Anti-CD40 antibodies and uses thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to CD40 and compositions containing the antibodies or antigen-binding fragments thereof. Furthermore, this invention relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof and host cells comprising the same, as well as related uses. In addition, this invention relates to therapeutic and detection uses of these antibodies or antigen-binding fragments thereof.

Description

Anti-CD40 antibodies and uses thereof Technical Field

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to CD40, and compositions containing such antibodies or antigen-binding fragments. Furthermore, the invention relates to nucleic acids encoding such antibodies or antigen-binding fragments, host cells containing such antibodies or antigen-binding fragments, and related uses. Furthermore, the invention relates to therapeutic and detection uses of such antibodies or antigen-binding fragments. Background Art

[0002] CD40, also known as TNFRSF5, is a member of the TNF receptor superfamily and is expressed on a variety of cell types, including immune B cells, antigen-presenting cells (APCs), and some tumor cells. The CD40 precursor is a 297aa type I transmembrane glycoprotein composed of an N-terminal signal peptide (20aa), an extracellular domain (193aa), a transmembrane domain (22aa), and a cytoplasmic domain (62aa). The extracellular domain contains four CDRs and a total of 22 cysteines. CD40 is highly glycosylated, with a molecular weight estimated from the amino acid sequence of 28kD, but the molecular weight after glycosylation is 40-50kD.

[0003] The cognate ligand of CD40 is CD154 (TNFSF5 / CD40L), a 39 kDa type II transmembrane protein. CD40L expression is normally induced and restricted to cells of the hematopoietic system, such as platelets, granulocytes, activated T cells, activated B cells, and activated natural killer (NK) cells, but is also weakly expressed on endothelial cells and smooth muscle cells.

[0004] Numerous studies have confirmed that CD40-CD40L plays a crucial role in the function of CD8 cytotoxic T lymphocytes (CTLs) in immune responses and is required for adaptive immune responses.

[0005] CD40 is widely expressed on monocytes and their mature progeny DCs, macrophages, and B cells, and plays an important role in the function of immune cells. Monocytes are innate immune precursor cells with high plasticity. They have the ability to differentiate into multiple cell types, such as myeloid-derived suppressor cells (MDSCs), macrophages, and DCs. CD40 signaling is an important trigger for the monocyte maturation process, primarily driving differentiation into macrophages and DCs of the M1 lineage. The binding of CD40 to the DC cell surface promotes the production of cytokines and chemokines, induces the expression of co-stimulatory molecules, and promotes cross-presentation of antigens. One of the main functions of CD40L is to enhance antigen presentation to T cells by activating DC cells. This step is called "permission," and it increases the interaction between DCs and T cells by upregulating surface proteins such as CD54 and CD86, thereby activating the latter.

[0006] B cells are also targets of CD40L activity. Interactions between B cells and activated CD40L-expressing T cells increase the expression of MHC-II and co-stimulatory molecules such as CD80 or CD86, inducing Ig class switching in B cells. Activated B cells migrate to lymphoid organs, where they present antigens to T cells. CD40-activated DCs and B cells support immune responses by releasing immunostimulatory cytokines and chemokines such as IL-12p70, CXCL10, and TNFα. Furthermore, CD40-activated B cells can induce antigen-specific CD8+ T cells by promoting the secretion of cytokines such as TNFα and IFNγ. Several studies have demonstrated that in vitro activated CD40-expressing B cells are fully functional antigen-presenting B cells, and that subsequent adoptive cell transfer (ACT) therapy with these cells can enhance anti-tumor efficacy.

[0007] Because CD40 plays a key role in anti-tumor immune responses, various strategies for inducing CD40 signals have been extensively studied and explored. Current drug development targeting CD40 can be divided into activation (agonists) or inhibition (antagonists) to address cancer environments that require enhanced immune activity and autoimmune disease environments that require suppression of the immune system, respectively. However, as a single-agent therapy, CD40 antibodies have only shown modest activity, with tumor objective response rates below 20%. This is especially true in "cold tumors" with insufficient immune responses or in tumors where immune cells have not yet been highly infiltrated. As a result, many CD40 monoclonal antibody projects have stalled.

[0008] Given the important role of CD40 in tumor immune responses, CD40 is considered an attractive target for tumor immunotherapy. Therefore, there is a need in the field to develop new CD40 antibodies, especially CD40 antibodies with different epitopes, blocking and non-blocking binding to the ligand CD40L. These antibodies have different application potentials in tumor immunity and autoimmune diseases, and can be combined with clinical combination therapies for disease treatment, especially cancer treatment.

[0009] Summary of the Invention

[0010] After extensive research, the inventors of this application have screened and obtained a series of fully human anti-CD40 antibodies with high binding affinity to CD40, cross-reactivity with human and cynomolgus monkey CD40, and the ability to block or non-block the binding of human CD40 to its ligand CD40L. Based on this, this application also provides compositions containing these antibodies or antigen-binding fragments thereof, nucleic acids encoding these antibodies or antigen-binding fragments thereof, host cells containing these antibodies, and related uses.

[0011] Antibodies or antigen-binding fragments thereof

[0012] Therefore, in one aspect, the present application provides an antibody or antigen-binding fragment thereof that can specifically bind to CD40, wherein the antibody or antigen-binding fragment thereof comprises:

[0013] (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0014] (a) a VH CDR1 having a structure selected from the group consisting of:

[0015] FTFX1X2YX3MH (SEQ ID NO:76), GSISSYYWS (SEQ ID NO:6);

[0016] (b) a VH CDR2 having a structure selected from the group consisting of:

[0017] VIX4YX5X6X7X8KYYAX9SVKG(SEQ ID NO:77),GISWSSX 10 SIX 11 YADSVKG (SEQ ID NO:78), SIYYSGSTNYNPSLKS (SEQ ID NO:22);

[0018] (c) a VH CDR3 having a structure selected from the group consisting of:

[0019] ARDTSRGHDI (SEQ ID NO:23), AKDPTATTGARGYFDL (SEQ ID NO:24), ARDANYYKWHPY (SEQ ID NO:25);

[0020] and / or,

[0021] (2) The following three light chain variable region (VL) CDRs:

[0022] (d) a VL CDR1 having a structure selected from the group consisting of:

[0023] X 12 ASQSVSX 13 X 14 YLA (SEQ ID NO: 79), X 15 ASX 16 X 17 ISSWLA (SEQ ID NO: 7);

[0024] (e) a VL CDR2 having a structure selected from the group consisting of:

[0025] GASX 18 X 19 X 20 X 21(SEQ ID NO:8), AASX 22 LX 23 S (SEQ ID NO: 9);

[0026] (f) a VL CDR3 having a structure selected from the group consisting of:

[0027] X 24 X 25 YX 26 DYPPFT (SEQ ID NO: 10), QQX 27 X 28 SX 29 PPT (SEQ ID NO: 11);

[0028] in,

[0029] X1 is selected from the group consisting of (i) amino acid residues S, E, G, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0030] X2 is selected from the group consisting of (i) amino acid residues S, K, D, T and (ii) amino acid residues that are conservative substitutions relative to (i);

[0031] X3 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0032] X4 is selected from the group consisting of (i) amino acid residues S, H, and (ii) amino acid residues that are conservative substitutions relative to (i);

[0033] X5 is selected from the group consisting of (i) amino acid residues E, H, and (ii) amino acid residues that are conservative substitutions relative to (i);

[0034] X6 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0035] X7 is selected from the group consisting of (i) amino acid residues S, E, T, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0036] X8 is selected from the group consisting of (i) amino acid residues N, S, I, K and (ii) amino acid residues that are conservative substitutions relative to (i);

[0037] X9 is selected from (i) amino acid residues D, E and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0038] X 10 selected from (i) amino acid residues G, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0039] X 11selected from (i) amino acid residues G, H and (ii) amino acid residues that are conservative substitutions relative to (i);

[0040] X 12 selected from (i) amino acid residues R, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0041] X 13 selected from (i) amino acid residues Y, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0042] X 14 selected from the group consisting of (i) amino acid residues S, D, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0043] X 15 selected from (i) amino acid residues R, E and (ii) amino acid residues that are conservative substitutions relative to (i);

[0044] X 16 selected from (i) amino acid residues Q, K and (ii) amino acid residues that are conservative substitutions relative to (i);

[0045] X 17 selected from (i) amino acid residues G, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0046] X 18 An amino acid residue selected from (i) S, T, A and (ii) a conservative substitution relative to (i);

[0047] X 19 selected from (i) amino acid residues R, L and (ii) amino acid residues that are conservative substitutions relative to (i);

[0048] X 20 selected from (i) amino acid residues N, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0049] X 21 selected from (i) amino acid residues T, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0050] X 22 selected from (i) amino acid residues S, Y and (ii) amino acid residues that are conservative substitutions relative to (i);

[0051] X 23 selected from (i) amino acid residues E, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0052] X 24selected from the group consisting of (i) amino acid residues S, G, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0053] X 25 selected from the group consisting of (i) amino acid residues E, Q, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0054] X 26 selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0055] X 27 selected from (i) amino acid residues R, H, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0056] X 28 An amino acid residue selected from (i) S, L, A and (ii) a conservative substitution relative to (i);

[0057] X 29 Selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservative substitutions relative to (i).

[0058] (ADI-47754 family)

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0060] (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0061] (a) VH CDR1 having the structure shown as FTFX1X2YX3MH (SEQ ID NO: 76);

[0062] (b) VH CDR2 having the structure shown in VIX4YX5X6X7X8KYYAX9SVKG (SEQ ID NO: 77);

[0063] (c) VH CDR3 having the structure shown as ARDTSRGHDI (SEQ ID NO: 23);

[0064] and / or,

[0065] (2) The following three light chain variable region (VL) CDRs:

[0066] (d) VL CDR1, which has X 12 ASQSVSX 13 X 14 The structure shown in YLA (SEQ ID NO: 79);

[0067] (e) VL CDR2, which has a 18 X 19 X 20 X 21 (SEQ ID NO: 8);

[0068] (f) VL CDR3, which has X 24 X 25 YX 26 The structure shown in DYPPFT (SEQ ID NO: 10);

[0069] in,

[0070] X1 is selected from the group consisting of (i) amino acid residues S, E, G and (ii) amino acid residues that are conservative substitutions relative to (i);

[0071] X2 is selected from (i) amino acid residues S, K and (ii) amino acid residues that are conservative substitutions relative to (i);

[0072] X3 is selected from the group consisting of (i) amino acid residue G and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0073] X4 is selected from the group consisting of (i) amino acid residues S, H, and (ii) amino acid residues that are conservative substitutions relative to (i);

[0074] X5 is selected from the group consisting of (i) amino acid residues E, H, and (ii) amino acid residues that are conservative substitutions relative to (i);

[0075] X6 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0076] X7 is selected from the group consisting of (i) amino acid residues S, E, T, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0077] X8 is selected from the group consisting of (i) amino acid residues N, S, I, K and (ii) amino acid residues that are conservative substitutions relative to (i);

[0078] X9 is selected from (i) amino acid residues D, E and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0079] X 12 selected from (i) amino acid residues R, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0080] X 13 selected from (i) amino acid residues Y, S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0081] X 14 selected from the group consisting of (i) amino acid residues S, D, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0082] X 18 An amino acid residue selected from (i) S, T, A and (ii) a conservative substitution relative to (i);

[0083] X 19 selected from (i) amino acid residues R, L and (ii) amino acid residues that are conservative substitutions relative to (i);

[0084] X 20 selected from (i) amino acid residues N, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0085] X 21 selected from (i) amino acid residues T, N and (ii) amino acid residues that are conservative substitutions relative to (i);

[0086] X 24 selected from the group consisting of (i) amino acid residues S, G, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0087] X 25 selected from the group consisting of (i) amino acid residues E, Q, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0088] X 26 Selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i).

[0089] In certain embodiments, X1 is selected from amino acid residues S, E, G; X2 is selected from amino acid residues S, K; X3 is amino acid residue G; X4 is selected from amino acid residues S, H; X5 is selected from amino acid residues E, H; X6 is selected from amino acid residues G, A; X7 is selected from amino acid residues S, E, T, V; X8 is selected from amino acid residues N, S, I, K; X9 is selected from amino acid residues D, E; X 12 Selected from amino acid residues R, Q; X 13 Selected from amino acid residues Y, S; X 14 Selected from amino acid residues S, D, N; X 18 Selected from amino acid residues S, T, A; X 19 Selected from amino acid residues R, L; X 20 Selected from amino acid residues N, A; X 21 Selected from amino acid residues T, N; X 24 Selected from amino acid residues S, G, Q; X 25 Selected from amino acid residues E, Q, D; X26 Selected from amino acid residues A, S.

[0090] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 as shown in any one of SEQ ID NOs: 1-3; a VH CDR2 as shown in any one of SEQ ID NOs: 12-18, 82; and a VH CDR3 as shown in SEQ ID NO: 23; a VL CDR1 as shown in any one of SEQ ID NOs: 37-40; a VL CDR2 as shown in any one of SEQ ID NOs: 45-50; and a VL CDR3 as shown in any one of SEQ ID NOs: 54-57.

[0091] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0092] (1) VH CDR1 as set forth in SEQ ID NO: 1; VH CDR2 as set forth in SEQ ID NO: 12; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 37; VL CDR2 as set forth in SEQ ID NO: 45; and VL CDR3 as set forth in SEQ ID NO: 54;

[0093] (2) VH CDR1 as set forth in SEQ ID NO: 1; VH CDR2 as set forth in SEQ ID NO: 13; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 37; VL CDR2 as set forth in SEQ ID NO: 45; and VL CDR3 as set forth in SEQ ID NO: 54;

[0094] (3) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 14; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 38; VL CDR2 as set forth in SEQ ID NO: 46; and VL CDR3 as set forth in SEQ ID NO: 55;

[0095] (4) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 15; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 47; and VL CDR3 as set forth in SEQ ID NO: 56;

[0096] (5) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 16; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 48; and VL CDR3 as set forth in SEQ ID NO: 57;

[0097] (6) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 17; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 40; VL CDR2 as set forth in SEQ ID NO: 49; and VL CDR3 as set forth in SEQ ID NO: 57;

[0098] (7) VH CDR1 as set forth in SEQ ID NO: 3; VH CDR2 as set forth in SEQ ID NO: 18; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 38; VL CDR2 as set forth in SEQ ID NO: 50; and VL CDR3 as set forth in SEQ ID NO: 54;

[0099] or,

[0100] (8) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 82; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 48; and VL CDR3 as set forth in SEQ ID NO: 57.

[0101] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region. For example, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene; for example, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene.

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0103] (1) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0104] (i) a sequence as shown in any one of SEQ ID NOs: 26-32, 83;

[0105] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 26-32, 83; or

[0106] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 26-32, 83;

[0107] and / or,

[0108] (2) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0109] (i) a sequence as shown in any one of SEQ ID NOs: 61-66;

[0110] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 61-66; or

[0111] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 61-66.

[0112] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising a sequence as shown in any one of SEQ ID NOs: 26-32, 83 or a variant thereof, and a VL comprising a sequence as shown in any one of SEQ ID NOs: 61-66 or a variant thereof;

[0113] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In certain embodiments, the substitutions are conservative substitutions.

[0114] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 26 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 61 or a variant thereof.

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 27 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 61 or a variant thereof.

[0116] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 28 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 62 or a variant thereof.

[0117] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 29 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 63 or a variant thereof.

[0118] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 30 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 64 or a variant thereof.

[0119] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 31 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 65 or a variant thereof.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 32 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 66 or a variant thereof.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 83 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 64 or a variant thereof.

[0122] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In certain embodiments, the substitutions are conservative substitutions.

[0123] In certain embodiments, the antibody or antigen-binding fragment thereof is capable of blocking the binding of human CD40 to its ligand CD40L.

[0124] (ADI-47720 family)

[0125] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0126] (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0127] (a) VH CDR1 having the structure shown as FTFX1X2YX3MH (SEQ ID NO: 76);

[0128] (b) VH CDR2, which has GISWSSX 10 SIX 11 The structure shown in YADSVKG (SEQ ID NO: 78);

[0129] (c) VH CDR3 having the structure shown as AKDPTATTGARGYFDL (SEQ ID NO: 24);

[0130] and / or,

[0131] (2) The following three light chain variable region (VL) CDRs:

[0132] (d) VL CDR1, which has X 15 ASX 16 X17 The structure shown in ISSWLA (SEQ ID NO: 7);

[0133] (e) VL CDR2, which has 22 LX 23 The structure shown in S (SEQ ID NO: 9);

[0134] (f) VL CDR3, which has 27 X 28 SX 29 The structure shown in PPT (SEQ ID NO: 11);

[0135] in,

[0136] X1 is selected from (i) an amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0137] X2 is selected from the group consisting of (i) amino acid residues D, T and (ii) amino acid residues that are conservative substitutions relative to (i);

[0138] X3 is selected from (i) amino acid residue A and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0139] X 10 selected from (i) amino acid residues G, D and (ii) amino acid residues that are conservative substitutions relative to (i);

[0140] X 11 selected from (i) amino acid residues G, H and (ii) amino acid residues that are conservative substitutions relative to (i);

[0141] X 15 selected from (i) amino acid residues R, E and (ii) amino acid residues that are conservative substitutions relative to (i);

[0142] X 16 selected from (i) amino acid residues Q, K and (ii) amino acid residues that are conservative substitutions relative to (i);

[0143] X 17 selected from (i) amino acid residues G, V and (ii) amino acid residues that are conservative substitutions relative to (i);

[0144] X 22 selected from (i) amino acid residues S, Y and (ii) amino acid residues that are conservative substitutions relative to (i);

[0145] X 23 selected from (i) amino acid residues E, Q and (ii) amino acid residues that are conservative substitutions relative to (i);

[0146] X 27 selected from (i) amino acid residues R, H and (ii) amino acid residues that are conservative substitutions relative to (i);

[0147] X 28 selected from (i) amino acid residues S, A and (ii) amino acid residues that are conservative substitutions relative to (i);

[0148] X 29 is selected from (i) amino acid residue F and (ii) an amino acid residue that is a conservative substitution relative to (i).

[0149] In certain embodiments, X1 is an amino acid residue D; X2 is selected from amino acid residues D, T; X3 is an amino acid residue A; X 10 Selected from amino acid residues G, D; X 11 Selected from amino acid residues G, H; X 15 Selected from amino acid residues R, E; X 16 Selected from amino acid residues Q, K; X 17 Selected from amino acid residues G, V; X 22 Selected from amino acid residues S, Y; X 23 Selected from amino acid residues E, Q; X 27 Selected from amino acid residues R, H; X 28 Selected from amino acid residues S, A; X 29 is the amino acid residue F.

[0150] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NOs: 4 or 5; a VH CDR2 as set forth in any one of SEQ ID NOs: 19-21; and a VH CDR3 as set forth in SEQ ID NO: 24; a VL CDR1 as set forth in any one of SEQ ID NOs: 41-43; a VL CDR2 as set forth in any one of SEQ ID NOs: 51-53; and a VL CDR3 as set forth in SEQ ID NOs: 58 or 59.

[0151] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0152] (1) VH CDR1 as set forth in SEQ ID NO:4; VH CDR2 as set forth in SEQ ID NO:19; and VH CDR3 as set forth in SEQ ID NO:24; VL CDR1 as set forth in SEQ ID NO:41; VL CDR2 as set forth in SEQ ID NO:51; and VL CDR3 as set forth in SEQ ID NO:58;

[0153] (2) VH CDR1 as set forth in SEQ ID NO:4; VH CDR2 as set forth in SEQ ID NO:20; and VH CDR3 as set forth in SEQ ID NO:24; VL CDR1 as set forth in SEQ ID NO:42; VL CDR2 as set forth in SEQ ID NO:52; and VL CDR3 as set forth in SEQ ID NO:59;

[0154] or,

[0155] (3) VH CDR1 as set forth in SEQ ID NO: 5; VH CDR2 as set forth in SEQ ID NO: 21; and VH CDR3 as set forth in SEQ ID NO: 24; VL CDR1 as set forth in SEQ ID NO: 43; VL CDR2 as set forth in SEQ ID NO: 53; and VL CDR3 as set forth in SEQ ID NO: 59.

[0156] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region. For example, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene; for example, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene.

[0157] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0158] (1) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0159] (i) a sequence as shown in any one of SEQ ID NOs: 33-35;

[0160] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 33-35; or

[0161] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 33-35;

[0162] and / or,

[0163] (2) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0164] (i) a sequence as shown in any one of SEQ ID NOs: 67-69;

[0165] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 67-69; or

[0166] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 67-69.

[0167] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising a sequence as shown in any one of SEQ ID NOs: 33-35 or a variant thereof, and a VL comprising a sequence as shown in any one of SEQ ID NOs: 67-69 or a variant thereof;

[0168] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In certain embodiments, the substitutions are conservative substitutions.

[0169] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 33 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 67 or a variant thereof.

[0170] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 34 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 68 or a variant thereof.

[0171] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 35 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 69 or a variant thereof.

[0172] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In certain embodiments, the substitutions are conservative substitutions.

[0173] In certain embodiments, the antibody or antigen-binding fragment thereof does not block the binding of human CD40 to its ligand CD40L.

[0174] (ADI-47741 family)

[0175] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0176] (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0177] (a) VH CDR1 having the structure shown as GSISSYYWS (SEQ ID NO: 6);

[0178] (b) VH CDR2 having the structure shown as SIYYSGSTNYNPSLKS (SEQ ID NO: 22);

[0179] (c) VH CDR3 having the structure shown as ARDANYYKWHPY (SEQ ID NO: 25);

[0180] and / or,

[0181] (2) The following three light chain variable region (VL) CDRs:

[0182] (d) VL CDR1, which has X 12 ASQSVSX 13 X 14 The structure shown in YLA (SEQ ID NO: 79);

[0183] (e) VL CDR2, which has a 18 X 19 X 20 X 21 (SEQ ID NO: 8);

[0184] (f) VL CDR3, which has 27 X28 SX 29 The structure shown in PPT (SEQ ID NO: 11);

[0185] in,

[0186] X 12 selected from (i) an amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0187] X 13 selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0188] X 14 selected from (i) amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0189] X 18 selected from (i) amino acid residue S and (ii) amino acid residues that are conservative substitutions relative to (i);

[0190] X 19 is selected from (i) an amino acid residue R and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0191] X 20 selected from (i) amino acid residue A and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0192] X 21 selected from (i) amino acid residue T and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0193] X 27 selected from (i) amino acid residue V and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0194] X 28 selected from (i) an amino acid residue L and (ii) an amino acid residue that is a conservative substitution relative to (i);

[0195] X 29 is selected from (i) amino acid residue Y and (ii) an amino acid residue that is a conservative substitution relative to (i).

[0196] In certain embodiments, X 12 is the amino acid residue R; X 13 is the amino acid residue S; X 14 is the amino acid residue D; X 18 is the amino acid residue S; X 19 is the amino acid residue R; X 20 is the amino acid residue A; X 21is the amino acid residue T; X 27 is the amino acid residue V; X 28 is the amino acid residue L; X 29 is the amino acid residue Y.

[0197] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NO:6; a VH CDR2 as set forth in SEQ ID NO:22; and a VH CDR3 as set forth in SEQ ID NO:25; a VL CDR1 as set forth in SEQ ID NO:44; a VL CDR2 as set forth in SEQ ID NO:45; and a VL CDR3 as set forth in SEQ ID NO:60.

[0198] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region. For example, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene; for example, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene.

[0199] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0200] (1) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0201] (i) the sequence shown in SEQ ID NO: 36;

[0202] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 36; or

[0203] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 36;

[0204] and / or,

[0205] (2) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0206] (i) a sequence as shown in any one of SEQ ID NO: 70;

[0207] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 70; or

[0208] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:70.

[0209] In certain embodiments, the antibody or antigen-binding fragment thereof comprises

[0210] a VH comprising the sequence shown in SEQ ID NO: 36 or a variant thereof, and a VL comprising the sequence shown in SEQ ID NO: 70 or a variant thereof;

[0211] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In certain embodiments, the substitutions are conservative substitutions.

[0212] In certain embodiments, the antibody or antigen-binding fragment thereof is capable of blocking the binding of human CD40 to its ligand CD40L.

[0213] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0214] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; and / or,

[0215] The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having up to 20 conservative substitutions of amino acids compared to the sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0216] In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function compared to the wild-type sequence from which it is derived.

[0217] In other embodiments, the variant of the heavy chain constant region (CH) may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function, etc. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or chemically modifying it, for example, by altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function (e.g., reducing or enhancing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0218] In certain embodiments, the antibody or its antigen-binding fragment has ADCC activity. In certain embodiments, the antibody or its antigen-binding fragment comprises a mutated (e.g., amino acid substitution) or chemically modified Fc region, wherein the mutated or chemically modified Fc region provides increased ADCC activity compared to the wild-type Fc region. In certain embodiments, the antibody or its antigen-binding fragment is produced by expression in a host cell with low or no fucosylation activity, and the resulting antibody has low fucosylation or no fucosylation, thereby increasing its ADCC activity. Such host cells can be mammalian cells that lack expression of a gene encoding a fucosyltransferase (e.g., FUT8), such as CHO cells.

[0219] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0220] In certain exemplary embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO:155.

[0221] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

[0222] In certain exemplary embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO:156.

[0223] In certain embodiments, the antibody or antigen-binding fragment thereof, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single-domain antibody (sdAb); and / or the antibody is a chimeric antibody, a bispecific antibody or a multispecific antibody.

[0224] In certain embodiments, the antibody or antigen-binding fragment thereof has cross-reactivity with human and cynomolgus monkey CD40.

[0225] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0226] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0227] Isolated nucleic acid molecules

[0228] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof as described above.

[0229] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0230] carrier

[0231] In another aspect, the present application also provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0232] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0233] host cells

[0234] On the other hand, the application also provides a host cell comprising a nucleic acid molecule or a vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0235] Preparation method

[0236] On the other hand, the present application also provides a method for preparing the antibody or antigen-binding fragment thereof as described above, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0237] Therapeutic uses

[0238] In another aspect, the present application also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0239] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0240] In another aspect, the present application also provides the use of the above-described antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or host cell for preparing a medicament for activating CD40 in a subject, increasing immune cell activity, enhancing immune response, and / or preventing and / or treating tumors or infections.

[0241] In certain embodiments, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells.

[0242] In certain embodiments, the immune response is a CD40-mediated immune response.

[0243] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0244] In certain embodiments, the tumor is selected from colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.

[0245] In certain embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0246] In certain embodiments, the subject is a mammal, such as a human.

[0247] In certain embodiments, the antibodies or antigen-binding fragments thereof are used alone or in combination with another pharmaceutically active agent.

[0248] On the other hand, the present application also provides a method for enhancing the immune response in a subject, and / or preventing and / or treating tumors or infections; the method comprises: administering an effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition as described above to a subject in need thereof.

[0249] In certain embodiments, the immune response is a CD40-mediated immune response.

[0250] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0251] In certain embodiments, the tumor is selected from colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.

[0252] In certain embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0253] In certain embodiments, the subject is a mammal, such as a human.

[0254] The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0255] The antibody of the application or its Fab or pharmaceutical composition of the present invention can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technical personnel should understand that route of administration and / or mode will change according to intended purpose. In certain embodiments, antibody of the present invention or its Fab or pharmaceutical composition are given by intravenous injection or push injection.

[0256] Detection purpose

[0257] In another aspect, the present application also provides a conjugate comprising the antibody or antigen-binding fragment thereof as described above, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0258] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0259] In another aspect, the present application also provides a kit comprising the antibody or antigen-binding fragment or conjugate thereof as described above.

[0260] In certain embodiments, the kit comprises a conjugate as described above.

[0261] In certain embodiments, the kit comprises an antibody or antigen-binding fragment thereof as described above, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0262] In another aspect, the present application also provides a method for detecting the presence or level of CD40 in a sample, comprising using the antibody, antigen-binding fragment, or conjugate thereof as described above. In certain embodiments, the method is used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0263] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0264] In certain embodiments, the methods comprise use of a conjugate as described above.

[0265] In certain embodiments, the method comprises using an antibody or antigen-binding fragment thereof as described above, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).

[0266] In certain embodiments, the method comprises: (1) contacting the sample with an antibody, or antigen-binding fragment thereof, or conjugate thereof; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of CD40 or cells expressing CD40.

[0267] In another aspect, the present application provides use of the antibody or antigen-binding fragment or conjugate thereof as described above in preparing a detection reagent for detecting the presence or level of CD40 in a sample.

[0268] In certain embodiments, the detection reagent detects the presence or level of CD40 in a sample by a method as described above for detecting the presence or level of CD40 in a sample.

[0269] In certain embodiments, the sample is a cell sample (eg, immune cell) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).

[0270] Definition of terms

[0271] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0272] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0273] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0274] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. The allocation of amino acids within each region or domain can be based on the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0275] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0276] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are identified by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are determined by the numbering method as described in the "Sequence analysis" section on page 11 of Lu X, Nobrega RP, Lynaugh H, et al. Deamidation and isomerization liability analysis of 131 clinical-stage antibodies. MAbs. 2019 Jan; 11(1): 45-57. doi: 10.1080 / 19420862.2018.1548233., the entire contents of which are incorporated herein by reference; wherein, CDRs L1-L3 refer to positions 24-34, 50-56, 89-97, respectively, and CDRs H1-H3 refer to positions 27-35, 50-65 and 93-102, respectively; the positions refer to the positions of Kabat.

[0277] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0278] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0279] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0280] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as humans; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0281] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0282] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0283] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0284] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0285] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0286] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

[0287] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen.

[0288] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0289] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody, and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0290] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0291] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0292] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0293] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0294] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0295] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0296] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0297] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0298] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0299] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0300] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0301] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0302] As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a tumor, infection, or autoimmune disease, or is at risk of suffering from the above diseases.

[0303] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infection, or an autoimmune disease) refers to an amount sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0304] Advantageous Effects of the Invention

[0305] The present invention provides a fully human anti-CD40 antibody, which has high binding affinity to CD40, has cross-reactivity with human and cynomolgus monkey CD40, and can block or non-block the binding of CD40 to its ligand CD40L. BRIEF DESCRIPTION OF THE DRAWINGS

[0306] Figure 1 shows the binding activity of the parental anti-CD40 candidate molecules to human CD40-CHO cells.

[0307] Figure 2 shows the activation of CD40 reporter gene activity by the parental anti-CD40 candidate molecule in the absence of bead cross-linking.

[0308] Figure 3 shows the activation of CD40 reporter gene activity by the parental anti-CD40 candidate molecule in the presence of beads cross-linking.

[0309] Figure 4 shows the binding activity of progeny anti-CD40 candidate molecules to human CD40 CHO cells.

[0310] Figure 5 shows the binding activity of progeny anti-CD40 candidate molecules to monkey CD40 CHO cells.

[0311] Figure 6 shows that progeny anti-CD40 candidate molecules activate CD40 reporter gene activity in the presence of anti-Fc cross-linking.

[0312] Figure 7 shows that progeny anti-CD40 candidate molecules activate CD40 reporter gene activity in the absence of anti-Fc cross-linking.

[0313] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0314] Sequence information

[0315] A description of the sequences involved in this application is provided in the table below.

[0316] Table 1: Sequence information DETAILED DESCRIPTION

[0317] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0318] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0319] Example 1: Preparation of antibodies

[0320] 1.1 Screening of fully human anti-CD40 antibodies using yeast display technology

[0321] Based on the yeast antibody display library (Adimab), eight natural synthetic libraries were amplified according to existing methods (WO2009036379; WO2010105256; WO2012009568), with the diversity of each library reaching 1x10 9 Briefly, the first two rounds of screening used the Miltenyi MACS system for magnetic bead cell sorting. First, yeast cells (~1x10 10 Each cell / library was incubated in FACS wash buffer (phosphate buffered saline with 0.1% BSA) for 15 minutes at room temperature. The buffer contained 10 nM biotinylated human CD40-Fc fusion protein antigen (purchased from ACRO, Catalog No. CD0-H5253, where the amino acid sequence of human CD40 is shown as SEQ ID NO:71). The cells were washed once with 50 mL of pre-chilled FACS wash buffer, resuspended in 40 mL of buffer, and 500 μL of streptavidin magnetic beads (Miltenyi LS) were added and incubated at room temperature for 15 minutes. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 5 mL of FACS wash buffer. The cell suspension was then applied to a Miltenyi LS column. After loading, the column was washed three times with 3 mL of FACS wash buffer. The Miltenyi LS column was removed from the magnetic area and eluted with 5 mL of growth medium. The eluted yeast cells were collected and grown overnight at 37°C.

[0322] The next round of sorting was performed using flow cytometry: approximately 1x10 8 Yeast cells were washed three times with FACS buffer and incubated at room temperature in the presence of 100 nM biotinylated CD40 monomer protein (purchased from Acro, Catalog No. CD0-H5228, wherein the amino acid sequence of CD40 is shown as SEQ ID NO: 71) or 10 nM CD40-Fc fusion protein. The culture medium was discarded, and the cells were washed twice with FACS wash buffer. The cells were then mixed with LC-FITC (FITC-labeled anti-human immunoglobulin kappa light chain antibody, purchased from Southern Biotech) (1:100 dilution) and SA-633 (streptavidin-633, Molecular Probes) (1:500 dilution) or SA-PE (streptavidin-PE, Sigma) (1:50 dilution) and incubated at 4°C for 15 minutes. The cells were then eluted twice with pre-chilled FACS wash buffer, resuspended in 0.4 mL of buffer, and transferred to a separate tube with a filter. Cells were sorted using FACS ARIA (BD Biosciences).

[0323] Yeast cells expressing anti-CD40 antibodies obtained through screening were induced with shaking at 30°C for 48 hours to secrete the full-length anti-CD40 antibody. After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 minutes, and the supernatant was harvested. The anti-CD40 antibodies in the supernatant were purified using Protein A and eluted with pH 2.0 acetic acid to yield the anti-CD40 antibodies. This screening yielded the anti-CD40 antibodies ADI-47754, ADI-47720, and ADI-47741.

[0324] 1.2 Affinity optimization of anti-human CD40 antibody

[0325] To obtain higher affinity anti-human CD40 antibodies, we optimized antibodies ADI-47754 and ADI-47720 by, but not limited to, the following methods.

[0326] 1.3 CDRH1 / CDRH2 screening

[0327] The CDRH3 gene of the parent molecule was constructed into 1x10 8 The first two rounds of screening used the MACS method, and the third and fourth rounds used the FACS method to increase the affinity of the antibody-antigen binding and screen for high-affinity antibodies.

[0328] Yeast cells expressing anti-CD40 antibodies were cultured with shaking at 30°C for 48 hours to produce CD40 antibodies. After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 minutes, and the supernatant was harvested. The anti-CD40 antibodies in the supernatant were purified using Protein A and eluted with pH 2.0 acetic acid to yield the anti-CD40 antibodies. The corresponding fab fragments were purified using papain and KappaSelect (GE Healthcare).

[0329] 1.4 VHmut screening

[0330] The method introduces mutations into the heavy chain region using conventional mismatch PCR. During the PCR process, 1 μM of the highly mutagenic base analogs dPTP and 8-oxo-dGTP is used to increase the base mismatch probability to approximately 0.01 bp.

[0331] The product obtained by mismatched PCR was constructed into a vector containing the heavy chain constant region by homologous recombination. In this way, under the selection pressure including CD40 antigen titer, unlabeled antigen competition, and competition with parental antibody, a library capacity of 1x10 7 The secondary library was screened for three rounds by FACS, and the corresponding antibodies were expressed in yeast.

[0332] The relationship between each antibody progeny and the parent antibody after affinity maturation is shown in Table 2.

[0333] Table 2. Relationship between progeny and parent of the anti-CD40 molecule of the present invention

[0334] 1.5 Antibody Expression and Purification

[0335] The sequences and numbers of the anti-human CD40 antibodies involved in this application are shown in Table 3, which includes the parent antibody ADI-47741 not mentioned in Table 2.

[0336] The antibodies used in the examples were expressed and purified in HEK293 cells. The specific procedure was as follows: HEK293 cells were transfected with a pcDNA3.1 vector carrying the heavy and light chains of the antibody (wherein the vector encoded the heavy chain constant region amino acid sequence as shown in SEQ ID NO: 72, and the light chain constant region amino acid sequence as shown in SEQ ID NO: 73) using chemical transfection. The cells were cultured at 37°C, 8% CO₂ for 7 days. The cell suspension was collected and centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected and purified by Protein A. The antibody purity was tested by SEC, and the endotoxin content was also controlled.

[0337]

[0338]

[0339]

[0340] Example 2: Antibody affinity detection

[0341] The biofilm optical interferometry technique (ForteBio) was used to determine the binding dissociation constant (K) of the antibody of the present invention to human and cynomolgus monkey CD40. D ), and whether it blocks the binding of human CD40 to its ligand (human CD40L), wherein the amino acid sequence of human CD40 is shown in SEQ ID NO:71, the amino acid sequence of cynomolgus monkey CD40 is shown in SEQ ID NO:74, and the amino acid sequence of human CD40L is shown in SEQ ID NO:75. Fortebio affinity determination was performed according to existing methods (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013. 5(2): p. 270-8).

[0342] The monovalent affinity of candidate antibody Fab fragments for human and cynomolgus macaque CD40-Fc proteins was measured: the sensor was equilibrated offline in assay buffer for 20 minutes, followed by 120 seconds of online measurement to establish a baseline. CD40-Fc antigen was then loaded onto the AHQ sensor to a thickness of 1 nm for affinity measurement. The loaded sensor was incubated in 100 nm of Fab solution until plateau phase, and then the sensor was transferred to assay buffer for at least 2 minutes for dissociation rate measurement. Kinetic analysis was performed using a 1:1 binding model.

[0343] To measure the bivalent affinity of intact antibodies for human and cynomolgus macaque CD40-Fc proteins, the sensor was equilibrated offline in assay buffer for 20 minutes, followed by an online assay for 120 seconds to establish a baseline. The intact CD40 antibody was then loaded onto the AHQ sensor to a thickness of 1 nm for affinity measurement. The loaded sensor was then incubated with a high concentration of irrelevant intact antibody for 10 minutes to saturate the Fc binding sites on the AHQ sensor. The blocked sensor was then exposed to 100 nm CD40-Fc antigen until plateau phase was reached. The sensor was then transferred to assay buffer for at least 2 minutes for off-rate measurement. Kinetic analysis was performed using a 1:1 binding model.

[0344] In the above determination method, the K of each parent antibody molecule and its variant molecule is D The values ​​are shown in Table 4:

[0345] The results in the above charts show that: (1) After affinity maturation, the monovalent affinity of each progeny molecule to human CD40 protein was significantly improved compared to the parent molecule. (2) After affinity maturation, the monovalent affinity of each progeny molecule to cynomolgus monkey CD40 protein was significantly improved compared to the parent molecule.

[0346] Table 4. K values ​​of each parent antibody molecule and its variant molecules D value Note: PF = Poor Fit, NB = Non-Binder under the conditions of this assay, NA = Not Available

[0347] Example 3: Binding of the parental anti-CD40 antibody to CHO cells overexpressing human CD40

[0348] The binding activity of the parental clone of the anti-CD40 antibody to human CD40 overexpressed on CHO cells was determined by flow cytometry. The amino acid sequences of the light and heavy chains of the control antibody CP870893-IgG2 are shown in SEQ ID NOs: 80 and 81, respectively.

[0349] Specifically, CHO-S cells overexpressing human CD40 (CHO-huCD40 cells) were generated by pressure screening by transfecting the human CD40 cDNA pCHO1.0 vector (Invitrogen) cloned into MCS. The expanded overexpressing cells were adjusted to an appropriate cell density and added to a 96-well flow cytometer. After centrifugation, the sample to be tested was added in a gradient dilution and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, and the fluorescent secondary antibody diluted to the appropriate concentration was added. The cells were incubated at 4°C for 30 minutes and washed twice with PBS. The cells were resuspended in PBS, detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. The results are shown in Figure 1.

[0350] The following conclusions can be drawn from the experimental results: the parent anti-CD40 antibodies ADI-47754, ADI-47720 and ADI-47741 can all have good single-site binding activity to CHO cells overexpressing human CD40.

[0351] Example 4: Activation of human CD40 signaling pathway activity by the parental anti-CD40 antibody

[0352] Based on the reporter gene assay, the activity of the NFkB-Luc2P reporter gene in the CD40 downstream signaling pathway was detected by the anti-CD40 antibody parental clone under conditions of Protein G Magnetic Beads cross-linking or not.

[0353] Specifically, U2OS cells overexpressing human CD40 were generated by transfection and cloning into the NFkB-Luc2P vector (Promega, Cat. No. 11501ES03) and pressure screening. The parent cloned antibody was added to 0.25 μL / well of Pierce TM Protein G Magnetic Beads (Thermo Scientific, Cat. No. 88847) were added to NFkB-Luc2P-U2OS cells and incubated for 4 hours in a cell culture incubator to examine CD40 downstream signaling activity induced by bead cross-linking. Alternatively, antibodies alone were added to NFkB-Luc2P-U2OS cells at the corresponding concentrations and incubated for 4 hours in a cell culture incubator to examine CD40 downstream signaling activity induced by antibodies alone without bead cross-linking.

[0354] The experimental results indicate that the parental clones ADI-47754, ADI-47720, and ADI-47741 failed to activate CD40 downstream signaling pathways without Fc crosslinking. However, they all activated CD40 downstream signaling pathways with beads crosslinking, and signaling activity was enhanced for all antibodies with beads crosslinking. In contrast, the positive control antibody CP-870893-G2-WT activated CD40 downstream signaling pathways independently of anti-Fc crosslinking. Detailed results are shown in Tables 5-7 and Figures 2 and 3.

[0355] Table 5. Activity of the parental anti-CD40 antibody in activating the CD40 signaling pathway

[0356] Table 6 EC50 values ​​of CD40-NFkB reporter gene assay (without beads cross-linking)

[0357] Table 7 EC50 values ​​of CD40-NFkB reporter gene assay (with beads cross-linking)

[0358] Example 5: Binding of progeny anti-CD40 antibodies to CHO cells overexpressing human / monkey CD40

[0359] Based on the results of Example 4, we optimized the affinity of ADI-47720, ADI-47741, and ADI-47754, which rely on beads cross-linking to activate CD40 reporter gene activity, to produce the antibodies shown in Table 2.

[0360] ADI-47720, ADI-47741, ADI-47754 and their progeny antibodies ADI-55136, ADI-55140, ADI-55142, ADI-55144-2, ADI-55145, ADI-55147, ADI-55164 and ADI-55168 were selected for the study of binding activity to human / monkey CD40 overexpressing cells.

[0361] Specifically, CHO-S cells overexpressing human / monkey CD40 (CHO-huCD40 cells) were generated by pressure screening by transfecting the human / monkey CD40 cDNA pCHO1.0 vector (Invitrogen) cloned into the MCS. The expanded overexpressing cells were adjusted to an appropriate cell density and added to a 96-well flow cytometer. After centrifugation, the sample to be tested was added in a gradient dilution and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, and the corresponding fluorescent secondary antibody diluted to the appropriate concentration was added. The cells were incubated at 4°C for 30 minutes and washed twice with PBS. The cells were resuspended in PBS and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated.

[0362] The following conclusions can be drawn from the test results: compared with the parent antibody, the offspring anti-CD40 antibodies have a certain improvement in binding activity to human / monkey CD40-overexpressing cells. The results are shown in Tables 8-9 and Figures 4 and 5.

[0363] Table 8 EC50 values ​​of human CD40 CHO cell binding assay

[0364] Table 9 EC50 values ​​of monkey CD40 CHO cell binding assay

[0365] Example 6: Progeny anti-CD40 antibodies activate human CD40 signaling pathway activity

[0366] The reporter gene assay was used to detect the activity of the NFkB-Luc2P reporter gene in the CD40 downstream signaling pathway activated by the parent and progeny anti-CD40 antibodies under conditions of cross-linking with or without F(ab)2 fragment goat anti-human IgG-Fc (Jackson, Cat. No. 109-036-098).

[0367] Specifically, Jurkat cells overexpressing human CD40 were generated by transfection and cloning into the NFkB-Luc2P vector (Promega, Catalog No. 11501ES03) and pressure screening. The parent and progeny cloned antibodies were diluted 3-fold at a final concentration of 200 nM, and 11 concentrations were added to CD40-NFkB-Jurkat cells along with F(ab)2 fragment goat anti-human IgG-Fc (Thermo Scientific, Catalog No. 88847) at a 1:2 molar ratio. The cells were incubated for 6 hours in a cell culture incubator to assess CD40 downstream signaling activity induced by anti-Fc fragment crosslinking. Alternatively, CD40-NFkB-Jurkat cells were incubated for 6 hours with only the corresponding concentrations of antibody alone, without anti-Fc fragment crosslinking, to assess CD40 downstream signaling activity induced by the antibody alone.

[0368] The experimental results are shown in Figures 6 and 7. It can be seen from the experimental results that the parent antibodies ADI-47720, ADI-47741, ADI-47754 and the progeny antibodies ADI-55142, ADI-55144-2, ADI-55147, ADI-55164, and ADI-55168 cannot activate the CD40 downstream signaling pathway in the absence of anti-Fc cross-linking, but can activate the CD40 downstream signaling pathway to varying degrees in the presence of anti-Fc cross-linking; while the positive control antibody CP-870893-G2-WT can activate the CD40 downstream signaling pathway independently of anti-Fc cross-linking. In addition, as a non-cross-linking-dependent CD40 agonist, the control antibody CP-870893-G2-WT has been reported to cause severe liver toxicity in clinical practice, and its clinical trials have been suspended.

[0369] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CD40, wherein the antibody or antigen-binding fragment thereof comprises: (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs): (a) a VH CDR1 having a structure selected from the group consisting of: FTFX1X2YX3MH (SEQ ID NO:76), GSISSYYWS (SEQ ID NO:6); (b) a VH CDR2 having a structure selected from the group consisting of: VIX4YX5X6X7X8KYYAX9SVKG(SEQ ID NO:77),GISWSSX 10 SIX 11 YADSVKG(SEQ ID NO:78),SIYYSGSTNYNPSLKS(SEQ ID NO:22); (c) a VH CDR3 having a structure selected from the group consisting of: ARDTSRGHDI (SEQ ID NO:23), AKDPTATTGARGYFDL (SEQ ID NO:24), ARDANYYKWHPY (SEQ ID NO:25); and / or, (2) The following three light chain variable region (VL) CDRs: (d) a VL CDR1 having a structure selected from the group consisting of: X 12 ASQSVSX 13 X 14 YLA(SEQ ID NO:79),X 15 ASX 16 X 17 ISSWLA(SEQ ID NO:7); (e) a VL CDR2 having a structure selected from the group consisting of: GASX 18 X 19 X 20 X 21 (SEQ ID NO:8),AASX 22 LX 23 S(SEQ ID NO:9); (f) a VL CDR3 having a structure selected from the group consisting of: X 24 X 25 YX 26 DYPPFT(SEQ ID NO:10),QQX 27 X 28 SX 29 PPT(SEQ ID NO:11); in, X1 is selected from the group consisting of (i) amino acid residues S, E, G, D and (ii) amino acid residues that are conservative substitutions relative to (i); X2 is selected from the group consisting of (i) amino acid residues S, K, D, T and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i); X4 is selected from the group consisting of (i) amino acid residues S, H, and (ii) amino acid residues that are conservative substitutions relative to (i); X5 is selected from the group consisting of (i) amino acid residues E, H, and (ii) amino acid residues that are conservative substitutions relative to (i); X6 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i); X7 is selected from the group consisting of (i) amino acid residues S, E, T, V and (ii) amino acid residues that are conservative substitutions relative to (i); X8 is selected from the group consisting of (i) amino acid residues N, S, I, K and (ii) amino acid residues that are conservative substitutions relative to (i); X9 is selected from (i) amino acid residues D, E and (ii) an amino acid residue that is a conservative substitution relative to (i); X 10 selected from (i) amino acid residues G, D and (ii) amino acid residues that are conservative substitutions relative to (i); X 11 selected from (i) amino acid residues G, H and (ii) amino acid residues that are conservative substitutions relative to (i); X 12 selected from (i) amino acid residues R, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 13 selected from (i) amino acid residues Y, S and (ii) amino acid residues that are conservative substitutions relative to (i); X 14 selected from the group consisting of (i) amino acid residues S, D, N and (ii) amino acid residues that are conservative substitutions relative to (i); X 15 selected from (i) amino acid residues R, E and (ii) amino acid residues that are conservative substitutions relative to (i); X 16 selected from (i) amino acid residues Q, K and (ii) amino acid residues that are conservative substitutions relative to (i); X 17 selected from (i) amino acid residues G, V and (ii) amino acid residues that are conservative substitutions relative to (i); X 18 An amino acid residue selected from (i) S, T, A and (ii) a conservative substitution relative to (i); X 19 selected from (i) amino acid residues R, L and (ii) amino acid residues that are conservative substitutions relative to (i); X 20 selected from (i) amino acid residues N, A and (ii) amino acid residues that are conservative substitutions relative to (i); X 21 selected from (i) amino acid residues T, N and (ii) amino acid residues that are conservative substitutions relative to (i); X 22 selected from (i) amino acid residues S, Y and (ii) amino acid residues that are conservative substitutions relative to (i); X 23 selected from (i) amino acid residues E, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 24 selected from the group consisting of (i) amino acid residues S, G, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 25 selected from the group consisting of (i) amino acid residues E, Q, D and (ii) amino acid residues that are conservative substitutions relative to (i); X 26 selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i); X 27 selected from (i) amino acid residues R, H, V and (ii) amino acid residues that are conservative substitutions relative to (i); X 28 An amino acid residue selected from (i) S, L, A and (ii) a conservative substitution relative to (i); X 29 Selected from (i) amino acid residues F, Y and (ii) amino acid residues that are conservative substitutions relative to (i).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs): (a) VH CDR1 having the structure shown as FTFX1X2YX3MH (SEQ ID NO: 76); (b) VH CDR2 having the structure shown in VIX4YX5X6X7X8KYYAX9SVKG (SEQ ID NO: 77); (c) VH CDR3 having the structure shown as ARDTSRGHDI (SEQ ID NO: 23); and / or, (2) The following three light chain variable region (VL) CDRs: (d) VL CDR1, which has X 12 ASQSVSX 13 X 14 The structure shown in YLA (SEQ ID NO: 79); (e) VL CDR2, which has a 18 X 19 X 20 X 21 (SEQ ID NO: 8); (f) VL CDR3, which has X 24 X 25 YX 26 The structure shown in DYPPFT (SEQ ID NO: 10); in, X1 is selected from the group consisting of (i) amino acid residues S, E, G and (ii) amino acid residues that are conservative substitutions relative to (i); X2 is selected from (i) amino acid residues S, K and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is selected from the group consisting of (i) amino acid residue G and (ii) an amino acid residue that is a conservative substitution relative to (i); X4 is selected from the group consisting of (i) amino acid residues S, H, and (ii) amino acid residues that are conservative substitutions relative to (i); X5 is selected from the group consisting of (i) amino acid residues E, H, and (ii) amino acid residues that are conservative substitutions relative to (i); X6 is selected from the group consisting of (i) amino acid residues G, A and (ii) amino acid residues that are conservative substitutions relative to (i); X7 is selected from the group consisting of (i) amino acid residues S, E, T, V and (ii) amino acid residues that are conservative substitutions relative to (i); X8 is selected from the group consisting of (i) amino acid residues N, S, I, K and (ii) amino acid residues that are conservative substitutions relative to (i); X9 is selected from (i) amino acid residues D, E and (ii) an amino acid residue that is a conservative substitution relative to (i); X 12 selected from (i) amino acid residues R, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 13 selected from (i) amino acid residues Y, S and (ii) amino acid residues that are conservative substitutions relative to (i); X 14 selected from the group consisting of (i) amino acid residues S, D, N and (ii) amino acid residues that are conservative substitutions relative to (i); X 18 An amino acid residue selected from (i) S, T, A and (ii) a conservative substitution relative to (i); X 19 selected from (i) amino acid residues R, L and (ii) amino acid residues that are conservative substitutions relative to (i); X 20 selected from (i) amino acid residues N, A and (ii) amino acid residues that are conservative substitutions relative to (i); X 21 selected from (i) amino acid residues T, N and (ii) amino acid residues that are conservative substitutions relative to (i); X 24 selected from the group consisting of (i) amino acid residues S, G, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 25 selected from the group consisting of (i) amino acid residues E, Q, D and (ii) amino acid residues that are conservative substitutions relative to (i); X 26 selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservative substitutions relative to (i); Preferably, X1 is selected from amino acid residues S, E, G; X2 is selected from amino acid residues S, K; X3 is amino acid residue G; X4 is selected from amino acid residues S, H; X5 is selected from amino acid residues E, H; X6 is selected from amino acid residues G, A; X7 is selected from amino acid residues S, E, T, V; X8 is selected from amino acid residues N, S, I, K; X9 is selected from amino acid residues D, E; X 12 Selected from amino acid residues R, Q; X 13 Selected from amino acid residues Y, S; X 14 Selected from amino acid residues S, D, N; X 18 Selected from amino acid residues S, T, A; X 19 Selected from amino acid residues R, L; X 20 Selected from amino acid residues N, A; X 21 Selected from amino acid residues T, N; X 24 Selected from amino acid residues S, G, Q; X 25 Selected from amino acid residues E, Q, D; X 26 Selected from amino acid residues A, S.

3. The antibody or antigen-binding fragment thereof of claim 2, comprising: A VH CDR1 as set forth in any one of SEQ ID NOs: 1-3; a VH CDR2 as set forth in any one of SEQ ID NOs: 12-18, 82; and a VH CDR3 as set forth in SEQ ID NO: 23; a VL CDR1 as set forth in any one of SEQ ID NOs: 37-40; a VL CDR2 as set forth in any one of SEQ ID NOs: 45-50; and a VL CDR3 as set forth in any one of SEQ ID NOs: 54-57; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) VH CDR1 as set forth in SEQ ID NO: 1; VH CDR2 as set forth in SEQ ID NO: 12; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 37; VL CDR2 as set forth in SEQ ID NO: 45; and VL CDR3 as set forth in SEQ ID NO: 54; (2) VH CDR1 as set forth in SEQ ID NO: 1; VH CDR2 as set forth in SEQ ID NO: 13; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 37; VL CDR2 as set forth in SEQ ID NO: 45; and VL CDR3 as set forth in SEQ ID NO: 54; (3) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 14; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 38; VL CDR2 as set forth in SEQ ID NO: 46; and VL CDR3 as set forth in SEQ ID NO: 55; (4) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 15; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 47; and VL CDR3 as set forth in SEQ ID NO: 56; (5) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 16; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 48; and VL CDR3 as set forth in SEQ ID NO: 57; (6) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 17; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 40; VL CDR2 as set forth in SEQ ID NO: 49; and VL CDR3 as set forth in SEQ ID NO: 57; (7) VH CDR1 as set forth in SEQ ID NO: 3; VH CDR2 as set forth in SEQ ID NO: 18; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 38; VL CDR2 as set forth in SEQ ID NO: 50; and VL CDR3 as set forth in SEQ ID NO: 54; or (8) VH CDR1 as set forth in SEQ ID NO: 2; VH CDR2 as set forth in SEQ ID NO: 82; and VH CDR3 as set forth in SEQ ID NO: 23; VL CDR1 as set forth in SEQ ID NO: 39; VL CDR2 as set forth in SEQ ID NO: 48; and VL CDR3 as set forth in SEQ ID NO:

57.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising: (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs): (a) VH CDR1 having the structure shown as FTFX1X2YX3MH (SEQ ID NO: 76); (b) VH CDR2, which has GISWSSX 10 SIX 11 The structure shown in YADSVKG (SEQ ID NO: 78); (c) VH CDR3 having the structure shown as AKDPTATTGARGYFDL (SEQ ID NO: 24); and / or, (2) The following three light chain variable region (VL) CDRs: (d) VL CDR1, which has X 15 ASX 16 X 17 The structure shown in ISSWLA (SEQ ID NO: 7); (e) VL CDR2, which has 22 LX 23 The structure shown in S (SEQ ID NO: 9); (f) VL CDR3, which has 27 X 28 SX 29 The structure shown in PPT (SEQ ID NO: 11); in, X1 is selected from (i) an amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i); X2 is selected from the group consisting of (i) amino acid residues D, T and (ii) amino acid residues that are conservative substitutions relative to (i); X3 is selected from (i) amino acid residue A and (ii) an amino acid residue that is a conservative substitution relative to (i); X 10 selected from (i) amino acid residues G, D and (ii) amino acid residues that are conservative substitutions relative to (i); X 11 selected from (i) amino acid residues G, H and (ii) amino acid residues that are conservative substitutions relative to (i); X 15 selected from (i) amino acid residues R, E and (ii) amino acid residues that are conservative substitutions relative to (i); X 16 selected from (i) amino acid residues Q, K and (ii) amino acid residues that are conservative substitutions relative to (i); X 17 selected from (i) amino acid residues G, V and (ii) amino acid residues that are conservative substitutions relative to (i); X 22 selected from (i) amino acid residues S, Y and (ii) amino acid residues that are conservative substitutions relative to (i); X 23 selected from (i) amino acid residues E, Q and (ii) amino acid residues that are conservative substitutions relative to (i); X 27 selected from (i) amino acid residues R, H and (ii) amino acid residues that are conservative substitutions relative to (i); X 28 selected from (i) amino acid residues S, A and (ii) amino acid residues that are conservative substitutions relative to (i); X 29 selected from (i) amino acid residue F and (ii) an amino acid residue that is a conservative substitution relative to (i); Preferably, X1 is an amino acid residue D; X2 is selected from amino acid residues D and T; X3 is an amino acid residue A; X 10 Selected from amino acid residues G, D; X 11 Selected from amino acid residues G, H; X 15 Selected from amino acid residues R, E; X 16 Selected from amino acid residues Q, K; X 17 Selected from amino acid residues G, V; X 22 Selected from amino acid residues S, Y; X 23 Selected from amino acid residues E, Q; X 27 Selected from amino acid residues R, H; X 28 Selected from amino acid residues S, A; X 29 is the amino acid residue F.

5. The antibody or antigen-binding fragment thereof of claim 4, comprising: VH CDR1 as set forth in SEQ ID NOs: 4 or 5; VH CDR2 as set forth in any one of SEQ ID NOs: 19-21; and VH CDR3 as set forth in SEQ ID NO: 24; VL CDR1 as set forth in any one of SEQ ID NOs: 41-43; VL CDR2 as set forth in any one of SEQ ID NOs: 51-53; and VL CDR3 as set forth in SEQ ID NOs: 58 or 59; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) VH CDR1 as set forth in SEQ ID NO:4; VH CDR2 as set forth in SEQ ID NO:19; and VH CDR3 as set forth in SEQ ID NO:24; VL CDR1 as set forth in SEQ ID NO:41; VL CDR2 as set forth in SEQ ID NO:51; and VL CDR3 as set forth in SEQ ID NO:58; (2) VH CDR1 as set forth in SEQ ID NO:4; VH CDR2 as set forth in SEQ ID NO:20; and VH CDR3 as set forth in SEQ ID NO:24; VL CDR1 as set forth in SEQ ID NO:42; VL CDR2 as set forth in SEQ ID NO:52; and VL CDR3 as set forth in SEQ ID NO:59; or (3) VH CDR1 as shown in SEQ ID NO: 5; VH CDR2 as shown in SEQ ID NO: 21; and VH CDR3 as shown in SEQ ID NO: 24; VL CDR1 as shown in SEQ ID NO: 43; the VL CDR2 shown in SEQ ID NO:53; and the VL CDR3 shown in SEQ ID NO:

59.

6. The antibody or antigen-binding fragment thereof of claim 1, comprising: VH CDR1 as set forth in SEQ ID NO:6; VH CDR2 as set forth in SEQ ID NO:22; and VH CDR3 as set forth in SEQ ID NO:25; VL CDR1 as set forth in SEQ ID NO:44; VL CDR2 as set forth in SEQ ID NO:45; and VL CDR3 as set forth in SEQ ID NO:

60.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a framework region of a human immunoglobulin; For example, the antibody or its antigen-binding fragment comprises a framework region contained in the amino acid sequence encoded by human germline antibody genes; for example, the antibody or its antigen-binding fragment comprises: a heavy chain framework region contained in the amino acid sequence encoded by human heavy chain germline genes, and / or a light chain framework region contained in the amino acid sequence encoded by human light chain germline genes.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: (1) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) a sequence as shown in any one of SEQ ID NOs: 26-36, 83; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 26-36, 83; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 26-36, 83; and / or, (2) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (i) a sequence as shown in any one of SEQ ID NOs: 61-70; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 61-70; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 61-70; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising a sequence as shown in any one of SEQ ID NOs: 26-32, 83 or a variant thereof, and, VL of the sequence shown in any one of SEQ ID NOs:61-66 or a variant thereof; or, (b) a VH comprising a sequence as shown in any one of SEQ ID NOs: 33-35 or a variant thereof, and a VL comprising a sequence as shown in any one of SEQ ID NOs: 67-69 or a variant thereof; in, The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 26 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 61 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 27 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 61 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 28 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 62 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 29 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 63 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 30 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 64 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 31 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 65 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 32 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 66 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 33 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 67 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 34 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 68 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 35 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 69 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 36 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 70 or a variant thereof; For example, the antibody or antigen-binding fragment thereof comprises a VH having a sequence as shown in SEQ ID NO: 83 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 64 or a variant thereof; Wherein, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, further comprising a constant region derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3 or IgG4); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single-domain antibody (sdAb); and / or the antibody is a chimeric antibody, a bispecific antibody or a multispecific antibody.

11. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the heavy chain variable region and / or light chain variable region thereof.

12. A vector comprising the nucleic acid molecule according to claim 11; preferably, the vector is a cloning vector or an expression vector.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising culturing the host cell according to claim 13 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier and / or excipient.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the isolated nucleic acid molecule according to claim 11, the vector according to claim 12, or the host cell according to claim 13 for preparing a medicament for activating CD40, increasing immune cell activity, enhancing immune response, and / or preventing and / or treating tumors or infections in a subject; Preferably, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells; Preferably, the immune response is a CD40-mediated immune response; Preferably, the subject is a mammal, such as a human; Preferably, the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent.

17. A method for enhancing an immune response in a subject, and / or preventing and / or treating a tumor or infection; the method comprising: administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 15 to a subject in need thereof; Preferably, the immune response is a CD40-mediated immune response; Preferably, the subject is a mammal, such as a human.

18. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a detectable label linked to the antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

19. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the conjugate according to claim 18; Preferably, the kit comprises the conjugate of claim 18; Preferably, the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

20. A method for detecting the presence or level of CD40 in a sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the conjugate according to claim 18; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 18; Preferably, the method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the conjugate according to claim 18 in the preparation of a detection reagent for detecting the presence or level of CD40 in a sample; Preferably, the detection reagent detects the presence or level of CD40 in a sample by the method of claim 20; Preferably, the sample is a cell sample (eg, immune cell) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).

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