Anti-CD40 antibodies and their uses

Abstract

This invention relates to anti-CD40 antibodies or antigen-binding fragments thereof that specifically recognize and weakly activate CD40 molecules in humans, cynomolgus monkeys, and mice, as well as immunoconjugates, pharmaceutical compositions, and combination products comprising said anti-CD40 antibodies or antigen-binding fragments thereof. The invention also relates to nucleic acids encoding said anti-CD40 antibodies or antigen-binding fragments thereof and host cells comprising said nucleic acids, and methods for preparing said anti-CD40 antibodies or antigen-binding fragments thereof. Furthermore, the invention relates to the use of said anti-CD40 antibodies or antigen-binding fragments thereof in the prevention or treatment of tumors or infectious diseases in subjects.

Description

[0001] This invention relates to anti-CD40 antibodies or antigen-binding fragments thereof. Specifically, this invention relates to anti-CD40 antibodies or antigen-binding fragments thereof that specifically recognize and weakly activate CD40 molecules in humans, cynomolgus monkeys, and mice, as well as immunoconjugates, pharmaceutical compositions, and combination products comprising said anti-CD40 antibodies or antigen-binding fragments thereof. This invention also relates to nucleic acids encoding said anti-CD40 antibodies or antigen-binding fragments thereof and host cells comprising said nucleic acids, and methods for preparing said anti-CD40 antibodies or antigen-binding fragments thereof. This invention further relates to the use of said anti-CD40 antibodies or antigen-binding fragments thereof in the prevention or treatment of tumors or infectious diseases in subjects. Background Technology

[0002] Clinical trials of CD40 agonists (e.g., selicrelumab, dacetuzumab, APX005M, etc.) have shown clinical activity in multiple indications (Hassan SB et al., Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol 2014; 36:96–104). However, these studies have also shown that these CD40 agonists are associated with adverse events leading to dose-limiting toxicity. The most common adverse events include cytokine release syndrome (CRS) and hepatotoxicity. Currently, clinical trials commonly use CD40 agonists with normal Fc function. These agonists can be divided into two categories based on their agonistic activity. Some of these agonists are highly toxic because they can non-specifically overactivate the immune response, making them unsuitable as therapeutic agents. Others require crosslinking to form immunologically active anti-CD40 monoclonal antibodies. These antibodies can generate a local immune enhancement response by activating CD40-expressing immune cells in a limited and controllable manner, thereby killing tumor cells within a limited range.

[0003] In recent years, the Therapeutic Index (TI) has increasingly become an important indicator for evaluating drug safety. TI compares the amount of drug that produces a therapeutic effect to the amount that causes toxicity. This means that at the dose administered to a patient, a higher therapeutic index and lower toxicity indicate a higher safety profile. However, in the current technology, the known therapeutic indices of anti-CD40 antibodies are relatively low. At actual doses administered to patients, the ratio of the immune cell-killing activity generated by the antibody at the lesion site to the non-specific agonistic activity (e.g., hepatotoxicity) caused at other sites is not high. Therefore, there is a need in the art to develop new CD40 agonists that can provide sufficient immune stimulation while reducing the adverse events caused by traditional CD40 agonists.

[0004] Furthermore, in the development of new CD40 agonists, preclinical animal model evaluation is a crucial step in assessing drug safety and therapeutic efficacy. Rodents (e.g., mice) are widely used to establish animal models of human diseases. However, anti-CD40 antibodies that do not cross-react with mouse CD40 (e.g., selicrelumab developed by Roche) need to be used in mouse tumor models transgenic with the human CD40 gene to reflect their in vivo efficacy and safety. There is also an urgent need to develop agonistic anti-CD40 antibodies that simultaneously exhibit cross-reactivity with mouse CD40 in testing the efficacy of bispecific anti-CD40 antibodies in mouse models. Invention Overview

[0005] This invention provides a highly therapeutic index agonist anti-CD40 antibody that can provide sufficient immune stimulation in subjects while mitigating the adverse effects of non-specific immune activation caused by CD40 activation. It possesses one or more of the following properties:

[0006] (1) Binds to and activates CD40 with high affinity, for example, with about 10 -7 M to approximately 10 -10 M binds to CD40, such as human CD40, cynomolgus monkey CD40 and mouse CD40, as measured by the ForteBio kinetic binding assay;

[0007] (2) Enhance the binding between CD40 and CD40L;

[0008] (3) Activate antigen-presenting cells by binding to CD40 expressed on antigen-presenting cells, for example, said antigen-presenting cells include dendritic cells (DCs), B cells, monocytes, macrophages;

[0009] (4) Inducing B cells expressing CD40 to express CD95;

[0010] (5) It significantly enhances B cell-mediated immune responses when cross-linking effects are formed;

[0011] (6) It hardly enhances or only slightly enhances B cell-mediated immune responses when no cross-linking effect is formed.

[0012] The anti-CD40 antibody of the present invention has a complete human sequence and is a human antibody. It is anticipated that the anti-CD40 antibody of the present invention will have minimal immunogenicity in human subjects, induce fewer anti-drug antibodies (ADA), thereby minimizing ADA-related clearance of the therapeutic anti-CD40 antibody, and that the therapeutic anti-CD40 antibody will be well-tolerated in human subjects.

[0013] In some embodiments, the anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises three complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises three complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, wherein:

[0014] (a) LCDR1 contains the amino acid sequence shown in SEQ ID NO:1 or a variant of SEQ ID NO:1 with no more than 2 or no more than 1 amino acid variation;

[0015] (b) LCDR2 contains a variant of any amino acid sequence selected from SEQ ID NO:2, 5, 6, 7, or any amino acid sequence selected from SEQ ID NO:2, 5, 6, 7, with no more than 2 or no more than 1 amino acid variation;

[0016] (c) LCDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:3, 4, or any amino acid sequence of SEQ ID NO:3, 4, with no more than two or no more than one amino acid variation.

[0017] (d)HCDR1 contains a variant of any amino acid sequence selected from SEQ ID NO:8, 12, 14, 20, or any amino acid sequence selected from SEQ ID NO:8, 12, 14, 20, with no more than 2 or no more than 1 amino acid variation;

[0018] (e)HCDR2 comprises a variant of any amino acid sequence selected from SEQ ID NO:9, 21, or any amino acid sequence of SEQ ID NO:9, 21 with no more than 2 or no more than 1 amino acid variation;

[0019] (f) HCDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:10, 11, 13, 15, 16, 17, 18, 19, or any amino acid sequence selected from SEQ ID NO:10, 11, 13, 15, 16, 17, 18, 19, with no more than two or no more than one amino acid variation.

[0020] The amino acid changes mentioned therein refer to the addition, deletion, or substitution of amino acids.

[0021] In some embodiments, the anti-CD40 antibody of the present invention comprises

[0022] (i) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10;

[0023] (ii) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:4 or SEQ ID NO:4, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10;

[0024] (iii) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:11 or SEQ ID NO:11;

[0025] (iv) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:5 or SEQ ID NO:5, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:12 or SEQ ID NO:12, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10;

[0026] (v) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:13 or SEQ ID NO:13;

[0027] (vi) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:6 or SEQ ID NO:6, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:14 or SEQ ID NO:14, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10;

[0028] (vii) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:15 or SEQ ID NO:15;

[0029] (viii) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:16 or SEQ ID NO:16;

[0030] (ix) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:17 or SEQ ID NO:17;

[0031] (x) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:18 or SEQ ID NO:18;

[0032] (xi) LCDR1 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and LCDR3 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3; and HCDR1 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, HCDR2 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and LCDR3 containing no more than 2 or 1 amino acid variations of the amino acid sequence SEQ ID NO:19 or SEQ ID NO:19; or

[0033] (xii) LCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, LCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:7 or SEQ ID NO:7, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, as well as HCDR1 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:20 or SEQ ID NO:20, HCDR2 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:21 or SEQ ID NO:21, and LCDR3 containing no more than 2 or no more than 1 amino acid variation of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10.

[0034] In some embodiments, the anti-CD40 antibody of the present invention comprises:

[0035] LCDR1 containing the amino acid sequence RSSQGIRSSLA (SEQ ID NO:1), LCDR2 containing the amino acid sequence GX1SSLX2X3 (SEQ ID NO:56), and LCDR3 containing the amino acid sequence QQLX4SFPST (SEQ ID NO:57), wherein X1 is A or G; X2 is E or L; X3 is G or V; X4 is N or A; and

[0036] HCDR1 containing amino acid sequences such as GFTX5GSYEMX6 (SEQ ID NO:58), HCDR2 containing amino acid sequences such as YISSX7GETTD (SEQ ID NO:59), and HCDR2 containing amino acid sequences such as DVFFFX8X9SX 10 X 11 X 12 X 13 HCDR3 of AYGMDV (SEQ ID NO:60), wherein X5 is F, A, or P; X6 is N or D; X7 is S or A; X8 is D or S; X9 is S or P; X 10 For G or R; X 11 For D, P, S, or F; X 12 For P, N, or R; X 13 It can be G or H.

[0037] In some specific embodiments, the anti-CD40 antibody of the present invention comprises:

[0038] (i) LCDR1 containing amino acid sequence SEQ ID NO:1, LCDR2 containing amino acid sequence SEQ ID NO:2 and LCDR3 containing amino acid sequence SEQ ID NO:3, and HCDR1 containing amino acid sequence SEQ ID NO:8, HCDR2 containing amino acid sequence SEQ ID NO:9 and LCDR3 containing amino acid sequence SEQ ID NO:10.

[0039] (ii) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:2 and LCDR3 containing the amino acid sequence SEQ ID NO:4, and HCDR1 containing the amino acid sequence SEQ ID NO:8, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:10.

[0040] (iii) LCDR1 containing amino acid sequence SEQ ID NO:1, LCDR2 containing amino acid sequence SEQ ID NO:2 and LCDR3 containing amino acid sequence SEQ ID NO:3, and HCDR1 containing amino acid sequence SEQ ID NO:8, HCDR2 containing amino acid sequence SEQ ID NO:9 and LCDR3 containing amino acid sequence SEQ ID NO:11.

[0041] (iv) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:5 and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:12, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:10.

[0042] (v) LCDR1 containing amino acid sequence SEQ ID NO:1, LCDR2 containing amino acid sequence SEQ ID NO:2 and LCDR3 containing amino acid sequence SEQ ID NO:3, and HCDR1 containing amino acid sequence SEQ ID NO:8, HCDR2 containing amino acid sequence SEQ ID NO:9 and LCDR3 containing amino acid sequence SEQ ID NO:13.

[0043] (vi) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:6 and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:14, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:10.

[0044] (vii) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:2 and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:8, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:15.

[0045] (viii) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:2 and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:8, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:16.

[0046] (ix) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:2 and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:8, HCDR2 containing the amino acid sequence SEQ ID NO:9 and LCDR3 containing the amino acid sequence SEQ ID NO:17.

[0047] (x) LCDR1 containing amino acid sequence SEQ ID NO:1, LCDR2 containing amino acid sequence SEQ ID NO:2 and LCDR3 containing amino acid sequence SEQ ID NO:3, and HCDR1 containing amino acid sequence SEQ ID NO:8, HCDR2 containing amino acid sequence SEQ ID NO:9 and LCDR3 containing amino acid sequence SEQ ID NO:18.

[0048] (xi) LCDR1 containing the amino acid sequence SEQ ID NO:1, LCDR2 containing the amino acid sequence SEQ ID NO:2, and LCDR3 containing the amino acid sequence SEQ ID NO:3, and HCDR1 containing the amino acid sequence SEQ ID NO:8, HCDR2 containing the amino acid sequence SEQ ID NO:9, and LCDR3 containing the amino acid sequence SEQ ID NO:19; or

[0049] (xii) LCDR1 containing amino acid sequence SEQ ID NO:1, LCDR2 containing amino acid sequence SEQ ID NO:7 and LCDR3 containing amino acid sequence SEQ ID NO:3, and HCDR1 containing amino acid sequence SEQ ID NO:20, HCDR2 containing amino acid sequence SEQ ID NO:21 and LCDR3 containing amino acid sequence SEQ ID NO:10.

[0050] In some embodiments, the anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein:

[0051] (i) The light chain variable region contains any amino acid sequence from SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains any amino acid sequence from SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55; or

[0052] (ii) The light chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55.

[0053] In some embodiments, the anti-CD40 antibody of the present invention comprises:

[0054] (i) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:25;

[0055] (ii) Contains the light chain variable region of amino acid sequence SEQ ID NO:29 and the heavy chain variable region of amino acid sequence SEQ ID NO:25;

[0056] (iii) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:31;

[0057] (iv) Contains a light chain variable region of amino acid sequence SEQ ID NO:33 and a heavy chain variable region of amino acid sequence SEQ ID NO:35;

[0058] (v) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:37;

[0059] (vi) Contains the light chain variable region of amino acid sequence SEQ ID NO:39 and the heavy chain variable region of amino acid sequence SEQ ID NO:41;

[0060] (vii) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:43;

[0061] (viii) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:45;

[0062] (ix) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:47;

[0063] (x) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:49;

[0064] (xi) Contains the light chain variable region of amino acid sequence SEQ ID NO:23 and the heavy chain variable region of amino acid sequence SEQ ID NO:51; or

[0065] (xii) Contains the light chain variable region of amino acid sequence SEQ ID NO:53 and the heavy chain variable region of amino acid sequence SEQ ID NO:55.

[0066] Preferably, the anti-CD40 antibody or antigen-binding fragment is a fully human antibody.

[0067] In some embodiments, the anti-CD40 antibody of the present invention is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is a human IgG1 or human IgG4 antibody.

[0068] In some embodiments, the anti-CD40 antibody of the present invention is an antigen-binding fragment, including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab, and diabody antibody.

[0069] In some embodiments, the anti-CD40 antibody of the present invention has its glycosylation site in the CH2 domain of the immunoglobulin Fc region removed, for example, by mutating the N297 residue in the CH2 domain of the human IgG Fc region to remove the glycosylation site, for example, by changing the N297 residue to Gly, Ala, Gln, Asp or Glu, preferably by changing the N297 residue to Gln.

[0070] In a second aspect, the present invention provides a method for preparing the anti-CD40 antibody of the present invention, the method comprising culturing a host cell in which a nucleic acid encoding the anti-CD40 antibody of the present invention or an expression vector containing said nucleic acid is introduced under conditions suitable for expressing a nucleic acid encoding the anti-CD40 antibody of the present invention, isolating said anti-CD40 antibody, and optionally further comprising recovering said anti-CD40 antibody from said host cell.

[0071] In a third aspect, the present invention provides an immunoconjugate comprising the anti-CD40 antibody of the present invention and other substances, such as cytotoxic agents.

[0072] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the anti-CD40 antibody or immunoconjugate of the present invention, and optionally a pharmaceutical excipient.

[0073] In some embodiments, the present invention provides a pharmaceutical composition comprising the anti-CD40 antibody or immunoconjugate of the present invention, as well as other therapeutic agents and optionally pharmaceutical excipients; preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies), and cytotoxic agents.

[0074] In some embodiments, the present invention provides a combination product comprising the anti-CD40 antibody or immunoconjugate of the present invention, and one or more other therapeutic agents, such as chemotherapeutic agents, cytotoxic agents, other antibodies, preferably anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0075] In a fifth aspect, the present invention provides a method for preventing or treating tumors or infectious diseases in a subject or individual, comprising administering to the subject an effective amount of the anti-CD40 antibody, immune conjugate, pharmaceutical composition, or combination product of the present invention.

[0076] In some embodiments, the anti-CD40 antibody, immune conjugate, pharmaceutical composition, or combination product of the present invention prevents or treats cancer, such as cancer expressing CD40 molecules or cancer not expressing CD40 molecules; or the anti-CD40 antibody, immune conjugate, pharmaceutical composition, or combination product of the present invention prevents or treats infectious diseases such as bacterial infections, viral infections, fungal infections, or protozoan infections, preferably said infectious diseases are chronic infections in which the subject or individual has a weakened immune system.

[0077] In a sixth aspect, the present invention provides a kit for detecting CD40 in a sample, the kit comprising the anti-CD40 antibody of the present invention for performing the following steps:

[0078] (a) Contacting the sample with the anti-CD40 antibody of the present invention; and

[0079] (b) Detecting the formation of the complex between the anti-CD40 antibody and CD40; optionally, the anti-CD40 antibody is detectably labeled.

[0080] This allows us to determine the CD40 expression level in samples from subjects or individuals. Brief description of the attached diagram

[0081] Combine with the following appendix Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0082] Figure 1The graph shows the binding of CD40 antigen P17-Fc to two control antibodies, dasizumab and bleslumab. An isotype of human IgG1 antibody was used as a negative control. The horizontal axis in the graph represents antibody concentration.

[0083] Figure 2 The diagram shows a comparison of the binding of unlabeled P17-Fc and biotin-labeled P17-Fc to two control antibodies, dasizumab and Bleslumab, respectively. In the figure, "P17-Fc-CD40-ble" represents the binding of unlabeled P17-Fc to Bleslumab (also referred to as CD40-Ble or CD40-ble in the text); "P17-Fc-CD40-dac" represents the binding of unlabeled P17-Fc to dasizumab (also referred to as CD40-Dac or CD40-dac in the text).

[0084] Figure 3 The image shows the ELISA results of a phage library after three rounds of panning for a human natural antibody phage display library. In the figure, "VCSM13" indicates "VCSM13 helper phage," obtained from Stratagene (USA).

[0085] Figure 4 The graph shows the binding affinity of the C8 clone CD40 Fab molecule, screened from a human natural antibody phage display library, to CD40. Bleslumab monoclonal antibody was used as a positive control, and isotype human IgG1 antibody was used as a negative control. The horizontal axis in the graph represents antibody concentration.

[0086] Figure 5A The binding of the C8 clone's anti-CD40 Fab molecule to human CD40, which is stably expressed on CHO cells, was demonstrated.

[0087] Figure 5B The cross-reactivity of the anti-CD40 Fab molecule of the C8 clone with cynomolgus CD40 stably expressed on CHO cells was demonstrated.

[0088] Figure 6A The study demonstrated the affinity of the full-length anti-CD40 candidate antibody for human CD40, which is stably expressed on CHO cells.

[0089] Figure 6B The study demonstrated the affinity of the full-length anti-CD40 candidate antibody for cynomolgus monkey CD40, which is stably expressed on CHO cells.

[0090] Figure 6C The affinity of the full-length anti-CD40 candidate antibody for mouse CD40 stably expressed on CHO cells was demonstrated. An isotype human IgG1 antibody was used as a negative control.

[0091] Figure 7 The effect of full-length anti-CD40 antibody on the binding of CD40L to CD40-expressing CHO cells was demonstrated.

[0092] Figure 8A and Figure 8B An assay of iDC activation induced by anti-CD40 antibody was shown, which was demonstrated by IL-12 secretion as measured by ELISA and CD83 expression as measured by FACS.

[0093] Figure 9 The study showed that anti-CD40 antibody induces CD95 expression on Ramos cells, as measured by FACS.

[0094] Figure 10A The final tumor volume of the Ramos cell mouse tumor model after administration of anti-CD40 antibody is shown.

[0095] Figure 10B The final tumor weight of the Ramos cell mouse tumor model after administration of anti-CD40 antibody is shown.

[0096] Figure 10C The changes in tumor volume over time in a Ramos cell mouse tumor model after administration of anti-CD40 antibody are shown. NS in the figure indicates no significant difference; compared with the PBS group, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

[0097] Figure 10D The final tumor weight after administration of anti-CD40 antibody in the MC-38 cell tumor model is shown.

[0098] Figure 11A Non-specific binding of the affinity variant full-length candidate antibody to CHO-K cells was demonstrated.

[0099] Figure 11B Non-specific binding of the affinity variant full-length candidate antibody to HEK293 cells was demonstrated.

[0100] Figure 12A and Figure 12B The affinity variant of the full-length candidate antibody was demonstrated to have affinity for human CD40, which is stably expressed on CHO cells.

[0101] Figure 12C and Figure 12D The affinity of the mature full-length candidate antibody to mouse CD40 stably expressed on CHO cells was demonstrated.

[0102] Figure 13A and Figure 13BThe study demonstrates how, in the presence of a cross-linking agent, the full-length candidate antibody for the C8 molecule and its affinity variants induces CD95 expression on Ramos cells, as measured by FACS.

[0103] Figure 13C and Figure 13D The study demonstrates that, in the absence of a cross-linking agent, full-length candidate antibodies containing the C8 molecule and its affinity variants induce CD95 expression on Ramos cells, as measured by FACS.

[0104] Figure 14A - Figure 14D This demonstrates the ability of the C8 molecule and its affinity variants, in the presence of a cross-linking agent, to induce the expression of the NF-κB luciferase reporter gene.

[0105] Figure 15A - Figure 15D This demonstrates the ability of the C8 molecule and its affinity variant full-length candidate antibodies to induce NF-κB luciferase reporter gene expression in the absence of a cross-linking agent. Invention Details

[0106] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0107] I. Definition

[0108] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0109] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0110] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0111] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0112] The term "CD40-expressing cell" refers to any cell that expresses the CD40 molecule, including but not limited to antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, macrophages, and monocytes. CD40 is also expressed on other cell types, such as epithelial cells, endothelial cells, and platelets. CD40 expression has also been demonstrated in a variety of tumor cells, including B-cell lymphoma and renal cell carcinoma. In one specific embodiment, CD40-expressing cells include CD40-expressing cell lines, such as Jurkat cells, Raji cells, Ramos cells, and Daudi cells. In another embodiment, CD40-expressing cells are tumor cells or cancer cells. In yet another embodiment, CD40-expressing cells include B cells, NK cells, and T cells found to infiltrate tumors, also known as tumor-infiltrating lymphocytes.

[0113] The term "antigen-presenting cell" or "APC" refers to a cell that displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptors (TCRs). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblast-like cells, and dendritic cells (DCs) derived from monocytes.

[0114] The term “antigen presentation” refers to the process by which APCs capture antigens and make them recognizable by T cells, for example, by processing antigens as components of MHC-I and / or MHC-II conjugates.

[0115] MHC molecules include two types: MHC class I and MHC class II. MHC class I molecules present antigens to specific CD8+ T cells, while MHC class II molecules present antigens to specific CD4+ T cells. Exogenous antigens delivered to APCs primarily bind to MHC class II molecules. Conversely, endogenous antigens delivered to APCs primarily bind to MHC class I molecules.

[0116] The term "T cell-mediated response" refers to any response mediated by T cells, including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0117] The term “antibody” is used in the broadest sense herein to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.

[0118] An "antibody fragment" or "antigen-binding fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.

[0119] The term "cross-reactivity" refers to the ability of the antibodies of the present invention to bind to CD40 from different species. For example, the antibodies of the present invention that bind to human CD40 can also bind to CD40 from another species. Cross-reactivity is measured by detecting specific reactivity with purified CD40 antigens or binding to or otherwise functionally interacting with physiological CD40-expressing cells in a binding assay (e.g., SPR, ELISA). Methods for determining cross-reactivity include, for example, using Octet... TM Biototal Interferometry (BLI) using QKe instruments or Biacore™ 2000SPR instruments (Biacore AB, Uppsala, Sweden). TM Surface plasmon resonance (SPR) analysis or flow cytometry.

[0120] The complementarity-determining region (CDR) or CDR is a region within the antibody's variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. Heavy chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (http: / / imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0121] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0122] The CDR can also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDRs in the examples of this invention). In one embodiment, the CDR of the antibody of this invention is located according to an AbM numbering scheme.

[0123] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the AbM numbering system.

[0124] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, or AbM may be substituted with conserved amino acid residues.

[0125] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, utilizing a human antibody library or other human antibody encoding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0126] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0127] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6) th (ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to provide antigen binding specificity.

[0128] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biomembrane interferometry, or other conventional binding assays known in the art.

[0129] The term "co-stimulatory molecule" refers to cell surface molecules and their ligands that provide co-stimulatory signals for the complete activation of T cells or B cells. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.

[0130] The term "cytokine" is a general term for proteins released by one group of cells that act as intercellular mediators to another cell. Examples of such cytokines include lymphokines, monokines, interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, and IL-15; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors, including gamma interferon.

[0131] "Immune conjugates" are antibodies conjugated to one or more other substances (including but not limited to cytotoxic agents or markers).

[0132] The terms “inducing” and “increasing” used in inducing CDC or ADCC refer to stimulating a specific direct cell-killing mechanism. For example, in one embodiment, the anti-CD40 antibody of the present invention induces ADCC in CD40-expressing cells, resulting in at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% lysis of tumor cells or cells infected by viruses, bacteria, etc.

[0133] The term "excitation" refers to the increase in certain parameters (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, this term includes substances that increase the activity of a given molecule (e.g., CD40) by at least 5%, 10%, 20%, 30%, 40%, or more. Therefore, the excitation effect does not have to be 100%.

[0134] The “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be coupled with a binding domain (e.g., antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.

[0135] "Effective functions" refer to biological activities that can be attributed to the Fc region of an antibody and vary with antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0136] The term "Fc receptor" refers to an Fc receptor that, upon binding to the Fc region of an immunoglobulin, triggers a signal transduction event that stimulates the cell carrying the receptor to perform effector functions. Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0137] The term "effective amount" refers to such an amount or dose of the anti-CD40 antibody, conjugate, or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health condition; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0138] "Therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective amount of an antibody or antibody fragment, or its conjugate or composition, can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or antibody fraction to elicit the desired response in the individual. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment, or its conjugate or composition, are less than the therapeutically beneficial effects. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0139] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0140] The "therapeutic index" (TI) is usually the ratio of the median lethal dose (LD50) to the median effective dose (ED50), and is an indicator of drug safety.

[0141] The terms “individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human being.

[0142] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and liquid tumors.

[0143] The terms “cancer” and “cancerous” refer to physiological disorders in mammals where cell growth is not regulated.

[0144] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0145] "Tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion weakens, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0146] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

[0147] The term "chronic infection" refers to an infection in which the infectious agent (e.g., pathogens such as viruses, bacteria, protozoa such as parasites, fungi, or the like) has induced an immune response in the infected host but has not yet been cleared or eliminated from the host as in the case of an acute infection. Chronic infections can be persistent, latent, or slow-growing.

[0148] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of probes or antibodies by reacting with another directly labeled reagent. Examples of indirect labeling include the detection of a first antibody using a fluorescently labeled second antibody and the end labeling of a biotinylated DNA probe, making it detectable with a fluorescently labeled streptomycin protein.

[0149] "Isolated" anti-CD40 antibody refers to a component that has been separated from its native environment. In some embodiments, the anti-CD40 antibody is purified to a purity exceeding 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B848: 79-87 (2007).

[0150] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include those contained in cells that normally contain them, but which are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations. "Isolated nucleic acids encoding anti-CD40 antibodies" refers to one or more nucleic acid molecules encoding a chain or fragment thereof of an anti-CD40 antibody, including such nucleic acid molecules in a single or separate vector, and such nucleic acid molecules present at one or more locations within a host cell.

[0151] The following is a calculation of sequence identity between sequences.

[0152] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0153] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0154] Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0155] Additionally or alternatively, the nucleic acid and protein sequences described herein may be further used as “query sequences” to perform searches against public databases, for example, to identify other family member sequences or related sequences.

[0156] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0157] The term "pharmaceutical excipients" refers to diluents, adjuvants, carriers, excipients, or stabilizers that are applied together with active substances.

[0158] When used herein, "treatment" means to slow, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay disease development or to slow disease progression.

[0159] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0160] The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

[0161] "Chemotherapy agents" include chemical compounds useful in the treatment of cancer, including but not limited to antitumor agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; antiestrogens; antiandrogens; and nonsteroidal antiandrogens, etc. Examples of chemotherapeutic agents are those disclosed in WO2015 / 153513 or WO2016 / 028672 or WO2015 / 138920.

[0162] As used in this article, the term "immunomodulator" refers to natural or synthetic active agents or drugs that inhibit or modulate immune responses. Immune responses can be humoral or cellular. Immunomodulators include inhibitors of immune checkpoint molecules and activators of co-stimulatory molecules.

[0163] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Examples of cytotoxic agents can be found in those disclosed in WO2015 / 153513, WO2016 / 028672 or WO2015 / 138920.

[0164] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of the nucleic acid to which they are effectively linked. Such vectors are referred to herein as "expression vectors."

[0165] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells. Host cells are any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0166] "Subject / Patient Sample" refers to a collection of cells, tissues, or body fluids obtained from a patient or subject. The source of tissue or cell samples can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds naturally occurring and not contaminated with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples in this document include, but are not limited to, tumor biopsies, fine-needle aspirations, bronchoalveolar lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples.

[0167] II. The anti-CD40 antibody of the present invention

[0168] This invention provides a new generation of anti-human CD40 antibodies that can reduce or avoid adverse events caused by conventional CD40 agonists.

[0169] The anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises three complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises three complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, wherein:

[0170] (a) LCDR1 contains the amino acid sequence shown in SEQ ID NO:1 or a variant of SEQ ID NO:1 with no more than 2 or no more than 1 amino acid variation;

[0171] (b) LCDR2 contains a variant of any amino acid sequence selected from SEQ ID NO:2, 5, 6, 7, or any amino acid sequence selected from SEQ ID NO:2, 5, 6, 7, with no more than 2 or no more than 1 amino acid variation;

[0172] (c) LCDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:3, 4, or any amino acid sequence of SEQ ID NO:3, 4, with no more than two or no more than one amino acid variation.

[0173] (d)HCDR1 contains a variant of any amino acid sequence selected from SEQ ID NO:8, 12, 14, 20, or any amino acid sequence selected from SEQ ID NO:8, 12, 14, 20, with no more than 2 or no more than 1 amino acid variation;

[0174] (e)HCDR2 comprises a variant of any amino acid sequence selected from SEQ ID NO:9, 21, or any amino acid sequence of SEQ ID NO:9, 21 with no more than 2 or no more than 1 amino acid variation;

[0175] (f) HCDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:10, 11, 13, 15, 16, 17, 18, 19, or any amino acid sequence selected from SEQ ID NO:10, 11, 13, 15, 16, 17, 18, 19, with no more than two or no more than one amino acid variation.

[0176] The amino acid change mentioned therein is the addition, deletion, or substitution of an amino acid; for example, the amino acid change is a conserved amino acid substitution.

[0177] In some embodiments, the anti-CD40 antibody of the present invention comprises:

[0178] LCDR1 containing the amino acid sequence RSSQGIRSSLA (SEQ ID NO:1), LCDR2 containing the amino acid sequence GX1SSLX2X3 (SEQ ID NO:56), and LCDR3 containing the amino acid sequence QQLX4SFPST (SEQ ID NO:57), wherein X1 is A or G; X2 is E or L; X3 is G or V; X4 is N or A; and

[0179] HCDR1 containing amino acid sequences such as GFTX5GSYEMX6 (SEQ ID NO:58), HCDR2 containing amino acid sequences such as YISSX7GETTD (SEQ ID NO:59), and HCDR2 containing amino acid sequences such as DVFFFX8X9SX 10 X 11 X 12 X 13 HCDR3 of AYGMDV (SEQ ID NO:60), wherein X5 is F, A, or P; X6 is N or D; X7 is S or A; X8 is D or S; X9 is S or P; X 10 For G or R; X 11 For D, P, S, or F; X 12 For P, N, or R; X 13 It can be G or H.

[0180] In some embodiments, the anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein:

[0181] (i) The light chain variable region contains any amino acid sequence from SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains any amino acid sequence from SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55; or

[0182] (ii) The light chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55.

[0183] In some embodiments, the anti-CD40 antibody of the present invention comprises an immunoglobulin Fc region. In some embodiments, the anti-CD40 antibody of the present invention is linked to the immunoglobulin Fc region at the C-terminus of its Fab fragment, optionally via an amino acid linker, for example, via an amino acid linker of length between 1 and 20 amino acids. In some embodiments, the amino acid linker is at least 90% glycine and / or serine amino acids. In some embodiments, the Fc region is derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1. In some embodiments, the Fc region is derived from human IgG1.

[0184] In some embodiments of the invention, the amino acid changes described herein include the substitution, addition, or deletion of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, and more preferably, conservative substitutions.

[0185] In some embodiments, the amino acid changes described in this invention occur in regions outside the CDR (e.g., in the FR). In some embodiments, the substitution is a conservative substitution. A conservative substitution means that an amino acid is substituted by another amino acid within the same class, such as an acidic amino acid substituted by another acidic amino acid, a basic amino acid substituted by another basic amino acid, or a neutral amino acid substituted by another neutral amino acid.

[0186] In some implementations, the anti-CD40 antibodies provided herein are modified to increase or decrease their degree of glycosylation. The addition or deletion of glycosylation sites in anti-CD40 antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. Fc glycosylation can also be altered when the anti-CD40 antibody contains an Fc region. In some applications, removing unwanted glycosylation site modifications can be useful, for example, removing fucose modifications to enhance antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC).

[0187] In some embodiments, the anti-CD40 antibody of the present invention has its glycosylation site in the CH2 domain of the immunoglobulin Fc region removed, for example, by mutating the N297 residue in the CH2 domain of the human IgG Fc region to remove the glycosylation site, for example, by changing the N297 residue to Gly, Ala, Gln, Asp or Glu, preferably by changing the N297 residue to Gln.

[0188] The anti-CD40 antibody of the present invention is capable of binding to CD40 with high affinity, for example, at about 10... -7 M to approximately 10 -10 M binds to CD40, such as human CD40, cynomolgus monkey CD40, and mouse CD40, as measured by the ForteBio kinetic binding assay. In some embodiments, the anti-CD40 antibody of the present invention binds at approximately 0.5 × 10⁻⁶. －8 M to 2×10 －8 M binds to the human CD40 antigen with affinity. The anti-CD40 antibody of this invention exhibits cross-reactivity with cynomolgus monkey CD40 and mouse CD40.

[0189] The anti-CD40 antibody of the present invention enhances the immune response to antigens (e.g., tumor-associated antigens (TAAs)) by enhancing B cell-mediated immune responses, B cell activation, and / or cytokine production.

[0190] In one embodiment, the anti-CD40 antibody of the present invention enhances the immune response to an antigen and induces antibody-dependent cytotoxicity (ADCC) against CD40-expressing cells (e.g., CD40-expressing tumor cells). Additionally, the anti-CD40 antibody of the present invention induces apoptosis, as measured by increased expression of CD95.

[0191] In another embodiment, the anti-CD40 antibody of the present invention can enhance the immune response to antigens without inducing antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of CD40-expressing cells. Therefore, the anti-CD40 antibody of the present invention can also work against tumor cells that do not express CD40 or against infectious diseases by enhancing the immune response to antigens, for example by enhancing the B cell-mediated immune response.

[0192] In one embodiment, the anti-CD40 antibody of the present invention is modified to include a constant region without effector function. For example, when the anti-CD40 antibody of the present invention includes an Fc region, the effector function of the anti-CD40 antibody of the present invention can be reduced or eliminated by mutating the N297 residue of the glycosylation site in the Fc region to Gly, Ala, Gln, Asp, or Glu, etc. Additionally, when the anti-CD40 antibody of the present invention includes an Fc region, modifications that reduce the binding affinity of the anti-CD40 antibody of the present invention to FcγRIIIA (CD16A) can also be included in the Fc region to reduce or eliminate the effector function induced by the Fc region. In one embodiment, the modification is in the CH2 domain of the Fc region, for example at position 329 (EU number) of the heavy chain (e.g., P329G). In one embodiment, the anti-CD40 antibody of the present invention includes amino acid substitutions at positions 234 and 235 (EU number) of the heavy chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (also referred to as the "LALA mutation").

[0193] In one embodiment, the anti-CD40 antibody of the present invention can enhance the immune response regardless of antibody binding to an Fc receptor. For example, the anti-CD40 antibody of the present invention can exhibit potent CD40 agonistic characteristics without cross-linking with an Fc receptor such as FcγR. These agonistic characteristics are determined, for example, by measuring increased B cell activity and / or increased B cell activation.

[0194] In another embodiment, the anti-CD40 antibody of the present invention enhances the binding of CD40 to CD40L (CD154) on CD40-expressing cells. In a specific embodiment, the anti-CD40 antibody of the present invention enhances the binding of soluble CD40L to CD40-expressing cells by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In a specific embodiment, for example, as measured by FACS, biological layer interferometry (BLI), or Biacore, the anti-CD40 antibody enhances the binding of soluble CD40L to CD40-expressing cells by at least about 50%.

[0195] In some embodiments, the anti-CD40 antibody of the present invention induces CD95 expression when incubated with Ramos cells. In some embodiments, the anti-CD40 antibody of the present invention increases B cell proliferation when incubated with human B cells. In some embodiments, the anti-CD40 antibody of the present invention increases IL-12 secretion and CD83 expression when incubated with dendritic cells.

[0196] The anti-CD40 antibody of the present invention is not expected to cause dose-limiting toxicity, cytokine release syndrome (CRS), or hepatotoxicity associated with elevated levels of circulating liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GLDH) after administration to subjects, or the adverse reactions are mild.

[0197] III. Immunoconjugates

[0198] The present invention also relates to the anti-CD40 antibody of the present invention (“immunoconjugate”) conjugated with other substances. In some embodiments, the other substances are, for example, therapeutic agents (such as cytotoxic agents). Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to: radioactive isotopes; growth inhibitors; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents.

[0199] Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immune conjugates also include, for example, WO2015 / 153513 or WO2015 / 138920.

[0200] The anti-CD40 antibody of the present invention can also be linked to a solid support, which is particularly useful for immunoassays or the purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0201] In some embodiments, the immune conjugate is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the immune conjugate is used to prevent or treat infections, such as chronic infections, including bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0202] IV. The nucleic acid of the present invention and the host cell containing it.

[0203] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned anti-CD40 antibodies or fragments thereof or any strand thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0204] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55.

[0205] The present invention also covers nucleic acids that hybridize under stringent conditions with or encode polypeptide sequences having one or more amino acid substitutions (e.g., conserved substitutions), deletions, or insertions compared to the following nucleic acids: nucleic acids comprising nucleic acid sequences encoding amino acid sequences selected from any one of SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55; or nucleic acids comprising nucleic acid sequences encoding amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acid sequences selected from any one of SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55.

[0206] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, kinases, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is a pcDNA3.3 vector.

[0207] Once the expression vector or DNA sequence for expression has been prepared, it can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells.

[0208] Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0209] In one embodiment, a host cell comprising the polynucleotides of the present invention is provided. In some embodiments, a host cell comprising the expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for antibody preparation. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*. The host cell can also be a eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as a host. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O. In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0210] V. Production and purification of the anti-CD40 antibody of the present invention

[0211] In one embodiment, the present invention provides a method for preparing an anti-CD40 antibody, wherein the method includes culturing a host cell containing a nucleic acid encoding the anti-CD40 antibody or an expression vector containing the nucleic acid under conditions suitable for expressing a nucleic acid encoding the anti-CD40 antibody, and optionally isolating the anti-CD40 antibody. In one embodiment, the method further includes recovering the anti-CD40 antibody from the host cell (or host cell culture medium).

[0212] To recombinantly generate the anti-CD40 antibody of the present invention, the nucleic acid encoding the anti-CD40 antibody of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the anti-CD40 antibody of the present invention.

[0213] The anti-CD40 antibody of the present invention, prepared as described herein, can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the anti-CD40 antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0214] VI. The method for determining the activity of the anti-CD40 antibody of the present invention

[0215] The anti-CD40 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the anti-CD40 antibodies of the present invention can be tested, for example by known methods such as ELISA, Western blotting, etc. Binding to CD40 can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, the binding of the anti-CD40 antibodies of the present invention to CD40 is determined using SPR or biofilm layer interference.

[0216] The present invention also provides an assay for identifying biologically active anti-CD40 antibodies. Biological activity may include, for example, binding to cell surface CD40 (e.g., human CD40, monkey CD40, mouse CD40), enhancing CD40 / CD40L binding, activating antigen-presenting cells, inducing CD95 expression in CD40-expressing tumor cells, and enhancing B cell-mediated immune responses.

[0217] Cells used for any of the above in vitro assays include naturally expressed CD40 or modified CD40-expressing cell lines. The modified CD40-expressing cell line is a cell line that does not normally express CD40 but expresses CD40 after being transfected with DNA encoding CD40.

[0218] It is understood that the immunoconjugate of the present invention can be used to replace the anti-CD40 antibody for any of the above-described assays.

[0219] VII. Pharmaceutical compositions and pharmaceutical preparations

[0220] In some embodiments, the present invention provides compositions comprising any anti-CD40 antibody or its immunoconjugate described herein, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-CD40 antibody or its immunoconjugate of the present invention, and a combination of one or more other therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators, preferably anti-PD-1 or anti-PD-L1 antibodies).

[0221] In some embodiments, the composition is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the composition is used to prevent or treat infections, such as chronic infections, including bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0222] The present invention also includes compositions comprising anti-CD40 antibodies or their immunoconjugates (including pharmaceutical compositions or pharmaceutical formulations) and / or compositions comprising polynucleotides encoding anti-CD40 antibodies (including pharmaceutical compositions or pharmaceutical formulations). These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0223] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Pharmaceutical carriers suitable for this invention can be sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For information on the use and applications of excipients, see also "Handbook of Pharmaceutical Excipients," 5th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago. If desired, the compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin.

[0224] Pharmaceutical formulations containing the antiCD40 antibody described herein can be prepared by mixing the antiCD40 antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0225] The pharmaceutical compositions or formulations of the present invention may further comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anticancer or anti-infective active ingredients, such as chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, etc. The active ingredients are suitably combined in amounts effective for the intended use.

[0226] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the anti-CD40 antibody of the present invention, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0227] VIII. Combination products or reagent kits

[0228] In some embodiments, the present invention also provides a combination product comprising the anti-CD40 antibody or antigen-binding fragment of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs, or immunomodulators, etc.). In some embodiments, other antibodies include, for example, anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0229] In some embodiments, the combined product is used for the prevention or treatment of tumors. In some embodiments, the tumor is cancer, etc.

[0230] In some regimens, two or more components of the combined product may be administered to the subject sequentially, separately, or simultaneously.

[0231] In some embodiments, the present invention also provides a kit comprising the anti-CD40 antibody, pharmaceutical composition, immunoconjugate, or combination product of the present invention, and optionally a package insert with instructions for administration.

[0232] In some embodiments, the present invention also provides pharmaceutical articles comprising the anti-CD40 antibody of the present invention, pharmaceutical compositions, immunoconjugates, or combination products, optionally including a packaging insert for instructions of administration.

[0233] IX. Uses of the anti-CD40 antibody of the present invention

[0234] In one aspect, the present invention relates to a method for modulating an immune response in an individual. The method comprises administering to a subject an effective amount of the anti-CD40 antibody disclosed herein, or a pharmaceutical composition, immunoconjugate, or combination product comprising said anti-CD40 antibody, thereby modulating an immune response in the subject. In one embodiment, the therapeutically effective amount of the anti-CD40 antibody, pharmaceutical composition, immunoconjugate, or combination product disclosed herein restores, enhances, stimulates, or increases the immune response in the subject.

[0235] In some embodiments, the present invention relates to a method for enhancing CD40 activity, enhancing the binding of CD40 to CD40L, and inducing the secretion of cytokines such as IL-12 in an individual, the method comprising administering to the subject an effective amount of the anti-CD40 antibody disclosed herein or a pharmaceutical composition or immunoconjugate or combination product containing therein.

[0236] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of the anti-CD40 antibody disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising therein. In some embodiments, the tumor is a tumor immune escape. In some embodiments, the tumor is cancer.

[0237] In another aspect, the present invention relates to a method for inducing antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of the anti-CD40 antibody disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising the same.

[0238] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient with or at risk of having the diseases described herein). In one embodiment, the subject has or is at risk of having the diseases described herein (e.g., a tumor or infectious disease as described herein). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is immunocompromised due to an infection or is at risk of being immunocompromised due to an infection.

[0239] In some implementations, the tumors described herein, such as cancers, include, but are not limited to, solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions.

[0240] Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas) affecting multiple organ systems, such as those invading the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), pancreas, genitourinary tract (e.g., kidney, bladder epithelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Squamous cell carcinomas include malignant tumors such as those in the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, for example, advanced melanoma. In one embodiment, the cancer is lymphoma, renal cell carcinoma, non-small cell lung cancer, liver cancer, pancreatic cancer, colonic adenocarcinoma, and breast cancer. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention.

[0241] Non-limiting examples of preferred cancers for treatment include lymphoma (e.g., diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma), breast cancer (e.g., metastatic breast cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC), such as stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (e.g., multiple myeloma), leukemia (e.g., chronic myeloid leukemia), skin cancer (e.g., melanoma (e.g., stage III or IV melanoma) or Merkel cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), spinal dysplasia syndrome, bladder cancer (e.g., transitional cell carcinoma), kidney cancer (e.g., renal cell carcinoma, such as clear cell renal cell carcinoma, such as advanced or metastatic clear cell renal cell carcinoma), and colon cancer. In addition, refractory or recurrent malignancies (e.g., pancreatic cancer) can be treated with the anti-CD40 antibodies described herein or pharmaceutical compositions or immunoconjugates or combination products containing them.

[0242] In some embodiments, the anti-CD40 antibody of the present invention generates a cascade effect by binding to CD40 on the surface of B cells or antigen-presenting cells, thereby activating T cells and thus treating tumors.

[0243] In some embodiments, for tumors that overexpress CD40 on their surface, the anti-CD40 antibody of the present invention treats tumors by ADCC, CTL and / or inducing tumor cell apoptosis.

[0244] In some embodiments, the antiCD40 antibody of the present invention, or an immunoconjugate or composition thereof, or a combination product thereof, may delay the onset of the disease and / or disease-related symptoms.

[0245] In some embodiments, the preventive or treatment methods described herein also include administration to the subject or individual in combination of the anti-CD40 antibody or pharmaceutical composition or immunoconjugate or combination product disclosed herein, and one or more other therapies, such as modes of treatment and / or other therapeutic agents.

[0246] In some implementations, the treatment modality includes surgery (e.g., tumor resection); radiotherapy (e.g., external beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed at a pre-selected target or organ), or focused irradiation, etc. Focused irradiation can be selected from stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy. Focused irradiation can have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays), for example, as described in WO 2012 / 177624.

[0247] Radiation therapy can be administered through one or a combination of several methods, including but not limited to external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole-body radiation therapy, radiotherapy, and permanent or transient interstitial brachytherapy.

[0248] In some implementations, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0249] Other exemplary antibodies include, but are not limited to, inhibitors of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, anti-CEACAM, or anti-LAG-3); antibodies that stimulate immune cells (e.g., agonistic GITR antibodies or CD137 antibodies), etc. Preferably, the other antibodies are selected from anti-PD-1 antibodies and / or anti-PD-L1 antibodies. More preferably, the anti-PD-1 antibody is nivolumab from BMS or pembrolizumab from Merck; the anti-PD-L1 antibody is atezolizumab from Roche, avelumab co-developed by Merck KGaA and Pfizer, or durvalumab from AstraZeneca.

[0250] In some embodiments, the immunomodulator is an activator or agonist of the co-stimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from agonists of the following molecules (e.g., agonistic antibodies or their antigen-binding fragments, or soluble fusions): OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands.

[0251] The combination therapy of the present invention covers both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, the anti-CD40 antibody or immune conjugate of the present invention may be administered before, simultaneously with, and / or after the administration of other therapies.

[0252] In one implementation, the administration of the anti-CD40 antibody and the administration of other therapies (e.g., treatment modalities or therapeutic agents) occur within each other within about one month, or about one, two, or three weeks, or about 1, 2, 3, 4, 5, or 6 days.

[0253] The anti-CD40 antibody of the present invention (and pharmaceutical compositions or immunoconjugates comprising it) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment requires, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including but not limited to single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0254] For the prevention or treatment of disease, the appropriate dosage of the anti-CD40 antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of anti-CD40 antibody, the severity and progression of the disease, whether the anti-CD40 antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the anti-CD40 antibody, and the judgment of the attending physician. The anti-CD40 antibody is appropriately administered to the patient as a single treatment or after a series of treatments. The dosage of the anti-CD40 antibody and the treatment regimen can be determined by a skilled technician.

[0255] It is understood that the immune conjugates or compositions or combination products of the present invention can be used to replace the anti-CD40 antibody for any of the above-described prevention or treatment.

[0256] X. Methods and compositions for diagnosis and detection

[0257] In some embodiments, any anti-CD40 antibody provided herein can be used to detect the presence of CD40 in a biological sample. The term "detection," as used herein, includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, or ELISA assays. In some embodiments, the biological sample is blood, serum, or other bodily fluid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from hyperplastic or cancerous lesions.

[0258] In one embodiment, an anti-CD40 antibody is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of CD40 in a biological sample is provided. In some embodiments, the method includes detecting the presence of the CD40 protein in a biological sample. In some embodiments, CD40 is human CD40. In some embodiments, the method includes contacting a biological sample with an anti-CD40 antibody as described herein under conditions that allow the anti-CD40 antibody to bind to CD40, and detecting whether a complex is formed between the anti-CD40 antibody and CD40. The formation of the complex indicates the presence of CD40. This method can be in vitro or in vivo. In one embodiment, the anti-CD40 antibody is used to select subjects suitable for treatment with the anti-CD40 antibody, for example, where CD40 is a biomarker used for selecting said subjects.

[0259] In one embodiment, the anti-CD40 antibody of the present invention can be used to diagnose cancer or tumors, such as to evaluate (e.g., monitor) the treatment or progression of the disease described herein (e.g., hyperplastic or cancerous disease), its diagnosis, and / or staging. In some embodiments, a labeled anti-CD40 antibody is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C 125 I, 3 H and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labeling, phage labeling, stable free radicals, etc.

[0260] In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-CD40 antibody. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin-fixed and paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine-needle aspirate.

[0261] In some implementations, CD40 is detected before treatment, for example, before starting treatment or before a treatment after a treatment interval.

[0262] In some embodiments, a method for treating a tumor or infection is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample containing cancer cells) for the presence of CD40, thereby determining a CD40 value; comparing the CD40 value with a control value (e.g., the value of CD40 in a sample from a healthy individual); and if the CD40 value is less than the control value, administering to the subject a therapeutically effective amount of an anti-CD40 antibody (e.g., the anti-CD40 antibody described herein) optionally in combination with one or more other therapies, thereby treating the tumor or infection.

[0263] It is understood that the various embodiments described in the various parts of this invention, such as diseases, therapeutic agents, treatment methods, and administration, are also applicable to embodiments in other parts of this invention, or can be combined with embodiments in other parts. The embodiments described in the various parts of this invention, such as the properties, uses, and methods applicable to anti-CD40 antibodies, are also applicable to compositions, conjugates, combination products, and kits containing anti-CD40 antibodies.

[0264] XI. Sequence of an exemplary anti-CD40 antibody of the present invention

[0265] The sequence of an exemplary anti-CD40 antibody of the present invention is shown in the table below.

[0266] Table 1. CDR sequences in the variable domain (VL) of the exemplary antibody light chain of the present invention (defined by AbM numbering).

[0267]

[0268] Table 2. CDR sequences in the variable domain (VH) of the exemplary antibody heavy chain of the present invention (defined by AbM numbering).

[0269]

[0270]

[0271] Table 3. Variable region nucleotide and amino acid sequences of exemplary antibodies of the present invention.

[0272]

[0273]

[0274]

[0275]

[0276] The following embodiments are described to aid in understanding the invention. It is not intended, and should not be construed in any way, as limiting the scope of the invention. Example

[0277] Example 1: Preparation of anti-CD40 antibody

[0278] 1.1. Preparation of P17-Fc antigen

[0279] The extracellular domain of human CD40 (CD40 ECD) (Uniprot: positions 21-193 of P25942) gene fragment was fused to the N-terminus of the DNA sequence of human IgG1 Fc fragment, and the nucleotide sequence encoding the CD40 ECD-Fc fusion protein was synthesized and cloned into the multiple cloning site of the pcDNA3.3 (Invitrogen) vector to obtain the expression vector for expressing the CD40 ECD-Fc fusion protein, which is also referred to in this paper as the P17-Fc fusion protein expression vector.

[0280] The P17-Fc fusion protein expression vector was introduced into CHO-K cells (Thermo Fisher) for eukaryotic expression (detailed preparation steps can be found in the book *Therapeutic Fc-Fusion Proteins* (Steven M. Chamow et al., pp. 45-62, "Methods of Production for Fc-Fusion Proteins") to obtain the P17-Fc fusion protein. The immunogenicity of the prepared P17-Fc fusion protein was verified by measuring the binding of the P17-Fc fusion protein to the anti-CD40 antibody dacetuzumab (see EP1885399B1, hereinafter referred to as CD40-Dac) and Bleslumab (see US20100234578, hereinafter referred to as CD40-Ble) using standard ELISA. Figure 1 As shown, the P17-Fc fusion protein exhibits affinity for dasizumab and Bleslumab.

[0281] 1.2. Biotinylation of the P17-Fc fusion protein

[0282] Use EZ-Link according to the manufacturer's instructions. TM Sulfo-NHS-LC-Biotinylation Kit (EZ-Link) TM The Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher, catalog number: 21435) was used to label the P17-Fc fusion protein with calculated amounts of EZ-LinkSulfo-NHS-LC-Biotin. The amount of biotin-labeled P17-Fc fusion protein (also referred to herein as "P17-Fc-Biotin" or "Biotinized P17-Fc fusion protein") was measured using a biotin quantification kit (Thermo Fisher, 28005).

[0283] Furthermore, the conformation of the biotinylated P17-Fc fusion protein was verified by ELISA assays against dasizumab and Bleslumab using the same method as in Example 1.1.

[0284] like Figure 2 As shown, compared with the binding of the unbiotinylated P17-Fc fusion protein to dasizumab and Bleslumab, the biotinylated P17-Fc fusion protein showed similar binding to both anti-CD40 antibodies.

[0285] IL-23-Fc-biotin (IL-23 sequence source: Uniprot: Q9NPF7) was prepared using a similar method and used to remove Fc-bound phages in subsequent antibody library screening.

[0286] The P17-His protein (the sequence of P17 is the same as that of Uniprot: P25942, positions 21-193) was prepared using a similar method, and its C-terminus has 6 histidine tags.

[0287] 1.3. Antibody library screening

[0288] The construction and screening methods for the fully human antibody library are described in patent application CN202010236256.8. The biotinylated P17-Fc fusion protein of Example 1.1 was used to screen the fully human antibody phage display library to obtain anti-CD40 Fab antibody fragments that specifically bind to human CD40 and are displayed on phages.

[0289] The specific screening method is as follows: The antibody library was screened using the magnetic bead method. Before each round of panning the fully human antibody phage display library using the biotinylated P17-Fc fusion protein of Example 1.1, the antibody library was panned with IL-23-Fc-biotin from Example 1.1 to remove phages binding to Fc. Then, biotinylated P17-Fc fusion protein was used to screen for phage monoclonals that specifically bind to human CD40 protein. After washing to remove non-specifically adsorbed phages, the collected phages were amplified using E. coli SS320 cells and used for the next round of panning.

[0290] In each round of selection, the ratio of input phage to output phage is calculated and used as an indicator of CD40-specific phage enrichment.

[0291] ELISA analysis of phage libraries also confirmed the enrichment of CD40 in each library. After three rounds of panning, phages binding to CD40 were highly enriched, such as... Figure 3 As shown.

[0292] The selected phage Fab was tested for binding to P17-Fc-biotin and IL23-Fc-biotin. Phage clones showing specific affinity for P17-Fc-biotin were selected, and their DNA was extracted and sequenced.

[0293] A phage clone C8 containing a unique Fab sequence was obtained, and its encoding nucleotide and amino acid sequences are shown below. The nucleotide sequence encoding the C8 Fab light chain variable region (C8-WT-VL) is also shown.

[0294] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGACTGCATCTGTCGGAGACAGAGTCACCATTACTTGCCGGTCAAGTCAGGGCATTCGCAGTTCTTTAGCCTGGTATCAACAGAAACCAGGGAAACCTCCTAAGCTCCTGATCTATGGTGCCTCCAGTTTG GAAGGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGCTCAATAGTTTCCCGTCCACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA(SEQ ID NO:22)

[0295] Amino acid sequence encoding the light chain variable region (C8-WT-VL) of C8 Fab

[0296] DIQLTQSPSSLTASVGDRVTITCRSSQGIRSSLAWYQQKPGKPPKLLIYGASSLEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSFPSTFGQGTKLEIK(SEQ ID NO:23)

[0297] Nucleotide sequence encoding the heavy chain variable region (C8-WT-VH) of C8 Fab

[0298] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACGGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGGTAGTTATGAAATGAATTGGGTCCGCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTCTCATATATAAGTAGCAGTGGTGAAACGACCGACTACGCAGACTCTGTG AAGGGCCGCTTCACCGTCTCCAGAGACAACAGTAAGAATTTACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGACGTGTTTTTTTTTGATAGTAGTGGTGACAATGGGGCCTACGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCATCA(SEQ ID NO:24)

[0299] Amino acid sequence encoding the heavy chain variable region (C8-WT-VH) of C8 Fab

[0300] EVQLLESGGGLVRPGGSLRLSCAASGFTFGSYEMNWVRQAPGKGLEWVSYISSSGETTDYADSVKGRFTVSRDNSKNLLYLQLEGMNSLRAEDTAVYYCARDVFFFDSSGDNGAYGMDVWGQGTMVTVSS(SEQ ID NO:25)

[0301] The nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the phage C8 clone were ligated into the pcDNA3.3 vector, respectively, and expressed and purified as Fab fragments in CHO-K cells (Thermo Fisher). The affinity of the Fab candidate molecule for P17-Fc was detected by ELISA, and the results are as follows: Figure 4 As shown.

[0302] Depend on Figure 4 It can be seen that the EC50 value of C8 clone Fab binding to CD40 is approximately 0.4764 μg / mL, while the EC50 value of the positive control Bleslumab monoclonal antibody binding to CD40 is approximately 0.04701 μg / mL.

[0303] Furthermore, to test the affinity of anti-CD40 Fab for CD40 expressed on the cell surface, CHO-K cells expressing human CD40 (Uniprot ID P25942) and CHO-K cells expressing cynomolgus monkey CD40 (Uniprot ID G7PG38) were prepared. Specifically, the coding sequence of human CD40 (Uniprot ID P25942) was cloned into the multiple cloning site of the pcDNA3.3 (Invitrogen) vector to obtain an expression vector expressing human CD40; this expression vector was then introduced into CHO-K cells (Thermo Fisher) for eukaryotic expression, resulting in CHO-K cells expressing human CD40 on the cell surface (hereinafter referred to as "CHO-K-huCD40 cells"). Similarly, CHO-K cells expressing cynomolgus monkey CD40 on the cell surface (hereinafter referred to as "CHO-K-cynoCD40 cells") were obtained.

[0304] CHO-K-huCD40 cells or CHO-K-cynoCD40 cells were injected at a dose of 1.0 × 10⁻⁶. 5Cells were seeded per well in 96-well plates, and diluted C8 anti-CD40 Fab candidate molecules were added. After incubation at 4°C for 30 minutes, cells were washed, and 100 μL of APC-labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch Inc., catalog number: 109-136-097, Allophycocyanin (APC) AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, F(ab')2 fragment specific) was added, followed by incubation at 4°C for 30 minutes. Cells were then washed, and the binding of the C8 anti-CD40 Fab candidate molecules to CD40 molecules expressed on the cell surface was detected by flow cytometry (Beckman Coulter).

[0305] Figure 5A Different concentrations of C8 clone anti-CD40 Fab molecules showed binding affinity to CHO-K-huCD40 cells, and this binding affinity was C8 Fab concentration-dependent within the C8 Fab concentration range of 3.2 μg / mL to 80 μg / mL.

[0306] Figure 5B The study showed that the C8 clone anti-CD40 Fab molecule also exhibited binding affinity to CHO-K-cynoCD40 cells, with a C8 Fab concentration of 80 μg / mL. This demonstrates that the C8 clone Fab molecule exhibits cross-reactivity with cynomolgus monkey CD40.

[0307] 1.4. Expression, purification, and concentration determination of full-length antibodies

[0308] The C8 clone Fab candidate molecule from Example 1.3 was used to construct a full-length antibody, wherein the sequence of human IgG1 Fc was linked to the C-terminus of the heavy chain sequence of the Fab molecule.

[0309] Specifically, the obtained nucleotide sequences encoding the variable region of the antibody heavy chain and the variable region of the antibody light chain were constructed into a modified eukaryotic expression vector plasmid pcDNA3.3 (Invitrogen) containing light and heavy chain constant region fragments, respectively. Following the manufacturer's instructions, the full-length heavy and light chains of the antibody were co-expressed in CHO-K cells using the ExpiCHO transient expression system (Thermo Fisher, A29133). The antibodies were then purified by protein A affinity chromatography to obtain the C8 clone anti-CD40 full-length antibody.

[0310] The purified full-length antibody was analyzed for concentration using A280 absorbance and its quality was verified by SDS-PAGE, differential scanning fluorometry (DSF), and size exclusion chromatography (SEC). After passing quality control, the antibody was aliquoted and stored at -80°C. Specific methods are described in patent application number CN 202010236256.8.

[0311] The amino acid sequence encoding the light chain (C8-WT-LC) of the full-length C8 antibody.

[0312] DIQLTQSPSSLTASVGDRVTITCRSSQGIRSSLAWYQQKPGKPPKLLIYGASSLEGGVPSRFSGSGSGTDFTLTI

[0313] SSLQPEDFATYYCQQLNSFPSTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:26)

[0314] The amino acid sequence encoding the heavy chain (C8-WT-HC) of the full-length C8 antibody.

[0315] EVQLLESGGGLVRPGGSLRLSCAASGFTFGSYEMNWVRQAPGKGLEWVSYISSSGETTDYADSVKGRFTVSRDNSKNLLYLQLEGMNSLRAEDTAVYYCARDVFFFDSSGDNGAYG MDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:27)

[0316] Example 2: Cell-based functional assay of anti-CD40 antibody

[0317] 2.1. CD40 binding characteristics and species cross-reactivity of candidate full-length anti-CD40 antibodies

[0318] To verify the binding activity and cross-reactivity of the full-length anti-CD40 antibody prepared in Example 1.4 of this invention to CD40, CHO-K-huCD40 cells or CHO-K-cynoCD40 cells were cultured at 1.0 × 10⁻⁶ cells / cells. 5 Cells were seeded per well in a 96-well plate. 100 μL of a diluted anti-CD40 candidate antibody prepared in Example 1.4 was added to each well. After incubation at 4°C for 30 min, cells were washed, and 100 μL of APC-labeled anti-human IgG secondary antibody (Allophycocyanin (APC) Affini Pure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch Inc., Catalog No.: 109-135-098) was added, followed by incubation at 4°C for another 30 min. Cells were then washed, and the binding of the full-length anti-CD40 antibody to the CD40 molecules expressed on the cells was detected by flow cytometry. Results are as follows: Figure 6A and Figure 6B As shown.

[0319] Depend on Figure 6A and Figure 6B It is evident that after constructing the full-length antibody from the C8 clone Fab fragment, the full-length C8 clone anti-CD40 antibody still retains the affinity of the C8 clone Fab fragment for human CD40 and the cross-reactivity with cynomolgus monkey CD40.

[0320] Similar to Example 1.3, CHO-K cells expressing full-length mouse CD40 (Uniprot ID P27512) (hereinafter also referred to as "CHO-K-mouseCD40 cells") were prepared. Using CHO-K-mouseCD40 cells, the cross-reactivity of the candidate C8 full-length anti-CD40 antibody prepared in Example 1.4 with mouse CD40 was tested. CHO-K-mouseCD40 cells were cultured at 1.0 × 10⁻⁶ cells / cells. 5Cells were seeded per well in 96-well plates. 100 μL of diluted anti-CD40 candidate antibody and two control antibodies (dasizumab and APX005) were added to each well. After incubation at 4°C for 30 minutes, cells were washed and FITC-labeled anti-human IgG Fcγ secondary antibody (affinity-purified F(ab')2 fragmented goat anti-human IgG secondary antibody that specifically reacts with the Fcγ fragment, Jackson Immunoresearch, catalog number: 109-006-098) was added, followed by incubation at 4°C for 30 minutes. Cells were then washed and analyzed by flow cytometry. Results are shown below. Figure 6C As shown.

[0321] Depend on Figure 6C As can be seen, the full-length C8 clone antibody molecule showed significant binding activity to mouse CD40, while the control antibodies dasizumab and APX005 did not show cross-reactivity against mouse CD40.

[0322] 2.2. Effect of candidate anti-CD40 antibodies on the binding of CD40 to CD40L

[0323] To test the effect of the full-length anti-CD40 antibody prepared in Example 1.4 on the binding of CD40 to CD40L, CHO-K cells expressing human CD40 (i.e., CHO-K-huCD40 cells) were used at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded into 96-well plates, and 100 μL of diluted full-length anti-CD40 antibody or isotype control (i.e., purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502)) was added to each well. After incubation at 4°C for 30 minutes, the cells were washed.

[0324] CD40L (ACRO Biosystems, catalog number CDL-H5248) was biotinylated as described in Example 1. The biotinylated CD40L protein was diluted to 200 ng / mL, and 100 μL of the diluted biotinylated CD40L (also referred to herein as CD40L-Biotin) was added to a 96-well plate. After incubation at 4°C for 30 min, the cells were washed, and then 100 μL of PE-labeled streptavidin (eBioscience catalog number 12-4317-87) was added, followed by incubation at 4°C for another 30 min. The cells were then washed and analyzed by flow cytometry.

[0325] like Figure 7As shown, the candidate antibody C8 clone exhibited enhanced activity in binding CD40 to CD40L at concentrations ranging from 0.01 to 20 μg / mL, indicating its potential as an anti-CD40 antibody with the potential to become a CD40 agonist. The control antibody Bleslumab significantly inhibited the binding of CD40 to CD40L, while dasizumab did not affect the binding of CD40 to CD40L at lower concentrations but inhibited the binding of CD40 to CD40L at higher concentrations.

[0326] 2.3. Assay for activation of immature dendritic cells mediated by candidate anti-CD40 antibodies

[0327] Immature dendritic cells (iDCs) are activated by anti-CD40 antibodies and then transformed into mature DCs. In this embodiment, the activation activity of iDCs mediated by candidate antibodies is evaluated by testing the expression of IL-12 cytokine and CD83 marker secreted by mature DCs.

[0328] Monocytes prepared from human peripheral blood were developed into iDCs by adding 1000 U / mL rhGM-CSF (PeproTech catalog number: 300-03-50) and 500 U / mL rhIL-4 (PeproTech catalog number: 200-04-50) to RPMI 1640 medium supplemented with 10% FCS, and then cultured at 5.0 × 10⁻⁶ ppm. 4 One iDC cell per well was seeded into a 6-well plate. The candidate full-length anti-CD40 antibody, each control antibody, and dilution of CD40L (ACRO Biosystems, catalog number CDL-H5248) were added to the iDC cells and cultured for three days. The culture medium and cells were then collected.

[0329] IL-12 secretion was measured in the collected culture medium by ELISA or flow cytometry, and the expression of CD83 markers was measured in the collected cells.

[0330] like Figure 8A and Figure 8B As shown, the full-length C8 clone antibody exhibited significant and dose-dependent iDC activation activity. In this respect, similar to dasizumab, the full-length C8 clone antibody is a weak CD40 agonist. As a control, CD40L resulted in significant IL-12 secretion.

[0331] 2.4. CD95 induction in Ramos cells mediated by candidate anti-CD40 antibodies

[0332] Ramos cells are human lymphoma cells that naturally express CD40 molecules on their cell surface. To test whether anti-CD40 antibodies bind to CD40 on Ramos cells and activate downstream CD40 signaling pathways, Ramos cells (ATCC No.: CRL-1596) were injected with 1.0 × 10⁻⁶ antibodies. 5 One cell / well is seeded into each well of a microtiter plate containing RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (hereinafter also referred to as "complete RPMI 1640 medium"). Diluted anti-CD40 antibody or CD40L (10 μg / mL for the first well, serially diluted 2-fold, 4 concentration points; see details for specific concentrations) is then added. Figure 9 (As shown) The sample was added to Ramos cells and incubated overnight at 37°C. Then, the expression of CD95 induced on the surface of Ramos cells was measured by flow cytometry.

[0333] like Figure 9 As shown, compared with hIgG1 as a negative control, the full-length C8 clone antibody showed dose-dependent CD95-inducing activity and weak agonistic activity.

[0334] Example 3: In vivo efficacy test of anti-CD40 antibody

[0335] 3.1. Anti-CD40 agonist antibody inhibits tumor growth in a mouse CDX model of Ramos cells.

[0336] A cell-derived xenograft (CDX) model is a tumor model constructed by transplanting a tumor cell line into nude mice or NSG mice. In this embodiment, a mouse CDX model was constructed by transplanting Ramos cells subcutaneously into mice, and the anti-tumor efficacy of a candidate anti-CD40 antibody in this mouse CDX model was tested.

[0337] Six-week-old female nude mice (Vitolliwa Balb / c Nude) were subcutaneously injected with 5 million human lymphoma Ramos cells on day 0 (defined as the day of tumor cell inoculation, and day 2 as day 1). When the average tumor volume reached 250 mmHg... 3 Eight animals per group were administered C8 anti-CD40 full-length antibody to all mice via intraperitoneal injection at doses of 0.2 mg / kg, 1 mg / kg, or 5 mg / kg (hereinafter, mg / kg is abbreviated as mpk). The administration was three times a week (TIW) for two weeks. Tumor volume was measured before each injection, and tumor weight was measured two weeks after the first administration. The final tumor volume and tumor weight are shown below. Figure 10A and Figure 10B Changes in tumor volume are shown in Figure 10C .

[0338] Compared with the control antibody APX005M (also referred to as APX or APX005 in this article) (see US20170246297, Apexigen), the full-length C8 anti-CD40 antibody showed similar anti-tumor efficacy.

[0339] The TGI (tumor growth inhibition rate) of each antibody group is shown in Table 4.

[0340] Table 4: TGI of APX005 and C8 anti-CD40 full-length antibodies

[0341]

[0342] 3.2. Anti-CD40 agonist antibody inhibits tumor growth in a mouse model of MC-38 cells.

[0343] MC38 is a mouse colon adenocarcinoma cell line that highly expresses mouse p53 protein (mp53) and does not express CD40 molecules.

[0344] The antitumor efficacy of the candidate antiCD40 antibody was tested in a mouse subcutaneous transplantation model of MC-38 cells.

[0345] Six-week-old female mice (Vitamin B1 C57BL / 6) were subcutaneously injected with 1 million mouse colon adenocarcinoma cells (MC-38 cells) (National Experimental Cell Resource Sharing Platform, catalog number 3111C0001CCC000523) into the axilla. When the average tumor volume reached 80-100 mm... 3 Mice (n=8 per group) were administered C8 anti-CD40 antibody via intraperitoneal injection at doses of 1.5 mg / kg, 5 mg / kg, or 15 mg / kg (hereinafter abbreviated as "mpk") three times a week for three weeks. Tumor volume was measured before each injection, and tumor weight was measured three weeks after the first administration. The final tumor weight is shown in [Figure / Table / Reference]. Figure 10D .

[0346] Depend on Figure 10D As can be seen, compared with the negative control PBS, the anti-CD40 agonist antibody C8 molecule exhibited anti-tumor efficacy at gradient concentrations.

[0347] Example 4. Affinity variants of C8 clone anti-CD40 antibody

[0348] In order to obtain the altered affinity of the full-length antibody of the C8 clone for the CD40 antigen, this embodiment further designed affinity variants and determined the function of the full-length antibody of the C8 clone.

[0349] 4.1. Construction of Affinity Variant Phage Display Library

[0350] First, the CDR and frame region of C8-WT-VL and C8-WT-VH obtained in Example 1.3 were defined using the AbM numbering method. Then, a series of primers were designed to perform single-point or three-point consecutive mutations on their CDRs to construct affinity variant phage display libraries.

[0351] 4.2 Screening of affinity variant phage display libraries

[0352] The screening of affinity variant phage display libraries employs immunotube screening (i.e., solid-phase screening). Immunotubes are coated with the antigen protein P17-His (prepared using a method similar to that in Example 1). The screening process involves incubating, washing, and eluting the antigen-bound immunotubes and the affinity variant phage display library, undergoing 2-3 rounds of panning. This allows for the enrichment of a large number of specific monoclonal antibodies against the antigen. For specific implementation methods of screening C8 affinity variants for this phage display library, please refer to the immunotube screening method in patent application number 202010236256.8.

[0353] After screening the phage display library for C8 affinity variants, numerous candidate antibodies capable of highly specific binding to the P17-His antigen were obtained at the ELISA level. Based on the binding affinity of the Fab molecules, the top 11 candidate molecules with the highest binding affinity were selected for full-length antibody construction. The sequence of human IgG1 Fc silent (N297Q mutation) (i.e., huIgG1(N297Q)) was ligated to the heavy chain sequence of the Fab molecule to obtain a fully human IgG1 (N297Q) antibody with all light chains in the κ type.

[0354] 4.3. Preparation of full-length antibodies from affinity variant candidate clones

[0355] The specific methods are as described in Example 1.4, "Expression, Purification, and Concentration Determination of Full-Length Antibodies." C8-1, C8-2, C8-3, C8-4, C8-5, C8-6, C8-7, C8-8, C8-9, C8-10, and C8-11 antibodies were obtained. The variable region nucleotide and amino acid sequences of the C8 parental antibody and each variant antibody are shown in Table 3.

[0356] 4.4. Non-specific binding assay of affinity variant candidate antibodies

[0357] The relationship between the non-specific binding of the obtained affinity variant candidate antibodies to different cells and the antibody concentration used was preliminarily investigated. HEK293 cells and CHO-K cells are both cells that do not naturally express CD40 molecules on their cell surface; therefore, antibodies that specifically bind to CD40 will not specifically bind to HEK293 cells and CHO-K cells. The positive control used in this experiment was the F4AM4 antibody described in patent CN202010825379.5. The F4AM4 antibody is a fully human antibody, and its light and heavy chain sequences are shown in Table 5 as F4AM4-LC and F4AM4-HC, respectively.

[0358] In the experiment, HEK293 (ATCC:CRL-3216) and CHO-K (ATCC:CCL-61) cells were respectively fed with 1.0 × 10⁻⁶ cells. 5 Cells were seeded per well in 96-well plates. 100 μL of serially diluted anti-CD40 candidate antibody prepared in Example 4.3 was added to each well. After incubation at 4°C for 60 min, cells were washed five times with FACS buffer (PBS + 5% FBS + 2% BSA), and 100 μL of PE-labeled anti-human IgG-Fc secondary antibody (Goat F(ab')2 Anti-Human IgG-Fc(PE), pre-adsorbed, Abcam, catalog number: ab98596) was added, followed by incubation at 4°C for another 30 min. Cells were then washed and analyzed by flow cytometry.

[0359] like Figure 11A and 11B As shown, higher concentrations of the C8-8 clone exhibited nonspecific binding in both HEK293 and CHO-K cells; C8-6 showed some nonspecific binding with HEK293 at a high concentration (100 μg / mL), while other candidate clones and IgG1 did not bind nonspecifically to either cell line. Therefore, in subsequent experiments, candidate molecules that did not bind nonspecifically will be used for further testing.

[0360] Table 5. Light and heavy chain sequences of positive control antibodies.

[0361]

[0362] 4.5. Cell-based functional assays of affinity variant candidate antibodies

[0363] 4.5.1. CD40 binding and cross-reactivity of affinity variant candidate antibodies

[0364] To verify the binding activity and cross-reactivity of the affinity variant antibody in Example 4.3, artificially constructed CHO-K cells expressing full-length human CD40 (CHO-K-huCD40 cells) were used at 1.0 × 10⁻⁶.5 Cells were seeded per well in a 96-well plate. 100 μL of the diluted anti-CD40 candidate antibody prepared in Example 4.3 was added to each well. After incubation at 4°C for 30 min, the cells were washed five times with FACS buffer (PBS + 5% FBS + 2% BSA), and 100 μL of APC-labeled anti-human IgG secondary antibody was added, followed by incubation at 4°C for another 30 min. The cells were then washed and analyzed by flow cytometry.

[0365] like Figure 12A and Figure 12B As shown, after constructing the full-length antibody from the affinity variant Fab, all candidate antibodies exhibited affinity for human CD40, comparable to the control antibody APX005 (APX005 was used as the control antibody in the C8 antibody affinity variant experiments because it showed good efficacy against pancreatic cancer in the Phase II clinical trial results; the APX005 sequence was obtained from Apexigen Inc. US20120301488 and prepared by Sanyou Bio).

[0366] The cross-reactivity of the affinity variant candidate antibody prepared in Example 4.3 with mouse CD40 was tested using artificially constructed CHO-K cells expressing full-length mouse CD40 (Uniprot#P27512) (CHO-K-mouseCD40 cells). The CHO-K cells expressing mouse CD40 were cultured at 1.0 × 10⁻⁶ cells per cell line. 5 Cells were seeded per well into 96-well plates. 100 μL of diluted affinity variant candidate antibody and control antibody (APX005) were added to each well. After incubation at 4°C for 30 min, cells were washed and FITC-labeled anti-human IgG Fcγ (AffiniPure F(ab')2Fragment Goat Anti-Human IgG, Fcγfragmentspecific, Jackson Immunoresearch, Catalog No.: 109-006-098) was added and incubated at 4°C for 30 min. Cells were then washed and analyzed by flow cytometry.

[0367] like Figure 12C and Figure 12D As shown, C8-6 exhibited better mouse CD40 antigen binding activity than the parent C8-WT. C8-1, C8-2, C8-7, C8-8, C8-9, C8-10, and C8-11 all showed binding activity with mouse CD40 antigen.

[0368] 4.5.2. CD95 induction in Ramos cells mediated by affinity variant candidate antibodies (with crosslinking agents, i.e., crosslinking effect).

[0369] To test whether the affinity variant candidate antibody, in the presence of a cross-linking agent, binds to CD40 on Ramos cells and activates downstream CD40 signaling pathways, Ramos cells (ATCC No.: CRL-1596) were subjected to a cross-linking agent at a concentration of 1.0 × 10⁻⁶. 5 10 cells / well were seeded into complete RPMI 1640 medium. Diluted affinity variant candidate antibody, control antibody APX005, and CD40L (20 μg / mL for the first well, serially diluted 3-fold, 8 concentration spots) were added to Ramos cells. Simultaneously, a cross-linking agent (AffiniPure F(ab')2Fragment Goat Anti-Human IgG, Fcγfragment specific, Jackson Immunoresearch, catalog number: 109-006-098) was added at a concentration of 20 μg / mL, and the cells were incubated overnight at 37°C. CD95 expression was then measured by flow cytometry.

[0370] like Figure 13A and Figure 13B As shown, all affinity variant antibodies exhibited CD95-inducing activity comparable to APX005, with no significant difference.

[0371] 4.5.3. CD95 induction in Ramos cells mediated by affinity variant candidate antibodies (without crosslinking agents, i.e., no crosslinking effect)

[0372] To test whether the affinity variant candidate antibody could bind to CD40 on Ramos cells and activate downstream CD40 signaling pathways without a cross-linking agent, Ramos cells (ATCC No.: CRL-1596) were loaded with 1.0 × 10⁻⁶ antibodies. 5 One cell / well was seeded into complete RPMI 1640 medium. Diluted affinity variant candidate antibody, control antibody APX005, and CD40L (20 μg / mL for the first well, serially diluted 3-fold, 8 concentration spots) were added to Ramos cells and incubated overnight at 37°C. CD95 expression was then measured by flow cytometry.

[0373] like Figure 13C and Figure 13DAs shown, all affinity variant antibodies exhibited weaker CD95-inducing activity than APX005 and CD40L, with C8-2, C8-6, C8-9, C8-10, and C8-11 showing approximately 10 times weaker CD95-inducing activity than APX005. This indicates that, without a cross-linking agent, the candidate molecules of this invention exhibit significantly lower CD40 molecule activation capacity compared to APX005, and can be expected to have significantly fewer toxic side effects while weakly activating CD40 molecules. Currently, the main adverse event of anti-CD40 agonist antibodies undergoing clinical trials is hepatotoxicity. This is because liver cells express a large number of FcR receptors on their surface, while the agonist antibodies used in clinical trials (such as APX005) often have normal Fc function and can form a crosslinking effect with FcR receptors on the surface of liver cells, thereby non-specifically activating CD40-expressing immune cells on the surface of liver cells, leading to hepatotoxicity and a low therapeutic index. Analysis of the mechanism of cross-linking effect reveals that the cross-linking effect causing hepatotoxicity is due to the binding of Fc receptors. If the cross-linking effect of Fc receptors can be controlled to significantly enhance the activation effect at the lesion site, hepatotoxicity can be controlled and the therapeutic index can be improved. This places high demands on the difference in agonistic activity of antibody drug molecules with and without cross-linking effect.

[0374] To maximize the ratio of efficacy to toxicity, this invention aims to improve the therapeutic index by amplifying the difference in agonistic activity with and without the crosslinking effect. Specifically, it seeks to generate a significant immune activation response when a crosslinking effect is formed, and to generate little or no immune activation when no crosslinking effect is present. As can be seen in this embodiment, compared to the APX005 molecule, the difference in immune cell agonistic activity with and without a crosslinking agent is relatively small, meaning the final drug efficacy-to-toxicity ratio is smaller. However, the candidate antibody of this invention can maximize the efficacy-to-toxicity ratio with and without a crosslinking agent, maximizing the activation of immune cells within the tumor microenvironment while avoiding hepatotoxicity and other toxic side effects caused by non-specific activation of immune cells at non-tumor sites. If a bispecific antibody is constructed using the candidate antibody of this invention, this characteristic can be utilized to achieve high-intensity specific activation of immune cells at the lesion site, while achieving low agonistic activity at sites such as hepatocytes where a crosslinking effect cannot be formed. This achieves the goal of specifically killing tumor cells without causing hepatotoxicity or other adverse events, effectively improving the therapeutic index and safety.

[0375] 4.5.4. Preparation of Jurkat NF-κB luciferase reporter gene stable transgenic cell lines

[0376] First, the vector pGL4.32[Luc2p / NF-κB-RE / Hygro] plasmid (Promega, catalog number E8491) was electroporated into Jurkat cells using an Invitrogen Neon™ Transfection System (MP922947). TIB-152 TM After electroporation, the resulting cells were transferred to RPMI containing 10% FBS (Gibco, 15140-141) and no antibiotics. Cells were cultured in 1640 medium (Hyclone, SH30243.01) and seeded into 6-well cell culture dishes for 48 hours. Then, cells were aliquoted into 96-well cell culture plates at an average density of 1500 cells / well. Hygromycin B (Yuanpei, S160J7) was added to a final concentration of 500 μg / mL for selection. Cell line clonal growth was observed after 2-3 weeks, and cell lines that formed clones were transferred to 24-well plates. After cell culture expansion, a portion of clones were transferred to 96-well white-background plates (Corning, 3610) for stimulation with phorbol ester (10 ng / ml) and iomycin (1 nM). Cells were incubated at 37°C in a 5% CO2 incubator for 6 hours, followed by the addition of Bright-Lite substrate (Vazyme, DD1204-03) and microplate readings using a Spectramax microplate reader. After reading the signal values ​​using i3x, the expression levels of NF-κB in different clones were evaluated from Jurkat cell lines that highly expressed the NF-κB gene. Jurkat NF-κB luciferase reporter gene stable cell clones with high NF-κB expression levels were cryopreserved for later use.

[0377] 4.5.5. Construction of Jurkat CD40 / NF-κB luciferase reporter gene cell line

[0378] In this embodiment, the constructed Jurkat CD40 / NF-κB luciferase reporter gene cell line was used to screen candidate antibody molecules capable of activating downstream signaling activity of CD40. The Jurkat NF-κB luciferase reporter gene stably transfected cell line prepared in Example 4.5.4 was used, and the full-length expression gene sequence of CD40 (Uniprot Gene ID: P25942) was stably transfected from this cell line for screening of monoclonal cell lines. Recombinant CD40L protein was added to this cell line culture system, which activated the transcription and expression of the intracellular NF-κB luciferase reporter gene by binding to CD40, and the catalytic substrate of luciferase was added to generate a fluorescent signal. The preparation process of the Jurkat CD40 / NF-κB luciferase reporter gene cell line is as follows:

[0379] Construction of the full-length plasmid expressing human CD40 (Met1-Gln277): A DNA fragment containing human CD40 protein was synthesized using gene synthesis technology and cloned into the pLVX-Puro expression vector (Clontech, 632164). The plasmid was introduced into *E. coli* via chemical transformation. Single *E. coli* clones were picked and sequenced to obtain the correct plasmid clone. Plasmid extraction was performed, and sequencing was repeated for confirmation. Electroporation: Jurkat cells prepared in Example 4.5.4 were cultured in Gibco's RPMI 1640 serum-free medium (catalog number: 11875085). One day before electroporation, cells were passaged to 2 × 10⁶ cells / mL. 5 The next day, using an Invitrogen electroporation kit (MPK10096) and electroporator (MP922947), the constructed plasmid was transduced into Jurkat NF-κB luciferase reporter gene stable cell lines. The electroporated cells were transferred to RPMI 1640 medium and cultured at 37°C for 48 hours. Cell seeding after electroporation: Jurkat cells were seeded at 1000 cells / well into 96-well plates, and puromycin was added to a final concentration of 2 μg / mL. The plates were then cultured at 37°C in a CO2 incubator. After 14 days, RPMI 1640 medium with 2 μg / mL puromycin was added again. Clone selection, cell expansion culture, and FACS identification: Single-cell clones grown from the 96-well plates were picked and transferred to 24-well plates for further expansion culture. Cell lines stably transfected with human CD40 were then identified using FACS.

[0380] 4.5.6. Candidate antibodies with affinity variants activate CD40 downstream NF-κB luciferase reporter gene signaling activity (with cross-linking agents, i.e., crosslinking effect exists).

[0381] In this embodiment, to test whether the affinity variant candidate antibody can activate the CD40 signaling pathway in the presence of a cross-linking agent, the Jurkat CD40 / NF-κB luciferase reporter gene cell line prepared in Example 4.5.5 was used as material to detect the ability of the candidate molecule to bind to CD40 and thereby activate the expression of the downstream NF-κB luciferase reporter gene. The specific implementation method is as follows:

[0382] The parental C8 molecule and affinity variant candidate antibody, along with the control antibody APX005, were serially diluted in RPMI 1640 medium (10 μg / mL for the first well, 3-fold serial dilutions, 8 concentration points). A 10 μg / mL cross-linking agent (AffiniPure F(ab')2FragmentGoat Anti-Human IgG, Fcγfragment specific, Jackson Immunoresearch, catalog number: 109-006-098) was added to each well of a 96-well plate for premixing. 50 μL of the antibody and cross-linking agent premix was added to each well. Jurkat CD40 / NF-κB luciferase reporter cell lines were revived, and cells passaged 2-4 times and in good growth condition were used for experiments. Cells were washed and resuspended in RPMI 1640 medium, counted, and the cell density was adjusted to 2 × 10⁶ cells / well. 6 Cells / mL were added at a rate of 50 μL per well to a 96-well cell culture plate containing a premix of antibody and cross-linking agent, and incubated at 37°C for 6 h. After incubation, 30 μL of Bright-Lite luciferase substrate (Vazyme, DD1204-03) was added to each well, and the plate was shaken for 5 min before detecting the fluorescence value of the 96-well plate.

[0383] like Figure 14A , Figure 14B , Figure 14C and Figure 14D As shown, in the presence of a cross-linking agent, the maternal C8 molecule and all affinity variant antibodies exhibited comparable activation of CD40 downstream NF-κB luciferase reporter gene signaling activity to APX005, with no significant difference.

[0384] 4.5.7. Candidate antibodies with affinity variants activate CD40 downstream NF-κB luciferase reporter gene signaling activity (without crosslinking agents, i.e., no crosslinking effect).

[0385] In this embodiment, to test whether the affinity variant candidate antibody can activate the CD40 signaling pathway without a cross-linking agent, the Jurkat CD40 / NF-κB luciferase reporter gene cell line was used as material to detect the ability of the candidate molecule to bind to CD40 and thereby activate the expression of the downstream NF-κB luciferase reporter gene. The specific implementation method is as follows:

[0386] The parental C8 molecule and its affinity variants, along with the control antibody APX005, were serially diluted in RPMI 1640 medium (10 μg / mL for the first well, 3-fold serial dilutions, 8 concentration points). 50 μL of each diluted candidate antibody and control antibody was added to each well of a 96-well plate. The Jurkat CD40 / NF-κB luciferase reporter cell line was revived, and cells passaged 2-4 times and in good growth condition were used for experiments. Cells were washed and resuspended in RPMI 1640 medium, counted, and then the cell density was adjusted to 2 × 10⁶ cells / well. 6 Cells / mL were added at a rate of 50 μL per well to a 96-well cell culture plate containing antibody, and incubated at 37°C for 6 h. After culture, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the plate was shaken for 5 min before the fluorescence value of the 96-well plate was measured.

[0387] like Figure 15A , Figure 15B , Figure 15C and Figure 15D As shown, without a cross-linking agent, APX005 exhibits relatively strong activation activity of the CD40 downstream NF-κB luciferase reporter gene signaling pathway. In contrast, affinity mutant candidate antibodies, except for C8-7 and C8-9 which show some activation ability, have no activation ability. It can be expected that these molecules, while weakly activating CD40 molecules, will have significantly less toxicity than APX005. Figure 14A - Figure 14D and Figure 15A - Figure 15D Comparing the activation of the CD40 signaling pathway by various candidate molecules, in the presence or absence of the crosslinking effect, the activation of the CD40 signaling pathway by the parent C8 molecule and various affinity variant candidate molecules is basically the same. Figure 13A - Figure 13D The activation of B cells was consistent with that of Ramos cells, which also verifies the results of the B cell (Ramos cell) activation assay from a molecular mechanism perspective. The results show that the B cell activation induced by the anti-CD40 antibody in this invention is achieved through activation of the CD40 signaling pathway.

[0388] 4.6. Affinity kinetics detection of candidate antibodies for affinity variants

[0389] In this embodiment, the affinity of the C8 parent antibody (C8-WT) and the affinity variant antibody to the human CD40 antigen (P17-His) was detected using a Fortebio Octet RED96 instrument.

[0390] Materials Preparation: Weigh 1g of BSA, measure 500μL of Tween 20, and add it to 1000mL of 1×PBS. Mix well. Filter and aliquot for storage. Pipette 0.1mL of 0.1M pH 2.0 glycine solution into 0.9mL of ultrapure water and mix well. Dilute the antibody with KB buffer to 10μg / mL, and dilute the antigen with KB buffer to a series of concentration gradients: 40nM, 20nM, 10nM, 5nM, and 0nM.

[0391] Experimental Procedure: After pre-humidifying the Protein A sensor in the dark for at least 10 minutes, begin testing the sample plate (GreinierBio, PN655209). Once the test is successful, proceed according to the preset procedure. Specifically, for sample plate 1, add 200 μL / well of KB buffer to columns 1, 10, and 12; add 0.01 M pH 2.0 glycine solution to column 11; add the prepared sample solution to columns 2-8 (one sample per 5 wells); and add P17-His solution to column 9 in descending order of concentration. See Table 6 for detailed data results.

[0392] Table 6. Affinity test results of C8 parental antibody (C8-WT) and affinity variant antibody.

[0393] Antibody name KD(M) ka(1 / Ms) kd(1 / s) <![CDATA[R 2 ]]> APX005 6.30E-09 1.98E+06 1.24E-02 0.973 C8-WT 1.93E-08 8.53E+05 1.64E-02 0.974 C8-1 1.27E-08 1.16E+06 1.48E-02 0.969 C8-2 1.19E-08 9.32E+05 1.11E-02 0.968 C8-3 4.51E-09 2.46E+06 1.11E-02 0.972 C8-4 5.34E-09 1.36E+06 7.25E-03 0.969 C8-5 8.23E-09 9.94E+05 8.18E-03 0.982 C8-6 9.04E-09 1.09E+06 9.84E-03 0.99 C8-7 1.24E-08 8.95E+05 1.11E-02 0.99 C8-8 1.03E-08 1.07E+06 1.10E-02 0.982 C8-9 9.27E-09 1.21E+06 1.12E-02 0.987 C8-10 6.82E-09 1.24E+06 8.47E-03 0.984 C8-11 9.24E-09 1.60E+06 1.48E-02 0.976

[0394] As shown in Table 6, the C8 parental antibody (C8-WT) and the affinity variant antibody have a ratio of approximately 0.5 × 10⁻⁶. －8 M to 2×10 －8 M has the affinity to bind to the human CD40 antigen, and all affinity variants of the antibody have improved affinity compared to the C8 parent antibody (C8-WT), making them affinity-mature antibodies.

Claims

1. An anti-CD40 antibody or antigen-binding fragment, comprising a light chain variable region comprising three complementarity determining regions, LCDR1, LCDR2, and LCDR3, respectively, and a heavy chain variable region comprising three complementarity determining regions, HCDR1, HCDR2, and HCDR3, respectively, wherein: (i) the LCDR1 of the anti-CD40 antibody or antigen-binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the LCDR3 is the amino acid sequence of SEQ ID NO: 10; (ii) the LCDR1 of the anti-CD40 antibody or antigen-binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 4, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 10; (iii) the LCDR1 of the anti-CD40 antibody or antigen-binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 11; (iv) the LCDR1 of the anti-CD40 antibody or antigen-binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 15; (v) the LCDR1 of the anti-CD40 antibody or antigen-binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 16; ​ ​ ​ ​ ​ (vi) the LCDR1 of the anti-CD40 antibody or antigen binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 17; (vii) the LCDR1 of the anti-CD40 antibody or antigen binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 18; (viii) the LCDR1 of the anti-CD40 antibody or antigen binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 2, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 8, the HCDR2 is the amino acid sequence of SEQ ID NO: 9, and the HCDR3 is the amino acid sequence of SEQ ID NO: 19; or (ix) the LCDR1 of the anti-CD40 antibody or antigen binding fragment is the amino acid sequence of SEQ ID NO: 1, the LCDR2 is the amino acid sequence of SEQ ID NO: 7, and the LCDR3 is the amino acid sequence of SEQ ID NO: 3, and the HCDR1 is the amino acid sequence of SEQ ID NO: 20, the HCDR2 is the amino acid sequence of SEQ ID NO: 21, and the HCDR3 is the amino acid sequence of SEQ ID NO:

10.

2. The anti-CD40 antibody or antigen binding fragment of claim 1, comprising a light chain variable region and a heavy chain variable region, which are: (i) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 25; (ii) a light chain variable region of the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 25; (iii) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 31; (iv) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 43; (v) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 45; (vi) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 47; (vii) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 49; (viii) a light chain variable region of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region of the amino acid sequence of SEQ ID NO: 51; or (ix) a light chain variable region of the amino acid sequence of SEQ ID NO: 53 and a heavy chain variable region of the amino acid sequence of SEQ ID NO:

55.

3. The anti-CD40 antibody or antigen-binding fragment of claim 2, wherein the anti-CD40 antibody or antigen-binding fragment is a fully human antibody.

4. The anti-CD40 antibody or antigen-binding fragment of claim 3, which is an IgGl, IgG2, IgG3, or IgG4 antibody.

5. The anti-CD40 antibody or antigen-binding fragment of claim 4, which is an IgGl or IgG4 antibody.

6. The anti-CD40 antibody or antigen-binding fragment of claim 5, which is a human IgGl or human IgG4 antibody.

7. The anti-CD40 antibody or antigen-binding fragment of claim 2, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, diabody.

8. The anti-CD40 antibody or antigen-binding fragment of any one of claims 1 to 7, which has one or more of the following properties: (1) about 10 -7 M to about 10 -10 M Kd binding to human CD40, cynomolgus CD40 and mouse CD40 as measured by ForteBio Kinetic Binding Assay; (2) enhances the binding of CD40 to CD40L; (3) activates antigen presenting cells by binding to CD40 expressed on the antigen presenting cells including dendritic cells, B cells, monocytes, macrophages; (4) induces CD95 expression by B cells expressing CD40; (5) enhances B cell-mediated immune responses; (5) significantly enhances B cell-mediated immune responses when cross-linking effect is formed; (6) hardly or weakly enhances B cell-mediated immune responses when cross-linking effect is not formed.

9. The anti-CD40 antibody or antigen-binding fragment of any one of claims 1 to 7, wherein a glycosylation site in the CH2 domain of the immunoglobulin Fc region is eliminated.

10. The anti-CD40 antibody or antigen-binding fragment of claim 9, wherein the N297 residue in the CH2 domain of the human IgG Fc region is mutated to eliminate the glycosylation site.

11. The anti-CD40 antibody or antigen-binding fragment of claim 10, wherein the N297 residue is changed to Gly, Ala, Gin, Asp, or Glu.

12. The anti-CD40 antibody or antigen-binding fragment of claim 11, wherein the N297 residue is changed to Gin.

13. An isolated nucleic acid encoding the anti-CD40 antibody or antigen-binding fragment of any one of claims 1 to 12.

14. A vector comprising the nucleic acid of claim 13.

15. The vector of claim 14, wherein the vector is an expression vector.

16. The vector of claim 15, wherein the expression vector is a pcDNA3.3 vector.

17. A host cell comprising the nucleic acid of claim 13 or the vector of any one of claims 14 to 16.

18. The host cell of claim 17, wherein the host cell is prokaryotic or eukaryotic.

19. The host cell of claim 18, wherein the host cell is selected from the group consisting of an E. coli cell, a yeast cell, a mammalian cell.

20. The host cell of claim 19, wherein the host cell is a 293 cell or a CHO cell.

21. A method of making the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12, the method comprising culturing the host cell of any one of claims 17 to 20 under conditions suitable for expression of the nucleic acid encoding the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12, and recovering the anti-CD40 antibody or antigen binding fragment from the host cell.

22. An immunoconjugate comprising the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 and a further agent, wherein the further agent is a cytotoxic agent.

23. A pharmaceutical composition comprising the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 or the immunoconjugate of claim 22, and a pharmaceutically acceptable excipient.

24. A pharmaceutical composition comprising the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 or the immunoconjugate of claim 13, and a further therapeutic agent, and a pharmaceutically acceptable excipient.

25. The pharmaceutical composition of claim 24, wherein the further therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibody, cytotoxic agent.

26. A combination product comprising the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 or the immunoconjugate of claim 22, and one or more further therapeutic agents.

27. The combination product of claim 26, wherein the one or more further therapeutic agents are selected from the group consisting of a chemotherapeutic agent, cytotoxic agent, other antibody.

28. Use of the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 or the immunoconjugate of claim 22 for the manufacture of a medicament for the prevention or treatment of a tumor in a subject, wherein the tumor is selected from the group consisting of lymphoma and colon adenocarcinoma.

29. A kit for detecting CD40 in a sample, the kit comprising the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12 for performing the following steps: (a) contacting a sample with the anti-CD40 antibody or antigen binding fragment of any one of claims 1 to 12; and (b) detecting the formation of a complex between the anti-CD40 antibody or antigen binding fragment and CD40; optionally, the anti-CD40 antibody or antigen binding fragment is detectably labeled. ​ ​

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