Multispecific antibodies and uses thereof

Abstract

The present disclosure relates to anti-CLEC5A (C-type lectin domain family 5 member A) antibodies and antigen binding fragments thereof. The present disclosure relates to an antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain (TAA) that specifically binds to a tumor-associated antigen and a second antigen-binding domain that specifically binds to CLEC5A and an Fc region. The present invention also relates to an antibody or an antigen-binding fragment thereof comprising a second antigen-binding domain that specifically binds to an autoimmune disease target and that specifically binds to CLEC5A, and an Fc region.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on PCT application number PCT / CN2023 / 107143 and application date July 13, 2023, PCT application number PCT / CN2024 / 086867 and application date April 9, 2024, CN application number 202311183154.4 and application date September 14, 2023, CN application number 202311436666.7 and application date October 31, 2023, and CN application number 202311532388.5 and application date November 16, 2023, and claims its priority. The contents of all applications are hereby introduced into this application as a whole. Technical Field

[0003] The present invention relates to anti-CLEC5A (C-type lectin domain family 5 member A) antibodies, antigen-binding fragments thereof, antibody-drug conjugates derived therefrom and uses thereof. The present invention also relates to anti-CLEC5A multispecific antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof), and antibody-drug conjugates derived therefrom. Background Art

[0004] Human C-type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-binding lectin-1 (MDL-1), is a type II transmembrane protein expressed exclusively in the myeloid lineage, including macrophages, monocytes, neutrophils, and dendritic cells (DCs). As a pattern recognition receptor, CLEC5A transmits signals into the cytoplasm by non-covalently binding to the adaptor protein DAP12. Phosphorylation of DAP12 subsequently initiates a Syk kinase-based signaling cascade, leading to macrophage activation and the release of chemokines and proinflammatory cytokines, including IL-6, TNF, CCL3, and CXCL8.

[0005] CLEC5A can trigger myeloid cell-related immune responses and is associated with a variety of infections and inflammatory diseases. In flavivirus infections, especially dengue and Japanese encephalitis virus infections, CLEC5A promotes the production of high levels of proinflammatory cytokines and chemokines, and anti-CLEC5A monoclonal antibodies or CLEC5A inhibitors can reverse disease progression, indicating that CLEC5A is a promising therapeutic target for flavivirus infections. In addition, similar to some autoimmune diseases, high levels of CLEC5A are found in active rheumatoid arthritis, and CLEC5A activators increase proinflammatory cytokine levels. CLEC5A is also a key factor in the occurrence and progression of cancer. In high-grade severe ovarian cancer (HGSOC), gastric cancer, and glioma, abnormally high expression of CLEC5A is significantly associated with reduced overall survival.

[0006] Bispecific antibodies are artificial proteins that can bind to two different types of antigens or two different antigenic epitopes at the same time. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of Catumaxomab (anti-EpCAM and anti-CD3) and Blinatumomab (anti-CD19 and anti-CD3) has become an important milestone in the development of bispecific antibodies.

[0007] Because bispecific antibodies have multiple uses, there is a need to continue developing various bispecific antibody-based therapeutic approaches.

[0008] Considering the important role of CLEC5A in the immune system, it is necessary to develop therapeutic agents targeting CLEC5A, especially bispecific antibodies targeting CLEC5A. Summary of the invention

[0009] The present invention relates to anti-CLEC5A antibodies, antigen-binding fragments thereof and uses thereof. The present invention also relates to multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to a tumor-associated antigen (TAA) and human C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the antibody or antigen-binding fragment thereof has an enhanced Fc. In some embodiments, the antibody or antigen-binding fragment thereof has an increased binding affinity to the FcγRIIa receptor and / or the FcγRIIIa receptor. In some embodiments, the antibody or antigen-binding fragment thereof has a non-functional Fc.

[0010] Compared to the reference antibodies used in the present invention (e.g., DX244), the anti-CLEC5A antibodies described herein are not "classic agonists". For example, the anti-CLEC5A antibodies described herein can mediate phagocytosis of macrophages (similar to DX244), but with greater potency and very low levels of cytokine release. In addition, the TAA / CLEC5A bispecific antibodies described herein have been shown to mediate myeloid cell (e.g., monocytes and macrophages) killing of target cells expressing different TAAs at very low E:T ratios and produce very low cytokine release. Without being bound by theory, it is conceivable that the anti-CLEC5A antibodies and TAA / CLEC5A bispecific antibodies described herein can be used to prepare potential myeloid cell engagers with high efficacy and good safety.

[0011] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A (C-type lectin domain family 5 member A), comprising: a heavy chain variable region (VH), comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL), comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; in some embodiments, the selected VH CDR 1, 2 and 3 amino acid sequences and the selected VL CDR 1, 2 and 3 amino acid sequences are one of the following:

[0012] (1) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, and 7, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively;

[0013] (2) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, and 7, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively;

[0014] (3) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, and 17, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively;

[0015] (4) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, and 17, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively;

[0016] (5) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, and 27, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively;

[0017] (6) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, and 27, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively;

[0018] (7) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, and 37, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively;

[0019] (8) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, and 37, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively;

[0020] (9) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively;

[0021] (10) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively;

[0022] (11) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively;

[0023] (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively;

[0024] (13) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively;

[0025] (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively;

[0026] (15) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, 77, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; and

[0027] (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

[0028] In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 3, 5, 7, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 8-10, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 13, 15, 17, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 18-20, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 23, 25, 27, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 28-30, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 33, 35, 37, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 38-40, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 43, 45, 47, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 48-50, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 53, 55, 57, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 58-60, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 63, 65, 67, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 68-70, respectively. In some embodiments, according to the Kabat definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 73, 75, 77, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 78-80, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 4, 6, 7, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 8-10, respectively.In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 14, 16, 17, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 18-20, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 24, 26, 27, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 28-30, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 34, 36, 37, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 38-40, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 44, 46, 47, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 48-50, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 54, 56, 57, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 58-60, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 64, 66, 67, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, 3 listed in SEQ ID NOs: 68-70, respectively. In some embodiments, according to the Chothia definition, VH comprises the amino acid sequences of CDRs 1, 2, and 3 listed in SEQ ID NOs: 74, 76, and 77, respectively, and VL comprises the amino acid sequences of CDRs 1, 2, and 3 listed in SEQ ID NOs: 78-80, respectively.

[0029] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to a selected VL sequence. In some embodiments, the selected VH sequence and the selected VL sequence are one of the following:

[0030] (1) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;

[0031] (2) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22;

[0032] (3) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32;

[0033] (4) the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42;

[0034] (5) the selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52;

[0035] (6) the selected VH sequence is SEQ ID NO: 61, and the selected VL sequence is SEQ ID NO: 62;

[0036] (7) the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72;

[0037] (8) the selected VH sequence is SEQ ID NO: 81, and the selected VL sequence is SEQ ID NO: 82;

[0038] (9) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84;

[0039] (10) the selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86;

[0040] (11) the selected VH sequence is SEQ ID NO: 87, and the selected VL sequence is SEQ ID NO: 88;

[0041] (12) the selected VH sequence is SEQ ID NO: 89, and the selected VL sequence is SEQ ID NO: 90; and

[0042] (13) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92.

[0043] In some embodiments, the VH comprises the sequence of SEQ ID NO: 11 and the VL comprises the sequence of SEQ ID NO: 12. In some embodiments, the VH comprises the sequence of SEQ ID NO: 21 and the VL comprises the sequence of SEQ ID NO: 22. In some embodiments, the VH comprises the sequence of SEQ ID NO: 31 and the VL comprises the sequence of SEQ ID NO: 32. In some embodiments, the VH comprises the sequence of SEQ ID NO: 41 and the VL comprises the sequence of SEQ ID NO: 42. In some embodiments, the VH comprises the sequence of SEQ ID NO: 51 and the VL comprises the sequence of SEQ ID NO: 52. In some embodiments, the VH comprises the sequence of SEQ ID NO: 61 and the VL comprises the sequence of SEQ ID NO: 62. In some embodiments, the VH comprises the sequence of SEQ ID NO: 71 and the VL comprises the sequence of SEQ ID NO: 72. In some embodiments, the VH comprises the sequence of SEQ ID NO: 81 and the VL comprises the sequence of SEQ ID NO: 82. In some embodiments, the VH comprises the sequence of SEQ ID NO: 83 and the VL comprises the sequence of SEQ ID NO: 84. In some embodiments, the VH comprises the sequence of SEQ ID NO: 85 and the VL comprises the sequence of SEQ ID NO: 86. In some embodiments, the VH comprises the sequence of SEQ ID NO: 87 and the VL comprises the sequence of SEQ ID NO: 88. In some embodiments, the VH comprises the sequence of SEQ ID NO: 89 and the VL comprises the sequence of SEQ ID NO: 90. In some embodiments, the VH comprises the sequence of SEQ ID NO: 91 and the VL comprises the sequence of SEQ ID NO: 92.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds human, mouse or monkey CLEC5A.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof can mediate phagocytosis of macrophages (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% target cell killing compared to a reference antibody (e.g., DX244).

[0046] In some embodiments, the antibody or antigen-binding fragment thereof can induce low levels of cytokine (e.g., IL-6 or TNFα) release (e.g., less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to cytokine release induced by a reference antibody (e.g., DX244).

[0047] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A. In some embodiments, the antibody or antigen-binding fragment thereof can mediate phagocytosis of macrophages. In some embodiments, the antibody or antigen-binding fragment thereof can mediate phagocytosis of macrophages (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% target cell killing effect compared to a reference antibody (e.g., DX244), and / or in some embodiments, the antibody or antigen-binding fragment thereof can induce low levels of cytokine (e.g., IL-6 or TNFα) release (e.g., less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%).

[0048] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof comprising:

[0049] i) a first antigen binding domain that specifically binds to a first antigen, in some embodiments, the first antigen is a tumor associated antigen (TAA); and

[0050] ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A).

[0051] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof comprising:

[0052] i) a first antigen binding domain that specifically binds to a first antigen, which in some embodiments is an autoimmune disease target; and

[0053] ii) a second antigen binding domain that specifically binds to CLEC5A.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof has one or more of the following effects:

[0055] i) the antibody or antigen-binding fragment thereof can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages (e.g., M0, M1 and / or M2 macrophages));

[0056] ii) the antibody or antigen-binding fragment thereof can mediate killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T (effector cell:target cell) ratio, optionally, in some embodiments, the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1, or less than 10:1); and

[0057] iii) the antibody or antigen-binding fragment thereof can mediate low levels of cytokine (e.g., IL-6 or TNFα) release by myeloid cells (e.g., monocytes and / or macrophages), for example, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the level induced by a reference antibody.

[0058] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2). In some embodiments, the second antigen binding domain is a single-chain fragment variable (scFv) domain, and in some embodiments, VH2 and VL2 are connected by a first linker. In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain by a second linker. In some embodiments, the antibodies or antigen binding fragments thereof described herein further comprise an Fc region. In some embodiments, the C-terminus of the VH1 of the first antigen binding domain is connected to the Fc region, optionally through the CH1 domain. In some embodiments, the C-terminus of the VH1 of the first antigen binding domain is connected to the N-terminus of the Fc region, and the N-terminus of the second antigen binding domain is connected to the C-terminus of the Fc region. In some embodiments, the antibody comprises a first heavy chain, the heavy chain comprising VH1; a first light chain, the light chain comprising VL1; a second heavy chain, the heavy chain comprising VH2; a second light chain, the light chain comprising VL2. In some embodiments, the first heavy chain comprises one or more knob mutations; the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations; the second heavy chain comprises one or more knob mutations.

[0059] In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1. In some embodiments, the Fc region comprises one or more of the following amino acid residues (all numberings are according to EU numbering):

[0060] (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332;

[0061] (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid (E) at position 332;

[0062] (3) Alanine (A) at position 236;

[0063] (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid (E) at position 332;

[0064] (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332;

[0065] (6) aspartic acid (D) at position 293; leucine (L) at position 330 and glutamic acid (E) at position 332; and

[0066] (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329;

[0067] Optionally, the Fc region is aglycosylated.

[0068] In some embodiments, the tumor-associated antigen (TAA) is HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycocoll, globulinyl-calcium amide (GB3Cer / CD77), gangliosides (GD2, GD3 and GM2), B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD19 , WT-L, B7H3, TEM8, CD22, LI-CAM, ROR-I, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cells, CA242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-I receptor, IGF-I, IgGI, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1, MUC1, mucin Canag, N-glycosylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R A, TWEAK, phosphatidylserine, prostate cancer cells, Rankl, Ron, SCH 900105, SDC1, SLAMF7, TAG-72, Tenascin C, TGF B Beta 2. TGF-B, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or Vimentin.

[0069] In some embodiments, the target of autoimmune disease is CD79b, CD38, ACHE, BAFF, BTK, CCL2, CD19, CD20, CD25, CD40, CD52, CD80, CD86, ETAR, ETBR, FCGRT, GM-CSF, JAK1, IFNAR, IFNB1, IFNG, IgE, IgG Fc, IL1A, IL1B, IL-2, IL-4, IL-5, IL-6, IL6R, IL7, IL-12, IL-13, IL-17, IL-18, IL-21, IL-22, IL-23, Integrin, ITG-A4B1, ITG-A4B7, ITG-AVB6, TL1A, TNF-α, TNF-β, TNFSF13B, TSLP, TYK2, VEGFR.

[0070] In some embodiments, the TAA is HER2.

[0071] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 100, 102, 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105-107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0072] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 101, 103, 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105-107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences and the selected VL2 CDR 1, 2, 3 amino acid sequences are selected from one of the sequences described herein.

[0073] In some embodiments, the TAA or autoimmune disease target is CD79b. In some embodiments,

[0074] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 200, 202, 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205-207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0075] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 201, 203, 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205-207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein.

[0076] In some embodiments, TAA is EGFR. In some embodiments,

[0077] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 126, 128, 130, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 131-133, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein;

[0078] (2) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 127, 129, 130, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 131-133, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein;

[0079] (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 136, 138, 140, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 141-143, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0080] (4) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 137, 139, 140, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 141-143, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences and the selected VL2 CDR 1, 2, 3 amino acid sequences are selected from one of the sequences described herein.

[0081] In some embodiments, the TAA is EpCAM. In some embodiments,

[0082] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 150, 152, 154, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 155-157, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0083] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 151, 153, 154, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 155-157, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein.

[0084] In some embodiments, the TAA or autoimmune disease target is GPRC5D.

[0085] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 164, 166, 168, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 169-171, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0086] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 165, 167, 168, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 169-171, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein.

[0087] In some embodiments, the TAA or autoimmune disease target is BCMA.

[0088] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 176, 178, 180, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 181-183, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0089] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 177, 179, 180, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 181-183, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein.

[0090] In some embodiments, the TAA or autoimmune disease target is CD38. In some embodiments,

[0091] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 188, 190, 192, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 193-195, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein; or

[0092] (2) The selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 189, 191, 192, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 193-195, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequence and the selected VL2 CDR 1, 2, 3 amino acid sequence are selected from one of the sequences described herein.

[0093] In one aspect, the invention relates to antibodies or antigen-binding fragments that cross-compete with the antibodies or antigen-binding fragments described herein.

[0094] In one aspect, the present disclosure relates to nucleic acids comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein.

[0095] In one aspect, the present disclosure relates to a vector comprising a nucleic acid described herein. In one aspect, the present disclosure relates to a cell comprising a vector described herein.

[0096] In one aspect, the present disclosure relates to a method of reducing the growth rate of a tumor, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0097] In one aspect, the present invention relates to a method of killing tumor cells, comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0098] In one aspect, the present disclosure relates to a method of increasing an immune response in a subject, comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0099] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0100] In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer and / or liver cancer. In some embodiments, the cancer is multiple myeloma, B cell lymphoma, diffuse large B cell lymphoma, acute B cell leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma.

[0101] In some embodiments, the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, and / or an anti-PD-L1 antibody.

[0102] In one aspect, the present invention relates to a method for treating a subject with an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or its antigen-binding fragment described herein. In certain embodiments, the autoimmune disease is selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related diseases, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Gao'an disease, cold agglutinin disease, warm autoimmune hemolytic anemia and anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) and microscopic polyangiitis (MPA). In certain embodiments, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus and / or rheumatoid arthritis.

[0103] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0104] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by amino acids from a single polypeptide, or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides) that are capable of specifically binding to one or more different antigens (e.g., effector antigens or tumor antigens). In some examples, the antigen binding domain can bind to an antigen or epitope with similar specificity and affinity as a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or fragment thereof. In some embodiments, the antigen binding domain is formed by VH-VL. In some embodiments, the antigen binding domain can include an alternative scaffold. In some embodiments, the antigen binding domain is VHH. In some embodiments, the antigen binding domain may include the non-limiting examples of antigen binding domains described herein. In some examples, the antigen binding domain can bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0105] As used herein, the term "antibody" refers to any antigen binding molecule that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs of an immunoglobulin light chain or any one of the three CDRs of an immunoglobulin heavy chain) and can specifically bind to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may include the Fc region of a human antibody. The term "antibody" also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0106] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen binding fragment comprises at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of a light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0107] As used herein, the term "multispecific antibody" is an antibody comprising two or more different antigen binding domains, which specifically bind to two or more different epitopes together. Two or more different epitopes can be epitopes on the same antigen (e.g., a single polypeptide present on the cell surface), or epitopes on different antigens (e.g., different proteins present on the same cell surface or on different cell surfaces). In some aspects, a multispecific antibody binds to two different epitopes (i.e., "bispecific antibodies"). In some aspects, a multispecific antibody binds to three different epitopes (i.e., "trispecific antibodies"). In some aspects, a multispecific antibody binds to four different epitopes (i.e., "tetraspecific antibodies"). In some aspects, a multispecific antibody binds to five different epitopes (i.e., "pentaspecific antibodies"). Each binding specificity can exist in any suitable valence state. Non-limiting examples of multispecific antibodies are described herein.

[0108] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be located on the same antigen or on different antigens.

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0110] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1A Shown is the gating strategy used to assess immune cell subsets in human PBMCs.

[0112] Figure 1B Shown is the expression of CLEC5A by immune cell subsets in human PBMCs.

[0113] Figure 2A Shown is the gating strategy for assessing immune cell subsets in human solid tumors.

[0114] Figure 2B-2C CLEC5A expression in tumor-associated myeloid cells was shown.

[0115] Figure 3A The gating strategy used to characterize non-neutrophil myeloid cells is shown.

[0116] Figure 3B Demonstrating the characteristics of tumor-associated macrophages in human solid tumors.

[0117] Figure 3C-3D Shown is the expression of CLEC5A in tumor-associated macrophages in human solid tumors.

[0118] Figure 4 The binding affinity of chimeric anti-CLEC5A antibodies to human macrophages is shown.

[0119] Figure 5 Showing M1 macrophage-mediated TNFα release in culture plates coated with chimeric anti-CLEC5A antibody.

[0120] Figure 6 The binding affinity of the chimeric anti-CLEC5A antibody to mouse CLEC5A is shown.

[0121] Figure 7A-7B The BLI binding results of chimeric anti-CLEC5A antibodies to human CLEC5A are shown.

[0122] Figures 8A-8E Binding of humanized anti-CLEC5A antibodies to human CLEC5A as determined by ELISA is shown.

[0123] Figures 9A-9F Schematic diagram showing an example of a TAA / CLEC5A bispecific antibody.

[0124] Fig.10 Binding of the HER2 / CLEC5A bispecific antibody to human macrophages is shown.

[0125] Fig.11 Binding of the TAA / CLEC5A bispecific antibody to human macrophages is shown.

[0126] Fig.12 Binding of the CD79b / CLEC5A bispecific antibody to human macrophages is shown.

[0127] Fig.13 Binding of the TAA / CLEC5A bispecific antibody to human CLEC5A is shown.

[0128] Fig.14 Binding of the HER2 / CLEC5A bispecific antibody to human HER2 is shown.

[0129] Fig.15 Binding of the EGFR / CLEC5A bispecific antibody to human EGFR is shown.

[0130] Fig.16Binding of EpCAM / CLEC5A bispecific antibody to DLD-1 cells is shown.

[0131] Fig.17 Binding of the CD79b / CLEC5A bispecific antibody to Ramos cells is shown.

[0132] Figures 18A-18C It was shown that the HER2 / CLEC5A bispecific antibody mediated the killing of SK-BR-3 cells by M0 macrophages (E:T ratio of 5:1).

[0133] Fig.19 The HER2 / CLEC5AFc bispecific antibody was shown to mediate the killing of SK-BR-3 cells by both M1 and M2 macrophages (E:T ratio of 5:1).

[0134] Fig. 20 The release of INF-γ was shown when HER2 / CLEC5A antibody mediated the killing of SK-BR-3 cells by M1 and M2 macrophages (E:T ratio of 5:1).

[0135] Figures 21A-21C Shown in culture medium ( Fig.21A ),plasma( Fig.21B ) or hIgG1( Fig. 21C ), HER2 / CLEC5A bispecific antibody mediated the killing of SK-BR-3 cells by M0 macrophages.

[0136] Fig. 22 It was shown that EGFR / CLEC5A bispecific antibody mediated the killing of DLD-1 cells by M0 macrophages.

[0137] Fig.23 It was shown that EpCAM / CLEC5A bispecific antibody mediated the killing of DLD-1 cells by M0 macrophages.

[0138] Fig.24 Shown are the expression levels of EpCAM in different tumor cell lines.

[0139] Figures 25A-25F It was shown that EpCAM / CLEC5A bispecific antibody mediated the killing of cancer cells by M0 macrophages.

[0140] Fig.26 Different myeloid cell engagers were shown to mediate killing of multiple myeloma cancer cells by PBMCs.

[0141] Fig. 27 CD79b expression levels in malignant lymphoma cell lines and B cells from healthy PBMC donors are shown.

[0142] Figures 28A-28C It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (experiment 1).

[0143] Figures 29A-29B It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (experiment 2).

[0144] Figures 30A-30C It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (experiment 3).

[0145] Figures 31A-31B The IL-6 level is shown when CD79b / CLEC5A bispecific antibody mediates the killing of Ramos cells.

[0146] Fig.32 The TNFα level is shown when CD79b / CLEC5A bispecific antibody mediates killing of Ramos cells.

[0147] Figures 33A-33C It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (experiment 1).

[0148] Figures 34A-34D It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (experiment 2).

[0149] Figures 35A-35B It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (experiment 3).

[0150] Fig.36 It was shown that CD79b / CLEC5A bispecific antibody mediated the release of TNFα when PBMCs killed endogenous B cells.

[0151] Figures 37A-37F The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M0 macrophages (experiment 1).

[0152] Figures 38A-38D The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines during the killing of Ramos cells by M0 macrophages (experiment 1).

[0153] Figures 39A-39B The CD79b / CLEC5A bispecific antibody was shown to mediate killing of Ramos cells by M0 macrophages at different E:T ratios (experiment 2).

[0154] Figures 40A-40B The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines when M0 macrophages killed Romas cells (experiment 2).

[0155] Figures 41A-41C The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M0 macrophages (experiment 3).

[0156] Fig.42 The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines when M0 macrophages killed Ramos cells (experiment 3).

[0157] Fig.43 The survival rate of M0 macrophages was shown when CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M0 macrophages (experiment 3).

[0158] Figures 44A-44B The CD79b / CLEC5A bispecific antibody was shown to mediate killing of Daudi cells by M0 macrophages at different E:T ratios (experiment 1).

[0159] Figures 45A-45B The CD79b / CLEC5A bispecific antibody was shown to mediate killing of Daudi cells by M0 macrophages at different E:T ratios (experiment 2).

[0160] Figures 46A-46D It was shown that CD79b / CLEC5A bispecific antibody mediated cytokine release during the killing of Daudi cells by M0 macrophages.

[0161] Figures 47A-47B The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M1 macrophages (experiment 1).

[0162] Figures 48A-48B The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines during the killing of Ramos cells by M1 macrophages (experiment 1).

[0163] Fig.49 The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M1 macrophages (experiment 2).

[0164] Fig.50 The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines when M1 macrophages killed Ramos cells (experiment 2).

[0165] Fig.51The survival rate of M1 macrophages was shown when CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M1 macrophages (experiment 2).

[0166] Fig.52 It was shown that CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M1 macrophages.

[0167] Fig.53 It was shown that CD79b / CLEC5A bispecific antibody mediated the release of cytokines when M1 macrophages killed Daudi cells.

[0168] Figures 54A-54D The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M2 macrophages (experiment 1).

[0169] Figures 55A-55D The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines when M2 macrophages killed Ramos cells (experiment 1).

[0170] Fig.56 The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by M2 macrophages (experiment 2).

[0171] Fig.57 The CD79b / CLEC5A bispecific antibody was shown to mediate the release of cytokines when M2 macrophages killed Ramos cells (experiment 2).

[0172] Fig.58 The survival rate of M2 macrophages was shown when CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M2 macrophages (experiment 2).

[0173] Fig.59 It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M2 macrophages at different E:T ratios.

[0174] Fig.60 It was shown that CD79b / CLEC5A bispecific antibody mediated the release of cytokines when M2 macrophages killed Daudi cells.

[0175] Figures 61A-61B It was shown that CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by monocytes.

[0176] Figures 62A-62B It was shown that CD79b / CLEC5A bispecific antibody mediated the release of cytokines when monocytes killed Ramos cells.

[0177] Figures 63A-63B The survival rate of monocytes was shown when CD79b / CLEC5A bispecific antibody mediated monocyte killing of Ramos cells.

[0178] Figures 64A-64B It was shown that the CD79b / CLEC5A bispecific antibody mediated the killing of B cells by monocytes.

[0179] Figures 65A-65B It was shown that CD79b / CLEC5A bispecific antibody mediated the release of cytokines during monocyte killing of B cells.

[0180] Figures 66A-66B The survival rate of monocytes was shown when CD79b / CLEC5A bispecific antibody mediated monocyte killing of B cells. DETAILED DESCRIPTION

[0181] Detailed Description

[0182] A multispecific antibody or an antigen-binding fragment thereof is an artificial protein that can bind to two or more different epitopes simultaneously (e.g., bind to two different antigens). In some embodiments, the multispecific antibody is a bispecific antibody. A bispecific antibody or an antigen-binding fragment thereof can have two arms. Each arm can have a heavy chain variable region and a light chain variable region to form an antigen-binding domain (or antigen-binding region). The two arms can bind to two different antigens. In some embodiments, an additional antigen-binding domain can be added to a monoclonal antibody (e.g., added to the C-terminus of a light chain or a heavy chain).

[0183] The present invention provides several anti-CLEC5A antibodies, antigen-binding fragments thereof, and methods of using these anti-CLEC5A antibodies and antigen-binding fragments to inhibit tumor growth, treat cancer, and treat autoimmune diseases.

[0184] The present invention relates to a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof, which comprises a first antigen-binding domain that specifically binds to a tumor-associated antigen and a second antigen-binding domain that specifically binds to CLEC5A. Human C-type lectin domain family 5 member A (CLEC5A)

[0185] Human C-type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-binding lectin-1 (MDL-1), is a type II transmembrane protein. C-type lectins are characterized by a common structural C-type lectin domain that can bind to Ca 2+ Binds glycans and non-glycan ligands in a Ca-independent manner. 2+CTLDs that bind glycans in a protein-dependent manner are called “carbohydrate recognition domains” (CRDs). The myeloid C-type lectin CLEC5A is a spleen tyrosine kinase (Syk)-coupled type II membrane protein that contains a C-terminal CTLD and a short N-terminal cytoplasmic domain. Among the 15 groups of C-type lectins, CLEC5A belongs to group V (NK cell receptor family), which includes CLEC7A, CLEC5A, CLEC2, CLEC1, NK receptors (e.g., NKG2D, NKRP1 family, NKG2 family, CD69 and CD94), mast cell-associated functional antigen (MAFA), osteoclast inhibitory lectin, and CD72. Similar to NKG2D, CLEC5A signals through the ITAM-containing DNAX-activating protein 12 (DAP12) when phosphorylated by Src upon activation.

[0186] Human CLEC5A mRNA encodes a 165-residue polypeptide with an N-terminal signal peptide (aa1–22), followed by a short intracellular cytoplasmic domain (aa23–56), a transmembrane domain (aa57–70), and an extracellular domain (aa71–165). The transmembrane domain contains a positively charged amino acid Lys-58, which recruits DAP10 and DAP12 to bind to CLEC5A upon activation. CLEC5A is primarily expressed in myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells, and is further upregulated by interferon-γ (IFN-γ). In addition, CLEC5A expression is controlled by the PU.1 transcription factor, a central regulator of myeloid cell differentiation. CLEC5A expression is upregulated by nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting that CLEC5A is regulated by oxidative stress.

[0187] X-ray crystal structures revealed that CLEC5A is a homodimeric protein when bound to dengue virus serotypes. In addition, the CLEC5A crystal structure showed conformational flexibility, indicating that CLEC5A can adopt multiple conformations in vivo and that its conformation is ligand-dependent.

[0188] NK receptors recognize stress-related self-antigens and are essential for immune surveillance, while group V spleen tyrosine kinase-coupled C-type lectins in myeloid cells recognize a variety of exogenous and endogenous antigens and are involved in host defense, sterile inflammation, platelet activation, and development. Studies have shown that CLEC5A can interact with glycan moieties on dengue virus (DV), Japanese encephalitis virus (JEV), and influenza A virus (IAV). In addition, CLEC5A has been found to interact with N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid disaccharide (MurNAc) on Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus). In addition, CLEC5A plays a key role in the inflammatory response associated with collagen-induced rheumatoid arthritis and concanavalin A-induced liver inflammation. CLEC5A also interacts with exosomes released by activated platelets.

[0189] For a detailed review of CLEC5A and its functions, see Sung, Pei-Shan, Wei-Chiao Chang, and Shie-Liang Hsieh. "CLEC5A: a promiscuous pattern recognition receptor to microbes and beyond" Lectin in Host Defense Against Microbial Infections (2020): 57-73; Chen, Rui, et al., "A pan-cancer analysis reveals CLEC5A as a biomarker for cancer immunity and progNOis" Frontiers in Immunology 13 (2022): 831542; and Wang, Quhui, et al., "CLEC5A promotes the proliferation of gastric cancer cells by activating the PI3K / AKT / mTOR pathway" Biochemical and Biophysical Research Communications 524.3 (2020): 656-662; each of which is incorporated herein by reference in its entirety. Anti-CLEC5A antibodies and antigen-binding fragments

[0190] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to CLEC5A (e.g., human CLEC5A). The antibodies and antigen-binding fragments described herein are capable of binding to CLEC5A. These antibodies can be agonists or antagonists of CLEC5A-mediated signaling. In some embodiments, the antibodies and antigen-binding fragments can bind to the extracellular domain of human CLEC5A.

[0191] The present invention provides, for example, anti-CLEC5A antibodies 6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6, 9E11, chimeric antibodies thereof, and humanized antibodies thereof.

[0192] The CDR sequences of 3A7 and 3A7-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 3, 5, 7 according to the Kabat definition, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 8-10. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 4, 6, 7, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 8-10.

[0193] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 13, 15, 17 and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 18-20 according to the Kabat definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16, 17 and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18-20.

[0194] The CDR sequences of 6A5 and 6A5-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 23, 25, 27 according to the Kabat definition, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 28-30. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 24, 26, 27, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 28-30.

[0195] The CDR sequences of 6G9 and 6G9-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 33, 35, 37 according to the Kabat definition, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 38-40. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 34, 36, 37, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 38-40.

[0196] The CDR sequences of 7G10 and 7G10-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 43, 45, 47 according to the Kabat definition, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 48-50. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 44, 46, 47, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 48-50.

[0197] The CDR sequences of 9E11 and 9E11-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 53, 55, 57 according to the Kabat definition, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 58-60. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 54, 56, 57, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 58-60.

[0198] The CDR sequences of 13E6 and 13E6-derived antibodies include the CDR sequences of the heavy chain variable domain according to the Kabat definition as listed in SEQ ID NOs: 63, 65, 67, and the CDR sequences of the light chain variable domain as listed in SEQ ID NOs: 68-70. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 64, 66, 67, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 68-70.

[0199] The CDR sequences of 14A2 and 14A2-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 73, 75, 77 and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 78-80 according to the Kabat definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 74, 76, 77 and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 78-80.

[0200] The amino acid sequence of the heavy chain variable region of the 3A7 antibody is set forth in SEQ ID NO:11. The amino acid sequence of the light chain variable region of the 3A7 antibody is set forth in SEQ ID NO:12.

[0201] The amino acid sequence of the heavy chain variable region of the 5C7 antibody is listed in SEQ ID NO:21. The amino acid sequence of the light chain variable region of the 5C7 antibody is listed in SEQ ID NO:22.

[0202] The amino acid sequence of the heavy chain variable region of the 6A5 antibody is listed in SEQ ID NO:31. The amino acid sequence of the light chain variable region of the 6A5 antibody is listed in SEQ ID NO:32.

[0203] The amino acid sequence of the heavy chain variable region of the 6G9 antibody is listed in SEQ ID NO:41. The amino acid sequence of the light chain variable region of the 6G9 antibody is listed in SEQ ID NO:42.

[0204] The amino acid sequence of the heavy chain variable region of the 7G10 antibody is set forth in SEQ ID NO:51. The amino acid sequence of the light chain variable region of the 7G10 antibody is set forth in SEQ ID NO:51.

[0205] The amino acid sequence of the heavy chain variable region of the 9E11 antibody is listed in SEQ ID NO:61. The amino acid sequence of the light chain variable region of the 9E11 antibody is listed in SEQ ID NO:62.

[0206] The amino acid sequence of the heavy chain variable region of the 13E6 antibody is listed in SEQ ID NO:71. The amino acid sequence of the light chain variable region of the 13E6 antibody is listed in SEQ ID NO:72.

[0207] The amino acid sequence of the heavy chain variable region of the 14A2 antibody is listed in SEQ ID NO:81. The amino acid sequence of the light chain variable region of the 14A2 antibody is listed in SEQ ID NO:82.

[0208] Humanization percentage refers to the percentage identity of heavy chain or light chain variable region sequence and human antibody sequence in the International Immunogenetics Information System (IMGT) database. In certain embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. A detailed description of how to determine humanization percentage and how to determine hot matches is known in the art, and is described in, for example, Jones, et al. "The INNs and outs of antibody nonproprietary names." MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. Higher humanization percentages generally have a variety of advantages, for example, safer, more effective, more easily tolerated by human subjects, and / or less likely to produce side effects. In some embodiments, the variable region is fully human, e.g., derived from a human heavy chain immunoglobulin locus sequence (e.g., a recombination of human IGHV, human IGHD, and human IGHJ genes) and / or a human kappa chain immunoglobulin locus sequence (e.g., a recombination of human IGKV and human IGKJ genes).

[0209] The amino acid sequence of the heavy chain variable region of the humanized 3A7 antibody is listed in SEQ ID NO: 83. The amino acid sequence of the light chain variable region of the humanized 3A7 antibody is listed in SEQ ID NO: 84.

[0210] The amino acid sequence of the heavy chain variable region of the humanized 5C7 antibody is listed in SEQ ID NO: 85. The amino acid sequence of the light chain variable region of the humanized 5C7 antibody is listed in SEQ ID NO: 86.

[0211] The amino acid sequence of the heavy chain variable region of the humanized 6A5 antibody is listed in SEQ ID NO: 87. The amino acid sequence of the light chain variable region of the humanized 6A5 antibody is listed in SEQ ID NO: 88.

[0212] The amino acid sequence of the heavy chain variable region of the humanized 7G10 antibody is listed in SEQ ID NO: 89. The amino acid sequence of the light chain variable region of the humanized 7G10 antibody is listed in SEQ ID NO: 90.

[0213] The amino acid sequence of the heavy chain variable region of the humanized 13E6 antibody is listed in SEQ ID NO: 91. The amino acid sequence of the light chain variable region of the humanized 13E6 antibody is listed in SEQ ID NO: 92.

[0214] Also provided are amino acid sequences of the heavy chain variable region and the light chain variable region of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 83, 85, 87, 89, and 91. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 84, 86, 88, 90, 91, and 92. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence to jointly bind to CLEC5A.

[0215] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain 1, 2 or 3 heavy chain variable region CDRs selected from the following groups: SEQ ID NO: 3, 5, 7, SEQ ID NO: 4, 6, 7, SEQ ID NO: 13, 15, 17, SEQ ID NO: 14, 16, 17, SEQ ID NO: 23, 25, 27, SEQ ID NO: 24, 26, 27, SEQ ID NO: 33, 35, 37, SEQ ID NO: 34, 36, 37, SEQ ID NO: 43, 45, 47, SEQ ID NO: 44, 46, 47, SEQ ID NO: 53, 55, 57, SEQ ID NO: 54, 56, 57, SEQ ID NO: 63, 65, 67, SEQ ID NO: 64, 66, 67, SEQ ID NO: 73, 75, 77 and SEQ ID NO: 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 NO: 74, 76, 77; and / or contains 1, 2 or 3 light chain variable region CDRs selected from the following groups: SEQ ID NO: 8-10, SEQ ID NO: 18-20, SEQ ID NO: 28-30, SEQ ID NO: 38-40, SEQ ID NO: 48-50, SEQ ID NO: 58-60, SEQ ID NO: 68-70 and SEQ ID NO: 78-80.

[0216] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) with CDR 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR 1, 2, 3 amino acid sequences and the selected VL CDR 1, 2, 3 amino acid sequences are shown in Table 22.

[0217] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0218] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0219] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions or substitutions.

[0220] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 14 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 16 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0221] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions or substitutions.

[0222] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions or substitutions.

[0223] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 37 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0224] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 37 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0225] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions or substitutions.

[0226] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions or substitutions.

[0227] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 53 having zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 55 having zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 57 having zero, one or two amino acid insertions, deletions or substitutions.

[0228] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions or substitutions.

[0229] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 65 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions or substitutions.

[0230] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 64 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 66 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions or substitutions.

[0231] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 73 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 75 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 77 with zero, one or two amino acid insertions, deletions or substitutions.

[0232] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 74 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 76 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 77 with zero, one or two amino acid insertions, deletions or substitutions.

[0233] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions or substitutions.

[0234] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions or substitutions.

[0235] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions or substitutions.

[0236] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions or substitutions.

[0237] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions or substitutions.

[0238] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions or substitutions.

[0239] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 68 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 69 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 70 with zero, one or two amino acid insertions, deletions or substitutions.

[0240] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the following CDRs: SEQ ID NO: 78 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 79 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO: 80 with zero, one or two amino acid insertions, deletions or substitutions.

[0241] Insertions, deletions and substitutions can be performed within the CDR sequence, or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat definition. In some embodiments, the CDR is determined based on the Chothia definition. In some embodiments, the CDR is determined based on a combination of the Kabat definition and the Chothia definition.

[0242] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the CDR1 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises, or consists of, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence.

[0243] The present invention also provides an antibody or antigen-binding fragment thereof that binds to CLEC5A. The antibody or antigen-binding fragment thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12. In some embodiments, the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22. In some embodiments, the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32. In some embodiments, the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42. In some embodiments, the selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52. In some embodiments, the selected VH sequence is SEQ ID NO: 61 and the selected VL sequence is SEQ ID NO: 62. In some embodiments, the selected VH sequence is SEQ ID NO: 71 and the selected VL sequence is SEQ ID NO: 72. In some embodiments, the selected VH sequence is SEQ ID NO: 81 and the selected VL sequence is SEQ ID NO: 82. In some embodiments, the selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 84. In some embodiments, the selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 86. In some embodiments, the selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 88. In some embodiments, the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 90. In some embodiments, the selected VH sequence is SEQ ID NO: 91 and the selected VL sequence is SEQ ID NO: 92.

[0244] The present invention also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In certain aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

[0245] The present invention also provides antibodies or antigen binding fragments thereof that cross-compete with any antibody or antigen binding fragment described herein. Cross-competition assays are known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1gp120exterior envelope glycoprotein." Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present invention also provides antibodies or antigen binding fragments thereof that bind to the same epitope or region as any antibody or antigen binding fragment described herein. Epitope sorting assays are known in the art and are described, for example, in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which are incorporated herein by reference in its entirety.

[0246] The present invention also provides nucleic acids comprising polynucleotides encoding polypeptides comprising heavy immunoglobulin chains or light immunoglobulin chains. The heavy immunoglobulin chains or light immunoglobulin chains comprise CDRs or have sequences as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CLEC5A (e.g., human CLEC5A).

[0247] Anti-CLEC5A antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) and multispecific (e.g., bispecific) antibodies or antibody fragments thereof. Other antibodies provided herein include polyclonal antibodies, monoclonal antibodies, multimeric antibodies, multispecific antibodies (e.g., bispecific), humanized antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellular antibodies (i.e., intracellular antibodies) and antigen-binding fragments thereof. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0248] As long as the antibody fragments retain the affinity and specificity required for the full-length antibody, they are suitable for the provided methods. Therefore, the antibody fragments that bind to CLEC5A will retain the ability to bind to CLEC5A. Fv fragments are antibody fragments that contain complete antigen recognition and binding sites. The region consists of a dimer of a heavy chain and a light chain variable domain tightly bound, and its nature can be covalent, such as in scFv. It is in this structure that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In general, six CDRs or a subset thereof confer antigen binding specificity to antibodies. However, even a single variable domain (or half of an Fv containing only three CDRs for an antigen) can have the ability to recognize and bind to an antigen, although its affinity is generally lower than that of the entire binding site.

[0249] Single-chain Fv or scFv antibody fragments contain the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0250] The Fab fragment contains the variable domain and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment contains a pair of Fab fragments, which are usually covalently linked near their carboxyl termini by hinge cysteines. Other chemical couplings of antibody fragments are also known in the art.

[0251] The antibodies and antibody fragments of the present invention can be modified in the Fc region to provide desired effector functions or serum half-life. In some embodiments, the Fc region can be modified to silence or reduce complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

[0252] In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize endogenous CLEC5A or recombinant CLEC5A. In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize human CLEC5A (eg, the extracellular region of human CLEC5A).

[0253] Multispecific antibodies or antigen-binding fragments thereof

[0254] In one aspect, provided herein is an antibody or antigen binding fragment thereof, comprising: a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is a tumor associated antigen (TAA); and a second antigen binding domain that specifically binds to c-type lectin domain family 5 member A (CLEC5A). In some embodiments, the antibody or antigen binding fragment thereof comprises a fragment crystallizable region (Fc region). In some embodiments, the antibody or antigen binding fragment thereof is a bispecific antibody.

[0255] In some embodiments, the bispecific antibody or antigen-binding fragment thereof (e.g., anti-TAA / CLEC5A antibody) specifically binds to a tumor-associated antigen and CLEC5A, and such bispecific antibody has a modified or enhanced Fc region (e.g., an Fc region having a GAALIE mutation, a LALAPG mutation, a S293D+I332E mutation, a knob-in-hole (KIH) mutation, or a fucosylated Fc region).

[0256] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: a first heavy chain comprising a first heavy chain variable region (VH1); a first light chain comprising a first light chain variable region (VL1); and a second heavy chain comprising a second heavy chain variable region (VH2); and a second light chain comprising a second light chain variable region (VL2).

[0257] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain. In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv) or VHH. In some embodiments, the second antigen binding domain is a scFv or VHH.

[0258] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region comprising a LALAPG mutation: Alanine (A) at position 234; Alanine (A) at position 235; and Glycine (G) at position 329, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region, Alanine (A) at position 234; Alanine (A) at position 234; and Glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0259] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region comprising a GAALIE mutation: alanine (A) at position 236; leucine (L) at position 330; and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having a leucine (L) at position 330; and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 97.

[0260] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region with aspartic acid (D) at position 239; and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure with S239D+I332E mutations, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region, with aspartic acid (D) at position 239; and glutamic acid (E) at position 332, according to EU numbering.

[0261] In some embodiments, the multispecific antibodies described herein can be designed as IgG1 subtype structures with knob-in-hole (KIH) mutations, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the multispecific antibodies have a higher endocytosis rate than the corresponding monoclonal antibodies or control multispecific antibodies. In some embodiments, the antibodies or antigen-binding fragments thereof have increased binding affinity for FcγRIIa receptors and / or FcγRIIIa receptors.

[0262] In some embodiments, the multispecific antibodies described herein are designed to have a non-fucosylated Fc region. A non-fucosylated antibody is designed so that the oligosaccharides in the antibody Fc region do not have any fucose units. When the antibody is non-fucosylated, antibody-dependent cellular cytotoxicity (ADCC) increases.

[0263] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can be any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable domain (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0264] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain and VL1 is connected to the CL domain. Fig. 9C and Fig.9F A schematic diagram of the structure is shown.

[0265] Also provided herein is an antibody or antigen binding fragment thereof, comprising: a first antigen binding domain that specifically binds to a first antigen; and a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the second antigen binding domain that specifically binds to CLEC5A is connected to the light chain of the antibody or its antigen binding fragment. In some embodiments, the second antigen binding domain that specifically binds to CLEC5A is connected to the C-terminus of the light chain of the antibody or its antigen binding fragment. In some embodiments, the second antigen binding domain that specifically binds to CLEC5A is connected to the light chain of the antibody or its antigen binding fragment via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Fig. 9C A schematic diagram of this structure is shown.

[0266] Also provided herein are antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to a first antigen; and a second antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the C-terminus of the Fc region of the antibody or its antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the C-terminus of the Fc region of the antibody or its antigen-binding fragment via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Fig.9F A schematic diagram of this structure is shown.

[0267] Also provided herein is an antibody or an antigen-binding fragment thereof, comprising: a first chain comprising a first light chain and a first scFv or VHH; a second chain comprising a first heavy chain; a third chain comprising a second heavy chain; and a fourth chain comprising a second light chain and a second scFv or VHH, wherein the first light chain and the second light chain each comprise a VL having the same sequence (VL1), and the first heavy chain and the second heavy chain each comprise a VH having the same sequence (VH1). In some embodiments, the first and second scFv or VHH comprise the same amino acid sequence. In some embodiments, the first and second scFv or VHH each comprise VH2 and / or VL2. In some embodiments, the antibody or its antigen-binding fragment comprises an Fc region. In some embodiments, the first and second heavy chains comprise an Fc region. In some embodiments, VH1 and VL1 bind to the first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to the second antigen. In some embodiments, the second antigen is CLEC5A. Fig. 9C A schematic diagram of the structure is shown.

[0268] Also provided herein is an antibody or an antigen-binding fragment thereof, comprising: a first chain comprising a first light chain; a second chain comprising a first heavy chain and a first scFv or VHH; a third chain comprising a second heavy chain and a second scFv or VHH; and a fourth chain comprising a second light chain, wherein the first light chain and the second light chain each comprise a VL having the same sequence (VL1), and the first heavy chain and the second heavy chain each comprise a VH having the same sequence (VH1). In some embodiments, the first and second scFv or VHH comprise the same amino acid sequence. In some embodiments, the first and second scFv or VHH each comprise VH2 and / or VL2. In some embodiments, the antibody or its antigen-binding fragment comprises an Fc region. In some embodiments, the first and second heavy chains comprise an Fc region. In some embodiments, the first scFv or VHH is connected to the C-terminus of the Fc region of the first heavy chain. In some embodiments, the second scFv or VHH is connected to the C-terminus of the Fc region of the second heavy chain. In some embodiments, VH1 and VL1 are bound to the first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 are bound to the second antigen. In some embodiments, the second antigen is CLEC5A. Fig.9F A schematic diagram of this structure is shown in .

[0269] Also provided herein is an antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to a first antigen; and a second antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the heavy chain of the antibody or its antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the Fc region of the antibody or its antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the Fc region of the antibody or its antigen-binding fragment via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Fig.9D A schematic diagram of the structure is shown.

[0270] Also provided herein is an antibody or antigen-binding fragment thereof, comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1 and a first Fc region; and a third chain comprising an scFv or VHH, and a second Fc region. In some embodiments, the scFv or VHH comprises VH2 and / or VL2. In some embodiments, the first Fc region comprises one or more knob mutations, and the second Fc region comprises one or more hole mutations. In some embodiments, the first Fc region comprises one or more hole mutations, and the second Fc region comprises one or more knob mutations. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. Fig.9D A schematic diagram of the structure is shown.

[0271] Also provided herein is an antibody or an antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to a first antigen; a second antigen-binding domain that specifically binds to a first antigen; and a third antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the third antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the heavy chain of the antibody or its antigen-binding fragment. In some embodiments, the third antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the Fc region of the antibody or its antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is connected to the N-terminus of the Fc region of the antibody or its antigen-binding fragment via a linker described herein. In some embodiments, the first antigen and / or the second antigen is a TAA (e.g., HER2). In some embodiments, the first antigen and the second antigen are the same TAA. In some embodiments, the first antigen and the second antigen are different TAAs. Fig. 9B A schematic diagram of the structure is shown.

[0272] Also provided herein is an antibody or antigen-binding fragment thereof, comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1 and VL2; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1, VH2 and a first Fc region; and a third chain comprising an scFv or VHH and a second Fc region. In some embodiments, the scFv or VHH comprises VH3 and / or VL3. In some embodiments, the first Fc region comprises one or more knob mutations and the second Fc region comprises one or more hole mutations. In some embodiments, the first Fc region comprises one or more hole mutations and the second Fc region comprises one or more knob mutations. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the first antigen and / or the second antigen is TAA. In some embodiments, VH3 and VL3 bind to a third antigen. In some embodiments, the third antigen is CLEC5A. Fig. 9B A schematic diagram of this structure is shown.

[0273] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a first heavy chain comprising VH1 and a first light chain comprising VL1; and a second heavy chain comprising VH2 and a second light chain comprising VL2. Fig. 9A or Fig.9E A schematic diagram of this structure is shown. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations.

[0274] Also provided herein are antibodies or antigen-binding fragments thereof, comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1; a third chain comprising a second heavy chain, wherein the second heavy chain comprises VH2; and a fourth chain comprising a second light chain, wherein the second light chain comprises VL2. Fig. 9A A schematic diagram of this structure is shown. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. In some embodiments, the first heavy chain includes one or more knob mutations and the second heavy chain includes one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations and the second heavy chain includes one or more knob mutations.

[0275] Also provided herein is an antibody or antigen-binding fragment thereof, comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1; a third chain comprising a second heavy chain and an scFv or VHH, wherein the second heavy chain comprises VH2; and a fourth chain comprising a second light chain, wherein the second light chain comprises VL2. In some embodiments, the scFv or VHH comprises VH3 and / or VL3. Fig.9E A schematic diagram of the structure is shown. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the first antigen and / or the second antigen is a TAA. In some embodiments, the first antigen and the second antigen are the same TAA. In some embodiments, the first antigen and the second antigen are different TAAs. In some embodiments, VH3 and VL3 bind to a third antigen. In some embodiments, the third antigen is CLEC5A. In some embodiments, the first heavy chain includes one or more knob mutations and the second heavy chain includes one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations and the second heavy chain includes one or more knob mutations.

[0276] In some embodiments, CrossMab substitutions are introduced in the first light chain and the first heavy chain. In some embodiments, the CH1 domain of the first heavy chain is replaced by the light chain constant region (CL) of the first light chain, and the CL of the first light chain is replaced by the CH1 domain of the first heavy chain.

[0277] In some embodiments, CrossMab substitutions are introduced in the second light chain and the second heavy chain. In some embodiments, the CH1 domain of the second heavy chain is replaced by the light chain constant region (CL) of the second light chain, and the CL of the second light chain is replaced by the CH1 domain of the second heavy chain.

[0278] In some embodiments, the multispecific antibodies have a heavy chain sequence comprising a wild-type IgG1 Fc region (SEQ ID NO: 95). In some embodiments, the multispecific antibodies have a heavy chain sequence comprising an Fc region that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 95.

[0279] In some embodiments, the multispecific antibodies have a heavy chain sequence comprising an IgG1 Fc region with a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific antibodies have a heavy chain sequence comprising an Fc region that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96.

[0280] In some embodiments, the multispecific antibodies have a heavy chain sequence comprising an IgG1 Fc region (SEQ ID NO: 97) with optimized mutations. In some embodiments, the multispecific antibodies have a heavy chain sequence comprising an Fc region that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 97.

[0281] The present invention provides multispecific (eg, bispecific) anti-TAA / CLEC5A (eg, anti-HER2 / CLEC5A) antibodies and modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies, and human antibodies.

[0282] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies or antibody fragments thereof include a combination of anti-HER2 and anti-CLEC5A antigen binding domains shown in Table 22.

[0283] In certain embodiments, anti-TAA (for example, anti-HER2 / CLEC5A) antibody is a bispecific antibody. Bispecific antibodies can be prepared by designing the interface between a pair of antibody molecules, to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can include at least a portion of the CH3 domain of an antibody constant domain. In the method, one or more small amino acid side chains on the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains, a compensation "cavity" (for example, alanine or threonine) that is the same or similar to the large side chain size is formed on the interface of the second antibody molecule. This provides a mechanism for increasing the output of heterodimers so that it exceeds other unwanted final products, such as homodimers. The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0284] Any multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or antigen-binding fragment thereof described herein can be coupled to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). The binding of stabilizing molecules can increase the half-life of the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans) or prolong its biological activity.

[0285] Multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies can also be antibody variants (including derivatives and conjugates) or antibody fragments of multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies. Other multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies provided herein include polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellular antibodies (i.e., intrabodies), and antigen-binding fragments thereof. Multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the multispecific (eg, bispecific) anti-TAA / CLEC5A (eg, anti-HER2 / CLEC5A) antibody or antigen-binding fragment is an IgG (eg, the IgG1 Fc region is shown in SEQ ID NO: 95) antibody or antigen-binding fragment thereof.

[0286] Multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody fragments are suitable for use in the provided methods as long as they retain the desired affinity and specificity for TAA (e.g., HER2) and CLEC5A. Thus, multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody fragments will retain the ability to bind to TAA (e.g., HER2) and CLEC5A.

[0287] Tumor-associated antigens

[0288] Tumor-associated antigens (TAAs) are antigenic substances produced by tumor cells. It can trigger the host's immune response. Tumor antigens are useful tumor markers for identifying tumor cells through diagnostic tests and are also potential drug candidates for cancer treatment. Many tumor-associated antigens are known in the art (see, for example, Yu et al., Cancers (Basel). 2023Apr; 15(8): 2323; and Tong et al., Mol Cancer, 2022Nov 1; 21(1): 206, the entire contents of each document are incorporated herein by reference).

[0289] In some embodiments, the TAA is selected from the group consisting of HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycocoll, globulinyl-calcium amide (GB3Cer / CD77), gangliosides (GD2, GD3, and GM2), B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD19, W T-L, B7H3, TEM8, CD22, LI-CAM, ROR-I, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cells, CA242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-I receptor, IGF-I, IgGI, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1, MUC1, mucin Canag, N-glycosylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R A, TWEAK, phosphatidylserine, prostate cancer cells, Rankl, Ron, SCH 900105, SDC1, SLAMF7, TAG-72, Tenascin C, TGF B Beta 2. TGF-B, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or Vimentin.

[0290] In some embodiments, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA) and a second antigen-binding domain that specifically binds to CLEC5A. In some embodiments, the first antigen-binding domain specifically binds to HER2. In some embodiments, the TAA specifically bound by the first antigen-binding domain is selected from HER2, EGFR, EpCAM, CD79b, GPRC5D, BCMA, CD38, DLL3, CD70, GPC3, and mesothelin.

[0291] Anti-TAA / CLEC5A antibodies and antigen-binding fragments thereof

[0292] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have agonistic activity on the activation of macrophages. In some embodiments, the activation of macrophages after contact with the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the activation of macrophages not contacted with such antibodies. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have a LALAPG mutation (SEQ ID NO: 96) on the Fc region. In some embodiments, the anti-TAA / CLEC5A (eg, anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have an optimized mutation in the Fc region (SEQ ID NO: 97).

[0293] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have silent mutations (e.g., LALAPG mutations) in their Fc regions. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to target cells (e.g., HER2+ cancer cells) have silent mutations (e.g., LALAPG mutations) in their Fc regions.

[0294] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., by engaging CLEC5A), thereby mediating the killing of target cells (e.g., HER2+ cancer cells). In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that activate macrophages have a silent mutation (e.g., LALAPG mutation, SEQ ID NO: 96) on the Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein include a wild-type human IgG1 Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies that bind to macrophages described herein have an optimized mutation (SEQ ID NO: 97) on the Fc region.

[0295] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., by engaging CLEC5A) and induce macrophage-mediated killing of target cells. In some embodiments, macrophage-mediated killing results in killing of target cells (e.g., HER2+ cancer cells). In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total number of target cells are killed by mediating phagocytosis of macrophages.

[0296] In some embodiments, the TAA described herein is selected from HER2, EGFR, EpCAM, CD79b, GPRC5D, BCMA, CD38, DLL3, CD70, GPC3, and mesothelin.

[0297] Anti-HER2 / CLEC5A antibodies and antigen-binding fragments thereof

[0298] Receptor tyrosine protein kinase erbB-2 (HER2) is a human protein encoded by the ERBB2 gene. ERBB is the abbreviation for erythroid oncogene B, which was originally isolated from the avian genome. This human protein is also often referred to as HER2 (human epidermal growth factor receptor 2) or CD340 (cluster of differentiation 340).

[0299] HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. But in contrast to other members of the ERBB family, HER2 does not bind ligands directly. When HER2 concentrations are high, such as in cancer, HER2 activation is caused by heterodimerization or homodimerization with another ERBB member. Amplification or overexpression of the oncogene has been shown to play an important role in the occurrence and development of certain invasive breast cancers. In recent years, the protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0300] Detailed review comments on HER2 can be found, for example, in "ERBB2 erb-b2 receptor tyrosine kinase 2 [Homo sapiens (human)] -Gene-NCBI" on the NCBI website; "ERBB2".Genetics Home Reference; Barh D, Gunduz M (2015-01-22).Noninvasive Molecular Markers in Gynecologic Cancers.CRC Press.p.427.ISBN 9781466569393; and Hsu JL, Hung MC (2016). "The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer".Cancer and Metastasis Reviews.35(4):575–588.doi:10.1007 / s10555-016-9649-6, the entire contents of each document are incorporated herein by reference.

[0301] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to HER2 / CLEC5A (e.g., human HER2 / CLEC5A). In one aspect, the present invention provides anti-HER2 / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to HER2; and a second antigen-binding domain that specifically binds to CLEC5A.

[0302] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0303] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence, and the selected VL1 CDR 1, 2, 3 amino acid sequence are one of the following:

[0304] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 100, 102, and 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105, 106, and 107, respectively; and

[0305] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 101, 103, and 104, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 105, 106, and 107, respectively;

[0306] The second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, 3 amino acid sequence, and the selected VL2 CDR 1, 2, 3 amino acid sequence are one of the following: (1) a selected VH2 The amino acid sequences of CDR 1, 2, and 3 are listed in SEQ ID NOs: 3, 5, and 7, respectively, and the amino acid sequences of selected VL2 CDR 1, 2, and 3 are listed in SEQ ID NOs: 8, 9, and 10, respectively;

[0307] (2) the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, and 7, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8, 9, and 10, respectively;

[0308] (3) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0309] (4) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0310] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 100, 102 and 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105, 106 and 107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5 and 7, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8, 9 and 10, respectively.

[0311] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 101, 103 and 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105, 106 and 107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6 and 7, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8, 9 and 10, respectively.

[0312] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 100, 102 and 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105, 106 and 107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0313] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 101, 103 and 104, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 105, 106 and 107, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0314] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 108, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 109, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 11, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 12.

[0315] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 108, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 109, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 83, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 84.

[0316] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 108, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 109, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0317] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 108, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 109, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0318] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 108 and the selected VL sequence is SEQ ID NO: 109.

[0319] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0320] (1) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;

[0321] (2) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22;

[0322] (3) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; and

[0323] (4) The selected VH sequence is SEQ ID NO: 86, and the selected VL sequence is SEQ ID NO: 87.

[0324] In some embodiments, VH1 comprises VH1 CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL1 comprises VL1 CDR1, VL1 CDR2, and VL1 CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 108 and the selected VL sequence is SEQ ID NO: 109.

[0325] In some embodiments, VH2 comprises a VH2 CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0326] (1) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;

[0327] (2) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22;

[0328] (3) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; and

[0329] (4) The selected VH sequence is SEQ ID NO: 86, and the selected VL sequence is SEQ ID NO: 87.

[0330] In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-HER2 antibody trastuzumab. Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1 kappa monoclonal antibody that is a humanized version of the murine anti-HER2 antibody (4D5) that selectively binds to the extracellular domain of HER2 with high affinity (Kd = 5 nM) in cell-based assays (see, e.g., US Pat. Nos. 5,677,171; 5,821,337; 6,054,297; 6,165,464; 6,339,142; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 7,074,404; Coussens et al (1985) Science 230:1132-9; Slamon et al (1989) Science 244:707-12; Slamon et al (2001) New Engl. J. Med. 344:783-792).

[0331] In some embodiments, the second antigen binding domain is any of the antigen binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen binding domain is the antigen binding domain of the anti-CLEC5A antibodies 5C7 or 3A7, chimeric antibodies thereof, and humanized antibodies thereof.

[0332] The CDR sequences of 3A7 and 3A7-derived antibodies include the CDRs of the heavy chain variable domain, SEQ ID NOs: 3, 5 and 7, and the CDRs of the light chain variable domain, SEQ ID NOs: 8, 9 and 10, according to the Kabat definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 4, 6 and 7, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 8, 9 and 10.

[0333] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDRs of the heavy chain variable domain, SEQ ID NOs: 13, 15 and 17, and the CDRs of the light chain variable domain, SEQ ID NOs: 18, 19 and 20, according to the Kabat definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

[0334] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog HER2; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0335] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain.

[0336] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0337] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0338] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an IgG1 Fc region with an alanine (A) at position 234; an alanine (A) at position 234; and a glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0339] In some embodiments, the anti-HER2 / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having an alanine (A) at position 236; a leucine (L) at position 330; and a glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 97.

[0340] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an Fc region with an aspartic acid (D) at position 239 and a glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with S239D+I332E mutations, according to EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an IgG1 Fc region with an aspartic acid (D) at position 239 and a glutamic acid (E) at position 332, according to EU numbering.

[0341] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole mutation (KIH), which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-HER2 / CLEC5A antibodies have a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0342] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can adopt any suitable configuration. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0343] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain, and VL1 is connected to the CL domain. Fig. 9C A schematic diagram of this structure is shown.In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0344] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 118. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96).

[0345] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 119.

[0346] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 3A7(2+2)Fc-LALAPG" or "HER2 / 3A7(2+2A)Fc-silent" and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 118; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 119.

[0347] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 120. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96).

[0348] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121.

[0349] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 5C7(2+2)Fc-LALAPG" or "HER2 / 5C7(2+2A)Fc-silent" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 120; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121.

[0350] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 122. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97).

[0351] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 123.

[0352] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 5C7(2+2A) Fc-optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 122; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 123.

[0353] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 110. In some embodiments, the first heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain comprises one or more knob mutations.

[0354] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 112.

[0355] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 111. In some embodiments, the second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain comprises one or more hole mutations.

[0356] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 113.

[0357] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 3A7(1+1A)Fc-LALAPG" or "HER2 / 3A7(1+1A)Fc-Silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 110; and a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 112; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: NO:111 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:113; and the second light chain sequence is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:113.

[0358] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 114. In some embodiments, the first heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain comprises one or more hole mutations.

[0359] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 116.

[0360] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 115. In some embodiments, the second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain comprises one or more knob mutations.

[0361] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 117.

[0362] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 5C7(1+1A)Fc-LALAPG" or "HER2 / 5C7(1+1A)Fc-Silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 114; and a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 116; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 117. NO:115 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; and the second light chain sequence is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:117.

[0363] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may comprise a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0364] In some embodiments, knob-in-hole mutations are introduced into the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0365] The present invention also provides nucleic acids comprising polynucleotides encoding anti-HER2 / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-HER2 / CLEC5A antibody comprises a CDR as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to HER2 and / or CLEC5A.

[0366] Anti-EGFR / CLEC5A antibodies and antigen-binding fragments thereof

[0367] The epidermal growth factor receptor (EGFR in humans; ErbB-1; HER1) is a transmembrane protein that is a receptor for extracellular protein ligand members of the epidermal growth factor family (EGF family). The EGFR is a member of the ErbB receptor family, which consists of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many cancer types, mutations that affect EGFR expression or activity may lead to further cancer development. Defects in human EGFR and other receptor tyrosine kinase signaling are associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a variety of tumors. Blocking EGFR signaling, either by blocking the EGFR binding sites in the extracellular domain of the receptor or inhibiting the intracellular tyrosine kinase activity, can prevent the growth of tumors expressing EGFR and improve the patient's condition.

[0368] EGFR is a transmembrane protein that is activated by binding to its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFa). ErbB2 has no known direct activating ligand and may be constitutively activated or become active after heterodimerization with other family members such as EGFR. Upon activation by its growth factor ligands, EGFR switches from an inactive monomeric form to an active homodimer. EGFR dimerization stimulates its intrinsic intracellular protein tyrosine kinase activity. Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. This results in autophosphorylation of several tyrosine (Y) residues in the terminal domain of EGFRC. These include Y992, Y1045, Y1068, Y1148, and Y1173.

[0369] Autophosphorylation triggers downstream activation and signaling, which is carried out by several other proteins that bind to the phosphorylated tyrosine through their own phosphotyrosine-binding SH2 domains. These downstream signaling proteins initiate several signal transduction cascades, mainly the MAPK, Akt, and JNK pathways, leading to DNA synthesis and cell proliferation. Such proteins regulate phenotypes such as cell migration, adhesion, and proliferation. The kinase domain of EGFR can also cross-phosphorylate tyrosine residues of other receptors with which it clusters, and can itself be activated in this way.

[0370] Mutations that lead to overexpression of EGFR (called upregulation or amplification) are associated with a variety of cancers, including lung adenocarcinoma (40% of cases), anal cancer, glioblastoma (50%), and epithelial tumors of the head and neck (80-100%). These somatic mutations involving EGFR lead to its constant activation, resulting in uncontrolled cell division. In glioblastoma, a specific mutation of EGFR, called EGFRvIII, is frequently observed. Approximately 30% of epithelial cancers are associated with mutations, amplifications, or dysregulation of EGFR or a family member.

[0371] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to EGFR / CLEC5A (e.g., human EGFR / CLEC5A). In one aspect, the present invention provides anti-EGFR / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to EGFR; and a second antigen-binding domain that specifically binds to CLEC5A.

[0372] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0373] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0374] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0376] (1) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 126, 128, and 130, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 131, 132, and 133, respectively;

[0377] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 127, 129, and 130, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 131, 132, and 133, respectively;

[0378] (3) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 136, 138 and 140, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 141, 142 and 143, respectively; and

[0379] (4) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 137, 139, and 140, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 141, 142, and 143, respectively;

[0380] a second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence;

[0381] and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0382] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0383] (2) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0384] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 126, 128 and 130, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 131, 132 and 133, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0385] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 127, 129 and 130, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 131, 132 and 133, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0386] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 136, 138 and 140, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 141, 142 and 143, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0387] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 137, 139 and 140, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 141, 142 and 143, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0388] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 134, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 135, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0389] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 134, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 135, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0390] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 144, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0391] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 144, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0392] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 134 and the selected VL sequence is SEQ ID NO: 135. In some embodiments, the selected VH sequence is SEQ ID NO: 144 and the selected VL sequence is SEQ ID NO: 145.

[0393] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0394] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0395] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0396] In some embodiments, VH1 comprises VH1CDR1, VH CDR2 and VH CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; and VL1 comprises VL1CDR1, VL1CDR2 and VL1CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 134 and the selected VL sequence is SEQ ID NO: 135. In some embodiments, the selected VH sequence is SEQ ID NO: 144 and the selected VL sequence is SEQ ID NO: 145.

[0397] In some embodiments, VH2 comprises a VH2CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2CDR1, VL2CDR2, and VL2CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0398] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0399] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0400] In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-EGFR antibody cetuximab, panitumumab, nexitozumab or Eg-B4-VHH. In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-EGFR antibody amivantamab (EGFR1). In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-EGFR antibody nimotuzumab (EGFR2).

[0401] In some embodiments, the second antigen binding domain is any of the antigen binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen binding domain is the antigen binding domain of the anti-CLEC5A antibody 5C7, chimeric antibodies thereof, and humanized antibodies thereof.

[0402] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDR sequences of the heavy chain variable domain according to the Kabat definition as listed in SEQ ID NOs: 13, 15 and 17, and the CDR sequences of the light chain variable domain as listed in SEQ ID NOs: 18, 19 and 20. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

[0403] In some embodiments, the first antigen binding domain specifically binds to human, rabbit, mouse, monkey or dog EGFR; and / or the second antigen binding domain specifically binds to human, rabbit, mouse, monkey or dog CLEC5A.

[0404] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain. In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0405] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0406] In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 Fc region with an alanine (A) at position 234; an alanine (A) at position 234; and a glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0407] In some embodiments, the anti-EGFR / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having an alanine (A) at position 236; a leucine (L) at position 330; and a glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 97.

[0408] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an Fc region with alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with S239D+I332E mutations, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region with aspartic acid (D) at position 239, and glutamic acid (E) at position 332, according to EU numbering.

[0409] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole mutation (KIH), which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-EGFR / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0410] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can adopt any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0411] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain, and VL1 is connected to the CL domain. Fig. 9C A schematic diagram of this structure is shown.In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0412] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 146 or 148. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97).

[0413] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody comprises a light chain sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 147 or 149.

[0414] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody is referred to as "EGFR1 / 5C7 (2+2A) Fc-optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 146; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 147. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody is referred to as "EGFR2 / 5C7 (2+2A) Fc-optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 148; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 149.

[0415] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may comprise a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0416] In some embodiments, knob-in-hole mutations are introduced into the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0417] The present invention also provides nucleic acids comprising polynucleotides encoding anti-EGFR / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-EGFR / CLEC5A antibody comprises a CDR as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to EGFR and / or CLEC5A.

[0418] Anti-EpCAM / CLEC5A antibodies and antigen-binding fragments thereof

[0419] Epithelial cell adhesion molecule (EpCAM), also known as tumor-associated calcium signal transducer 1 (TACST-1), 17-1A and CD326, is a 40 kDa transmembrane glycoprotein that is highly expressed in epithelial cancers and expressed at lower levels in normal monolayer epithelia. The structure and function of EpCAM are described in the literature, for example: Schnell et al., Biochimica et Biophysica Acta-Biomembranes (2013), 1828(8): 1989-2001; Trzpis et al. Am J Pathol. (2007) 171(2): 386-395 and Baeuerle and Gires, Br. J. Cancer, (2007) 96: 417-423.

[0420] EpCAM is expressed on the basolateral membrane and plays a role in calcium-independent allogeneic cell adhesion. The mature EpCAM molecule consists of an N-terminus (processed to remove the 23 amino acid signal peptide), a 242 amino acid extracellular domain (containing an epidermal growth factor-like repeat region), a human thyroglobulin repeat region and a cysteine-poor region, a single-pass 23 amino acid transmembrane domain, and a 26 amino acid cytoplasmic domain at the C-terminus (containing two α-actinin binding sites and an NPXY internalization motif). EpCAM is frequently overexpressed in cancers of epithelial origin and is expressed by cancer stem cells, making it a molecule of great therapeutic and diagnostic significance. Due to its frequent and high expression in cancer and its metastasis, EpCAM can be used as a prognostic marker, a therapeutic target, and an anchoring molecule for circulating and disseminated tumor cells (CTCs / DTCs), which are considered to be the main source of metastatic cancer cells. The extracellular domain EpCAM can be cleaved to produce the soluble extracellular domain molecule EpEX and the intracellular molecule EpICD. EpICD has been shown to bind to other proteins to form nuclear complexes that upregulate the expression of genes that promote cell proliferation. EpCAM may also be involved in the epithelial to mesenchymal transition (EMT) and may promote the formation of large metastatic foci.

[0421] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to EpCAM / CLEC5A (e.g., human EpCAM / CLEC5A). In one aspect, the present invention provides anti-EpCAM / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to EpCAM; and a second antigen-binding domain that specifically binds to CLEC5A.

[0422] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0423] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0424] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0426] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 150, 152, and 154, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 155, 156, and 157, respectively; and

[0427] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 151, 153, and 154, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 155, 156, and 157, respectively;

[0428] the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0429] and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0430] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0431] (2) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0432] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 150, 152 and 154, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 155, 156 and 157, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0433] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 151, 153 and 154, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 155, 156 and 157, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0434] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 158, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 159, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0435] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:158, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:159, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:86.

[0436] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 158 and the selected VL sequence is SEQ ID NO: 159.

[0437] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0438] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0439] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0440] In some embodiments, VH1 comprises VH1 CDR1, VH1 CDR2 and VH1 CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; VL1 comprises VL1 CDR1, VL1 CDR2 and VL1 CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 158 and the selected VL sequence is SEQ ID NO: 159.

[0441] In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0442] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0443] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0444] In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-EpCAM antibody Solitomab.

[0445] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, chimeric antibodies thereof, and humanized antibodies thereof.

[0446] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDR sequences of the heavy chain variable domain according to the Kabat definition as listed in SEQ ID NOs: 13, 15 and 17, and the CDR sequences of the light chain variable domain as listed in SEQ ID NOs: 18, 19 and 20. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

[0447] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog EpCAM; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0448] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain. In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0449] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0450] In some embodiments, the multispecific (e.g., multispecific) anti-EpCAM / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 Fc region with an alanine (A) at position 234; an alanine (A) at position 234; and a glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0451] In some embodiments, the anti-EpCAM / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having an alanine (A) at position 236; a leucine (L) at position 330; and a glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 97.

[0452] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein are designed to have an Fc region comprising an alanine (A) at position 236 according to EU numbering; a leucine (L) at position 330; and a glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure, with S239D+I332E mutations according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region, comprising an aspartic acid (D) at position 239 according to EU numbering; and a glutamic acid (E) at position 332.

[0453] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole mutation (KIH), which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-EpCAM / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0454] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can adopt any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0455] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain, and VL1 is connected to the CL domain. Fig. 9C A schematic diagram of this structure is shown.In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region comprises a knob-to-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0456] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160 or 162. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97).

[0457] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody comprises a light chain sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161 or 163.

[0458] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody is referred to as "EpCAM / 5C7(2+2A) Fc-optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161.

[0459] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may comprise a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0460] In some embodiments, knob-in-hole mutations are introduced into the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0461] The present invention also provides nucleic acids comprising polynucleotides encoding anti-EpCAM / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-EpCAM / CLEC5A antibody comprises a CDR as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to EpCAM and / or CLEC5A.

[0462] Anti-GPRC5D / CLEC5A antibodies and antigen-binding fragments thereof

[0463] G protein coupled receptor family C group 5 member D (GPRC5D) is a protein encoded by the GPRC5D gene in humans. The protein encoded by the gene is a member of the G protein coupled receptor family.

[0464] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to GPRC5D / CLEC5A (e.g., human GPRC5D / CLEC5A). In one aspect, the present invention provides anti-GPRC5D / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to GPRC5D; and a second antigen-binding domain that specifically binds to CLEC5A.

[0465] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0466] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0467] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0469] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 164, 166 and 168, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 169, 170 and 171, respectively; and

[0470] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 165, 167, and 168, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 169, 170, and 171, respectively;

[0471] the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0472] and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0473] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0474] (2) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0475] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 164, 166 and 168, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 169, 170 and 171, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0476] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 165, 167 and 168, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 169, 170 and 171, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0477] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 172, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 173, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0478] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 172, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 173, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0479] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 172 and the selected VL sequence is SEQ ID NO: 173.

[0480] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0481] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0482] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0483] In some embodiments, VH1 comprises VH1 CDR1, VH1 CDR2 and VH1 CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; VL1 comprises VL1 CDR1, VL1 CDR2 and VL1 CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 172, and the selected VL sequence is SEQ ID NO: 173.

[0484] In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0485] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0486] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0487] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, chimeric antibodies thereof, and humanized antibodies thereof.

[0488] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDR sequences of the heavy chain variable domain according to the Kabat definition as listed in SEQ ID NOs: 13, 15 and 17, and the CDR sequences of the light chain variable domain as listed in SEQ ID NOs: 18, 19 and 20. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

[0489] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog GPRC5D; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0490] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain. In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0491] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0492] In some embodiments, the multispecific (e.g., multispecific) anti-GPRC5D / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 Fc region comprising an alanine (A) at position 234, an alanine (A) at position 234, and a glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0493] In some embodiments, the anti-GPRC5D / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having an alanine (A) at position 236; a leucine (L) at position 330; and a glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 97.

[0494] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein are designed to have an Fc region comprising an alanine (A) at position 236 according to EU numbering; a leucine (L) at position 330; and a glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure, with S239D+I332E mutations according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region, comprising an aspartic acid (D) at position 239 according to EU numbering; and a glutamic acid (E) at position 332.

[0495] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole mutation (KIH), which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-GPRC5D / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0496] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can adopt any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0497] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain, and VL1 is connected to the CL domain. Fig. 9C A schematic diagram of this structure is shown.In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region comprises a knob-to-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0498] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 174. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97).

[0499] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody comprises a light chain sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 175.

[0500] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody is referred to as "GPRC5D / 5C7(2+2A)Fc-optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 174; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 175.

[0501] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may comprise a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0502] In some embodiments, knob-in-hole mutations are introduced into the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0503] The present invention also provides nucleic acids comprising polynucleotides encoding anti-GPRC5D / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-GPRC5D / CLEC5A antibody comprises a CDR as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to GPRC5D and / or CLEC5A.

[0504] Anti-BCMA / CLEC5A antibodies and antigen-binding fragments thereof

[0505] B-cell maturation antigen (BCMA or BCM), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a human protein encoded by the TNFRSF17 gene. BCMA is a cell surface receptor of the TNF receptor superfamily that recognizes B-cell activating factor (BAFF). Serum B-cell maturation antigen (sBCMA) is the cleaved form of BCMA, which is present at low levels in the serum of normal patients and is generally present at high levels in patients with multiple myeloma (MM).

[0506] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to BCMA / CLEC5A (e.g., human BCMA / CLEC5A). In one aspect, the present invention provides anti-BCMA / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to BCMA; and a second antigen-binding domain that specifically binds to CLEC5A.

[0507] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0508] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0509] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0511] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 176, 178 and 180, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 181, 182 and 183, respectively; and

[0512] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 177, 179, and 180, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 181, 182, and 183, respectively;

[0513] the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0514] and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are one of the following:

[0515] (1) the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0516] (2) The selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0517] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 176, 178 and 180, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 181, 182 and 183, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0518] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 177, 179 and 180, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 181, 182 and 183, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0519] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:184, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:185, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22.

[0520] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 184, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 185, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0521] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 184 and the selected VL sequence is SEQ ID NO: 185.

[0522] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0523] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0524] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0525] In some embodiments, VH1 comprises VH1 CDR1, VH1 CDR2 and VH1 CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; VL1 comprises VL1 CDR1, VL1 CDR2 and VL1 CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 184, and the selected VL sequence is SEQ ID NO: 185.

[0526] In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0527] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0528] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0529] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, chimeric antibodies thereof, and humanized antibodies thereof.

[0530] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDR sequences of the heavy chain variable domain according to the Kabat definition as listed in SEQ ID NOs: 13, 15 and 17, and the CDR sequences of the light chain variable domain as listed in SEQ ID NOs: 18, 19 and 20. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

[0531] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog BCMA; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0532] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain. In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0533] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0534] In some embodiments, the multispecific (e.g., multispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with a LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 Fc region comprising an alanine (A) at position 234, an alanine (A) at position 234, and a glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0535] In some embodiments, the anti-BCMA / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region comprising alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 97.

[0536] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an Fc region comprising an alanine (A) at position 236 according to EU numbering; a leucine (L) at position 330; and a glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure, with S239D+I332E mutations according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region, comprising an aspartic acid (D) at position 239 according to EU numbering; and a glutamic acid (E) at position 332.

[0537] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein can be designed as an IgG1 subtype structure with a knob-in-hole mutation (KIH), which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-BCMA / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0538] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can adopt any suitable structure. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0539] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain, and VL1 is connected to the CL domain. Fig. 9C A schematic diagram of this structure is shown.In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0540] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 174. In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97).

[0541] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 187.

[0542] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody is referred to as "BCMA / 5C7 (2+2A) Fc-optimized" and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 186; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 187.

[0543] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may comprise a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0544] In some embodiments, knob-in-hole mutations are introduced into the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0545] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-BCMA / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-BCMA / CLEC5A antibody comprises a CDR as shown in Table 22. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to BCMA and / or CLEC5A.

[0546] Anti-CD38 / CLEC5A antibodies and antigen-binding fragments thereof

[0547] CD38 (Cluster of Differentiation 38), also known as cyclic ADP ribose hydrolase, is a glycoprotein present on the surface of many immune cells (leukocytes), including CD4+, CD8+, B lymphocytes, and natural killer cells. CD38 also plays a role in cell adhesion, signal transduction, and calcium signaling. In humans, the CD38 protein is encoded by the CD38 gene located on chromosome 4. CD38 is a paralog of CD157, which is also located on human chromosome 4 (4p15).

[0548] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to CD38 / CLEC5A (e.g., human CD38 / CLEC5A). In one aspect, the present invention provides anti-CD38 / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to CD38; and a second antigen-binding domain that specifically binds to CLEC5A.

[0549] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0550] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3; and

[0551] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0552] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 188, 190 and 192, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 193, 194 and 195, respectively; and

[0553] (2) the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 189, 191, and 192, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 193, 194, and 195, respectively;

[0554] the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0555] a second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR3,

[0556] wherein the selected VH2 CDR 1, 2 and 3 amino acid sequence and the selected VL2 CDR 1, 2 and 3 amino acid sequence are one of the following:

[0557] (1) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 13, 15, and 17, respectively.

[0558] VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively; and

[0559] (2) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 14, 16, and 17, respectively. The selected VL2

[0560] The amino acid sequences of CDRs 1, 2, and 3 are listed in SEQ ID NOs: 18, 19, and 20, respectively.

[0561] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 188, 190 and 192, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 193, 194 and 195, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0562] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 189, 191 and 192, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 193, 194 and 195, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0563] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 196, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 197, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 22.

[0564] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 196, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 197, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 86.

[0565] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 196 and the selected VL sequence is SEQ ID NO: 197.

[0566] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0567] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0568] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0569] In some embodiments, VH1 comprises VH1 CDR1, VH1 CDR2 and VH1 CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; VL1 comprises VL1 CDR1, VL1 CDR2 and VL1 CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 196 and the selected VL sequence is SEQ ID NO: 197.

[0570] In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0571] (1) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and

[0572] (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0573] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, chimeric antibodies thereof, and humanized antibodies thereof.

[0574] The CDR sequences of 5C7 and 5C7-derived antibodies include the CDRs of the heavy chain variable domain according to the Kabat definition, which are listed in SEQ ID NOs: 13, 15, and 17, respectively, and the CDRs of the light chain variable domain according to the Chothia definition, which are listed in SEQ ID NOs: 14, 16, and 17, respectively, and the CDRs of the light chain variable domain according to the Chothia definition, which are listed in SEQ ID NOs: 18, 19, and 20, respectively.

[0575] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CD38; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0576] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain.

[0577] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0578] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0579] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having an alanine (A) at position 234, an alanine (A) at position 235, and a glycine (G) at position 329 according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0580] In some embodiments, the anti-CD38 / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region with an alanine (A) at position 236, a leucine (L) at position 330, and a glutamic acid (E) at position 332 (by EU numbering). In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 97.

[0581] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with S239D+I332E mutations in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region with aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0582] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-in-hole (KIH) mutation, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-CD38 / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0583] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can be any suitable configuration. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0584] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain and VL1 is connected to the CL domain. Fig. 9C In some embodiments, the antibodies or antigen-binding fragments thereof described herein include a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A In some embodiments, the Fc region comprises a knob-to-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0585] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 198. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0586] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody comprises a light chain sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:199.

[0587] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody is referred to as "CD38 / 5C7 (2+2A) Fc optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 198; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 199.

[0588] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacer sequences (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0589] In some embodiments, knob-in-hole mutations are introduced in the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0590] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-CD38 / CLEC5A antibody, wherein the immunoglobulin heavy chain or immunoglobulin light chain in the anti-CD38 / CLEC5A antibody comprises a CDR as shown in Table 22, and when the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CD38 and / or CLEC5A.

[0591] Anti-CD79b / CLEC5A antibodies and antigen-binding fragments thereof

[0592] The CD79b molecule, immunoglobulin-associated beta, also known as CD79B (cluster of differentiation 79B), is a human gene. It is associated with agammaglobulinemia-6. The B lymphocyte antigen receptor is a multimeric complex that includes an antigen-specific component, surface immunoglobulin (Ig). The surface Ig is non-covalently associated with two other proteins, Ig-alpha and Ig-beta, which are required for the expression and function of the B cell antigen receptor. The gene encodes the Ig-beta protein of the B cell antigen component. Alternative splicing transcript variants encoding different isoforms have been described.

[0593] The present invention provides multispecific (e.g., bispecific) antibodies and antigen-binding fragments thereof that specifically bind to CD79b / CLEC5A (e.g., human CD79b / CLEC5A). In one aspect, the present invention provides anti-CD79b / CLEC5A multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, comprising: a first antigen-binding domain that specifically binds to CD79b; and a second antigen-binding domain that specifically binds to CLEC5A.

[0594] In some embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0595] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3; and

[0596] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3, wherein the selected VH1 CDR 1, 2, 3 amino acid sequence and the selected VL1 CDR 1, 2, and 3 amino acid sequence are one of the following:

[0597] (1) the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 200, 202, and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206, and 207, respectively; and

[0598] (2) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 201, 203, and 204, respectively.

[0599] The amino acid sequences of VL1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 205, 206, and 207, respectively;

[0600] the second heavy chain variable region (VH2) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0601] a second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR3,

[0602] wherein the selected VH2 CDR 1, 2 and 3 amino acid sequence and the selected VL2 CDR 1, 2 and 3 amino acid sequence are one of the following:

[0603] (1) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 13, 15, and 17, respectively. The selected VL2

[0604] The amino acid sequences of CDRs 1, 2, and 3 are listed in SEQ ID NOs: 18, 19, and 20, respectively;

[0605] (2) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 14, 16, and 17, respectively. The selected VL2

[0606] The amino acid sequences of CDRs 1, 2, and 3 are listed in SEQ ID NOs: 18, 19, and 20, respectively;

[0607] (3) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 3, 5, and 7, respectively. The amino acid sequences of the selected VL2 CDRs

[0608] The amino acid sequences of 1, 2, and 3 are listed in SEQ ID NOs: 8, 9, and 10, respectively;

[0609] (4) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 4, 6, and 17, respectively. The amino acid sequences of the selected VL2 CDRs

[0610] The amino acid sequences of 1, 2, and 3 are listed in SEQ ID NOs: 18, 19, and 20, respectively;

[0611] (5) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 63, 65, and 67, respectively. The selected VL2

[0612] The amino acid sequences of CDRs 1, 2, and 3 are listed in SEQ ID NOs: 68, 69, and 70, respectively;

[0613] (6) The amino acid sequences of the selected VH2 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 64, 66, and 67, respectively. The selected VL2

[0614] The amino acid sequences of CDRs 1, 2, and 3 are listed in SEQ ID NOs: 68, 69, and 70, respectively;

[0615] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 200, 202 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0616] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 201, 203 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16 and 17, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0617] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 200, 202 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5 and 7, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8, 9 and 10, respectively.

[0618] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 201, 203 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6 and 7, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8, 9 and 10, respectively.

[0619] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 200, 202 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65 and 67, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively.

[0620] In some embodiments, the selected VH1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 201, 203 and 204, respectively, and the selected VL1 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 205, 206 and 207, respectively; the selected VH2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 64, 66 and 67, respectively, and the selected VL2 CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68, 69 and 70, respectively.

[0621] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:21, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:22.

[0622] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:85, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:86.

[0623] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:11, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:12.

[0624] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:83, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:84.

[0625] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:71, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:72.

[0626] In some embodiments, the first heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:208, the first light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:209, the second heavy chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:91, and the second light chain variable region comprises a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:92.

[0627] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 208 and the selected VL sequence is SEQ ID NO: 209.

[0628] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0629] (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22;

[0630] (2) the selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86;

[0631] (3) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;

[0632] (4) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84;

[0633] (5) the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72;

[0634] (6) the selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92;

[0635] In some embodiments, VH1 comprises VH1 CDR1, VH1 CDR2 and VH1 CDR3 identical to VH CDR1, VH CDR2 and VH CDR3 of the selected VH sequence; VL1 comprises VL1 CDR1, VL1 CDR2 and VL1 CDR3 identical to VL CDR1, VL CDR2 and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 208 and the selected VL sequence is SEQ ID NO: 209.

[0636] In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and VL2 comprises a VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0637] (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22;

[0638] (2) the selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86;

[0639] (3) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12;

[0640] (4) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84;

[0641] (5) the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72;

[0642] (6) the selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92;

[0643] In some embodiments, the second antigen-binding domain is an antigen-binding domain of any one of the anti-CLEC5A antibodies, chimeric antibodies thereof, and humanized antibodies thereof described herein. In some embodiments, the second antigen-binding domain is an antigen-binding domain of the anti-CLEC5A antibodies 5C7, 3A7, 13E6, chimeric antibodies thereof, and humanized antibodies thereof.

[0644] The CDR sequences of 5C7 and 5C7-derived antibodies include: according to the Kabat definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NOs: 13, 15 and 17, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20, respectively. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20, respectively.

[0645] The CDR sequences of 3A7 and 3A7-derived antibodies include: according to the Kabat definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NOs: 3, 5 and 7, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 8, 9 and 10, respectively. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 4, 6 and 7, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 8, 9 and 10, respectively.

[0646] The CDR sequences of 13E6 and 13E6-derived antibodies include: According to the Kabat definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NOs: 63, 65 and 67, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 68, 69 and 70, respectively. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 64, 66 and 67, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 68, 69 and 70, respectively.

[0647] In some embodiments, the first antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CD79b; and / or the second antigen binding domain specifically binds human, rabbit, mouse, monkey or dog CLEC5A.

[0648] In some embodiments, the first antigen binding domain is a human or humanized antigen binding domain; and / or the second antigen binding domain is a human or humanized antigen binding domain.

[0649] In some embodiments, the first antigen binding domain is a single chain variable fragment (scFv); and / or the second antigen binding domain is a scFv.

[0650] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0651] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having an alanine (A) at position 234, an alanine (A) at position 235, and a glycine (G) at position 329 according to EU numbering. In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 96.

[0652] In some embodiments, the anti-CD79b / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region with an alanine (A) at position 236, a leucine (L) at position 330, and a glutamic acid (E) at position 332 (by EU numbering). In some embodiments, the Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 97.

[0653] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein are designed to have an Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure with S239D+I332E mutations in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region with aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0654] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-in-hole (KIH) mutation, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-CD79b / CLEC5A antibodies have a higher internalization rate than the corresponding monoclonal antibody or control bispecific antibody.

[0655] The first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody or antigen-binding fragment described herein can be any suitable configuration. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) connected by a first linker.

[0656] In some embodiments, the second antigen binding domain is connected to the C-terminus of the light chain of the first antigen binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen binding domain is connected to the Fc region. In some embodiments, VH1 is connected to the CH1 domain and VL1 is connected to the CL domain. Fig. 9C In some embodiments, the antibodies or antigen-binding fragments thereof described herein include a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Fig. 9A In some embodiments, the Fc region comprises a knob-in-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein may have Figures 9A-9F Any of the structures shown in .

[0657] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 210) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 210. Fig. 9A 97%, 98%, 99%, or 100% identical to SEQ ID NO: 211. Fig. 9A In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first light chain (in Fig. 9A 97%, 98%, 99%, or 100% identical to SEQ ID NO: 213. Fig. 9A ) sequence.

[0658] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1A)Fc silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 210; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 211; a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 212; NO:212 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; and the second light chain sequence is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:213.

[0659] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 227) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 227. Fig. 9A 97%, 98%, 99%, or 100% identical to SEQ ID NO: 228. Fig. 9AIn some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region (SEQ ID NO: 97) comprising an optimized mutation. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first light chain (in SEQ ID NO: 229) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 229. Fig. 9A 97%, 98%, 99%, or 100% identical to SEQ ID NO: 230. Fig. 9A ) sequence.

[0660] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1A)Fc optimized" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 227; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 228; a first light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 229; NO:229 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; and the second light chain sequence is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:230.

[0661] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 217) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 217. Fig. 9B97%, 98%, 99%, or 100% identical to SEQ ID NO: 218. Fig. 9B In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 219 ( Fig. 9B ) sequence.

[0662] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1A)Fc silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 217; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 218; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 219. NO: 219 is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0663] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 234) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 234. Fig. 9B 97%, 98%, 99%, or 100% identical to SEQ ID NO: 235. Fig. 9B In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 236 (shown as "H2" in FIG. 236). Fig. 9B ) sequence.

[0664] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1A)Fc optimized" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 234; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 235; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: NO:236 is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0665] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 223. Fig. 9C In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 224 (shown as "H" in FIG. 224). Fig. 9C The sequence is shown as "L" in FIG.

[0666] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2A)Fc silent" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 223; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 224.

[0667] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 240. Fig. 9C In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain (in SEQ ID NO: 241) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Fig. 9C The sequence is shown as "L" in FIG.

[0668] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2A)Fc optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 240; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 241.

[0669] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 214) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 214. Fig.9D 97%, 98%, 99%, or 100% identical to SEQ ID NO: 215. Fig.9D In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 216 (shown as "H2" in FIG. 216). Fig.9D ) sequence.

[0670] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1B)Fc silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 214; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 215; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 216. NO:216 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0671] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 231) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 231. Fig.9D 97%, 98%, 99%, or 100% identical to SEQ ID NO: 232. Fig.9D In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 233. Fig.9D ) sequence.

[0672] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1B)Fc optimized" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 231; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 232; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 233. NO: 233 is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0673] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 220) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 220. Fig.9E 97%, 98%, 99%, or 100% identical to SEQ ID NO: 221. Fig.9E In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 222 (shown as "H2" in FIG. 222). Fig.9E ) sequence.

[0674] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1B)Fc silent" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 220; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 221; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 222. NO: 222 is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0675] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a first heavy chain (in the range of 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 237) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 237. Fig.9E 97%, 98%, 99%, or 100% identical to SEQ ID NO: 238. Fig.9E In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain (SEQ ID NO: 239) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 239. Fig.9E ) sequence.

[0676] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1B)Fc optimized" and comprises a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 237; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 238; and a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 239. NO: 239 is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain sequence.

[0677] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 225. Fig.9F In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 226 (shown as "H" in FIG. 226). Fig.9F The sequence is shown as "L" in FIG.

[0678] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2B)Fc silent" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 225; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 226.

[0679] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 244. Fig.9F In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 245 (shown as "H" in FIG. 245). Fig.9FThe sequence is shown as "L" in FIG.

[0680] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 3A7(2+2B)Fc silent" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 244; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 245.

[0681] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 246. Fig.9F In some embodiments, the heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 247 (shown as "H" in FIG. 247). Fig.9F The sequence is shown as "L" in FIG.

[0682] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 13E6(2+2B)Fc silent" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 246; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 247.

[0683] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a heavy chain that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 242. Fig.9F In some embodiments, the heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain (in SEQ ID NO: 243) that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 243. Fig.9F The sequence is shown as "L" in FIG.

[0684] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2B)Fc optimized" and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 242; and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 243.

[0685] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer sequence. Various spacer sequences are known in the art, including but not limited to glycine serine (GS) spacer sequences (also referred to as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), wherein n represents an integer of at least 1. One skilled in the art will be able to select an appropriate spacer sequence.

[0686] In some embodiments, knob-in-hole mutations are introduced in the Fc region of a multispecific (eg, bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0687] The present invention also provides a nucleic acid comprising a polynucleotide encoding an anti-CD79b / CLEC5A antibody, wherein the immunoglobulin heavy chain or immunoglobulin light chain in the anti-CD79b / CLEC5A antibody comprises a CDR as shown in Table 22, and when the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CD79b and / or CLEC5A. Example

[0688] The present invention will be further described by the following examples, but these examples are not intended to limit the scope of the invention described in the claims.

[0689] Example 1: CLEC5A expression in immune cell subsets in human PBMCs

[0690] Evaluation of immune cell subsets in human PBMCs using a gating strategy Figure 1A PBMCs from healthy donors (Stanford Blood Center) were thawed and fixed with a viability dye (Zombie Aqua TM , BioLegend) and then labeled with a panel of specific antibodies against immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Bio-sciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences) and CLEC5A (283834, R&D Systems). After staining, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed, resuspended in FACS buffer (PBS (phosphate buffered saline) containing 2% heat-inactivated FBS (fetal bovine serum) and 0.05% BSA (bovine serum albumin)), and then analyzed by flow cytometry (Attune TM CytPik TM , Invitrogen) for analysis. Figure 1B As shown, CLEC5A is highly expressed in CD15+CD16+ neutrophils and CD14+CD16- classical monocytes. Some CLEC5A is also expressed in intermediate CD14+CD16+ and CD14-CD16+ non-classical monocytes.

[0691] Example 2: CLEC5A expression in tumor-associated myeloid cells in human solid tumors

[0692] Expression of CLEC5A was evaluated in tumor-associated myeloid cells of human solid tumors. Frozen human isolated tumor cells (Discovery LifeSciences) from six solid tumor indications (renal, lung, ovarian, colorectal, cholangiocarcinoma, and pancreatic) were thawed and stained with a fixable viability dye (Zombie Aqua TM , BioLegend) and then labeled with a panel of antibodies targeting immune cell subsets: CD45 (H130, Bio-Legend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), TREM2 (237920, R&D Systems), CD206 (15-2, BioLegend), and CLEC5A (283834, R&D Systems). After staining, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed, resuspended in FACS buffer (PBS containing 2% heat-inactivated FBS and 0.05% BSA), and analyzed by flow cytometry (Attune TM CytPik TM , Invitrogen). Gating strategy was used to evaluate immune cell subsets in human solid tumors ( Figure 2A ).like Figure 2B-2C As shown, in human tumors, the majority of neutrophils (80-98%, expressed as live / CD45+ / CD19- / CD3- / CD56- / CD15+ / CD16+) and non-neutrophil myeloid cells (>43%, expressed as live / CD45+ / CD19- / CD3- / CD56- / CD15-) were positive for CLEC5A. The highest expression of CLEC5A was observed in non-neutrophil myeloid cells. Little or no CLEC5A expression was observed in non-immune cells (live / CD45-), while some expression was observed in a subset of cells (<14%) in a mixture of cells (live / CD45+ / CD19+ / CD3+ / CD56+) of B cells, T cells, and NK cells.

[0693] Example 3: CLEC5A expression in tumor-associated macrophages (TAMs) in human solid tumors

[0694] The expression of CLEC5A was evaluated in tumor-associated macrophages (TAMs) from human solid tumors. Specifically, frozen human isolated tumor cells (Discovery Life Sciences) from six solid tumor indications (renal, lung, ovarian, colorectal, cholangiocarcinoma, and pancreatic) were thawed and stained with a fixable viability dye (Zombie Aqua TM , BioLegend) and then labeled with a panel of antibodies targeting immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, Bio-Legend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), TREM2 (237920, R&D Systems), CD206 (15-2, BioLegend), and CLEC5A (283834, R&D Systems). After staining, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed, resuspended in FACS buffer (PBS containing 2% heat-inactivated FBS and 0.05% BSA), and analyzed by flow cytometry (Attune TM CytPik TM , Invitrogen). A gating strategy was used to characterize non-neutrophil myeloid cells (live / CD45+ / CD19- / CD3- / CD56- / CD15-) in human isolated tumor cells ( Figure 3A ).like Figure 3B As shown in Figure 2, most (~30-60%) non-neutrophil myeloid cells were found to be TAMs due to their dual expression of CD11b and TREM2. Only a portion of TAMs expressed CD206 (>45% of CD11b+ cells). Figure 3C-3D As shown, CLEC5A was highly expressed in the majority (>70%) of TREM2+TAMs and CD206+TAMs.

[0695] Example 4: Generation of CLEC5A Antibodies

[0696] Immunization

[0697] To generate monoclonal antibodies against CLEC5A, two New Zealand white rabbits were immunized with human CLEC5A His-tagged protein according to a 63-day rabbit immunization schedule (Acro Biosystems, Cat#: CLA-H5243). During the first injection, 400 μg of CFA-emulsified antigen was injected subcutaneously on day 1, followed by 3 subcutaneous injections (200 μg of IFA-emulsified antigen) on days 7, 21, and 42, and a final intravenous booster injection was given on day 63.

[0698] Antibody titer

[0699] On days 28 and 49, 5 mL of serum was collected from each rabbit for titer detection. The antibody titer after immunization was determined by combining serially diluted serum with ELISA of human CLEC5A. Briefly, 96-well plates were coated with human CLEC5A (1 μg / mL) overnight at 4°C. The wells were blocked with PBS containing 1% BSA (pH 7.4, Fisher Scientific, Cat#: 21-040-CM) at room temperature for 1 hour. The samples (serum diluted 1:1000 and titrated 3 times) were added to the wells and incubated at room temperature for 1 hour, then washed with PBS. HRP donkey anti-rabbit IgG secondary antibody (BioLegend, Cat#: 406401) was then added to the wells and incubated at room temperature for 1 hour, then washed with PBS. The substrate was added to the wells and reacted for 5 minutes. Use an enzyme reader OD readings were measured at 450 nm.

[0700] Rabbit splenocyte isolation and B cell sorting

[0701] Rabbit spleens were collected on the fifth day after the last booster injection. Splenocytes were prepared in a sterile cell strainer placed at the bottom of a 100 mm sterile culture dish containing 20 mL RPMI + 1% penicillin / streptomycin (P / S). Sterile tweezers were used to transfer spleen tissue to the cell strainer. Specifically, the spleen was clamped with tweezers, cut into small pieces, and then pressed through the mesh of the cell strainer. The tissue fragments were washed with 10 mL RPMI + 1% P / S. Splenocytes were transferred from the culture dish to a new 50 mL conical tube. RPMI + 1% P / S was added to a final volume of 50 mL. The cells were centrifuged at 400 × g for 5 minutes, and the supernatant was aspirated. 13 mL ACK buffer (Gibco Cat #: A1049201) was added to resuspend the cells. The suspended cells were incubated at room temperature for 1 minute. RPMI + 1% P / S was added to a final volume of 50 mL. The cells were centrifuged at 400 × g for 5 minutes, and the supernatant was aspirated. Resuspend the cell pellet in RPMI + 10% FBS + 1% P / S. Centrifuge the cells at 400 × g for 5 minutes and aspirate the supernatant. Resuspend the pellet in 15 mL RPMI + 10% FBS + 1% P / S. Transfer the cells through a 100 μm cell strainer into a 50 mL conical tube to remove cell clumps. Then plate the spleen cells at the desired density (e.g., 4 × 10 7 cells / mL) were inoculated into appropriate culture medium for sorting. The remaining spleen cells were cultured at 6×10 7 The density of cells / vial (~1.8 ml) was frozen overnight in 90% serum + 10% DMSO at -80°C. The frozen cells were transferred to a liquid nitrogen tank for long-term storage.

[0702] For B cell isolation, freshly isolated or thawed splenocytes (~2 × 10 8 Splenocytes) were cultured overnight in B cell culture medium (RPMI-1640, 15% FBS, 1×HEPES, 1×2-ME (2-mercaptoethanol), 1% penicillin / streptomycin) and then sorted. The corresponding 96-well B cell culture plates were prepared the day before sorting. On the day of sorting, suspended and loosely attached splenocytes were collected by gently pipetting the culture medium onto the culture surface of the culture flask. The cells were then transferred to a conical tube and centrifuged at 400×g for 3 minutes. The cell pellet was washed twice with fluorescence-activated cell sorting (FACS) buffer (1×PBS+0.5% BSA). Biotinylated antigen was added at 5μg / mL (final concentration). The mixture was incubated at room temperature (RT) for 20 minutes. The staining mixture was then centrifuged at 400×g for 3 minutes, and the cells were resuspended in FACS buffer. The cells were transferred to a 1.5mL amber Eppendorf TMThe staining antibody mixture was then added to the cells. The staining mixture was incubated at 4°C for 15-30 minutes and then centrifuged at 400×g for 3 minutes. The cell pellet was washed twice with FACS buffer. The washed cell pellet was resuspended in 1×PBS + 1% FBS for 10 min. 7 The cells were resuspended at a concentration of 10 cells / mL. Antigen-specific single B cells were sorted into 96-well plates (20 plates per rabbit) using FACS. The 96-well B cell culture plates containing the sorted B cells were cultured at 37°C and 5% CO2 for 12 days.

[0703] ELISA screening of single B cell cultures, LEM supernatants, and purified antibodies

[0704] On the 8th day after sorting, 15 μL B cell culture supernatant was collected from each well for antigen-specific ELISA detection. In brief, the B cell culture supernatant was transferred to a human CLEC5A coated ELISA plate (384-well plate coated with CLEC5A antibody and blocked with BSA). The cells were incubated at room temperature for 1 hour and washed 3 times with PBS plus 0.05% Tween-20. Goat anti-rabbit IgG HRP+TMB substrate (VWR, Cat#: 5120-007) was used to detect antibodies. The B cell supernatant that met the OD450 cutoff value (>0.5 or 3 times higher than the pre-immune serum) was then selected.

[0705] 1920 supernatants from 40 well plates (96-well plate) were screened for human CLEC5A-specific antibodies in sorted B cells by preliminary ELISA. Approximately more than 200 human CLEC5A antigen-binding (ELISA OD cutoff 0.9) B cell clones were identified.

[0706] On day 12 after sorting, the B cell culture plate was centrifuged at 400×g for 3 minutes. The supernatant of positive clones (OD greater than the selected cutoff of 0.9 for antigen-specific ELISA) was collected and the cell pellet was stored in 100 μL DNA / RNA blocking solution (Zymo, Cat#: R1100-250) in a 250 μL PCR tube. The collected supernatant was subjected to additional testing to confirm ELISA and cell surface binding.

[0707] Heavy and light chain genes from positive clones were cloned into Linear Expression Modules (LEMs). The LEMs were then transfected into producer cells and culture supernatants were tested by ELISA as described.

[0708] Screening of cell surface CLEC5A specific antibodies

[0709] CLEC5A is mainly expressed in myeloid cells (monocytes, macrophages, neutrophils and dendritic cells). Peripheral monocytes were isolated from purified PBMCs using the EasySep Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058). Monocyte isolation was performed according to the supplier's instructions. Approximately 50,000 monocytes were incubated on ice for 60 minutes with undiluted B cell or LEM supernatant or serially diluted purified antibodies in FACS buffer (PBS + 0.5% BSA). After incubation, the cells were washed twice and detected with FITC-labeled donkey anti-rabbit IgG (BioLegend, Cat#: 406403) secondary antibody. For analysis, the mean fluorescence intensity (MFI of FITC) of each antibody was determined and plotted using GraphPadprism software (Version 9.4.1; GraphPad Software Inc). The top 20 clones that showed binding to human CLEC5A on the cell surface were selected and cloned into LEM. After testing, 8 clones that bound to human CLEC5A on the cell surface were selected (6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6 and 9E11). Finally, recombinant CLEC5A antibodies were expressed for further functional assays.

[0710] Example 5: Chimeric anti-CLEC5A antibodies and molecular structures

[0711] To express chimeric rabbit / human IgG1 antibodies, human IgG1 heavy chain constant region (hIgG1-Hc-LALAPG variant) and human light chain Kappa constant region (CL-kappa) were synthesized and cloned into pcDNA3.4 (GeneScript). pcDNA3.4 containing hIgG1-Hc-LALAPG of human IgG1 (pcDNA3.4-huIgG1-Hc-LALAPG) was further digested with EcoRI / NheI to clone the VH sequence. The VL sequence of the vector pcDNA3.4 (pcDNA3.4-huKappa-Lc) expressing human CL-κ was obtained by digestion with EcoRI / BsiWI. The VH and VL sequences of the selected eight rabbit anti-CLEC5A antibodies 6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6, and 9E11 and the VH and VL sequences of the reference antibody DX244 were synthesized by IDT, and the sequences were designed to overlap at the 5′ and 3′ ends to allow annealing and assembly with the ends of the corresponding vectors (NEB HiFi DNA Assembly). The assembled plasmid was transformed into competent E. coli cells ( 5-alpha), and the correct sequence was cloned according to the sequencing results (ElimBiopharm), and further cultured with LB containing carbenicillin (100 μg / mL) for plasmid purification (QIAGEN Plasmid Plus Kits). The plasmid was eluted with nuclease-free water (Sigma) and stored at -80°C.

[0712] Example 6: Transfection and purification of chimeric anti-CLEC5A antibodies

[0713] All antibody light and heavy chain coding sequences were generated by direct DNA synthesis and cloned into mammalian expression vectors. The cloned sequences were verified by Sanger sequencing. Chinese hamster ovary (CHO) cells were grown in CHOgro medium (Mirus) supplemented with 4 mM L-glutamine and 0.33% Poloxamer-188 and diluted to 4 × 10 with fresh cell culture medium at the time of transfection. 6 cells / mL. Every 4×10 6 Each cell was transfected with 1 μg of plasmid DNA by lipofection. The transfected cells were cultured in a shaking incubator at 32°C, 5% CO2, 70% humidity and 125 rpm.

[0714] Protein expression titer was measured every 5 days. Cell supernatant was collected by centrifugation 10-14 days after transfection and filtered through a 0.22 μm PES filter. The expressed antibody was purified by AF-r Protein A HC-650M resin, Tosoh) and the aggregates were removed by anion exchange resin (TOYOPEARL NH2-750F, Tosoh).

[0715] By Software-controlled HPLC (Dionex Purified antibodies were analyzed by 3000-RSUHPLC, ThermoFisher. Antibody purity and aggregation were analyzed using a size exclusion column TSKgel UP-SW3000, 2 μm, 4.6 mm ID×15 cm (Tosoh). All purified antibodies had a monomer purity of 97% or higher, were sterilized by filtration using a 0.22 μm PES filter and used for binding and functional assays.

[0716] Example 7: Binding of chimeric anti-CLEC5A antibodies to human macrophages

[0717] Evaluation of the ability of anti-CLEC5A antibodies (antibodies with silent Fc) to bind to human macrophages. CD14+ monocytes (using EasySep TMHuman monocyte enrichment kit (CD16 not removed, StemCell Technologies, Cat#: 19058) was purified from human PBMC donor #651) and differentiated into M0 macrophages after 7 days of action of 50ng / mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). Approximately 50,000 M0 macrophages were incubated with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) on ice for 30 minutes. After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and graphed using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown Figure 4 As shown in the table below, the selected eight anti-CLEC5A antibodies can bind to M0 macrophages (CLEC5A+) cells with different affinities.

[0718] Table 1: Binding of anti-CLEC5A antibodies to human macrophages

[0719] Anti-CLEC5A antibody Fc-silent IC50 binding (nM) 6A5 9.80 6G9 6.84 14A2 3.99 5C7 3.93 7G10 2.06 3A7 0.96 13E6 2.57 9E11 1.59 DX244 2.80 Negative control -

[0720] Example 8: TNFα Release by Chimeric Anti-CLEC5A Antibodies

[0721] Evaluation of TNFα release by anti-CLEC5A antibody. CD14+ monocytes (using EasySep TMHuman monocyte enrichment kit (non-CD16 depleted, StemCell Technologies, Cat#: 19058) purified from human PBMC donor #651) was differentiated with 50ng / mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 6 days, and then differentiated into M1 with 50ng / mL M-CSF and 50ng / mL IFN-… (StemCell Technologies, Cat#: 78020) for 24 hours. Anti-CLEC5A antibody was serially diluted and coated on 96-well plates at 4°C for 24 hours. After incubation, the culture plates were washed with PBS. Approximately 100,000 M1 macrophages were added to the anti-CLEC5A antibody-coated wells in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin / streptomycin). After 24 hours, the supernatant was collected and TNFα was measured using a human TNFα ELISA kit (R&D systems, Cat#: DY210-05) according to the supplier's instructions. For analysis, TNFα levels (in pg / mL) were determined and plotted using GraphPad Prism software (Version 9.4.1, GraphPad Software Inc.). Figure 5 As shown, all eight anti-CLEC5A antibodies exhibited moderate levels of TNFα release, which was lower than the benchmark DX244, suggesting that anti-CLEC5A antibodies may be safe.

[0722] Example 9: Binding of chimeric anti-CLEC5A antibodies to mouse CLEC5A

[0723] ELISA binding was used to evaluate whether the CLEC5A antibody with a silent Fc could cross-react with the recombinant mouse CLEC5A protein. Corning high-binding flat-bottom 96-well culture plates (Cat#: 3361) were coated with 1 μg / mL mouse CLEC5A (R&D Systems, Cat#: 8438-CL-050) and placed at 4°C for 48 hours. The protein antigen was aspirated and washed with 400 μL of wash buffer (PBS containing 0.05% 20) Wash the wells 3 times. Prepare the CLEC5A Fc-silent antibody as a primary antibody at 5 μg / mL and dilute it 3 times to 11 points (dilution concentration) with reagent buffer (PBS + 0.05% BSA). Add 100 μL of primary antibody to the corresponding wells and incubate at room temperature for 1 hour. Aspirate the primary antibody and wash with 400 μL of washing buffer (PBS containing 0.05% 20) Wash the wells 3 times. Dilute the secondary antibody BioLegend Donkey Anti-Human HRP (Cat#: 410902) with reagent buffer (PBS + 0.5% BSA) at 1:10,000, add 100 μL to each well, and incubate at room temperature for 30 minutes. Aspirate the secondary antibody and wash with 400 μL washing buffer (PBS containing 0.05% 20) Wash the wells 3 times. Add 100 μL Thermo 1-step TMB Turbo (Cat#: 34022) to each well and incubate at room temperature for 10 minutes in the dark. Add 100 μL Fisher 1N Sulfuric Acid (Cat#: SA212-2) stop solution to each well and read the data in the plate at 450 nm using a microplate reader. Use GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) to plot the OD450 of each antibody for analysis. Figure 6 As shown in the table below, anti-CLEC5A antibodies 6A5, 14A2, and 5C7 exhibit cross-reactivity with mouse CLEC5A.

[0724] Table 2: Binding of anti-CLEC5A antibodies to mouse CLEC5A

[0725] Anti-CLEC5A Antibody IC50 binding (nM) 6A5 0.1205 14A2 0.1844 5C7 0.1936 DX244 51.18 Negative control -

[0726] For BLI (biolayer interferometry) binding, 10 μg / mL anti-CLEC5A antibody was captured on an anti-human IgG Fc probe (Gator Bio, Cat#: 160024), and then the binding and dissociation to recombinant human CLEC5A, HisTag (ACRO Biosystems, Cat#: NC1-H52H4) was measured at a single concentration of 11 nM in PBS + 0.05% 20. Determine the absolute KD by applying a 1:1 binding model. BLI binding assay results are shown in Figure 7A-7B The results showed that the anti-CLEC5A antibodies 3A7 and 5C7 could bind to human CLEC5A with high affinity.

[0727] Example 10: Humanization of chimeric anti-CLEC5A antibodies and ELISA binding

[0728] The CDRs of the primary antibody were transplanted to the closest sequence identified by IgBLAST ( www.ncbi.nlm.nih.gov / igblast / ) and / or IMGT / DomainGapAlig( www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgiFour rabbit anti-human CLEC5A monoclonal antibody clones 3A7, 5C7, 6A5, 7G10 and 13E6 were humanized in selected human germline frameworks of rabbit frameworks recognized by ). Based on sequence similarity within the framework and CDRs, human germline IGHV, IGKV, IGHJ and IGKJ were selected. Certain human germline framework residues were backmutated to corresponding rabbit residues to maintain typical loop structures and light chain / heavy chain interfaces (Padlan E.A., Mol. Immunol., 1994, 31: 169; Foote J. and Winter G., JMB, 1992, 224: 487). By humanization, h3A7, h5C7, h6A5, h7G10 and h13E6 humanized antibodies were generated.

[0729] ELISA binding was performed to evaluate whether the humanized anti-CLEC5A antibodies were able to bind to the recombinant human CLEC5A protein compared to the corresponding chimeric parental clone. Corning high binding flat bottom 96-well culture plates (Cat#: 3361) were coated with 1 μg / mL human CLEC5A (ACROBiosystems, Cat#: CLA-H5243) and placed at 4°C for 48 hours. The protein antigen was aspirated and washed with 400 μL of wash buffer (0.05% 20) Wash the wells 3 times. Prepare the anti-CLEC5A antibody as a primary antibody at 5 μg / mL and serially dilute 3-fold to 11 points (dilution concentration) with reagent buffer (PBS + 0.05% BSA). Add 100 μL of primary antibody to the corresponding wells and incubate at room temperature for 1 hour. Aspirate the primary antibody and wash with 400 μL of washing buffer (0.05% 20) Wash the wells 3 times. Dilute the secondary antibody BioLegend Donkey Anti-Human HRP (Cat#: 410902) in reagent buffer (PBS + 0.5% BSA) at a ratio of 1:10,000, add 100 μL to each well, and incubate at room temperature for 30 minutes. Aspirate the secondary antibody and wash with 400 μL washing buffer (PBS with 0.05% 20) Wash the wells 3 times. Add 100 μL Thermo 1-step TMB Turbo (Cat#: 34022) to each well and incubate at room temperature in the dark for 10 minutes. Add 100 μL Shafer 1N Sulfuric Acid (Cat#: SA212-2) stop solution to each well and read at 450 nm using a microplate reader. Use GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) to plot the OD450 of each antibody and perform analysis. Figures 8A-8EAs shown in the table below, all humanized anti-CLEC5A antibodies showed similar affinity to human CLEC5A protein compared with the corresponding chimeric parental clones, indicating that the humanization of the above clones was successful.

[0730] Table 3: Binding of humanized anti-CLEC5A antibodies to human CLEC5A (ELISA)

[0731] Antibody cloning Chimera (ch) Humanization (h) 3A7 0.71 0.47 5C7 0.90 0.44 6A5 0.31 0.33 7G10 0.16 0.22 13E6 0.61 0..12

[0732] Example 11: Molecular construction of TAA / CLEC5A bispecific antibody

[0733] like Figures 9A-9F As shown, six different forms of TAA / CLEC5A bispecific antibodies were generated. According to the sequencing results (Elim Biopharm), competent E. coli ( 5-alpha) cells. The transformed cells were further cultured with LB containing carbenicillin (100 μg / mL) to purify the plasmid ( Plasmid Plus Kits). Plasmids were eluted in nuclease-free water (Sigma) and stored at -80°C.

[0734] CHO cells (ExpiCHO) were transfected with pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc containing paired VH and VL sequences. TM Expression system, Gibco) to express antibodies. ExpiCHO TM ExpiCHO TM The cells were cultured in expression medium and maintained at 0.3 to 6 × 10 cells / mL at 37°C, 125 rpm, 5% CO2, and 80% humidity according to the supplier's instructions. 6 In a 125 mL baffled flask, 3 × 10 6 Prepare 25 mL of fresh ExpiCHO from 1-day culture of 1 mL inoculum TM Cells (6×10 6 / mL, activity>95%). 1 mL of serum-free culture medium (OptiPRO TM SF...

Claims

1. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is cluster of differentiation 79b (CD79b); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate myeloid cells (e.g., monocytes and / or macrophages) (e.g., M0, M1, M1 and / or M2 macrophages)) killing of target cells; (2) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell: target cell), and optionally, the low E: T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10: 1; and (3) The antibody or antigen-binding fragment can induce myeloid cells (e.g., monocytes and / or macrophages) Low levels of cytokine (e.g., IL-6 or TNFα) release, e.g., less than 10% compared to a control antibody 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release.

2. The antibody or antigen-binding fragment of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 200, 202, and 204, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 205-207, respectively; or (2) The amino acid sequences of the selected VH1CDRs 1, 2, and 3 are listed in SEQ ID NOs: 201, 203, and 204, respectively. The amino acid sequences of selected VL1CDRs 1, 2, and 3 are listed in SEQ ID NOs: 205-207, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

3. The antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker, and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain contains one or more hole mutations; and the second heavy chain contains one or more knob mutations.

5. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a drug for treating a subject, The subject has a hematological tumor, preferably, the hematological tumor is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma; or The subject suffers from an autoimmune disease, preferably, the autoimmune disease is rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's disease, cold agglutinin disease, warm autoimmune hemolytic anemia and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), and / or microscopic polyangiitis (MPA).

6. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain, which specifically binds to a first antigen, wherein the first antigen is epithelial cell adhesion molecule (EpCAM); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages (e.g., M0, M1, M1 and / or M2 macrophages)); (2) the antibody or antigen-binding fragment can mediate killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell:target cell), optionally wherein the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10:1; and (3) The antibody or antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release from myeloid cells (e.g., monocytes and / or macrophages), for example, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release compared to a control antibody.

7. The antibody or antigen-binding fragment of claim 6, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 150, 152, and 154, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 155-157, respectively; or (2) The amino acid sequences of the selected VH1CDR 1, 2, and 3 are listed in SEQ ID NOs: 151, 153, and 154, respectively. The amino acid sequences of selected VL1CDRs 1, 2, and 3 are listed in SEQ ID NOs: 155-157, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

8. The antibody or antigen-binding fragment according to any one of claims 6-7, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

9. The antibody or antigen-binding fragment according to any one of claims 6 to 8, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker, and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain contains one or more hole mutations; and the second heavy chain contains one or more knob mutations.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 6 to 9 in the preparation of a drug for treating a subject, The subject has a solid tumor, preferably, the solid tumor is breast cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer, and / or liver cancer; or The subject suffers from malignant ascites and / or malignant pleural effusion.

11. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is epidermal growth factor receptor (EGFR); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages (e.g., M0, M1, M1 and / or M2 macrophages)); (2) the antibody or antigen-binding fragment can mediate killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell:target cell), optionally wherein the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10:1; and (3) The antibody or antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release from myeloid cells (e.g., monocytes and / or macrophages), for example, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release compared to a control antibody.

12. The antibody or antigen-binding fragment of claim 11, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 126, 128, and 130, respectively, and the amino acid sequences of the selected VL1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 131-133, respectively; (2) the amino acid sequences of the selected VH1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 127, 129, and 130, respectively, and the amino acid sequences of the selected VL1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 131-133, respectively; (3) The amino acid sequences of the selected VH1CDR 1, 2, and 3 are listed in SEQ ID NOs: 136, 138, and 140, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 141-143, respectively; or (4) the amino acid sequences of the selected VH1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 137, 139, and 140, respectively, and the amino acid sequences of the selected VL1 CDR 1, 2, and 3 are listed in SEQ ID NOs: 141-143, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

13. The antibody or antigen-binding fragment according to any one of claims 11-12, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

14. The antibody or antigen-binding fragment of any one of claims 11-13, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment according to claim 13, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain comprises one or more hole mutations; and the second heavy chain comprises one or more knob mutations.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 11 to 14 in the preparation of a drug for treating a subject, The subject has a solid tumor, preferably, the solid tumor is breast cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer, and / or liver cancer.

16. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is cluster of differentiation 38 (CD38); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate myeloid cells (e.g., monocytes and / or macrophages) (e.g., M0, M1, M1 and / or M2 macrophages)) killing of target cells; (2) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell: target cell), and optionally, the low E: T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10: 1; and (3) The antibody or antigen-binding fragment can induce myeloid cells (e.g., monocytes and / or macrophages) Low levels of cytokine (e.g., IL-6 or TNFα) release, e.g., less than 10% compared to a control antibody 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release.

17. The antibody or antigen-binding fragment of claim 16, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 188, 190, and 192, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 193-195, respectively; or (2) the amino acid sequences of the selected VH1CDR 1, 2, and 3 are listed in SEQ ID NOs: 189, 191, and 192, respectively, and the amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 193-195, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

18. The antibody or antigen-binding fragment according to any one of claims 16-17, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

19. The antibody or antigen-binding fragment of any one of claims 16-18, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment according to claim 13, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain comprises one or more hole mutations; and the second heavy chain comprises one or more knob mutations.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 16 to 19 in the preparation of a drug for treating a subject, The subject has a hematological tumor, preferably, the hematological tumor is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma; or The subject suffers from an autoimmune disease, preferably, the autoimmune disease is rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's disease, cold agglutinin disease, warm autoimmune hemolytic anemia and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), and / or microscopic polyangiitis (MPA).

21. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is G protein coupled receptor class C group 5 member D (GPRC5D); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate myeloid cells (e.g., monocytes and / or macrophages) (e.g., M0, M1, M1 and / or M2 macrophages)) killing of target cells; (2) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell: target cell), and optionally, the low E: T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10: 1; and (3) The antibody or antigen-binding fragment can induce myeloid cells (e.g., monocytes and / or macrophages) Low levels of cytokine (e.g., IL-6 or TNFα) release, e.g., less than 10% compared to a control antibody 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release.

22. The antibody or antigen-binding fragment of claim 21, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 164, 166, and 168, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 169-171, respectively; or (2) The amino acid sequences of the selected VH1CDR 1, 2, and 3 are listed in SEQ ID NOs: 165, 167, and 168, respectively. The amino acid sequences of selected VL1CDRs 1, 2, and 3 are listed in SEQ ID NOs: 169-171, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

23. The antibody or antigen-binding fragment according to any one of claims 21-22, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

24. The antibody or antigen-binding fragment of any one of claims 21-23, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment according to claim 13, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain comprises one or more hole mutations; and the second heavy chain comprises one or more knob mutations.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 21 to 24 for the preparation of a drug for treating a subject, The subject has a hematological tumor, preferably, the hematological tumor is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma; or The subject suffers from an autoimmune disease, preferably, the autoimmune disease is rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's disease, cold agglutinin disease, warm autoimmune hemolytic anemia and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), and / or microscopic polyangiitis (MPA).

26. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is B cell maturation antigen (BCMA); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate myeloid cells (e.g., monocytes and / or macrophages) (e.g., M0, M1, M1 and / or M2 macrophages)) killing of target cells; (2) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T ratio (effector cell: target cell), and optionally, the low E: T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10: 1; and (3) The antibody or antigen-binding fragment can induce myeloid cells (e.g., monocytes and / or macrophages) Low levels of cytokine (e.g., IL-6 or TNFα) release, e.g., less than 10% compared to a control antibody 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release.

27. The antibody or antigen-binding fragment of claim 26, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2); The VH1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR2, and the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, the VL1 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH1 CDR3, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDRs 1, 2, and 3 are listed in SEQ ID NOs: 176, 178, and 180, respectively. The amino acid sequences of the selected VL1CDR 1, 2, and 3 are listed in SEQ ID NOs: 181-183, respectively; or (2) The amino acid sequences of the selected VH1CDR 1, 2, and 3 are listed in SEQ ID NOs: 177, 179, and 180, respectively. The amino acid sequences of selected VL1CDRs 1, 2, and 3 are listed in SEQ ID NOs: 181-183, respectively; The VH2 comprises CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR2, and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, the VL2 comprises CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to the selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to the selected VH2 CDR3, wherein the selected VH2 CDR 1, 2 and 3 amino acid sequences and the selected VL2 CDR 1, 2 and 3 amino acid sequences are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

28. The antibody or antigen-binding fragment according to any one of claims 26-27, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; preferably, the Fc region is the Fc region of human IgG1; preferably, the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid at position 332 (E)； (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid at position 332 (E); and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated.

29. The antibody or antigen-binding fragment of any one of claims 26-28, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv), wherein VH2 and VL2 are connected by a first linker and the second antigen-binding domain is connected to the C-terminus of the light chain or the C-terminus of the Fc region by a second linker; Optionally, the antibody or antigen-binding fragment according to claim 13, wherein: (1) the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain comprises one or more hole mutations; and the second heavy chain comprises one or more knob mutations.

30. Use of the antibody or antigen-binding fragment thereof according to any one of claims 26 to 29 for the preparation of a drug for treating a subject, The subject has a hematological tumor, preferably, the hematological tumor is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma; or The subject suffers from an autoimmune disease, preferably, the autoimmune disease is rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's disease, cold agglutinin disease, warm autoimmune hemolytic anemia and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), and / or microscopic polyangiitis (MPA).

31. An antibody or antigen-binding fragment that binds CLEC5A (C-type lectin domain family 5 member A), comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3; and a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VCDR1, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3, wherein the selected VH CDR 1, 2 and 3 amino acid sequence and the selected VL CDR 1, 2 and 3 amino acid sequence are one of the following: (1) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, 47, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, 57, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, 67, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively.

32. The antibody or antigen-binding fragment of claim 31, wherein an antibody or antigen-binding fragment that binds to CLEC5A comprises: The heavy chain variable region (VH) comprises an amino acid sequence at least 90% identical to a selected VH, and the light chain variable region (VL) comprises an amino acid sequence at least 90% identical to a selected VL, wherein the selected VH and selected VL sequences are one of the following: (1) the selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (2) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (3) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; (4) the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42; (5) the selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52; (6) the selected VH sequence is SEQ ID NO: 61, and the selected VL sequence is SEQ ID NO: 62; (7) the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72; (8) the selected VH sequence is SEQ ID NO: 81, and the selected VL sequence is SEQ ID NO: 82; (9) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; (10) the selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86; (11) the selected VH sequence is SEQ ID NO: 87, and the selected VL sequence is SEQ ID NO: 88; (12) the selected VH sequence is SEQ ID NO: 89, and the selected VL sequence is SEQ ID NO: 90; (13) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO:

92.

33. An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is a tumor-associated antigen (TAA); and (ii) a second antigen-binding domain that specifically binds to CLEC5A, wherein the second antigen-binding domain is selected from the antibody or antigen-binding fragment of claims 1-2; or (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is an autoimmune disease target; and (ii) a second antigen binding domain, which specifically binds to CLEC5A.

34. The antibody or antigen-binding fragment of claim 33, which has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages (e.g., M0, M1, M1 and / or M2 macrophages)); (2) the antibody or antigen-binding fragment can mediate killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) having a low E:T ratio (effector cell:target cell), optionally wherein the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10:1; and (3) The antibody or antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release from myeloid cells (e.g., monocytes and / or macrophages), for example, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release compared to a control antibody.

35. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-34.

36. A vector comprising the nucleic acid of claim 35.

37. A cell comprising the vector of claim 36.

38. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, and a pharmaceutically acceptable carrier.

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