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:120.

[0178] In some embodiments, the anti-CD40 antibody comprises V L The V L Contains one, two or three LC-CDRs of the amino acid sequence SEQ ID NO:139.

[0179] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:120. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:139. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0180] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:120 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:120; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:139 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:139. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:120, and V L The V L It contains the amino acid sequence SEQ ID NO:139.

[0181] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:5; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; HC-CDR3, which contains the amino acid sequence SEQ ID NO:54; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; LC-CDR3, which contains the amino acid sequence SEQ ID NO:78; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0182] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:5; HC-CDR2, which contains the amino acid sequence SEQ ID NO:27; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:54; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62, LC-CDR2, which contains the amino acid sequence SEQ ID NO:67, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:78.

[0183] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:5, 27, and 54, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:62, 67, and 78, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:5, 27, and 54; and V L The V L It contains amino acid sequences SEQ ID NOs:62, 67 and 78.

[0184] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:121.

[0185] In some embodiments, the anti-CD40 antibody comprises V L The V LContains one, two or three LC-CDRs of the amino acid sequence SEQ ID NO:140.

[0186] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:121. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:140. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0187] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:121 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:121; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:140 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:140. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:121, and V L The V L It contains the amino acid sequence SEQ ID NO:140.

[0188] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:6; HC-CDR2, which contains the amino acid sequence SEQ ID NO:28; HC-CDR3, which contains the amino acid sequence SEQ ID NO:55; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:63; LC-CDR2, which contains the amino acid sequence SEQ ID NO:68; LC-CDR3, which contains the amino acid sequence SEQ ID NO:79; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0189] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:6; HC-CDR2, which contains the amino acid sequence SEQ ID NO:28; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:55; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:63, LC-CDR2, which contains the amino acid sequence SEQ ID NO:68, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:79.

[0190] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:6, 28, and 55, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:63, 68, and 79, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:6, 28, and 55; and V L The V L It contains amino acid sequences SEQ ID NOs:63, 68 and 79.

[0191] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:122.

[0192] In some embodiments, the anti-CD40 antibody comprises V L The V LContains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:141.

[0193] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:122. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:141. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0194] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:122 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:122; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:141 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:141. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:122, and V L The V L It contains the amino acid sequence SEQ ID NO:141.

[0195] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:7; HC-CDR2, which contains the amino acid sequence SEQ ID NO:29; HC-CDR3, which contains the amino acid sequence SEQ ID NO:56; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:64; LC-CDR2, which contains the amino acid sequence SEQ ID NO:69; LC-CDR3, which contains the amino acid sequence SEQ ID NO:80; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0196] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:7; HC-CDR2, which contains the amino acid sequence SEQ ID NO:29; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:56; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:64, LC-CDR2, which contains the amino acid sequence SEQ ID NO:69, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:80.

[0197] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:7, 29, and 56, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:64, 69, and 80, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:7, 29, and 56; and V L The V L It contains amino acid sequences SEQ ID NOs:64, 69 and 80.

[0198] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:123.

[0199] In some embodiments, the anti-CD40 antibody comprises V L The V LContains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:142.

[0200] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:123. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:142. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0201] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:123 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:123; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:142 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:142. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:123, and V L The V L It contains the amino acid sequence SEQ ID NO:142.

[0202] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:8; HC-CDR2, which contains the amino acid sequence SEQ ID NO:30; HC-CDR3, which contains the amino acid sequence SEQ ID NO:57; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:67; LC-CDR3, which contains the amino acid sequence SEQ ID NO:80; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0203] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:8; HC-CDR2, which contains the amino acid sequence SEQ ID NO:30; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:57; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62, LC-CDR2, which contains the amino acid sequence SEQ ID NO:67, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:80.

[0204] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:8, 30, and 57, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:62, 67, and 80, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:8, 30, and 57; and V L The V L It contains amino acid sequences SEQ ID NOs:62, 67 and 80.

[0205] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:124.

[0206] In some embodiments, the anti-CD40 antibody comprises V L The V LContains one, two or three LC-CDRs of the amino acid sequence SEQ ID NO:143.

[0207] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:124. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:143. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0208] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:124 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:124; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:143 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:143. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:124, and V L The V L It contains the amino acid sequence SEQ ID NO:143.

[0209] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:31; HC-CDR3, which contains the amino acid sequence SEQ ID NO:53; or the V H Variants of which contain up to about 5 amino acid substitutions in their HC-CDRs; and V L The V LContains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62; LC-CDR2, which contains the amino acid sequence SEQ ID NO:68; LC-CDR3, which contains the amino acid sequence SEQ ID NO:79; or the V L Variants of which contain up to about 5 amino acid substitutions in their LC-CDRs.

[0210] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:31; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:53; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:62, LC-CDR2, which contains the amino acid sequence SEQ ID NO:68, and LC-CDR3, which contains the amino acid sequence SEQ ID NO:79.

[0211] In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:4, 31, and 53, or the V H Variants containing substitutions of up to about 5 amino acids; and V L The V L Contains amino acid sequences SEQ ID NOs:62, 68, and 79, or the V L Variants of the antibody, wherein the variants contain substitutions of up to about 5 amino acids. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:4, 31, and 53; and V L The V L It contains amino acid sequences SEQ ID NOs:62, 68 and 79.

[0212] In some embodiments, the anti-CD40 antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:125.

[0213] In some embodiments, the anti-CD40 antibody comprises V L The V LContains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:144.

[0214] In some embodiments, the anti-CD40 antibody comprises V H It contains V as shown in the amino acid sequence SEQ ID NO:125. H Includes HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V as shown in the amino acid sequence SEQ ID NO:144. L It includes LC-CDR1, LC-CDR2 and LC-CDR3.

[0215] In some embodiments, the anti-CD40 antibody comprises: V H The V H Contains the amino acid sequence SEQ ID NO:125 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:125; and V L The V L The antibody comprises the amino acid sequence SEQ ID NO:144 or a variant thereof, said variant having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence SEQ ID NO:144. In some embodiments, the anti-CD40 antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:125, and V L The V L It contains the amino acid sequence SEQ ID NO:144.

[0216] In some embodiments, the amino acid substitutions described above are limited to the “Exemplary Substitutions” shown in Table 4 herein. In some embodiments, the amino acid substitutions are limited to the “Preferred Substitutions” shown in Table 4 herein.

[0217] In some embodiments, functional epitopes can be resolved using a combined alanine scanning technique. In this process, the combined alanine scanning technique can be used to identify amino acids in the CD40 protein essential for interaction with anti-CD40 antibodies. In some embodiments, the epitope is conformational, and the epitope can also be identified using the crystal structure of an anti-CD40 antibody that binds to the CD40 protein.

[0218] In some embodiments, this application provides an antibody that competitively binds to CD40 with any of the anti-CD40 antibodies described herein. In some embodiments, an antibody capable of competitively binding to an epitope on CD40 with any of the anti-CD40 antibodies described herein is provided. In some embodiments, an anti-CD40 antibody is provided that contains V H and V L The anti-CD40 antibody molecule binds to the same epitope, wherein the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125, and the V L Contains any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, an anti-CD40 antibody is provided, which, together with V H and V L The anti-CD40 antibody competitively binds to CD40, wherein the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125, and the V L It contains any of the amino acid sequences shown in SEQ ID NOs:126-144.

[0219] In some embodiments, a competition assay can be used to identify monoclonal antibodies that competitively bind to CD40 with the anti-CD40 antibody described herein. A competition assay can determine whether two antibodies bind to the same epitope by identifying identical or spatially overlapping epitopes or by one antibody competitively inhibiting the binding of another antibody to the antigen. In some embodiments, such a competitive antibody binds to the same epitope as the antibody described herein. Some exemplary competition assays include, but are not limited to, conventional assays as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving antibody-bound epitopes are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, if each antibody blocks 50% or more of the binding of another antibody, they are said to bind to the same epitope. In some embodiments, the antibody competing with the anti-CD40 antibody described herein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0220] Exemplary anti-CD40 antibody sequences are shown in Tables 2 and 3, where CDRs are numbered according to the Kabat definition. Those skilled in the art will recognize that various known algorithms (Kabat definition) exist for predicting CDR locations and defining the variable regions of the antibody light and heavy chains. CDRs containing antibodies as described herein, V H and / or V L Sequences, but antibodies based on prediction algorithms rather than those exemplified in the table below are also within the scope of this application.

[0221] Table 2. Exemplary anti-CD40 antibody CDR sequences

[0222]

[0223]

[0224]

[0225]

[0226] Table 3. Exemplary Sequences

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] CD40

[0233] CD40 is a 45-50 kDa type I membrane protein and a member of the TNFR superfamily. CD40 is expressed not only on antigen-presenting cells (such as B cells, dendritic cells (DCs), macrophages, and monocytes), but also on non-immune cells, such as epithelial, endothelial, and stromal cells (fibroblasts, myofibroblasts, synovial cells, stellate cells, etc.), platelets, and tumor cells. U, and P Libby. Cellular and Molecular Life Sciences: CMLS vol. 58, 1(2001): 4-43). CD40, as a transmembrane signal transducer, leads to the activation of intracellular kinases and transcription factors within the cell, participating in the regulation of a wide range of molecular and cellular processes, including the initiation and development of cellular and humoral adaptive immunity. Because the intracellular region of CD40 lacks intrinsic kinase activity, its signaling is primarily transduced through ligand-dependent recruitment of adaptor proteins from the TNF receptor-associated factor (TRAF) family. TRAF not only couples CD40 to intracellular signal transduction components but also triggers the dissociation of this signaling complex from CD40 and its release into the cytoplasm, where it activates proximal protein kinases as an E3 ubiquitin ligase. It can activate the mitogen-activated protein kinase (MAPK) pathway, including extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK, phosphatidylinositol 3-kinase (PI3K) cascade, transcription factor nuclear factor-κB (NF-κB), and signal transduction and transcription activator (STAT) (Loskog, Angelica SI, and Aristides G Eliopoulos. Seminars in immunology vol. 21, 5(2009): 301-7; and Matsuzawa, Atsushi et al. Science (New York, NY) vol. 321, 5889(2008): 663-8). These pathways work synergistically to regulate CD40 activity in a cell type and microenvironment-dependent manner.

[0234] The interaction between CD40 and CD40L triggers a variety of immune responses, including the expression of cellular stress-related genes and B cell activation (immunoglobulin class switching, germinal center formation, and cytokine production) (Belkhir, Re et al. Scandinavian journal of immunology vol. 79, 1(2014): 37-42; and Bensinger, William et al. British journal of haematology vol. 159, 1(2012): 58-66). The CD40-CD40L interaction also plays a crucial role in T cell activation and the upregulation of inflammatory response-related surface molecules and cytokines in chronic inflammatory diseases. These cytokines include interleukin (IL)-2, IL-12, TNF-α, interferon (IFN)-α, and IL-1 and IL-6 (Sidiropoulos, PI, and DT Boumpas. Lupus vol. 13, 5(2004): 391-7).

[0235] Given the important role of CD40–CD40L as a co-stimulatory molecule between B cells and T cells, there has been interest in the role of CD40–CD40L antagonists in inflammatory or autoimmune diseases such as psoriasis, lupus nephritis, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), diabetes, and immune rejection.

[0236] CD40L

[0237] CD40L is a 32-39 kDa type II transmembrane protein and a member of the TNF ligand superfamily (TNFSF, including TNF, lymphotoxin, CD27L, CD30L, and FasL). CD40L is generated by activated CD4+. + CD40L is expressed on T cells, activated B cells, and platelets, but under inflammatory conditions, it can also be induced to be expressed in monocytes, natural killer cells, mast cells, and basophils (Elgueta, Raul et al. Immunological reviews vol.229,1(2009):152-72). The soluble form of CD40L (sCD40L) has been reported to exhibit similar biological activity to the transmembrane form, triggering the CD40L / CD40 signaling cascade.

[0238] Therefore, neutralizing the binding of CD40 and CD40L could be a method for treating diseases and symptoms mediated by the CD40-CD40L pathway.

[0239] Full-length anti-CD40 antibody

[0240] In some embodiments, the anti-CD40 antibody is a full-length anti-CD40 antibody. In some embodiments, the full-length anti-CD40 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-CD40 antibody includes a constant IgG region, such as a constant region of IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the full-length anti-CD40 antibody includes a λ light chain constant region. In some embodiments, the full-length anti-CD40 antibody includes a κ light chain constant region. In some embodiments, the full-length anti-CD40 antibody is a full-length human anti-CD40 antibody. In some embodiments, the full-length anti-CD40 antibody includes a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-CD40 antibody includes an altered or otherwise modified Fc sequence such that it has enhanced effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0241] Therefore, for example, in some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, the anti-CD40 antibody specifically binding to CD40. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0242] In some embodiments, a full-length anti-CD40 antibody comprising an IgG2 constant region is provided, the anti-CD40 antibody specifically binding to CD40. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0243] In some embodiments, a full-length anti-CD40 antibody comprising an IgG3 constant region is provided, the anti-CD40 antibody specifically binding to CD40. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0244] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, the anti-CD40 antibody specifically binding to CD40. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0245] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs: 9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs: 32-57 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising SEQ ID NOs: 1-8 or variants thereof, the heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the heavy chain variable domain comprising: HC ... The amino acid sequences or variants thereof shown in SEQ ID NOs:58-64, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3), LC-CDR2, which contains amino acid sequences or variants thereof shown in SEQ ID NOs:65-69, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3); and LC-CDR3, which contains amino acid sequences or variants thereof shown in SEQ ID NOs:70-80, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3). In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0246] In some embodiments, a full-length anti-CD40 antibody comprising an IgG2 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs:9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs:32-57 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising SEQ ID NOs:9-31 or variants thereof, the heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:9-31 or variants thereof, the heavy chain variable domain comprising: HC-CDR2 ... The amino acid sequences or variants thereof shown in SEQ ID NOs:58-64, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3); LC-CDR2, which contains amino acid sequences or variants thereof shown in SEQ ID NOs:65-69, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3); and LC-CDR3, which contains amino acid sequences or variants thereof shown in SEQ ID NOs:70-80, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3). In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0247] In some embodiments, a full-length anti-CD40 antibody comprising an IgG3 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs:9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs:32-57 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising SEQ ID NOs:1-31 or variants thereof, the heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs:1-31 or variants thereof, the heavy chain variable domain comprising: HC ... The amino acid sequences shown in SEQ ID NOs:58-64 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3); LC-CDR2, which contains amino acid sequences shown in SEQ ID NOs:65-69 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3); and LC-CDR3, which contains amino acid sequences shown in SEQ ID NOs:70-80 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3). In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0248] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; HC-CDR2, comprising any of the amino acid sequences shown in SEQ ID NOs: 9-31 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and HC-CDR3, comprising any of the amino acid sequences shown in SEQ ID NOs: 32-57 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising SEQ ID NOs: 1-8 or variants thereof, the heavy chain variable domain comprising: HC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the variants comprising substitutions of up to about 3 (e.g., 1, 2, or 3) amino acids; and b) a light chain variable domain comprising: LC-CDR1, comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8 or variants thereof, the heavy chain variable domain comprising: HC-CDR2 ... The amino acid sequences shown in SEQ ID NOs:58-64 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3), LC-CDR2, which contains the amino acid sequences shown in SEQ ID NOs:65-69 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3), and LC-CDR3, which contains the amino acid sequences shown in SEQ ID NOs:70-80 or variants thereof, wherein the variants contain substitutions of up to about 3 amino acids (e.g., 1, 2, or 3). In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0249] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8, HC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 9-31, and HC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 32-57, or a variant of the heavy chain variable domain comprising a substitution of up to about 5 amino acids (e.g., 1, 2, 3, 4, or 5) in its HC-CDR sequence; and b) a light chain variable domain comprising: LC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, and HC ... The amino acid sequence shown in NOs:70-80, or a variant of the light chain variable domain, contains substitutions of up to about 5 amino acids (e.g., 1, 2, 3, 4, or 5) in its LC-CDR sequence. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0250] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises a) a heavy chain variable domain comprising: HC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs: 1-8, HC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 9-31, and HC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 32-57, or a variant of the heavy chain variable domain comprising a substitution of up to about 5 amino acids (e.g., 1, 2, 3, 4, or 5) in its HC-CDR sequence; and b) a light chain variable domain comprising: LC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs: 65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 58-64, and HC ... The amino acid sequence shown in NOs:70-80, or a variant of the light chain variable domain, contains substitutions of up to about 5 amino acids (e.g., 1, 2, 3, 4, or 5) in its LC-CDR sequence. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0251] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs:1-8, HC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs:9-31, and HC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs:32-57; and b) a light chain variable domain comprising: LC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs:58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs:65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs:70-80. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0252] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs:1-8, HC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs:9-31, and HC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs:32-57; and b) a light chain variable domain comprising: LC-CDR1 comprising any of the amino acid sequences shown in SEQ ID NOs:58-64, LC-CDR2 comprising any of the amino acid sequences shown in SEQ ID NOs:65-69, and LC-CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs:70-80. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0253] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:9, and HC-CDR3 comprising amino acid sequence SEQ ID NO:32; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:58, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0254] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:10, and HC-CDR3 comprising amino acid sequence SEQ ID NO:32; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0255] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:13, and HC-CDR3 comprising amino acid sequence SEQ ID NO:34; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0256] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:15, and HC-CDR3 comprising amino acid sequence SEQ ID NO:37; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:73. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0257] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:20, and HC-CDR3 comprising amino acid sequence SEQ ID NO:42; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0258] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising HC-CDR1 containing amino acid sequence SEQ ID NO:1, HC-CDR2 containing amino acid sequence SEQ ID NO:14, and HC-CDR3 containing amino acid sequence SEQ ID NO:43; and b) a light chain variable domain comprising: LC-CDR1 containing amino acid sequence SEQ ID NO:59, LC-CDR2 containing amino acid sequence SEQ ID NO:65, and LC-CDR3 containing amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0259] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:15, and HC-CDR3 comprising amino acid sequence SEQ ID NO:51; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0260] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:11, and HC-CDR3 comprising amino acid sequence SEQ ID NO:48; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:71. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0261] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:18, and HC-CDR3 comprising amino acid sequence SEQ ID NO:47; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:71. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0262] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:4, HC-CDR2 comprising amino acid sequence SEQ ID NO:27, and HC-CDR3 comprising amino acid sequence SEQ ID NO:53; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:62, LC-CDR2 comprising amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising amino acid sequence SEQ ID NO:78. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0263] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence SEQ ID NO:27, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:54; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:62, LC-CDR2 comprising the amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:78. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0264] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:6, HC-CDR2 comprising the amino acid sequence SEQ ID NO:28, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:55; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:63, LC-CDR2 comprising the amino acid sequence SEQ ID NO:68, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:79. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0265] In some embodiments, a full-length anti-CD40 antibody comprising a constant region of IgG1 is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:7, HC-CDR2 comprising amino acid sequence SEQ ID NO:29, and HC-CDR3 comprising amino acid sequence SEQ ID NO:56; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:64, LC-CDR2 comprising amino acid sequence SEQ ID NO:69, and LC-CDR3 comprising amino acid sequence SEQ ID NO:80. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0266] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:8, HC-CDR2 comprising the amino acid sequence SEQ ID NO:30, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:57; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:62, LC-CDR2 comprising the amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:80. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0267] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:4, HC-CDR2 comprising amino acid sequence SEQ ID NO:31, and HC-CDR3 comprising amino acid sequence SEQ ID NO:53; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:62, LC-CDR2 comprising amino acid sequence SEQ ID NO:68, and LC-CDR3 comprising amino acid sequence SEQ ID NO:79. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0268] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:9, and HC-CDR3 comprising amino acid sequence SEQ ID NO:32; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:58, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0269] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:10, and HC-CDR3 comprising amino acid sequence SEQ ID NO:32; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0270] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:13, and HC-CDR3 comprising amino acid sequence SEQ ID NO:34; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0271] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:15, and HC-CDR3 comprising amino acid sequence SEQ ID NO:37; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:73. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0272] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:20, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:42; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:59, LC-CDR2 comprising the amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0273] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:14, and HC-CDR3 comprising amino acid sequence SEQ ID NO:43; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0274] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:15, and HC-CDR3 comprising amino acid sequence SEQ ID NO:51; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:70. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0275] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:11, and HC-CDR3 comprising amino acid sequence SEQ ID NO:48; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:71. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0276] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:1, HC-CDR2 comprising amino acid sequence SEQ ID NO:18, and HC-CDR3 comprising amino acid sequence SEQ ID NO:47; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:59, LC-CDR2 comprising amino acid sequence SEQ ID NO:65, and LC-CDR3 comprising amino acid sequence SEQ ID NO:71. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0277] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:4, HC-CDR2 comprising amino acid sequence SEQ ID NO:27, and HC-CDR3 comprising amino acid sequence SEQ ID NO:53; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:62, LC-CDR2 comprising amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising amino acid sequence SEQ ID NO:78. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0278] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence SEQ ID NO:27, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:54; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:62, LC-CDR2 comprising the amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:78. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0279] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:6, HC-CDR2 comprising the amino acid sequence SEQ ID NO:28, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:55; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:63, LC-CDR2 comprising the amino acid sequence SEQ ID NO:68, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:79. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0280] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:7, HC-CDR2 comprising the amino acid sequence SEQ ID NO:29, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:56; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:64, LC-CDR2 comprising the amino acid sequence SEQ ID NO:69, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:80. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0281] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:8, HC-CDR2 comprising the amino acid sequence SEQ ID NO:30, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:57; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:62, LC-CDR2 comprising the amino acid sequence SEQ ID NO:67, and LC-CDR3 comprising the amino acid sequence SEQ ID NO:80. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0282] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 comprising amino acid sequence SEQ ID NO:4, HC-CDR2 comprising amino acid sequence SEQ ID NO:31, and HC-CDR3 comprising amino acid sequence SEQ ID NO:53; and b) a light chain variable domain comprising: LC-CDR1 comprising amino acid sequence SEQ ID NO:62, LC-CDR2 comprising amino acid sequence SEQ ID NO:68, and LC-CDR3 comprising amino acid sequence SEQ ID NO:79. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0283] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-125; and a light chain variable domain (V L ), the V LThe IgG1 comprises any of the amino acid sequences shown in SEQ ID NOs:126-144 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0284] In some embodiments, a full-length anti-CD40 antibody comprising an IgG2 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-125; and a light chain variable domain (V L ), the V L The IgG2 comprises any of the amino acid sequences shown in SEQ ID NOs:126-144 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0285] In some embodiments, a full-length anti-CD40 antibody comprising an IgG3 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V HContains any of the amino acid sequences shown in SEQ ID NOs:87-125 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-125; and a light chain variable domain (V L ), the V L The IgG3 comprises any of the amino acid sequences shown in SEQ ID NOs:126-144 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0286] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:87-125; and a light chain variable domain (V L ), the V LThe IgG4 comprises any of the amino acid sequences shown in SEQ ID NOs:126-144 or variants thereof, wherein the variants have at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0287] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V H Contains any of the amino acid sequences shown in SEQ ID NOs:87-125, and a light chain variable domain (V L ), the V L Contains any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0288] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: a heavy chain variable domain (V H ), the V HContains any of the amino acid sequences shown in SEQ ID NOs:87-125, and a light chain variable domain (V L ), the V L Contains any of the amino acid sequences shown in SEQ ID NOs:126-144. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0289] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:87 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:87; and V L It comprises the amino acid sequence SEQ ID NO:126 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0290] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:88 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:88; and V L It comprises the amino acid sequence SEQ ID NO:127 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:127. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0291] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:89 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:89; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0292] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:94 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:94; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0293] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:96 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:96; and V L It comprises the amino acid sequence SEQ ID NO:132 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:132. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0294] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:101 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:101; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0295] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:110 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:110; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0296] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:114 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:114; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0297] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:115 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:115; and V L It comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0298] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:117 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:117; and V L It comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0299] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:120 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:120; and V L It comprises the amino acid sequence SEQ ID NO:139 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:139. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0300] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:121 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:121; and V L It comprises the amino acid sequence SEQ ID NO:140 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:140. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0301] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:122 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:122; and V L It comprises the amino acid sequence SEQ ID NO:141 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:141. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0302] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:123 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:123; and V L It comprises the amino acid sequence SEQ ID NO:142 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:142. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0303] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:124 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:124; and V L It comprises the amino acid sequence SEQ ID NO:143 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:143. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0304] In some embodiments, a full-length anti-CD40 antibody comprising an IgG1 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:125 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:125; and V L It comprises the amino acid sequence SEQ ID NO:144 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:144. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0305] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:87 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:87; and V L It comprises the amino acid sequence SEQ ID NO:126 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0306] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:88 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:88; and V L It comprises the amino acid sequence SEQ ID NO:127 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:127. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0307] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:89 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:89; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0308] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:94 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:94; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0309] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:96 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:96; and V L It comprises the amino acid sequence SEQ ID NO:132 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:132. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0310] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:101 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:101; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0311] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:110 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:110; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0312] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:114 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:114; and V L It comprises the amino acid sequence SEQ ID NO:128 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:128. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0313] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:115 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:115; and V L It comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0314] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:117 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:117; and V L It comprises the amino acid sequence SEQ ID NO:129 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:129. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0315] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:120 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:120; and V L It comprises the amino acid sequence SEQ ID NO:139 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:139. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0316] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:121 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:121; and V L It comprises the amino acid sequence SEQ ID NO:140 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:140. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0317] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:122 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:122; and V L It comprises the amino acid sequence SEQ ID NO:141 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:141. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0318] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:123 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:123; and V L It comprises the amino acid sequence SEQ ID NO:142 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:142. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0319] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V H It comprises the amino acid sequence SEQ ID NO:124 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:124; and V L It comprises the amino acid sequence SEQ ID NO:143 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:143. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0320] In some embodiments, a full-length anti-CD40 antibody comprising an IgG4 constant region is provided, wherein the anti-CD40 antibody comprises: V HIt comprises the amino acid sequence SEQ ID NO:125 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:125; and V L It comprises the amino acid sequence SEQ ID NO:144 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence SEQ ID NO:144. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:148.

[0321] Combining affinity

[0322] Binding affinity is expressed as Kd, Koff, Kon, or Ka. As used herein, the term Koff refers to the rate constant for antibody dissociation from the antigen / antibody complex, determined by a kinetic selection device. The term Kon refers to the binding rate constant for antibody to bind to the antigen to form an antigen / antibody complex. The equilibrium dissociation constant Kd used herein refers to the dissociation constant for a specific antibody-antigen interaction, which is the antigen concentration required in the antibody molecule solution to occupy half of all antibody binding sites and reach equilibrium, equal to Koff / Kon. The determination of Kd assumes all binding molecules are in solution. For antibody-cell wall attachment, such as in yeast expression systems, the corresponding equilibrium dissociation rate constant is expressed as EC. 50 It is expressed as a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0323] The dissociation constant (Kd) can serve as an indicator of the affinity between an antibody moiety and an antigen. For example, a simple analysis can be performed using the Scatchard method with antibodies labeled with various markers and a Biacore instrument (manufactured by Amersham Biosciences), analyzing the interactions between biomolecules via surface plasmon resonance according to the user manual or accompanying kit. The Kd values ​​obtained using these methods are expressed in units of megohms (M). Antibodies that bind specifically to a target may have a Kd constant, for example, ≤10. -7 M, ≤10-8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 The Kd value of M.

[0324] The binding specificity of antibodies can be experimentally determined using methods known in the art. These methods include, but are not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0325] In some embodiments, the anti-CD40 antibody specifically binds to the CD40 target, and its Kd value is 10. -7 M to 10 -13 M (e.g., 10) -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Therefore, in some embodiments, the Kd value for binding between the anti-CD40 antibody and CD40 is 10. -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 - 12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10- 9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 - 13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 - 11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 M. In some embodiments, the Kd value for binding between the anti-CD40 antibody and CD40 is 10. -7 M to 10 -13 M.

[0326] In some embodiments, the Kd value of anti-CD40 antibody binding to a non-target is higher than the Kd value of anti-CD40 antibody binding to a target, and in some embodiments cited herein, the binding affinity of anti-CD40 antibody to a target (e.g., CD40) is higher than the binding affinity of anti-CD40 antibody to a non-target. In some embodiments, non-target refers to an antigen other than CD40. In some embodiments, the Kd value of anti-CD40 antibody (against CD40) binding to a non-CD40 target differs by at least 10-fold, for example, 10-100-fold, 100-1000-fold, or 10... 3 -10 4 times, 104 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0327] In some embodiments, the Kd value of the anti-CD40 antibody binding to the non-target is 10. -1 M to 10 -6 M (e.g., 10) -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target refers to a non-CD40 antigen. Therefore, in some embodiments, the Kd value for binding between the anti-CD40 antibody and the non-CD40 target is 10. -1 M to 10 -6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10 -4 M, 10 -1 M to 10 - 3 M, 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 -5 M, 1×10 -2 M to 5×10 -5 M, 10-2 M to 10 -4 M, 1×10 -2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 - 6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0328] In some embodiments, when it is mentioned that the anti-CD40 antibody specifically recognizes the CD40 target with high binding affinity and binds to non-targets with low binding affinity, the Kd value of the anti-CD40 antibody binding to the CD40 target is 10. -7 M to 10 -13 M (e.g., 10) -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -12 M), and the Kd value for binding to the non-target is 10. -1 M to 10 -6 M (e.g., 10) -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0329] In some embodiments, when referring to the specific recognition of CD40 by an anti-CD40 antibody, the binding affinity of the anti-CD40 antibody is compared with that of a control anti-CD40 antibody (e.g., ASKP1240). In some embodiments, the Kd value of the binding between the control anti-CD40 antibody and CD40 can be at least twice the Kd value of the binding between the anti-CD40 antibody and CD40 described in this application, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10-100 times, 100-1000 times, or 10 times. 3 -10 4 times.

[0330] Nucleic acid

[0331] Nucleic acid molecules encoding anti-CD40 antibodies are also considered. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-CD40 antibody is provided, including any of the full-length anti-CD40 antibodies described herein. In some embodiments, the nucleic acid (or set of nucleic acids) of the anti-CD40 antibody described herein may also include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His tag, a HA tag).

[0332] This paper also considers isolated host cells containing anti-CD40 antibodies, isolated nucleic acids encoding anti-CD40 antibody polypeptide components, or vectors containing nucleic acids encoding the anti-CD40 antibody polypeptide components described herein.

[0333] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize with nucleic acid sequences encoding the anti-CD40 antibody of this application at least under moderately stringent hybridization conditions.

[0334] This application also provides a vector into which the nucleic acid sequence of this application can be inserted.

[0335] In short, by inserting a natural or synthetic nucleic acid encoding an anti-CD40 antibody into a suitable expression vector, allowing the nucleic acid to be operatively linked to 5' and 3' regulatory elements, such as promoters (e.g., lymphocyte-specific promoters) and a 3' untranslated region (UTR), anti-CD40 antibodies (e.g., full-length anti-CD40 antibodies) can be expressed. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, a start sequence, and a promoter that regulate the expression of the target nucleic acid sequence.

[0336] The nucleic acids described in this application can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In some embodiments, this application also provides gene therapy vectors.

[0337] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and Cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0338] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0339] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus is then isolated and delivered in vivo or in vitro to the cells of a recipient. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as murine leukemia virus, because they can transduce non-dividing cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0340] Other promoter elements, such as enhancers, regulate transcription initiation frequency. They are typically located 30-110 bp upstream of the start site, although recent studies have found that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so the promoter retains its function even when elements interchange or move. In the thymidine kinase (TK) promoter, activity only begins to decline when the spacing between promoter elements increases to 50 bp.

[0341] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rouss sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, this application should not be limited to the use of only constitutive promoters; inducible promoters are also considered in this application. The use of inducible promoters provides a molecular switch that initiates the expression of the polynucleotide sequence to which it is operable when such expression is needed, and shuts it off when not needed. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0342] In some embodiments, expression of the anti-CD40 antibody is inducible. In some embodiments, the nucleic acid sequence encoding the anti-CD40 antibody is operatively linked to an inducible promoter, including any of the inducible promoters described herein.

[0343] Inducible promoters

[0344] The use of inducible promoters provides a molecular switch that initiates the expression of a polynucleotide sequence operatively linked to it when expression is desired, and shuts it off when expression is not desired. Exemplary inducible promoters applicable in eukaryotic cells include, but are not limited to, hormone regulatory elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand regulatory elements (see Spencer, D. M et al (1993) Science 262:1019-1024), and ionizing radiation regulatory elements (see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters applicable to in vivo or in vitro mammalian systems can be found in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system for expressing anti-CD40 antibodies is the Tet system. In some embodiments, the inducible promoter system for expressing anti-CD40 antibodies is the E. coli lac inhibitory system.

[0345] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter containing one or more Tet operon (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrous tetracycline, doxycycline (Dox), or an active analog thereof, the inducer causes the TetR to be released from the TetO, thereby leading to transcription. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0346] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, resulting in a large number of variants of a given nucleic acid sequence without any change in the amino acid sequence it encodes. However, many organisms exhibit differences in codon usage, also known as "codon preference" (i.e., a preference for using specific codons for a given amino acid). Codon preference is often associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, codon optimization can be used to tailor coding sequences derived from a specific species (e.g., prokaryotes) to improve their expression in different species (e.g., eukaryotes).

[0347] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-Off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcriptional activator protein (tTA), composed of TetR fused to the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of target nucleic acids under the transcriptional control of tetracycline-responsive promoter elements (TREs). TRE elements consist of a TetO sequence fused in tandem with a promoter (typically a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and the target gene cannot be expressed.

[0348] Conversely, in the Tet-On system, transcription is activated in the presence of Tc or Dox. The Tet-On system is based on the inverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein composed of a TetR repressor and a VP16 transcriptional activation domain. However, a four-amino acid change in the DNA-binding region of TetR alters the binding properties of rtTA, causing it to recognize only the tetO sequence on the target transgene TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can only activate the transcription of the TRE-regulated target gene in the presence of Dox.

[0349] Another inducible promoter system is the *E. coli* lac repressor system (see Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating the transcription of target polynucleotides operably linked to a promoter containing the lac operon (lacO). The lac repressor (lacR) binds to LacO, thereby preventing the transcription of the target polynucleotide. Expression of the target polynucleotide can be induced by suitable inducers, such as isopropyl-β-D-thiogalactopyranoside (IPTG).

[0350] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected by a viral vector. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection experiments. Both the selectable marker gene and the reporter gene may be side-mounted to a suitable regulatory sequence to enable its expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0351] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue; it encodes a polypeptide, and its expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is detected at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see, Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or obtained commercially. Typically, the smallest 5' flanking region that displays the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to reporter genes and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0352] In some embodiments, a nucleic acid encoding any of the full-length anti-CD40 antibodies described herein is provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of a full-length anti-CD40 antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector may be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).

[0353] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0354] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, gene gun methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells via calcium phosphate transfection.

[0355] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0356] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymeric complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in vivo and in vitro is a liposome (e.g., an artificial membrane capsule).

[0357] In the use of non-viral delivery systems, an exemplary delivery vector is the liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid can bind to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of a liposome, dispersed within a lipid bilayer of the liposome, linked to the liposome by linking molecules that bind to the liposome and oligonucleotides, embedded within the liposome, forming a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, suspended in lipids, contained in or mixed with micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions in solution are not limited to any particular structure. For example, they may exist in a bilayer structure, as micelles, or as a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0358] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cells. These experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" experiments, such as detecting the presence or absence of a specific polypeptide, for example, identification by immunological methods (ELISAs and Western blots) or by the experiments described herein, all of which fall within the scope of this application.

[0359] Preparation of anti-CD40 antibody

[0360] In some embodiments, the anti-CD40 antibody is a monoclonal antibody or is derived from a monoclonal antibody. In some embodiments, the anti-CD40 antibody includes V derived from a monoclonal antibody. H and V L Or a variant thereof. In some embodiments, the anti-CD40 antibody further comprises the CH1 and CL regions, or variants thereof, derived from a monoclonal antibody. The monoclonal antibody can be prepared using methods known in the art, including hybridoma cell methods, phage display methods, or methods using recombinant DNA. Furthermore, exemplary phage display methods are described herein and in the following examples.

[0361] In hybridoma cell methods, hamsters, mice, or other suitable host animals are typically immunized with an immunomodulator to induce the production or ability to produce antibodies that specifically bind to the immunomodulator. Alternatively, lymphocytes can be immunized in vitro. The immunomodulator may include a peptide or fusion protein of the target protein. Peripheral blood lymphocytes (PBLs) are typically used if human cells are desired, while spleen cells or lymph node cells are used if non-human mammalian cells are desired. The lymphocytes are fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibits the growth of HGPRT-deficient cells.

[0362] In some embodiments, the immortalized cell lines are efficiently fused, ensuring high-level and stable antibody expression through selected antibody-producing cells, and are sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell lines are mouse myeloma cell lines, obtainable from, for example, the Salk Cell Collection in San Diego, California, and the American Collection of Type Cultures in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines for the preparation of human monoclonal antibodies are also described.

[0363] The presence of monoclonal antibodies against the peptide can then be determined in the culture medium for hybridoma cells. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0364] After identifying the desired hybridoma cells, the target clone can be subcloned using limiting dilution and cultured using standard methods. Suitable media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites fluid.

[0365] Subclonal secreted monoclonal antibodies can be isolated or purified from culture medium or ascites using conventional immunoglobulin purification methods, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0366] In some embodiments, according to any of the anti-CD40 antibodies described herein, the anti-CD40 antibody comprises a sequence selected from an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening libraries of antibody fragment combinations with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods have been reviewed in, for example, Hoogenboome et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. Further descriptions can be found in al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004).

[0367] In some phage display methods, V is cloned separately using polymerase chain reaction (PCR). H and V LAll components of the gene are randomly recombined in a phage library, and then phages capable of binding antigens are screened, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv fragments or Fab fragments. Phages from immune-derived libraries provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared by cloning a non-rearranged V-gene fragment from stem cells, encoding the CDR3 hypervariable region using PCR primers containing random sequences, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Patent No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0368] The anti-CD40 antibody was prepared by screening a library for anti-CD40 antibody moieties that specifically bind to the target CD40 using phage display. The library may be a human scFv phage display library with at least 1 × 10⁻⁶ cells / mL. 9 (e.g., at least 1×10) 9 2.5×10 9 5×10 9 7.5×10 9 1×10 10 2.5×10 10 5×10 10 7.5×10 10 Or 1×10 11The library contains a variety of unique human antibody fragments. In some embodiments, the library is a natural human library constructed from DNA extracted from PMBCs and spleen of healthy subjects, containing all heavy and light chain subfamilies. In some embodiments, the library is a natural human library constructed from DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is completely randomized, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, R.M et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semi-synthetic human library is between 5 and 24 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0369] Phage clones with high affinity for the target CD40 can be screened by iterative binding of the phage to the target CD40, which is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phages and elution of specifically bound phages. The eluted phage clones are then used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Multiple rounds of panning (e.g., 2, 3, 4, 5, 6 or more rounds), such as solution panning, cell panning, or a combination of both, can be used to enrich phage clones that specifically bind to CD40. The specific binding of the enriched phage clones to the target CD40 can be detected by any method known in the art, including, for example, ELISA and FACS.

[0370] Another method for screening antibody libraries is to display proteins on the surface of yeast cells. Wittrup et al. (US Patents 6,699,658 and 6,696,251) developed a method for displaying yeast cells. In this yeast display system, one component includes a yeast lectin protein (Aga1) anchored to the yeast cell wall, and another component includes a second subunit of the lectin protein Aga2, which can bind to the Aga1 protein via disulfide bonds and thus be displayed on the yeast cell surface. The Aga1 protein is expressed by integrating the Aga1 gene into the yeast chromosome. A single-stranded variable fragment (scFv) library is fused with the Aga2 gene in a yeast display plasmid, and after transformation, the library is retained in the yeast due to the presence of additional nutrient markers. Both Aga1 and Aga2 proteins are expressed under the control of a galactose-inducible promoter.

[0371] Human Antibody V Gene Library (V H and V K The fragments were obtained by PCR using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998). PCR templates were derived from commercially available RNA or cDNA, including PBMCs, spleen, lymph nodes, bone marrow, and tonsils. Independent V... H and V K After PCR library merging, they were assembled into scFv form by overlap extension PCR (Sheets, MD et al, Proc. Natl. Acad. Sci. USA 95, 6157–61621998). To construct a yeast scFv display library, the obtained scFv PCR product was cloned into a yeast display plasmid in yeast by homologous recombination (Chao, G, et al, Nat Protoc. 2006; 1(2): 755-68. Miller KD, et al. Current Protocols in Cytometry 4.7.1-4.7.30, 2008).

[0372] Mammalian cell display systems can be used to screen for anti-CD40 antibodies, where the antibody portion is displayed on the cell surface and antibodies specifically targeting CD40 are isolated using antigen-directed screening methods (as described in US patent No. 7,732,195B2). Chinese hamster ovary (CHO) cell libraries displaying large amounts of human IgG antibody genes can be constructed and used to discover clones expressing high-affinity antibody genes. Another display system has been developed that uses alternative splicing to simultaneously display and secrete the same protein on the cell surface, where the displayed protein phenotype remains genotype-dependent, allowing for simultaneous characterization of the secreted soluble antibody in both biophysical and cell function-based analyses. This method overcomes many limitations of previous mammalian cell display methods, enabling direct screening and maturation of antibodies in full-length, glycosylated IgG forms (Peter M. Bowers, et al., Methods 2014, 65: 44-56). Transient expression systems are suitable for single-round antigen selection before antibody gene recovery, and are therefore most useful for selecting antibodies from smaller libraries. Stable exon vectors provide an attractive option. Exon vectors can be efficiently transfected and stably maintained at low copy numbers, thus allowing for multiple rounds of screening and the analysis of more complex antibody libraries.

[0373] The IgG library was constructed by linking germline sequences of the V gene fragment isolated from a group of human donors with rearranged (D)J regions. RNA collected from 2000 human blood samples was reverse transcribed into cDNA, and V was used... H and V K Specific primer amplification V H and V K The fragment was extracted and purified by gel extraction. V H and V K Fragments were subcloned into display vectors containing either the IgG1 or K constant region, and then electroporated or transduced into 293T cells to prepare IgG libraries. To prepare scFv antibody display libraries, V... H and V K The scFv is generated, then subcloned into a display vector, and then electroporated or transduced into 293T cells. It is well known that IgG libraries are constructed based on germline sequence V gene fragments isolated from a group of donors and rearranged (D)J regions; donors can be mice, rats, rabbits, or monkeys.

[0374] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibody described in this application can be easily isolated and sequenced using conventional methods, such as using oligonucleotide probes that specifically bind to genes encoding the light and heavy chains of murine antibodies. Hybridoma cells as described above or the CD40-specific phage clone of this application can serve as sources of this DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into host cells, such as simian COS cells, Chinese hamster ovary cancer (CHO) cells, or non-immunoglobulin-producing myeloma cells, to obtain monoclonal antibodies synthesized in recombinant host cells. The DNA can also be modified, for example, by replacing the human heavy and light chain constant regions with coding sequences and / or replacing homologous non-human sequences with frame regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequences of immunoglobulins to the coding sequences of non-immunoglobulin polypeptides. This non-immunoglobulin polypeptide can replace the constant region of the antibody in this application, or can replace an antigen-binding site in the variable domain of the antibody in this application, to form a chimeric bivalent antibody.

[0375] The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a recombinant expression method involving immunoglobulin light chains and modified heavy chains. Typically, the heavy chain is truncated at any position in the Fc region to prevent cross-linking between the heavy chains. Alternatively, relevant cysteine ​​residues are substituted with other amino acid residues or deleted to prevent cross-linking.

[0376] In vitro methods are also suitable for preparing monovalent antibodies. Digesting antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0377] Antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant regions. Fusion is preferably with immunoglobulin heavy chain constant regions, which include at least a partial hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the necessary light chain binding site is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion, and optionally DNA encoding the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism.

[0378] Humanized and whole-human antibodies

[0379] The anti-CD40 antibody (such as a full-length anti-CD40 antibody) can be a fully human antibody or a humanized antibody. The humanized form of the non-human (e.g., mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequence of the antibody), which typically includes a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins, immunoglobulin chains, or fragments thereof (receptor antibodies), wherein residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues having the desired specificity, affinity, and performance, such as mouse, rat, or rabbit CDRs. In some embodiments, human immunoglobulin Fv frame region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain amino acid residues that are neither part of the receptor antibody nor present in the introduced CDR or frame region sequence. Typically, humanized antibodies contain at least one, usually two, variable domains, where all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the frame regions are human immunoglobulin common sequences.

[0380] Typically, humanized antibodies contain one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are generally referred to as “shift-in” residues and typically originate from “shift-in” variable domains. According to some embodiments, humanization can be performed essentially as described by Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with rodent CDRs or CDR sequences. Thus, this “humanized” antibody portion (US Patent No. 4, 816, 567), which is essentially less than a complete human antibody, has its variable domain replaced by the corresponding sequence from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion, in which some CDR residues and possibly some framework region residues are replaced by residues from similar sites in rodent antibodies.

[0381] Fully human antibodies are an alternative to humanization. For example, it is currently possible to produce transgenic animals (e.g., mice) that generate a complete fully human antibody library after immunization without producing endogenous immunoglobulins. For instance, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice completely suppresses the production of endogenous antibodies. Transferring human germline immunoglobulin gene arrays into germline mutant mice can generate human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806,5,569,825,5,591,669,5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin gene loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that in humans in all aspects, including gene rearrangement, assembly, and antibody library formation. This method has been used in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and It is described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0382] Fully human antibodies can also be produced by activating B cells in vitro (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Techniques by Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991).

[0383] anti-CD40 antibody variant

[0384] In some embodiments, the amino acid sequences of the anti-CD40 antibody variants (e.g., full-length anti-CD40 antibodies) provided herein are also considered. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequence of the antibody variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. The final construction can be accomplished by any combination of amino acid residue deletions, insertions, and substitutions to give it the desired characteristics, such as antigen binding.

[0385] In some embodiments, anti-CD40 antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include hypervariable regions (HVRs) and frame regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products with desired activities, such as improved biological activity, maintenance / improvement of antigen-binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0386] Conservative alternatives are shown in Table 4 below.

[0387] Table 4 Conservative Substitution

[0388] Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine;Ile;Val;Met;Ala;Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0389] Amino acids are classified into different categories based on the properties of their side chains:

[0390] a. Hydrophobic amino acids: Norleucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile);

[0391] b. Neutral hydrophilic amino acids: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln);

[0392] c. Acidic amino acids: Aspartic acid (Asp), Glutamic acid (Glu);

[0393] d. Basic amino acids: Histidine (His), Lysine (Lys), Arginine (Arg);

[0394] e. Contains amino acids that affect chain direction: glycine (Gly) and proline (Pro);

[0395] f. Aromatic amino acids: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0396] Substitution of non-conservative amino acids involves replacing one class with another.

[0397] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated, the variant antibody is partially displayed on a phage, and screening for specific biological activities (e.g., HEK-Blue based). TM Variants of CD40L (the biological activity or binding affinity of NF-κB activation assays) can be used. Alterations (e.g., substitutions) in the HVRs region can be made to obtain improved HEK-Blue-based variants. TM The biological activity or antibody affinity of CD40L NF-κB activation assay can be determined by alterations in the "hotspot region" of HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic mat...

Claims

1. An isolated anti-CD40 antibody, comprising: V H The V H Contains: HC-CDR1, with the amino acid sequence SEQ ID NO: 1; HC-CDR2, with the amino acid sequence SEQ ID NO: 20; and HC-CDR3, with the amino acid sequence SEQ ID NO: 42; and V L The V L It includes: LC-CDR1, whose amino acid sequence is SEQ ID NO: 59; LC-CDR2, whose amino acid sequence is SEQ ID NO: 65; and LC-CDR3, whose amino acid sequence is SEQ ID NO:

70.

2. The isolated anti-CD40 antibody according to claim 1, comprising: V H Its amino acid sequence is SEQ ID NO: 101 or a variant thereof, said variant having at least 90% sequence identity with the amino acid sequence SEQ ID NO: 101; and V L Its amino acid sequence is SEQ ID NO: 128 or a variant thereof, the variant having at least 90% sequence identity with the amino acid sequence SEQ ID NO:

128.

3. The anti-CD40 antibody isolated according to claim 1, wherein the anti-CD40 antibody comprises an Fc fragment.

4. The anti-CD40 antibody isolated according to claim 3, wherein the anti-CD40 antibody is a full-length IgG antibody.

5. The anti-CD40 antibody isolated according to claim 4, wherein the anti-CD40 antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.

6. The anti-CD40 antibody isolated according to any one of claims 1-5, wherein the anti-CD40 antibody is a chimeric or humanized antibody.

7. The anti-CD40 antibody isolated according to any one of claims 1-2, wherein the CD40 antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb or double-chain antibody (diabody).

8. An isolated nucleic acid molecule encoding the anti-CD40 antibody according to any one of claims 1-7.

9. A carrier comprising the nucleic acid molecule of claim 8.

10. An isolated host cell comprising the anti-CD40 antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, or the vector of claim 9.

11. A method for preparing anti-CD40 antibody, comprising: a) Culture the host cells as described in claim 10 under conditions that enable effective expression of anti-CD40 antibodies; and b) Obtain the expressed anti-CD40 antibody from the host cell.

12. A pharmaceutical composition comprising an antiCD40 antibody according to any one of claims 1-7 and a pharmaceutically acceptable carrier, said pharmaceutical composition for treating a desired individual disease or condition.

13. Use of the anti-CD40 antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, the vector of claim 9, the isolated host cell of claim 10, the antibody prepared by the method of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating a desired individual disease or condition, wherein the disease or condition is Sjogren's disease or rheumatoid arthritis.

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