Anti-PD-1 / CD40 bispecific antibody and use thereof

Abstract

The present invention relates to a bispecific antibody or an antigen-binding fragment thereof capable of specifically binding to CD40 and PD-1, a pharmaceutical composition comprising same, and use thereof.

Description

Anti-PD-1 / CD40 bispecific antibodies and their uses

[0001] This application is based on the application with CN application number 202311466590.2 and application date November 3, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application belongs to the field of biomedicine technology. More specifically, the present application relates to a bispecific antibody or antigen-binding fragment thereof that can specifically bind to CD40 and PD-1, as well as a pharmaceutical composition containing the same and its application. Background Art

[0003] CD40, also known as TNFRSF5, is a member of the tumor necrosis factor receptor (TNFR) superfamily. It is expressed on antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, macrophages, classical and nonclassical monocytes, as well as a variety of non-immune cells, including platelets and endothelial cells, and several tumor cell types. The CD40 precursor contains 297 amino acids and is a type I transmembrane glycoprotein composed of an N-terminal signal peptide (20 amino acids), an extracellular domain (193 amino acids), a transmembrane domain (22 amino acids), and a cytoplasmic domain (62 amino acids). The extracellular domain contains four CDRs with a total of 22 cysteines. CD40 is highly glycosylated, and the molecular weight estimated from the amino acid sequence is 28 kD, but the molecular weight after glycosylation is 40-50 kD.

[0004] The cognate ligand of CD40 is CD154 (TNFSF5 / CD40L), a type II transmembrane protein with a molecular weight of approximately 39 kDa. CD40L expression is generally 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. Numerous studies have demonstrated that CD40-CD40L plays a crucial role in CD8 cytotoxic T lymphocyte (CTL) function in immune responses and is essential for adaptive immune responses.

[0005] CD40 is widely expressed on monocytes, their mature progeny dendritic cells (DCs), macrophages, and B cells, and plays a crucial role in immune cell function. Monocytes are innate immune precursor cells with a high degree of plasticity. They have the ability to differentiate into a variety of cell types, such as myeloid-derived suppressor cells (MDSCs), macrophages, and DCs. CD40 signaling is a key trigger for monocyte maturation, primarily driving differentiation into macrophages and DCs of the M1 lineage. CD40 binding to the DC cell surface promotes the production of cytokines and chemokines, induces the expression of co-stimulatory molecules, and promotes antigen cross-presentation. One of the primary functions of CD40L is to enhance antigen presentation by activating DCs. This step, called "permission," increases DC interaction with T cells by upregulating surface proteins such as CD54 and CD86, thereby activating the latter. However, the therapeutic window of most CD40 agonists is narrow, posing certain challenges to their development.

[0006] Programmed death 1 (PD-1, also known as CD279), is typically expressed on tumor-infiltrating lymphocytes. It can inhibit T cell activation and proliferation by binding to programmed death ligand 1 expressed on tumor cells and antigen-presenting cells, exerting a negative immunomodulatory effect and mediating tumor immune escape. The PD-1 pathway is a cornerstone of immune regulation, and inhibitory antibodies against PD-1 or PD-L1 (nivolumab, pembrolizumab, cemiplimab; atezolizumab, avelumab, and durvalumab) have been approved by the US Food and Drug Administration (FDA) for the treatment of various cancers. Despite these clinical successes, the majority of patients do not demonstrate a complete response, and many experience immune-related adverse events, highlighting the need for a better understanding of how to safely and effectively modulate the PD-1 pathway in patients.

[0007] Summary of the Invention

[0008] Based on extensive research, the inventors of this application have developed a bispecific antibody that specifically binds to PD-1 and CD40. The bispecific antibody provided herein can: block the PD-1 / PD-L1 signaling pathway, activate CD40 downstream signaling pathways (e.g., the NF-κB pathway) dependent on binding to PD-1, activate primary B cells, activate T cells in a mixed lymphocyte reaction system, and / or inhibit tumor growth in vivo.

[0009] In addition, the bispecific antibodies provided herein also have cross-binding activity with human CD40 and monkey CD40, as well as cross-binding activity with human PD-1 and monkey PD-1.

[0010] In certain embodiments, the bispecific antibodies provided herein are capable of simultaneously blocking the binding of PD-1 to PD-L1 and CD40 to CD40L. In certain embodiments, the bispecific antibodies provided herein are capable of blocking the binding of PD-1 to PD-L1 but not blocking the binding of CD40 to CD40L.

[0011] In certain embodiments, the bispecific antibodies provided herein have significant anti-tumor activity while being able to avoid systemic immune activation effects, thereby reducing toxic side effects.

[0012] Therefore, in one aspect, the present application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to CD40;

[0013] Among them, the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), and the first light chain variable region (VL) and the first heavy chain variable region (VH) together form a domain that can specifically bind to PD-1; the second antigen-binding domain comprises a second light chain variable region (VL) and a second heavy chain variable region (VH), and the second light chain variable region (VL) and the second heavy chain variable region (VH) together form a domain that can specifically bind to CD40.

[0014] In certain embodiments, the first antigen binding domain and the second antigen binding domain are each independently a scFv or a Fab.

[0015] In certain embodiments, the first light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:4; and / or the first heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3.

[0016] In certain embodiments, the first light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:40; and / or the first heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:39.

[0017] In certain embodiments, the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering systems.

[0018] In certain embodiments, the first light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; and / or the first heavy chain variable region (VH) comprises HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:15.

[0019] In certain embodiments, the first light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46; and / or the first heavy chain variable region (VH) comprises HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43.

[0020] In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0021] In certain embodiments of the bispecific antibodies of the present application, (a) the first light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, and / or the first heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof; or, (b) the first light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 40 or a variant thereof, and / or the first heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 39 or a variant thereof;

[0022] 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.

[0023] In certain embodiments, the second light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 23 or 33; and / or, the second heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 5 or 31.

[0024] In certain embodiments, the second light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 24 or 34; and / or, the second heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 6 or 32.

[0025] In certain embodiments, the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering systems.

[0026] In certain embodiments, the second light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27; and / or the second heavy chain variable region (VH) comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:7, HCDR2 of the amino acid sequence shown in SEQ ID NO:8, and HCDR3 of the amino acid sequence shown in SEQ ID NO:9.

[0027] In certain embodiments, the second light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30; and / or the second heavy chain variable region (VH) comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:10, HCDR2 of the amino acid sequence shown in SEQ ID NO:11, and HCDR3 of the amino acid sequence shown in SEQ ID NO:12.

[0028] In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0029] In certain embodiments of the bispecific antibodies of the present application, (a) the second light chain variable region (VL) comprises the amino acid sequence as shown in SEQ ID NO: 23 or 33 or a variant thereof, and / or the second heavy chain variable region (VH) comprises the amino acid sequence as shown in SEQ ID NO: 5 or 31 or a variant thereof; or, (b) the second light chain variable region (VL) comprises the amino acid sequence as shown in SEQ ID NO: 24 or 34 or a variant thereof, and / or the second heavy chain variable region (VH) comprises the amino acid sequence as shown in SEQ ID NO: 6 or 32 or a variant thereof;

[0030] 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.

[0031] In certain embodiments, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:23, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:5.

[0032] In certain embodiments, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:33, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:31.

[0033] In certain embodiments, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 24, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 6.

[0034] In certain embodiments, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:34, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:32.

[0035] In certain embodiments, the bispecific antibody comprises:

[0036] (1) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 4, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 3, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 23 or 33, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 5 or 31;

[0037] (2) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 4, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 3, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 24 or 34, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 6 or 32;

[0038] (3) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:40, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:39, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:23 or 33, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:5 or 31; or

[0039] (4) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:40, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:39, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:24 or 34, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:6 or 32.

[0040] In certain embodiments, the bispecific antibody comprises:

[0041] (1) the first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18, the first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15; the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO: 25, LCDR2 as shown in SEQ ID NO: 26, and LCDR3 as shown in SEQ ID NO: 27, and the second heavy chain variable region (VH) comprising HCDR1 with the amino acid sequence of SEQ ID NO: 7, HCDR2 with the amino acid sequence of SEQ ID NO: 8, and HCDR3 with the amino acid sequence of SEQ ID NO: 9;

[0042] (2) the first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18, the first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15; the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO: 28, LCDR2 as shown in SEQ ID NO: 29, and LCDR3 as shown in SEQ ID NO: 30, and the second heavy chain variable region (VH) comprising HCDR1 with the amino acid sequence shown in SEQ ID NO: 10, HCDR2 with the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 12;

[0043] (3) a first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46, a first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, a second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27, and a second heavy chain variable region (VH) comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and HCDR3 comprising the amino acid sequence of SEQ ID NO:9; or,

[0044] (4) a first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46, a first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, a second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30, and a second heavy chain variable region (VH) comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:10, HCDR2 comprising the amino acid sequence of SEQ ID NO:11, and HCDR3 comprising the amino acid sequence of SEQ ID NO:12.

[0045] In certain embodiments, the bispecific antibody comprises:

[0046] (1) comprising the first light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 4, the first heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 3, the second light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 23, and the second heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 5;

[0047] (2) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 33, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 31;

[0048] (3) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 24, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 6;

[0049] (4) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 34, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 32;

[0050] (5) the first light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 40, the first heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, the second light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 23, and the second heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5;

[0051] (6) the first light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 40, the first heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, the second light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 33, and the second heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31;

[0052] (7) the first light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 40, the first heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, the second light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 24, and the second heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6; or

[0053] (8) comprising the first light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:40, the first heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:39, the second light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:34, and the second heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:32.

[0054] In certain embodiments, the first antigen binding domain is Fab and the second antigen binding domain is scFv; alternatively, the first antigen binding domain is scFv and the second antigen binding domain is Fab.

[0055] In certain embodiments, the first antigen binding domain is a Fab and the second antigen binding domain is a scFv.

[0056] In certain embodiments, the bispecific antibody comprises two first antigen-binding domains and two second antigen-binding domains; wherein the two first antigen-binding domains are the same or different, and the two second antigen-binding domains are the same or different.

[0057] In certain embodiments, the bispecific antibody comprises peptide chain I and peptide chain II; wherein, peptide chain I comprises the first light chain variable region and the light chain constant region, and the peptide chain II comprises: the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region.

[0058] In certain embodiments, the peptide chain I comprises the first light chain variable region and the light chain constant region from N-terminus to C-terminus, and / or the peptide chain II comprises, from N-terminus to C-terminus: (i) the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region; or, (ii) the first heavy chain variable region, the heavy chain constant region, the second light chain variable region and the second heavy chain variable region.

[0059] In certain embodiments, the adjacent domains of the peptide chain I are optionally connected via a linker or not, and / or the adjacent domains of the peptide chain II are optionally connected via a linker or not.

[0060] In certain embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); preferably, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S) and / or alanine (A), for example (GGGGS) n or (GGGGA) n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 36 or 37.

[0061] In certain embodiments, the peptide chain II comprises, from N-terminus to C-terminus, the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region, and the second light chain variable region, wherein the heavy chain constant region and the second heavy chain variable region are connected by a peptide linker comprising one or more glycine (G) and / or alanine (S) (e.g., a peptide linker as shown in SEQ ID NO: 36); or, the peptide chain II comprises, from N-terminus to C-terminus, the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region, and the second light chain variable region, wherein the heavy chain constant region and the second heavy chain variable region are connected by a peptide linker comprising one or more glycine (G) and / or alanine (A) (e.g., a peptide linker as shown in SEQ ID NO: 36);

[0062] and / or,

[0063] The second heavy chain variable region and the second light chain variable region are connected via a peptide linker comprising one or more glycine (G) and / or serine (S) (eg, a peptide linker as shown in SEQ ID NO: 37).

[0064] In certain embodiments, the second light chain variable region and the second heavy chain variable region are capable of forming an intrachain disulfide bond therebetween.

[0065] In certain embodiments, (a) the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31; or, (b) the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34, and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32.

[0066] In certain embodiments, the heavy chain constant region is derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0067] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the CH2 domain (residues 231-340 of human IgG1, numbered according to the Eu numbering system), the CH3 domain (residues 341-447 of human IgG1, numbered according to the Eu numbering system) and / or the hinge region (residues 216-230, numbered according to the Eu numbering system) of the heavy chain constant region to alter one or more functional properties of the bispecific antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity.

[0068] In certain embodiments, one, two or more mutations (eg, amino acid substitutions) are introduced into the CH2 domain, CH3 domain, and / or hinge region of the heavy chain constant region, for example, to reduce or ablate the effector function of the Fc region.

[0069] In certain embodiments, the heavy chain constant region is selected from the heavy chain constant region of a wild-type human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4) or a variant thereof (e.g., a heavy chain constant region comprising a mutation or chemical modification); wherein the Fc domain comprised by the heavy chain constant region variant has altered (e.g., enhanced or reduced) effector function compared to the wild-type immunoglobulin heavy chain constant region from which it is derived; for example, the Fc domain comprised by the heavy chain constant region variant has reduced ADCC, ADCP and / or CDC activity compared to the wild-type immunoglobulin heavy chain constant region from which it is derived.

[0070] In certain embodiments, the heavy chain constant region is selected from the heavy chain constant region of wild-type human immunoglobulin IgG1 or a variant thereof (e.g., a heavy chain constant region comprising a mutation or chemical modification); wherein the heavy chain constant region variant comprises L234A, L235A and / or L237A substitution mutations compared to the wild-type immunoglobulin heavy chain constant region from which it is derived. In certain embodiments, the position of the mutation is determined according to the EU numbering system.

[0071] In certain embodiments, the Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO:19.

[0072] In certain embodiments, the light chain constant region is derived from a kappa or lambda light chain of a human immunoglobulin.

[0073] In certain embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:20.

[0074] In certain embodiments of the bispecific antibodies of the present application:

[0075] (a) the peptide chain I comprises the amino acid sequence shown in SEQ ID NO: 35 or a variant thereof, and / or the peptide chain II comprises the amino acid sequence shown in SEQ ID NO: 1 or 2 or a variant thereof; or

[0076] (b) the peptide chain I comprises the amino acid sequence shown in SEQ ID NO: 47 or a variant thereof, and / or the peptide chain II comprises the amino acid sequence shown in SEQ ID NO: 38 or a variant thereof;

[0077] 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; preferably, the substitutions are conservative substitutions;

[0078] In certain embodiments, the bispecific antibody comprises:

[0079] (1) a peptide chain I comprising the amino acid sequence shown in SEQ ID NO: 35, and a peptide chain II comprising the amino acid sequence shown in SEQ ID NO: 1;

[0080] (1) a peptide chain I comprising the amino acid sequence shown in SEQ ID NO: 35, and a peptide chain II comprising the amino acid sequence shown in SEQ ID NO: 2; or

[0081] (1) Peptide chain I comprising the amino acid sequence shown in SEQ ID NO: 47, and peptide chain II comprising the amino acid sequence shown in SEQ ID NO: 38.

[0082] In certain embodiments, the bispecific antibody comprises two of the peptide chains I and two of the peptide chains II.

[0083] In certain embodiments, the two peptide chains I comprised by the bispecific antibody are the same or different, and / or the two peptide chains II comprised by the bispecific antibody are the same or different.

[0084] In certain embodiments, the bispecific antibody comprises two identical peptide chains I and two identical peptide chains II.

[0085] The bispecific antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, a DNA molecule encoding the peptide chain of the bispecific antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the bispecific antibody of the present invention.

[0086] On the other hand, the application provides an isolated nucleic acid molecule or nucleic acid molecule group comprising the nucleotide sequence of a bispecific antibody as described above. According to the codon degeneracy in this area, in certain embodiments, the nucleotide sequence can be replaced according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.

[0087] It is easy to understand that the bispecific antibody of the present invention may be composed of one or more polypeptide chains, and the isolated nucleic acid molecule or nucleic acid molecule group encoding the bispecific antibody of the present invention is not limited in the number of nucleic acid molecule chains.

[0088] In certain embodiments, the bispecific antibody of the present invention comprises peptide chain I and peptide chain II as described above, and the isolated nucleic acid molecule or nucleic acid molecule group comprises a first nucleotide sequence encoding peptide chain I of the bispecific antibody of the present invention and a second nucleotide sequence encoding peptide chain II thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules.

[0089] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule or group of nucleic acid molecules as described above.

[0090] In certain embodiments, the vector is a cloning vector or an expression vector.

[0091] In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (eg, a mammal, such as a human).

[0092] It will be readily understood that the isolated nucleic acid molecule or nucleic acid molecule group described above may be present in any form within the vector. For example, when the isolated nucleic acid molecule or nucleic acid molecule group comprises multiple nucleotide sequences encoding different peptide chains, the multiple nucleotide sequences may be located on the same vector or on different vectors. The orientation, relative position, or linkage of the multiple nucleotide encoding sequences within the vector are not limited.

[0093] In certain embodiments, the bispecific antibody of the present invention comprises peptide chain I and peptide chain II as described above, and the vector comprises a first nucleotide sequence encoding peptide chain I of the bispecific antibody of the present invention and a second nucleotide sequence encoding peptide chain II thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vector molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different vector molecules, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0094] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or group of nucleic acid molecules, or the vector as described above.

[0095] The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-EBNA, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0096] In another aspect, the present application provides a method for preparing the bispecific antibody as described above, comprising culturing the host cell as described above under conditions allowing expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell culture.

[0097] The bispecific antibodies of the present invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of the bispecific antibody does not adversely affect its binding to PD-1 and CD40 (particularly human PD-1 and human CD40). Therefore, the bispecific antibodies of the present invention are also intended to include such derivatized forms. For example, the bispecific antibodies of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as detection reagents, pharmaceutical agents, and / or proteins or polypeptides capable of mediating the binding of the bispecific antibody to another molecule (e.g., avidin or polyhistidine tags).

[0098] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated moiety.

[0099] In certain embodiments, the conjugated moiety is selected from a therapeutic agent.

[0100] In certain embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life).

[0101] In another aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody, or isolated nucleic acid molecule or nucleic acid molecule group, or vector, or host cell as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0102] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0103] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity.

[0104] In certain embodiments, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0105] In another aspect, the present application provides use of the bispecific antibody, or isolated nucleic acid molecule or nucleic acid molecule group, or vector, or host cell, or pharmaceutical composition as described above in the preparation of a medicament, wherein the medicament is used for:

[0106] (1) increasing immune cell activity in vitro or in vivo in a subject (e.g., a human or monkey);

[0107] (2) enhancing an immune response in a subject (e.g., a human or monkey);

[0108] (3) preventing and / or treating tumors in subjects (e.g., humans or monkeys); and / or,

[0109] (4) Preventing and / or treating infection in a subject (eg, a human or monkey).

[0110] In certain embodiments, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells.

[0111] In certain embodiments, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma.

[0112] In certain embodiments, the tumor is a blood tumor, such as lymphoma or leukemia. In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; preferably, the non-Hodgkin's lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, Epstein-Barr virus-positive NK / T-cell lymphoma (nasal type), and B-cell non-Hodgkin's lymphoma.

[0113] 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.

[0114] In certain embodiments, the subject is a mammal, such as a human or a monkey.

[0115] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, eg, simultaneously, separately, or sequentially.

[0116] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity.

[0117] On the other hand, the present application provides a method for enhancing an immune response in a subject, and / or preventing and / or treating a tumor or infection; the method comprises: administering an effective amount of the bispecific antibody, or isolated nucleic acid molecule or nucleic acid molecule group, or vector, or host cell, or pharmaceutical composition as described above to a subject in need thereof.

[0118] In certain embodiments, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma.

[0119] In certain embodiments, the tumor is a blood tumor, such as lymphoma or leukemia. In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; preferably, the non-Hodgkin's lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, Epstein-Barr virus-positive NK / T-cell lymphoma (nasal type), and B-cell non-Hodgkin's lymphoma.

[0120] 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.

[0121] In certain embodiments, the subject is a mammal, such as a human or a monkey.

[0122] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, eg, simultaneously, separately, or sequentially.

[0123] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity.

[0124] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy can be applied simultaneously, separately, or sequentially with the bispecific antibody, isolated nucleic acid molecule or nucleic acid molecule group, vector, host cell, conjugate, or pharmaceutical composition of the present application.

[0125] The bispecific antibodies and pharmaceutical compositions of the present invention 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 route of administration and therapeutic use. The bispecific antibodies 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 bispecific antibody of the present invention into an appropriate solvent, and optionally, 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 ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0126] Furthermore, the bispecific antibodies of the present invention can be presented in pharmaceutical compositions in unit dosage form for ease of administration.

[0127] The bispecific antibodies and pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, preferred routes of administration / modes are parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Technicians will appreciate that routes of administration and / or modes will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibodies and pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0128] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" of the bispecific antibodies of the present invention. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the bispecific antibodies of the present invention may vary depending on factors such as the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the route of administration of the drug, and any other concurrently administered therapies.

[0129] In the present invention, the dosage regimen can be adjusted to obtain the optimal desired response (e.g., therapeutic response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation.

[0130] In the present invention, the subject may be a mammal, such as a human.

[0131] In another aspect, the present application also provides a conjugate comprising the bispecific antibody as described above and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0132] 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.

[0133] In another aspect, the present application also provides a kit comprising the bispecific antibody or conjugate as described above.

[0134] In certain embodiments, the kit comprises a conjugate as described above.

[0135] In certain embodiments, the kit comprises a bispecific antibody as described above, and a second antibody that specifically recognizes the antibody or its antigen-binding fragment. 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.

[0136] In another aspect, the present application also provides a method for detecting the presence or level of PD-1 and / or CD40 in a sample, comprising using a bispecific antibody or conjugate as described above. In certain embodiments, the method is used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

[0137] 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.

[0138] In certain embodiments, the methods comprise use of a conjugate as described above.

[0139] In certain embodiments, the method comprises using a bispecific antibody 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., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0140] In certain embodiments, the method comprises: (1) contacting the sample with a bispecific antibody or conjugate of the present invention; 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 PD-1 and / or CD40 or cells expressing PD-1 or CD40.

[0141] In another aspect, the present application provides use of the bispecific antibody or conjugate as described above in preparing a detection reagent for detecting the presence or level of PD-1 and / or CD40 in a sample.

[0142] In certain embodiments, the detection reagent detects the presence or level of PD-1 and / or CD40 in a sample by the method described above for detecting the presence or level of PD-1 and / or CD40 in a sample.

[0143] In certain embodiments, the sample is a cell sample from a subject (eg, a mammal, preferably a human or a monkey).

[0144] Definition of terms

[0145] 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.

[0146] 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."

[0147] 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.

[0148] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can be 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) light chains. 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 the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 consists 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 consists of a single domain, CL. The constant domain is not directly involved in antibody-antigen binding but exhibits various 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 be further subdivided into highly variable regions known as complementarity-determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL 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 / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; and Chothia et al. (1989) Nature 342:878-883.

[0149] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0150] 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 precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262:732-745, see also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)), the AHo numbering system (Honegger and Plückthun, A., J. Mol. Biol. 309:657-670 (2001)), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one 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).

[0151] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined 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 determined by the Kabat, Chothia, MacCallum, IMGT, AHo or AbM numbering systems.

[0152] 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.

[0153] 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.

[0154] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens (or epitopes), comprising two antibodies that have binding specificity for different antigens (or epitopes). The antigen binding domain is capable of binding to two different binding sites and / or target molecules. Each antigen binding domain comprised by a bispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv, Fab, scFab, or scFv). In some cases, each antigen binding domain is connected by a peptide linker.

[0155] As used herein, the term "Fc domain" refers to an antibody fragment formed by 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 bound via disulfide bonds. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0156] 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. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art peptide linkers are composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO: 37) may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0157] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains, which is generally composed of one peptide chain comprising VL and CL and another peptide chain comprising VH and CH1, however, those skilled in the art understand that the Fab domains can be arranged according to the above-mentioned natural orientation, but may also contain domain replacements or exchanges that promote correct VH and VL pairing (e.g., domain exchange in the Crossmab format).

[0158] As used herein, the terms "monoclonal antibody," "single antibody," and "mAb" have the same meaning and are used interchangeably, referring to an antibody or an antibody fragment from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for possible spontaneous natural mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, which typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" merely indicates the characteristic of the antibody as being obtained from a population of highly homologous antibodies and is not to be construed as requiring the antibody to be prepared by any particular method.

[0159] As used herein, the term "bispecific antibody" or "BsAb" refers to an antibody with two different binding domains that enable the bispecific antibody to simultaneously bind to two different antigens or two different epitopes of the same antigen.

[0160] 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 (KD) or the half-maximal effect concentration (EC) of the interaction. 50 ) determination.

[0161] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates. The "association rate constant" (k a or k on ) and the “dissociation rate constant” (k dis or k off ) Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio of the dissociation constant KD is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kD, and kD can be measured by any effective method. on and k dis In some embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Alternatively, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0162] 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.

[0163] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

[0164] 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, NSO cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0165] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. The algorithm of Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0166] The twenty conventional amino acids referred to herein are compiled according to 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.

[0167] 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 adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters 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. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. 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), etc. Stabilizers have the meanings 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.

[0168] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., tumor, pathogenic infection) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain beneficial or required clinical results. For the purposes of the present invention, beneficial or required clinical results 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 (no matter in part or in whole), no matter whether it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival period.

[0169] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (such as a human) suffers from a tumor or pathogen infection, or has a risk of suffering from the above diseases.

[0170] 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, a pathogenic infection) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor, a pathogenic infection); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop an existing disease or complication. 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.

[0171] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and is associated with the antibody and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of an antibody are known in the art, for example, by introducing mutations into the Fc region.

[0172] Advantageous Effects of the Invention

[0173] The present application provides bispecific antibodies that can specifically bind to PD-1 and CD40. The bispecific antibodies provided herein can: block the PD-1 / PD-L1 signaling pathway, activate CD40 downstream signaling pathways (e.g., NF-κB pathway) depending on binding to PD-1, activate primary B cells, activate T cells in a mixed lymphocyte reaction system, and / or inhibit tumor growth in vivo.

[0174] In addition, the bispecific antibodies provided herein also have cross-binding activity with human CD40 and monkey CD40, as well as cross-binding activity with human PD-1 and monkey PD-1.

[0175] In certain embodiments, the bispecific antibodies provided herein are capable of simultaneously blocking the binding of PD-1 to PD-L1 and CD40 to CD40L. In certain embodiments, the bispecific antibodies provided herein are capable of blocking the binding of PD-1 to PD-L1 but not blocking the binding of CD40 to CD40L.

[0176] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] FIG1A shows a schematic structural diagram of the anti-PD1-anti-CD40 (non-blocker) bispecific antibody of the present invention (corresponding to “Bispecific Antibody-1” in Table 2).

[0178] FIG1B shows a schematic structural diagram of the anti-PD1-anti-CD40 (blocker) bispecific antibody of the present invention (corresponding to “bispecific-2” in Table 2).

[0179] FIG1C shows a schematic structural diagram of the anti-PD1 (pembro) xCD40 bispecific antibody of the present invention (corresponding to “bispecific-3” in Table 2).

[0180] FIG2A shows the affinity of the anti-PD1-anti-CD40 bispecific antibody of the present invention to human / monkey PD-1 protein.

[0181] FIG2B shows the affinity of the anti-PD1-anti-CD40 bispecific antibody of the present invention to human / monkey CD40 protein.

[0182] FIG2C shows the affinity of the anti-PD1(pembro)xCD40 bispecific antibody of the present invention to human PD-1 / CD40 protein.

[0183] Figures 3A-3I show the binding activity of the anti-PD1-anti-CD40 bispecific antibodies of the present invention to CHO-S cells overexpressing human / monkey CD40 or PD-1 (or control CHO-S cells). The bispecific antibody "PD1xCD40" in Figure 3I corresponds to "Bispecific Antibody-1" in Table 2, and the bispecific antibody "PD1(Pembro)xCD40" corresponds to "Bispecific Antibody-3" in Table 2.

[0184] 4A-4B show the blocking activity of the anti-PD1-anti-CD40 bispecific antibody of the present invention in blocking the binding of CD40 to CD40L.

[0185] 5A-5B show the blocking activity of the anti-PD1-anti-CD40 bispecific antibody of the present invention in blocking the PD-L1 and PD-1 signaling pathways.

[0186] Figures 6A-6D show the activity of the anti-PD1-anti-CD40 bispecific antibody of the present invention in activating CD40 depending on the expression of PD-1.

[0187] Figures 7A-7D show the activity of the anti-PD1-anti-CD40 bispecific antibody of the present invention in activating primary B cells.

[0188] Figures 8A-8B show the experimental results of the anti-PD1-anti-CD40 bispecific antibodies of the present invention activating T cells to release IL-2 in a mixed lymphocyte reaction system. The bispecific antibody "PD1xCD40" in Figure 8B corresponds to "Bispecific Antibody-1" in Table 2, the bispecific antibody "PD1(Pembro)xCD40" corresponds to "Bispecific Antibody-3" in Table 2, and the anti-CD40 mAb corresponds to ADI-55164.

[0189] 9A-9C show the half-life of the anti-PD1-anti-CD40 bispecific antibodies of the present invention.

[0190] Figure 10A shows the in vivo efficacy test results of the anti-PD1-anti-CD40 bispecific antibody of the present invention in a mouse tumor model inoculated subcutaneously with A375 and human PBMC B-NDG B2M KO Plus.

[0191] Figure 10B shows the in vivo efficacy test results of the anti-PD1-anti-CD40 bispecific antibody of the present invention in a C57 mouse tumor model inoculated subcutaneously with B16F10 and human PBMC huPD-1 / CD40 double KI.

[0192] Note: In the drawings of this application, “PD1xCD40 (ADI-55164)” or “PD1xCD40 (non-blocker)” corresponds to “Dual Antibody-1” in Table 2; “PD1xCD40 (ADI-55147)” or “PD1xCD40 (blocker)” corresponds to “Dual Antibody-2” in Table 2; “PD1 (pembro)xCD40 (ADI-55164)” corresponds to “Dual Antibody-3” in Table 2; “PD-1mAb” or “anti-PD1” corresponds to ADI-54872; “CD40 mAb (blocker)” corresponds to ADI-55147; and “CD40 mAb (non-blocker)” corresponds to ADI-55164.

[0193] Sequence information

[0194] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0195] Table 1: Description of sequences DETAILED DESCRIPTION

[0196] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0197] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE. CULTURE) (RI Freshney, ed. (1987)).

[0198] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0199] Example 1. Cloning and expression of bispecific antibodies

[0200] 1.1 Structure of Antibody Constructs

[0201] In this example, two anti-PD-1 / CD40 bispecific antibodies were constructed, namely:

[0202] PD1xCD40 (non-blocker, ADI-55164): Composed of a non-blocker anti-CD40 antibody (i.e., anti-CD40 antibody ADI-55164 that has no blocking effect on the binding of CD40 and its ligand CD40L) and an anti-PD-1 antibody (ADI-54872). A schematic structural diagram of the bispecific antibody is shown in Figure 1A. The heavy chain of the bispecific antibody has the amino acid sequence set forth in SEQ ID NO: 1, and the light chain has the amino acid sequence set forth in SEQ ID NO: 35. The bispecific antibody comprises the heavy and light chains of the anti-PD-1 antibody (ADI-54872), wherein the heavy chain of the anti-PD-1 antibody comprises an Fc region derived from human IgG1 (in which the L234AL235AL237A mutations have been introduced to reduce Fc effector function), and the single-chain variable fragment (scFv) of the anti-CD40 monoclonal antibody ADI-55164 is linked to the C-terminus of the heavy chain.

[0203] PD1xCD40 (blocker, ADI-55147): consists of an anti-CD40 antibody with a blocker effect (i.e., anti-CD40 antibody ADI-55147 that blocks the binding of CD40 and its ligand CD40L) and an anti-PD-1 antibody (ADI-54872). The structural schematic is shown in Figure 1B. The heavy chain of the bispecific antibody has the amino acid sequence set forth in SEQ ID NO: 2, and the light chain has the amino acid sequence set forth in SEQ ID NO: 35. The bispecific antibody comprises the heavy and light chains of the anti-PD-1 antibody (ADI-54872). The heavy chain of the anti-PD-1 antibody comprises an Fc region derived from human IgG1 (in which the L234AL235AL237A mutations have been introduced to reduce Fc effector function), and the single-chain variable fragment (scFv) of the anti-CD40 monoclonal antibody ADI-55147 is linked to the C-terminus of the heavy chain.

[0204] PD1(pembro)xCD40(non-blocker, ADI-55164): It is composed of an anti-CD40 antibody with a non-blocker effect (i.e., an anti-CD40 antibody that has no blocking effect on the binding of CD40 and its ligand CD40L) and an anti-PD-1 antibody (Pembrolizumab, also referred to herein as pembro, whose heavy chain variable region and light chain variable region amino acid sequences are shown in SEQ ID NOs: 39 and 40, respectively). The schematic diagram of its structure is shown in FIG1C . The heavy chain of the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 38, and the light chain has the amino acid sequence shown in SEQ ID NO: 40. NO:47 shows the amino acid sequence, wherein the bispecific antibody comprises the heavy chain and light chain of the anti-PD-1 antibody (Pembrolizumab), wherein the Fc region comprised by the heavy chain of the anti-PD-1 antibody is an Fc region derived from human IgG1 (in which L234AL235AL237A mutations are introduced to reduce Fc effector function), and the single-chain variable fragment (scFv) of the anti-CD40 monoclonal antibody ADI-55164 is connected to the C-terminus of the heavy chain.

[0205] The structure / sequence information of the above three bispecific antibodies is shown in Table 2.

[0206] 1.2 Gene cloning and protein preparation

[0207] The above gene fragments were constructed into pCDNA3.1 vector. TM Using the Expression System kit (purchased from Thermo Fisher Scientific), the midi-prepared fusion protein expression plasmid was transformed into Expi-CHO cells according to the manufacturer's instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using Protein A magnetic beads (purchased from GenScript). The beads were resuspended in an appropriate volume of Binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the beads) and added to the sample to be purified. Incubate at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic rack (purchased from Beaver), the supernatant discarded, and the beads washed three times with Binding buffer. Add elution buffer (0.1 M sodium citrate, pH 3.2) at a volume 3-5 times the volume of the magnetic beads and shake at room temperature for 5-10 minutes. Place the beads back on the magnetic stand, collect the elution buffer, transfer it to a collection tube containing neutralization buffer (1 M Tris, pH 8.54), and mix thoroughly to complete the preparation.

[0208] Example 2: Bispecific Antibody Protein Level Affinity Detection

[0209] ForteBio affinity determination was performed according to the existing method (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated offline in the assay buffer for 30 min, then online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM antigen for 5 min, after which the sensor was transferred to PBS for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model. Among them, YH008 (see patent application WO2022078357A1) was a control anti-PD-1 / CD40 bispecific antibody, and its heavy chain and light chain amino acid sequences are shown in SEQ ID NOs: 48 and 49, respectively.

[0210] The results are shown in Figures 2A-2C. The results show that the bispecific antibodies can bind to human PD-1 and monkey PD-1 as well as human CD40 and monkey CD40 with strong affinity.

[0211] Example 3: Bispecific Antibody Cell-Level Binding

[0212] CHO-S cells overexpressing human or cynomolgus monkey CD40 and PD-1 were generated by transfection with the pCHO1.0 vector (Invitrogen) containing human CD40 and PD-1 cDNAs and cynomolgus monkey CD40 and PD-1 cDNAs cloned into MCS. The cell density of the expanded CHO-hCD40 and CHO-hPD-1 cells, CHO-cynoCD40 and CHO-cynoPD-1 cells was adjusted to 2×10 6cells / ml, 100 μL / well was added to each 96-well flow cytometer and centrifuged. The purified bispecific antibody was diluted with PBS, starting at 200 nM and diluted 3-fold for a total of 12 points. 100 μL / well of this diluted sample was added to the 96-well flow cytometer containing cells, incubated at 4°C for 30 minutes, and washed twice with PBS. 100 μL / well of Goat Anti-Human IgG-Fc (PE) (purchased from Abcam, ab98596) diluted 100-fold in PBS was added, incubated at 4°C for 30 minutes, and washed twice with PBS. 100 μL / well of cells was resuspended in PBS, analyzed on a CytoFlex (Bechman) flow cytometer, and the corresponding MFI was calculated. CP-870893 was used as an anti-CD40 positive control antibody; the amino acid sequences of its heavy and light chain variable regions are shown in SEQ ID NOs: 21 and 22, respectively. The CD40 mAb was ADI-55164.

[0213] In the assay experiment using the above method, the experimental results are shown in Figures 3A-3I , which show that the purified samples of the bispecific antibodies shown in Table 2 all had binding activity to CHO-S cells overexpressing human / monkey CD40 or PD-1, while there was no obvious non-specific binding to CHO-S cells that did not express PD-1 and CD40.

[0214] Example 4: Bispecific Antibody Blocks the Binding of CD40 and CD40L

[0215] The cell density of the expanded cultured Raji cells was adjusted to 2×10 6 100 μL / well of the diluted sample was added to a 96-well flow cytometry plate, centrifuged, and then used for subsequent use. The purified bispecific antibody was diluted in PBS, starting at 200 nM and diluted 3-fold for a total of 12 points. 60 μL / well of this diluted sample was added to a 96-well sample dilution plate. Simultaneously, 60 μL / well of biotinylated human CD40L protein (purchased from AcroBiosystems) was added to a final concentration of 500 ng / mL and incubated at 4°C for 30 minutes. 100 μL / well of the co-incubated sample was added to the 96-well flow cytometry plate containing cells, incubated at 4°C for 30 minutes, and washed twice with PBS. 100 μL / well of SA-PE antibody (purchased from Biolegend) diluted 10,000-fold in PBS was added to the plate, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells were resuspended in 100 μL / well of PBS and analyzed on a CytoFlex (Bechman) flow cytometer, and the corresponding MFI was calculated.

[0216] The results, as shown in Figures 4A-4B , show that one of the bispecific antibodies of the present invention (PD1xCD40 (ADI-55147)) blocked the binding of CD40 to CD40L, while the other (PD1xCD40 (ADI-55164)) did not. PD1(pembro)xCD40 (ADI-55164) also failed to block the binding of CD40 to CD40L.

[0217] Example 5: Bispecific Antibodies Blocking PD-L1 and PD-1 Signaling Pathways

[0218] CHO-K1-PD-L1 functional cells were digested and the cell density was adjusted. 100 μl / well was added to a 96-well white-bottom plate and allowed to adhere overnight. The next day, a Jurkat-PD-1 effector cell suspension was prepared and the sample to be tested was serially diluted in reaction medium. The white-bottom plate was removed, the culture supernatant aspirated, and 40 μl / well of the diluted sample was added to the white-bottom plate. Simultaneously, 40 μl / well of the Jurkat-PD-1-luc effector cell suspension was added. The cells were incubated at 37°C, 5% CO2 for 6 hours, during which time the Bio-Glo™ reagent was allowed to return to room temperature. After the incubation period, the cells were removed and equilibrated at room temperature for 5 minutes. 80 μl / well of the Bio-Glo™ reagent was added, and the fluorescence signal was read using a multi-microplate reader. The PD-1 / PD-L1 signaling pathway activity was detected using CHO-K1 hPD-L1 and Jurkat hPD-1NFAT-luciferase fluorescent reporter cells. The results are shown in Figures 5A-5B , which show that the bispecific antibody can block the PD-1 / PD-L1 signaling pathway in vitro, and the blocking activity is comparable to that of the PD-1 monoclonal antibody.

[0219] Example 6: Bispecific Antibodies Activate CD40 Signaling Detection Dependent on PD-1 Expression

[0220] Jurkat cells were co-transfected with a plasmid encoding human CD40 and a plasmid encoding the NF-κB luciferase reporter gene (purchased from Promega) to generate huCD40 Jurkat-NF-κB cells. huCD40 Jurkat-NF-κB cells were expanded and resuspended in 1640 complete medium to a cell count of 2 x 10^6 cells / mL. Antibody was diluted in culture medium, starting at 200 nM and serially diluted three-fold. 50 μL / well of the diluted solution was added to a sterile 96-well white-bottom plate (purchased from Nunc). Target cells were added and incubated at 50 μL / well for 30 minutes. huCD40-Jurkat-NFκB cells containing OKT-3 at a final concentration of 0.1 μg / mL were then added and incubated at 50 μL / well at 37°C in 5% CO2 overnight. Luciferase signal was detected.

[0221] The results, as shown in Figures 6A-6D, showed no significant signal activation in the bispecific antibody sample co-incubated with huCD40 Jurkat-NF-κB cells and CHO-S cells (which do not express PD-1). However, significant signal activation was observed in the co-incubation system of huCD40 Jurkat-NF-κB cells and CHO-S cells overexpressing human PD-1. This is due to the antibody aggregation effect caused by PD-1, which caused the CD40 on Jurkat cells bound by the other end of the antibody to also aggregate, activating the NF-κB pathway.

[0222] Example 7: Bispecific Antibody Activation Activity Detection (Primary B Cell Activation)

[0223] CHO-S cells overexpressing human PD-1 or CHO-S cells not expressing PD-1 were treated with mitomycin for 4 h, and the cell density was adjusted to 2 × 10 6 Cells / ml. Frozen human PBMCs (purchased from Shanghai Saili) were revived and B cells were isolated according to the EasySep human B cell enrichment kit instructions. The cell density was adjusted to 1*10^6 cells / mL using X-ViVOTM15 medium. HuPD-1-CHOS / HuPD-L1-CHOS and CHOS cells were collected (washed three times after treatment with mitomycin) and the cell density was adjusted to 1*10^6 cells / mL using X-ViVOTM15 medium. Antibodies were diluted in X-ViVOTM15 medium, starting at 200nM and serially diluted 3-fold at 50μL / well. Target cells were added at 50μL / well and incubated for 30 minutes. B cells were added at 50μL / well and incubated for 48 hours before detecting CD23 and CD86 expression.

[0224] The experimental results are shown in Figures 7A-7D. The bispecific antibody sample showed no obvious signal activation in the co-incubation system of B cells and CHO-S cells that do not express PD-1, but showed significant B cell activation in the co-incubation system of B cells and CHO-S cells that overexpress human PD-1.

[0225] Example 8: Bispecific Antibody Mixed Lymphocyte Reaction

[0226] PBMC cells (purchased from SAILY BIO, SLB-HPB) were revived and centrifuged, and PBMC were resuspended in 10 ml of X-VIVO-15 medium (purchased from LONZA). The cells were cultured in a cell culture incubator at 37° C. for 2 hours to adhere to the wall, and non-adherent cells were removed by aspiration. Add 10 ml of DC culture medium: X-VIVO-15 medium supplemented with 10 ng / ml GM-CSF (purchased from R&D) and 20 ng / ml IL-4 (purchased from R&D), culture for 3 days, add 5 ml of DC culture medium, continue culture until day 6, add DC maturation medium: X-VIVO-15 medium supplemented with 1000 U / ml TNF-α (purchased from R&D), 10 ng / ml IL-6 (purchased from R&D), 5 ng / ml IL-1β (purchased from R&D), 1 μM PGE2 (purchased from Tocris), culture for 2 days, collect mature DC cells, and adjust the cell density to 2 × 10 5 PBMCs from another donor (purchased from SAILY BIO, SLB-HPB) were revived, centrifuged, and resuspended in 10 ml of X-VIVO-15 medium. T cells were enriched using a T cell isolation kit (purchased from Stemcell) and resuspended in X-VIVO-15 medium to adjust the cell density to 2 × 10 6 Cells were mixed with the mature DCs collected above at a 1:1 ratio, and 100 μl / well was added to a 96-well U-bottom plate. Simultaneously, 100 μl / well of the bispecific antibody sample was diluted with X-VIVO-15 medium. The cells were cultured for 3 days, and the supernatant was collected. IL-2 expression was measured by ELISA (purchased from eBioscience).

[0227] The experimental results are shown in Figures 8A-8B , showing that the bispecific antibody sample can activate T cells to release IL-2 in a mixed lymphocyte reaction system.

[0228] Example 9: Half-life of bispecific antibodies in mice

[0229] Balb / c mice (half male and half female) were housed under a 12 / 12 hour light / dark cycle, maintained at a temperature of 24±2°C and a humidity of 40-70%, with free access to water and food. On the day of the experiment, the mice received a single tail vein injection of the bispecific antibody at a dose of 10 mg / kg. Blood was collected from the mouse orbital cavity at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration. Whole blood samples were incubated at 2-8°C for 30 minutes, and serum was collected by centrifugation at 12,000 rpm for 5 minutes. The serum was then centrifuged at 12,000 rpm for 5 minutes at 2-8°C and stored at -80°C. The serum was assayed for bispecific antibody levels by ELISA. The results are shown in Figures 9A-9C , which show that the half-lives of the bispecific antibody molecules in mice were approximately 133 hours (PD-1xCD40 (blocker), Figure 9A ), 238 hours (PD-1xCD40 (non-blocker), Figure 9B ), and 222 hours (PD-1 (pembro)xCD40 (non-blocker), Figure 9C ), respectively.

[0230] Example 10: In vivo pharmacodynamics study of bispecific antibodies

[0231] 1. In vivo pharmacodynamics study of the bispecific antibody in a subcutaneous inoculation of A375 and human PBMC B-NDG B2M KO Plus mouse tumor model

[0232] This study determined the antitumor activity of the bispecific antibody of the present invention in a B-NDG B2M KO Plus mouse tumor model in which A375 and human PBMC were co-inoculated subcutaneously. Specifically, an A375 tumor-bearing mouse model was established by subcutaneously inoculating A375+PBMC. The tumors were then grown to an average volume of approximately 300 mm. 3 The mice were divided into groups at approximately 12 h and treated with PBS, 7 mg / kg anti-CD40 monoclonal antibody, 7 mg / kg anti-PD-1 monoclonal antibody, 7 mg / kg anti-PD-1 monoclonal antibody combined with 7 mg / kg anti-CD40 monoclonal antibody, and 9.6 mg / kg anti-PD-1 / CD40 bispecific antibody by intraperitoneal injection, respectively. The tumor volume and body weight of the mice in each group were monitored every 2-3 days for 5 consecutive times. The dosage and method of administration are shown in Table 3.

[0233] Table 3: Dosage regimen for in vivo efficacy experiments

[0234] The results are shown in FIG10A , which show that the anti-PD-1 / CD40 bispecific antibody of the present invention has significant anti-tumor activity and is significantly superior to anti-PD-1 monoclonal antibody and the combination of anti-PD-1 monoclonal antibody and anti-CD40 monoclonal antibody.

[0235] 2. In vivo pharmacodynamics study of the bispecific antibody in a C57 mouse tumor model inoculated subcutaneously with B16F10 and human PBMC huPD-1 / CD40 double KI

[0236] First, a B16F10 tumor-bearing mouse model was established by subcutaneous inoculation of B16F10. The mice were divided into groups about seven days later and given intraperitoneal injections of PBS, 7 mg / kg anti-PD-1 monoclonal antibody, 7 mg / kg anti-PD-1 monoclonal antibody combined with 7 mg / kg anti-CD40 monoclonal antibody, 9.1 mg / kg YH008 BMK, and the anti-PD-1 / CD40 bispecific antibody PD1xCD40 (ADI55164) and PD1(Pembro)xCD40 (ADI55164). The tumor volume and body weight of mice in each group were monitored continuously every 2-3 days. The dosage and method of administration are shown in Table 4.

[0237] Table 4: Dosage regimen for in vivo efficacy experiments

[0238] The results are shown in Figure 10B. The bispecific antibodies PD1xCD40 (ADI55164) and PD1 (Pembro) xCD40 (ADI55164) of the present invention have significant anti-tumor activity and are significantly superior to anti-PD-1 monoclonal antibodies, the combination of anti-PD-1 monoclonal antibodies and anti-CD40 monoclonal antibodies, and YH008 BMK.

[0239] 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. A bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to CD40; in, The first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), and the first light chain variable region (VL) and the first heavy chain variable region (VH) together form a domain that can specifically bind to PD-1; the second antigen-binding domain comprises a second light chain variable region (VL) and a second heavy chain variable region (VH), and the second light chain variable region (VL) and the second heavy chain variable region (VH) together form a domain that can specifically bind to CD40.

2. The bispecific antibody according to claim 1, wherein The first antigen binding domain and the second antigen binding domain are each independently scFv or Fab.

3. The bispecific antibody of claim 1 or 2, wherein: (a) the first light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:4; and / or the first heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3; or, (b) the first light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:40; and / or the first heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:

39.

4. The bispecific antibody according to any one of claims 1 to 3, wherein: (a) the first light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18; and / or the first heavy chain variable region (VH) comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15; or, (b) the first light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46; and / or the first heavy chain variable region (VH) comprises HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43; Preferably, the CDRs are defined by the IMGT numbering system.

5. The bispecific antibody according to any one of claims 1 to 4, wherein: (a) the first light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, and / or the first heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof; or, (b) the first light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 40 or a variant thereof, and / or the first heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 39 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 with 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.

6. The bispecific antibody according to any one of claims 1 to 5, wherein: (a) the second light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 23 or 33; and / or the second heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 5 or 31; or, (b) the second light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 24 or 34; and / or the second heavy chain variable region (VH) comprises HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 6 or 32.

7. The bispecific antibody according to any one of claims 1 to 6, wherein: (a) the second light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27; and / or the second heavy chain variable region (VH) comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:7, HCDR2 of the amino acid sequence shown in SEQ ID NO:8, and HCDR3 of the amino acid sequence shown in SEQ ID NO:9; or, (b) the second light chain variable region (VL) comprises LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30; and / or the second heavy chain variable region (VH) comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:10, HCDR2 of the amino acid sequence shown in SEQ ID NO:11, and HCDR3 of the amino acid sequence shown in SEQ ID NO:12; Preferably, the CDRs are defined by the IMGT numbering system.

8. The bispecific antibody according to any one of claims 1 to 7, wherein: (a) the second light chain variable region (VL) comprises the amino acid sequence as shown in SEQ ID NO: 23 or 33 or a variant thereof, and / or the second heavy chain variable region (VH) comprises the amino acid sequence as shown in SEQ ID NO: 5 or 31 or a variant thereof; or, (b) the second light chain variable region (VL) comprises the amino acid sequence as shown in SEQ ID NO: 24 or 34 or a variant thereof, and / or the second heavy chain variable region (VH) comprises the amino acid sequence as shown in SEQ ID NO: 6 or 32 or a variant thereof; 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; preferably, the substitutions are conservative substitutions; For example, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 23, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 5; For example, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 33, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 31; For example, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 24, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 6; For example, the second light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:34, and / or the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:

32.

9. The bispecific antibody according to any one of claims 1 to 8, comprising: (1) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:4, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:3, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:23 or 33, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:5 or 31; (2) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:4, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:3, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:24 or 34, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:6 or 32; (3) a first light protein comprising LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 40 A first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 39, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 23 or 33, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 5 or 31; or, (4) a first light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:40, a first heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:39, a second light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:24 or 34, and a second heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:6 or 32.

10. The bispecific antibody according to any one of claims 1 to 9, comprising: (1) the first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18, the first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:15; the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27, and the second heavy chain variable region (VH) comprising HCDR1 with an amino acid sequence as shown in SEQ ID NO:7, HCDR2 with an amino acid sequence as shown in SEQ ID NO:8, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:9; (2) the first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18, the first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:15; the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30, and the second heavy chain variable region (VH) comprising HCDR1 with the amino acid sequence shown in SEQ ID NO:10, HCDR2 with the amino acid sequence shown in SEQ ID NO:11, and HCDR3 with the amino acid sequence shown in SEQ ID NO:12; (3) a first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46, a first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27, and the second heavy chain variable region (VH) comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and HCDR3 comprising the amino acid sequence of SEQ ID NO:9; or, (4) a first light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:44, LCDR2 as shown in SEQ ID NO:45, and LCDR3 as shown in SEQ ID NO:46, a first heavy chain variable region (VH) comprising HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, the second light chain variable region (VL) comprising LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30, and the second heavy chain variable region (VH) comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:10, HCDR2 comprising the amino acid sequence of SEQ ID NO:11, and HCDR3 comprising the amino acid sequence of SEQ ID NO:12; Preferably, the bispecific antibody comprises: (1) comprising the first light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:4, the first heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:3, the second light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:23, and the second heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:5; (2) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:33, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:31; (3) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:24, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:6; (4) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:4, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:3, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:34, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:32; (5) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:40, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:39, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:23, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:5; (6) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:40, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:39, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:33, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:31; (7) the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:40, the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:39, the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:24, and the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:6; or, (8) comprising the first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:40, comprising the first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:39, comprising the second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:34, and comprising the second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:

32.

11. The bispecific antibody according to any one of claims 1 to 10, wherein The first antigen binding domain is Fab, and the second antigen binding domain is scFv.

12. The bispecific antibody of claim 11, wherein The bispecific antibody comprises peptide chain I and peptide chain II; wherein, peptide chain I comprises the first light chain variable region and the light chain constant region, and peptide chain II comprises: the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region.

13. The bispecific antibody of claim 12, wherein: The peptide chain I comprises the first light chain variable region and the light chain constant region from N-terminus to C-terminus, and / or the peptide chain II comprises from N-terminus to C-terminus: (i) the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region; or, (ii) the first heavy chain variable region, the heavy chain constant region, the second light chain variable region and the second heavy chain variable region.

14. The bispecific antibody according to claim 12 or 13, wherein The adjacent domains of the peptide chain I are optionally connected via a linker or not, and / or the adjacent domains of the peptide chain II are optionally connected via a linker or not; Preferably, the linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); preferably, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S) and / or alanine (A), for example, (GGGGS) n or (GGGGA) n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 36 or 37; Preferably, the peptide chain II comprises the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region from the N-terminus to the C-terminus, wherein the heavy chain constant region and the second heavy chain variable region are linked by a peptide linker comprising one or more glycine (G) and / or alanine (S) (e.g., a peptide linker as shown in SEQ ID NO: 36). or, the peptide chain II comprises, from N-terminus to C-terminus, the first heavy chain variable region, the heavy chain constant region, the second heavy chain variable region and the second light chain variable region, wherein the heavy chain constant region and the second heavy chain variable region are connected by a peptide linker comprising one or more glycine (G) and / or alanine (A) (e.g., a peptide linker as shown in SEQ ID NO: 36); and / or, The second heavy chain variable region and the second light chain variable region are connected via a peptide linker comprising one or more glycine (G) and / or serine (S) (eg, a peptide linker as shown in SEQ ID NO: 37).

15. The bispecific antibody according to any one of claims 11 to 14, wherein The second light chain variable region and the second heavy chain variable region can form an intrachain disulfide bond connection; Preferably, (a) the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31; or, (b) the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34, and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

32.

16. The bispecific antibody according to any one of claims 12 to 15, wherein The heavy chain constant region is derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); Preferably, the heavy chain constant region is selected from the heavy chain constant region of a wild-type human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4) or a variant thereof (e.g., a heavy chain constant region comprising a mutation or chemical modification); wherein, compared to the wild-type immunoglobulin heavy chain constant region from which it is derived, the Fc domain contained in the heavy chain constant region variant has an altered (e.g., enhanced or reduced) effector function; for example, compared to the wild-type immunoglobulin heavy chain constant region from which it is derived, the Fc domain contained in the heavy chain constant region variant has reduced ADCC, ADCP and / or CDC activity; Preferably, the heavy chain constant region is selected from the heavy chain constant region of wild-type human immunoglobulin IgG1 or a variant thereof (e.g., a heavy chain constant region comprising a mutation or chemical modification); wherein, compared to the wild-type immunoglobulin heavy chain constant region from which it is derived, the heavy chain constant region variant comprises L234A, L235A and / or L237A substitution mutations; Preferably, the Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO:

19.

17. The bispecific antibody according to any one of claims 12 to 16, wherein The light chain constant region is derived from a κ or λ light chain of a human immunoglobulin; Preferably, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

20.

18. The bispecific antibody of any one of claims 12 to 17, wherein: (a) the peptide chain I comprises the amino acid sequence as shown in SEQ ID NO: 35 or a variant thereof, and / or the peptide chain II comprises the amino acid sequence as shown in SEQ ID NO: 1 or 2 or a variant thereof; or, (b) the peptide chain I comprises the amino acid sequence as shown in SEQ ID NO:47 or a variant thereof, and / or the peptide chain II comprises the amino acid sequence as shown in SEQ ID NO:38 or a variant thereof; 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; preferably, the substitutions are conservative substitutions; Preferably, the bispecific antibody comprises: (1) a peptide chain I comprising the amino acid sequence shown in SEQ ID NO: 35, and a peptide chain II comprising the amino acid sequence shown in SEQ ID NO: 1; (1) a peptide chain I comprising the amino acid sequence shown in SEQ ID NO: 35, and a peptide chain II comprising the amino acid sequence shown in SEQ ID NO: 2; or (1) A peptide chain I comprising the amino acid sequence shown in SEQ ID NO:47, and a peptide chain II comprising the amino acid sequence shown in SEQ ID NO:

38.

19. The bispecific antibody according to any one of claims 12 to 18, comprising two peptide chains I and two peptide chains II; Preferably, the two peptide chains I comprised in the bispecific antibody are the same or different, and / or the two peptide chains II comprised in the bispecific antibody are the same or different; Preferably, the bispecific antibody comprises the two identical peptide chains I and the two identical peptide chains II.

20. An isolated nucleic acid molecule or a group of nucleic acid molecules comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-19.

21. A vector comprising the isolated nucleic acid molecule or group of nucleic acid molecules according to claim 20; Preferably, the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located in different carrier molecules; Preferably, the vector is a cloning vector or an expression vector.

22. A host cell comprising the isolated nucleic acid molecule or group of nucleic acid molecules according to claim 20, or the vector according to claim 21.

23. A method for preparing the bispecific antibody of any one of claims 1 to 19, comprising culturing the host cell of claim 22 under conditions that allow expression of the bispecific antibody, and recovering the bispecific antibody from the culture of the cultured host cell.

24. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 19, or the isolated nucleic acid molecule or nucleic acid molecule group of claim 20, or the vector of claim 21, or the host cell of claim 22, and a pharmaceutically acceptable carrier and / or excipient.

25. The pharmaceutical composition of claim 24, wherein The pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.

26. Use of the bispecific antibody according to any one of claims 1 to 19, or the isolated nucleic acid molecule or nucleic acid molecule group according to claim 20, or the vector according to claim 21, or the host cell according to claim 22, or the pharmaceutical composition according to claim 24 or 25 in the preparation of a medicament for: (1) increasing immune cell activity in vitro or in vivo in a subject (e.g., a human or monkey); (2) enhancing an immune response in a subject (e.g., a human or a monkey); (3) preventing and / or treating tumors in a subject (e.g., a human or a monkey); and / or, (4) preventing and / or treating infection in a subject (e.g., a human or monkey); Preferably, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells; Preferably, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma; Preferably, the tumor is a blood tumor, such as lymphoma, leukemia; preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; preferably, the non-Hodgkin's lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, NK / T-cell lymphoma (nasal type) with Epstein-Barr virus positivity, and B-cell non-Hodgkin's lymphoma; Preferably, the infection is selected from viral infection, bacterial infection, fungal infection and parasitic infection; Preferably, the subject is a mammal, such as a human or a monkey.

27. The use of claim 26, wherein The bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as simultaneously, separately, or sequentially; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity.

28. 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 bispecific antibody of any one of claims 1 to 19, or the isolated nucleic acid molecule or nucleic acid molecule group of claim 20, or the vector of claim 21, or the host cell of claim 22, or the pharmaceutical composition of claim 24 or 25 to a subject in need thereof; Preferably, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma; Preferably, the tumor is a blood tumor, such as lymphoma, leukemia; preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; preferably, the non-Hodgkin's lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, NK / T-cell lymphoma (nasal type) with Epstein-Barr virus positivity, and B-cell non-Hodgkin's lymphoma; Preferably, the infection is selected from viral infection, bacterial infection, fungal infection and parasitic infection; Preferably, the subject is a mammal, such as a human or a monkey.

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