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 antibodies or antigen-binding fragments thereof comprising 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 and a Fc region. The present disclosure also relates to antibodies or antigen-binding fragments thereof comprising a first antigen-binding domain that specifically binds to an autoimmune disease target and a second antigen-binding domain that specifically binds to CLEC5A and a Fc region.

Description

[0001] This application is a divisional application. The original application was filed on July 12, 2024, with application number 202480032146.6 (international application number PCT / US2024 / 037706), entitled "Multispecific antibodies and their uses". Technical Field

[0002] This invention relates to anti-CLEC5A (member A of the C-type lectin domain family 5), its antigen-binding fragment, antibody-drug conjugates derived therefrom, and their uses. This invention also relates to anti-CLEC5A multispecific antibodies (e.g., bispecific antibodies or their antigen-binding fragments), and antibody-drug conjugates derived therefrom. Background Technology

[0003] 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 myeloid lineages, including macrophages, monocytes, neutrophils, and dendritic cells (DCs). As a pattern recognition receptor, CLEC5A transduces signals into the cytoplasm via non-covalent 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 pro-inflammatory cytokines, including IL-6, TNF, CCL3, and CXCL8.

[0004] CLEC5A can trigger myeloid cell-associated immune responses and is associated with a variety of infections and inflammatory diseases. In flavivirus infections, particularly dengue and Japanese encephalitis virus infections, CLEC5A promotes the production of high levels of pro-inflammatory cytokines and chemokines. Anti-CLEC5A monoclonal antibodies or CLEC5A inhibitors can reverse disease progression, suggesting that CLEC5A is a promising therapeutic target for flavivirus infections. Furthermore, similar to some autoimmune diseases, high levels of CLEC5A are found in active rheumatoid arthritis, and CLEC5A activators increase pro-inflammatory cytokine levels. CLEC5A is also a key factor in cancer development and progression. In high-grade severe ovarian cancer (HGSOC), gastric cancer, and glioma, abnormally high expression of CLEC5A is significantly associated with reduced overall survival.

[0005] Bispecific antibodies are artificial proteins that can simultaneously bind to two different types of antigens or two different antigenic epitopes. 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 target sites. The approval of Catumaxomab (anti-EpCAM and anti-CD3) and Blinatumomab (anti-CD19 and anti-CD3) marks a significant milestone in the development of bispecific antibodies.

[0006] Because bispecific antibodies have multiple applications, there is a need to continue developing various treatment methods based on bispecific antibodies.

[0007] Given the important role of CLEC5A in the immune system, it is necessary to develop therapeutics targeting CLEC5A, especially bispecific antibodies against CLEC5A. Summary of the Invention

[0008] This invention relates to anti-CLEC5A antibodies, their antigen-binding fragments, and their uses. The invention also relates to multispecific (e.g., bispecific) antibodies or their antigen-binding fragments, wherein the antibody or its antigen-binding fragment specifically binds to tumor-associated antigens (TAAs) and human C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the antibody or its antigen-binding fragment has an enhanced Fc. In some embodiments, the antibody or its antigen-binding fragment has increased binding affinity for FcγRIIa receptors and / or FcγRIIIa receptors. In some embodiments, the antibody or its antigen-binding fragment has a non-functional Fc.

[0009] Compared to the reference antibody (e.g., DX244) used in this invention, the anti-CLEC5A antibody described herein is not a “typical agonist.” For example, the anti-CLEC5A antibody described herein can mediate macrophage phagocytosis (similar to DX244), but with greater potency and very low cytokine release levels. Furthermore, the TAA / CLEC5A bispecific antibody described herein has been shown to mediate the killing of target cells expressing different TAAs by myeloid cells (e.g., monocytes and macrophages) at a very low E:T ratio, with very low cytokine release. Without being bound by theory, it is conceivable that the anti-CLEC5A antibody and TAA / CLEC5A bispecific antibody described herein can be used to prepare potential myeloid cell conjugates with high efficacy and good safety profiles.

[0010] On one hand, the present invention relates to an antibody or antigen-binding fragment that binds to CLEC5A (member A of the C-type lectin domain family 5), comprising: a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) including CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence; in some embodiments, the selected VH CDR... The amino acid sequences 1, 2, and 3, as well as the selected VL CDR amino acid sequences 1, 2, and 3, are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 3, 5, and 7, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8-10, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 4, 6, and 7, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8-10, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18-20, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18-20, respectively; (5) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 23, 25, and 27, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 28-30, respectively; (6) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 24, 26, and 27, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 28-30, respectively; (7) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 33, 35, and 37, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 38-40, respectively; (8) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 34, 36, and 37, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 38-40, respectively; (9) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 43, 45, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 48-50, respectively; (10) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 44, 46, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 48-50, respectively; (11) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 53, 55, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 58-60, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 58-60, respectively; (13) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 63, 65, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 68-70, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 68-70, respectively; (15) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 73, 75, and 77, respectively; and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 78-80, respectively; and (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 78-80, respectively.

[0011] In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 3, 5, and 7, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 8-10, respectively. In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 18-20, respectively. In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 23, 25, and 27, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 28-30, respectively. In some embodiments, according to the Kabat definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 33, 35, and 37, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 38-40, respectively. In some embodiments, according to the Kabat definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 43, 45, and 47, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 48-50, respectively. In some embodiments, according to the Kabat definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 53, 55, and 57, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 58-60, respectively. In some embodiments, according to Kabat's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 63, 65, and 67, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 68-70, respectively. In some embodiments, according to Kabat's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 73, 75, and 77, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 78-80, respectively. In some embodiments, according to Chothia's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 4, 6, and 7, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, listed in SEQ ID NO: 8-10, respectively.In some embodiments, according to Chothia's definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 18-20, respectively. In some embodiments, according to Chothia's definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 24, 26, and 27, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 28-30, respectively. In some embodiments, according to Chothia's definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 34, 36, and 37, respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 38-40, respectively. In some embodiments, according to Chothia's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 44, 46, and 47, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 48-50, respectively. In some embodiments, according to Chothia's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 54, 56, and 57, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 58-60, respectively. In some embodiments, according to Chothia's definition, VH contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 64, 66, and 67, respectively, and VL contains amino acid sequences of CDRs 1, 2, and 3, which are listed in SEQ ID NO: 68-70, respectively. In some embodiments, according to the definition of Chothia, VH contains amino acid sequences of CDR 1, 2, and 3, which are listed in SEQ ID NO: 74, 76, and 77, respectively, and VL contains amino acid sequences of CDR 1, 2, and 3, which are listed in SEQ ID NO: 78-80, respectively.

[0012] 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: (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; and (13) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92.

[0013] In some embodiments, VH comprises the sequence of SEQ ID NO: 11 and VL comprises the sequence of SEQ ID NO: 12. In some embodiments, VH comprises the sequence of SEQ ID NO: 21 and VL comprises the sequence of SEQ ID NO: 22. In some embodiments, VH comprises the sequence of SEQ ID NO: 31 and VL comprises the sequence of SEQ ID NO: 32. In some embodiments, VH comprises the sequence of SEQ ID NO: 41 and VL comprises the sequence of SEQ ID NO: 42. In some embodiments, VH comprises the sequence of SEQ ID NO: 51 and VL comprises the sequence of SEQ ID NO: 52. In some embodiments, VH comprises the sequence of SEQ ID NO: 61 and VL comprises the sequence of SEQ ID NO: 62. In some embodiments, VH comprises the sequence of SEQ ID NO: 71 and VL comprises the sequence of SEQ ID NO: 72. In some embodiments, VH comprises the sequence of SEQ ID NO: 81 and VL comprises the sequence of SEQ ID NO: 82. In some embodiments, VH comprises the sequence of SEQ ID NO: 83 and VL comprises the sequence of SEQ ID NO: 84. In some embodiments, VH comprises the sequence of SEQ ID NO: 85 and VL comprises the sequence of SEQ ID NO: 86. In some embodiments, VH comprises the sequence of SEQ ID NO: 87 and VL comprises the sequence of SEQ ID NO: 88. In some embodiments, VH comprises the sequence of SEQ ID NO: 89 and VL comprises the sequence of SEQ ID NO: 90. In some embodiments, VH comprises the sequence of SEQ ID NO: 91 and VL comprises the sequence of SEQ ID NO: 92.

[0014] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human, mouse, or monkey CLEC5A.

[0015] In some embodiments, the antibody or its antigen-binding fragment can mediate macrophage phagocytosis (e.g., having 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).

[0016] In some embodiments, the antibody or its antigen-binding fragment 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)).

[0017] In one aspect, the present invention relates to antibodies or antigen-binding fragments thereof that bind to CLEC5A. In some embodiments, the antibody or antigen-binding fragment thereof can mediate macrophage phagocytosis. In some embodiments, the antibody or antigen-binding fragment thereof can mediate macrophage phagocytosis (e.g., having a target cell killing effect of 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% 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% compared to cytokine release induced by a reference antibody (e.g., DX244)).

[0018] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, comprising: i) A first antigen-binding domain that specifically binds to a first antigen, wherein in some embodiments, the first antigen is a tumor-associated antigen (TAA); and ii) Specifically binds to the second antigen-binding domain of C-type lectin domain family 5 member A (CLEC5A).

[0019] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, comprising: i) A first antigen-binding domain that specifically binds to a first antigen, wherein in some embodiments, the first antigen is a target of an autoimmune disease; and ii) Specifically binds to the second antigen-binding domain of CLEC5A.

[0020] In some embodiments, the antibody or its antigen-binding fragment has one or more of the following functions: i) Antibodies or their antigen-binding fragments can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages (e.g., M0, M1 and / or M2 macrophages)); ii) The antibody or its antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T (effective 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 iii) The antibody or its antigen-binding fragment can mediate low levels of cytokine (e.g., IL-6 or TNFα) release in myeloid cells (e.g., monocytes and / or macrophages), for example, mediating low levels of cytokine release in myeloid cells at 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 the levels induced by the reference antibody.

[0021] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes 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 via a second linker. In some embodiments, the antibody or antigen-binding fragment thereof described herein further includes an Fc region. In some embodiments, the C-terminus of VH1 of the first antigen-binding domain is connected to the Fc region, optionally via a CH1 domain. In some embodiments, the C-terminus of 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 includes a first heavy chain containing VH1; a first light chain containing VL1; a second heavy chain containing VH2; and a second light chain containing VL2. In some embodiments, the first heavy chain contains one or more pestle mutations; the second heavy chain contains one or more mortar mutations.

[0022] 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 contains one or more of the following amino acid residues (all numbers are in EU format): (1) Alanine at position 236 (A); leucine at position 330 (L) and glutamic acid at position 332 (E); (2) Alanine at position 236 (A); Aspartic acid at position 293 (D); Leucine at position 330 (L) and Glutamic acid at position 332 (E); (3) Alanine at position 236 (A); (4) Alanine at position 236 (A); aspartic acid at position 293 (D); and glutamic acid at position 332 (E); (5) Aspartic acid at position 293 (D) and glutamic acid at position 332 (E); (6) Aspartic acid at position 293 (D); leucine at position 330 (L) and glutamic acid at position 332 (E); and (7) Alanine at position 234 (A); Alanine at position 235 (A) and glycine at position 329 (G); Alternatively, the Fc region is deglycosylated.

[0023] In some embodiments, tumor-associated antigens (TAAs) are HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycoglucan, globulin-based 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, and 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 gangliosides, glycoprotein 75, GPNMB, HGF, human scattering factor receptor kinase, IGF-I receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1, MUC1, mucin Canag, N-glycoside neuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-RA, TWEAK, phosphatidylserine, prostate cancer cells, Rank1, 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.

[0024] In some embodiments, the targets of autoimmune diseases are 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, and VEGFR.

[0025] In some embodiments, TAA is HER2. (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 100, 102, and 104, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 105-107, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 101, 103, and 104, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 105-107, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein.

[0026] In some embodiments, the target of the TAA or autoimmune disease is CD79b. (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 200, 202, and 204, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205-207, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 201, 203, and 204, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205-207, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein.

[0027] In some embodiments, TAA is EGFR. (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 126, 128, and 130, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 131-133, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 127, 129, and 130, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 131-133, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; (3) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 136, 138, and 140, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 141-143, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (4) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 137, 139, and 140, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 141-143, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0028] In some embodiments, TAA is EpCAM. In some embodiments, (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 150, 152, and 154, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 155-157, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 151, 153, and 154, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 155-157, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences and the selected VL2 CDR 1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0029] In some embodiments, the TAA or autoimmune disease target is GPRC5D. In some embodiments, (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 164, 166, and 168, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 169-171, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 165, 167, and 168, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 169-171, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein.

[0030] In some embodiments, the target of a TAA or autoimmune disease is BCMA. (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 176, 178, and 180, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 181-183, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 177, 179, and 180, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 181-183, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein.

[0031] In some embodiments, the target of the TAA or autoimmune disease is CD38. (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 188, 190, and 192, respectively; the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 193-195, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein; or (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 189, 191, and 192, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 193-195, respectively; the selected amino acid sequences of VH2 CDR 1, 2, and 3 and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are selected from one of the sequences described herein.

[0032] In one aspect, the present invention relates to antibody or antigen-binding fragments that cross-compete with the antibody or antigen-binding fragments described herein.

[0033] In one aspect, the present invention relates to nucleic acids comprising polynucleotides encoding antibodies or antigen-binding fragments thereof described herein.

[0034] In one aspect, the present invention relates to a vector comprising the nucleic acid described herein. In another aspect, the present invention relates to a cell comprising the vector described herein.

[0035] In one aspect, the present invention relates to a method for reducing the growth rate of tumors, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0036] In one aspect, the present invention relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0037] In one aspect, the present invention relates to a method for increasing the immune response of a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0038] In one aspect, the present invention relates to a method of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0039] In some embodiments, the cancer is a solid tumor or a hematologic malignancy. 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 bowel 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 chorioretinopathy, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma.

[0040] In some embodiments, the subject is further treated with effective amounts of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, and / or anti-PD-L1 antibody.

[0041] In one aspect, the present invention relates to a method of treating a subject suffering from an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Goyan disease, cold agglutinin disease, warm autoimmune hemolytic anemia, and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, polyangiitis-associated granulomatosis (GPA) (Wegener's granulomatosis) and microscopic polyangiitis (MPA). In some embodiments, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, and / or rheumatoid arthritis.

[0042] In one aspect, the present invention relates to pharmaceutical compositions comprising an antibody or an antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0043] As used herein, the term "antigen-binding domain" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide, or from two or more polypeptides (e.g., the same or different polypeptides)) capable of specifically binding to one or more different antigens (e.g., effector antigens or tumor antigens). In some examples, an antigen-binding domain may bind to an antigen or epitope with similar specificity and affinity to naturally occurring antibodies. In some embodiments, an antigen-binding domain may be an antibody or a fragment thereof. In some embodiments, an antigen-binding domain is formed by VH-VL. In some embodiments, an antigen-binding domain may include an alternative scaffold. In some embodiments, an antigen-binding domain is a VHH. In some embodiments, an antigen-binding domain may include non-limiting examples of antigen-binding domains described herein. In some instances, an antigen-binding domain may bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0044] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any one of the three CDRs of the immunoglobulin light chain or any one of the three CDRs of the immunoglobulin heavy chain) and capable of specifically binding 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, an antibody may comprise the Fc region of a human antibody. The term "antibody" also includes derivatives such as bispecific antibodies, single-chain antibodies, biantibodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0045] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment includes at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0046] As used herein, the term "multispecific antibody" is an antibody comprising two or more distinct antigen-binding domains that collectively and specifically bind to two or more distinct epitopes. The two or more distinct 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 respects, multispecific antibodies bind two distinct epitopes (i.e., "bispecific antibodies"). In some respects, multispecific antibodies bind three distinct epitopes (i.e., "trispecific antibodies"). In some respects, multispecific antibodies bind four distinct epitopes (i.e., "tetraspecific antibodies"). In some respects, multispecific antibodies bind five distinct epitopes (i.e., "pentaspecific antibodies"). Each binding specificity can exist in any suitable valence state. Non-limiting examples of multispecific antibodies are described herein.

[0047] 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.

[0048] 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 this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described 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 case of conflict, this specification (including definitions) shall prevail.

[0049] Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims. Attached Figure Description

[0050] Figure 1A This demonstrates a gating strategy for evaluating immune cell subsets in human PBMCs.

[0051] Figure 1B This shows the expression of CLEC5A in immune cell subsets in human PBMCs.

[0052] Figure 2A This demonstrates a gating strategy for assessing immune cell subsets in human solid tumors.

[0053] Figure 2B-2C The expression of CLEC5A was shown in tumor-associated myeloid cells.

[0054] Figure 3A This demonstrates a gating strategy used to characterize non-neutrophilic bone marrow cells.

[0055] Figure 3B This shows the characteristics of tumor-associated macrophages in human solid tumors.

[0056] Figure 3C-3D This shows the expression of CLEC5A in tumor-associated macrophages in human solid tumors.

[0057] Figure 4 The chimeric anti-CLEC5A antibody demonstrates its binding affinity to human macrophages.

[0058] Figure 5 This demonstrates M1 macrophage-mediated TNFα release in a culture plate coated with a chimeric anti-CLEC5A antibody.

[0059] Figure 6 This demonstrates the binding affinity of the chimeric anti-CLEC5A antibody to mouse CLEC5A.

[0060] Figures 7A-7B The results show the binding of the chimeric anti-CLEC5A antibody to the human CLEC5A BLI.

[0061] Figures 8A-8E The binding of the humanized anti-CLEC5A antibody to human CLEC5A, as determined by ELISA, is shown.

[0062] Figures 9A-9F A schematic diagram showing an example of a TAA / CLEC5A bispecific antibody.

[0063] Figure 10 The binding of the HER2 / CLEC5A bispecific antibody to human macrophages was demonstrated.

[0064] Figure 11 The binding of the TAA / CLEC5A bispecific antibody to human macrophages is shown.

[0065] Figure 12 The CD79b / CLEC5A bispecific antibody was shown to bind to human macrophages.

[0066] Figure 13 The TAA / CLEC5A bispecific antibody is shown to bind to human CLEC5A.

[0067] Figure 14 The HER2 / CLEC5A bispecific antibody is shown to bind to human HER2.

[0068] Figure 15 The EGFR / CLEC5A bispecific antibody was shown to bind to human EGFR.

[0069] Figure 16The binding of the EpCAM / CLEC5A bispecific antibody to DLD-1 cells was demonstrated.

[0070] Figure 17 The CD79b / CLEC5A bispecific antibody was shown to bind to Ramos cells.

[0071] Figures 18A-18C The study showed that the HER2 / CLEC5A bispecific antibody mediated the killing of SK-BR-3 cells by M0 macrophages (E:T ratio of 5:1).

[0072] Figure 19 The study showed that the HER2 / CLEC5AFc bispecific antibody mediated the killing of SK-BR-3 cells by M1 and M2 macrophages (E:T ratio of 5:1).

[0073] Figure 20 This demonstrates the release of INF-γ during the killing of SK-BR-3 cells by M1 and M2 macrophages mediated by HER2 / CLEC5A antibody (E:T ratio of 5:1).

[0074] Figures 21A-21C Displayed in culture medium ( Figure 21A ),plasma( Figure 21B ) or hIgG1 ( Figure 21C In the presence of HER2 / CLEC5A bispecific antibody, M0 macrophages mediated the killing of SK-BR-3 cells.

[0075] Figure 22 The study demonstrated that the EGFR / CLEC5A bispecific antibody mediated the killing of DLD-1 cells by M0 macrophages.

[0076] Figure 23 The study demonstrated that the EpCAM / CLEC5A bispecific antibody mediated the killing of DLD-1 cells by M0 macrophages.

[0077] Figure 24 This shows the expression levels of EpCAM in different tumor cell lines.

[0078] Figure 25A-25F The study demonstrated that the EpCAM / CLEC5A bispecific antibody mediated the killing of cancer cells by M0 macrophages.

[0079] Figure 26 This study demonstrates how different myeloid cell conjugates mediate the killing effect of PBMCs on multiple myeloma cancer cells.

[0080] Figure 27 This shows the expression level of CD79b in malignant lymphoma cell lines and B cells from healthy PBMC donors.

[0081] Figures 28A-28C The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (Experiment 1).

[0082] Figures 29A-29B The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (Experiment 2).

[0083] Figures 30A-30C The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by PBMCs (Experiment 3).

[0084] Figures 31A-31B This shows the IL-6 level during Ramos cell killing mediated by the CD79b / CLEC5A bispecific antibody.

[0085] Figure 32 This shows the TNFα level during Ramos cell killing mediated by the CD79b / CLEC5A bispecific antibody.

[0086] Figures 33A-33C The CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (Experiment 1).

[0087] Figures 34A-34D The CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (Experiment 2).

[0088] Figures 35A-35B The CD79b / CLEC5A bispecific antibody mediated the killing of endogenous B cells by PBMCs (Experiment 3).

[0089] Figure 36 The study showed that the CD79b / CLEC5A bispecific antibody mediated the release of TNFα during PBMC killing of endogenous B cells.

[0090] Figures 37A-37F The CD79b / CLEC5A bispecific antibody mediates the killing of Ramos cells by M0 macrophages (Experiment 1).

[0091] Figures 38A-38D This study demonstrates the release of cytokines mediated by the CD79b / CLEC5A bispecific antibody in the killing of Ramos cells by M0 macrophages (Experiment 1).

[0092] Figures 39A-39B The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M0 macrophages at different E:T ratios (Experiment 2).

[0093] Figures 40A-40B This study demonstrates the release of cytokines during the killing of Romas cells by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Experiment 2).

[0094] Figures 41A-41C The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M0 macrophages (Experiment 3).

[0095] Figure 42 This study demonstrates the release of cytokines during the killing of Ramos cells by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Experiment 3).

[0096] Figure 43 This shows the survival rate of M0 macrophages during the killing of Ramos cells by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Experiment 3).

[0097] Figures 44A-44B The CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M0 macrophages at different E:T ratios (Experiment 1).

[0098] Figures 45A-45B The CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M0 macrophages at different E:T ratios (Experiment 2).

[0099] Figures 46A-46D The study showed that the CD79b / CLEC5A bispecific antibody mediated the release of cytokines during the killing of Daudi cells by M0 macrophages.

[0100] Figures 47A-47B The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M1 macrophages (Experiment 1).

[0101] Figures 48A-48B This study demonstrates the release of cytokines mediated by the CD79b / CLEC5A bispecific antibody in the killing of Ramos cells by M1 macrophages (Experiment 1).

[0102] Figure 49 The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M1 macrophages (Experiment 2).

[0103] Figure 50 This demonstrates the release of cytokines during M1 macrophage killing of Ramos cells mediated by the CD79b / CLEC5A bispecific antibody (Experiment 2).

[0104] Figure 51This demonstrates the survival rate of M1 macrophages during M1 macrophage killing of Ramos cells mediated by the CD79b / CLEC5A bispecific antibody (Experiment 2).

[0105] Figure 52 The study demonstrated that the CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M1 macrophages.

[0106] Figure 53 The study demonstrated that the CD79b / CLEC5A bispecific antibody mediated the release of cytokines during the killing of Daudi cells by M1 macrophages.

[0107] Figures 54A-54D The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M2 macrophages (Experiment 1).

[0108] Figures 55A-55D This study demonstrates the release of cytokines mediated by the CD79b / CLEC5A bispecific antibody during M2 macrophage killing of Ramos cells (Experiment 1).

[0109] Figure 56 The CD79b / CLEC5A bispecific antibody mediated the killing of Ramos cells by M2 macrophages (Experiment 2).

[0110] Figure 57 This demonstrates the release of cytokines during M2 macrophage killing of Ramos cells mediated by the CD79b / CLEC5A bispecific antibody (Experiment 2).

[0111] Figure 58 This demonstrates the survival rate of M2 macrophages during M2 macrophage killing of Ramos cells mediated by the CD79b / CLEC5A bispecific antibody (Experiment 2).

[0112] Figure 59 The study showed that the CD79b / CLEC5A bispecific antibody mediated the killing of Daudi cells by M2 macrophages at different E:T ratios.

[0113] Figure 60 The study demonstrated that the CD79b / CLEC5A bispecific antibody mediated the release of cytokines during the killing of Daudi cells by M2 macrophages.

[0114] Figures 61A-61B The CD79b / CLEC5A bispecific antibody was shown to mediate the killing of Ramos cells by monocytes.

[0115] Figures 62A-62B The study demonstrated that the CD79b / CLEC5A bispecific antibody mediates the release of cytokines during the killing of Ramos cells by monocytes.

[0116] Figures 63A-63B This shows the monocyte survival rate during monocyte killing of Ramos cells mediated by the CD79b / CLEC5A bispecific antibody.

[0117] Figures 64A-64B The study demonstrated that the CD79b / CLEC5A bispecific antibody mediated the killing of B cells by monocytes.

[0118] Figures 65A-65B The study demonstrated that the CD79b / CLEC5A bispecific antibody mediates the release of cytokines during monocyte-to-B cell killing.

[0119] Figures 66A-66B This shows the monocyte survival rate during monocyte-to-B cell killing mediated by the CD79b / CLEC5A bispecific antibody. Detailed Implementation

[0120] A multispecific antibody or its antigen-binding fragment is an artificial protein capable of simultaneously binding to two or more different epitopes (e.g., binding to two different antigens). In some embodiments, a multispecific antibody is a bispecific antibody. A bispecific antibody or its antigen-binding fragment may have two arms. Each arm may have a heavy chain variable region and a light chain variable region, forming an antigen-binding domain (or antigen-binding region). The two arms may bind to two different antigens. In some embodiments, an additional antigen-binding domain may be added to the monoclonal antibody (e.g., added to the C-terminus of the light or heavy chain).

[0121] This invention provides several anti-CLEC5A antibodies, their antigen-binding fragments, and methods for using these anti-CLEC5A antibodies and antigen-binding fragments to inhibit tumor growth, treat cancer, and treat autoimmune diseases.

[0122] The present invention relates to multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, which include a first antigen-binding domain that specifically binds to tumor-associated antigens and a second antigen-binding domain that specifically binds to CLEC5A.

[0123] Human C-type lectin domain family 5 member A (CLEC5A) 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 C-type lectin domain, which allows them to express Ca2+ lectins as a protein. 2+ It binds to both glycan and non-glycan ligands in a non-dependent manner. Using Ca... 2+The CTLD that binds to glycans in a glycan-dependent manner is called the "carbohydrate recognition domain" (CRD). Myeloid C-type lectin CLEC5A is a type II membrane protein coupled to a splenic tyrosine kinase (Syk), containing 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, upon activation and phosphorylation by Src, signals via the ITAM-containing DNAX activator protein 12 (DAP12).

[0124] Human CLEC5Am RNA 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, upon activation, recruits DAP10 and DAP12 to bind to CLEC5A. CLEC5A is primarily expressed in myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells, and is further upregulated by interferon-γ (IFN-γ). Furthermore, 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-associated factor 2 (Nrf2), suggesting that CLEC5A is regulated by oxidative stress.

[0125] X-ray crystal structure analysis revealed that CLEC5A is a homodimeric protein when it binds to dengue virus serotypes. Furthermore, the CLEC5A crystal structure exhibits conformational flexibility, indicating that CLEC5A can adopt multiple conformations in vivo, and that its conformation is ligand-dependent.

[0126] NK receptors recognize stress-associated autoantigens and are crucial for immune surveillance, while group V spleen tyrosine kinases in myeloid cells, coupled with C-type lectins, recognize a variety of exogenous and endogenous antigens and participate in host defense, aseptic inflammation, platelet activation, and development. Studies have shown that CLEC5A can interact with the glycan moieties on dengue virus (DV), Japanese encephalitis virus (JEV), and influenza A virus (IAV). Furthermore, 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 inflammatory responses associated with collagen-induced rheumatoid arthritis and concanavalin A-induced liver inflammation. CLEC5A also interacts with exosomes released by activated platelets.

[0127] For a detailed review of CLEC5A and its functions, please 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 these articles is incorporated herein by reference in its entirety.

[0128] Anti-CLEC5A antibody and antigen-binding fragment This invention provides antibodies that specifically bind to CLEC5A (e.g., human CLEC5A) and antigen-binding fragments thereof. 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 domains of human CLEC5A.

[0129] The present invention provides, for example, anti-CLEC5A antibodies 6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6, 9E11, their chimeric antibodies and humanized antibodies thereof.

[0130] The CDR sequences of 3A7 and 3A7-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 3, 5, and 7, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 8-10. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 4, 6, and 7, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 8-10.

[0131] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 13, 15, and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 18-20. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 14, 16, and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 18-20.

[0132] The CDR sequences of 6A5 and 6A5-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 23, 25, and 27, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 28-30. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 24, 26, and 27, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 28-30.

[0133] The CDR sequences of 6G9 and 6G9-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 33, 35, and 37, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 38-40. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 34, 36, and 37, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 38-40.

[0134] The CDR sequences of 7G10 and 7G10-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 43, 45, and 47, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 48-50. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 44, 46, and 47, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 48-50.

[0135] The CDR sequences of 9E11 and 9E11-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 53, 55, and 57, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 58-60. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 54, 56, and 57, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 58-60.

[0136] The CDR sequences of 13E6 and 13E6-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 63, 65, and 67, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 68-70. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 64, 66, and 67, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 68-70.

[0137] The CDR sequences of 14A2 and 14A2-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 73, 75, and 77, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 78-80. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 74, 76, and 77, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 78-80.

[0138] The amino acid sequence of the variable region of the heavy chain of the 3A7 antibody is listed in SEQ ID NO: 11. The amino acid sequence of the variable region of the light chain of the 3A7 antibody is listed in SEQ ID NO: 12.

[0139] The amino acid sequence of the variable region of the heavy chain of the 5C7 antibody is listed in SEQ ID NO: 21. The amino acid sequence of the variable region of the light chain of the 5C7 antibody is listed in SEQ ID NO: 22.

[0140] The amino acid sequence of the variable region of the heavy chain of the 6A5 antibody is listed in SEQ ID NO: 31. The amino acid sequence of the variable region of the light chain of the 6A5 antibody is listed in SEQ ID NO: 32.

[0141] The amino acid sequence of the variable region of the heavy chain of the 6G9 antibody is listed in SEQ ID NO: 41. The amino acid sequence of the variable region of the light chain of the 6G9 antibody is listed in SEQ ID NO: 42.

[0142] The amino acid sequence of the variable region of the heavy chain of the 7G10 antibody is listed in SEQ ID NO: 51. The amino acid sequence of the variable region of the light chain of the 7G10 antibody is listed in SEQ ID NO: 51.

[0143] The amino acid sequence of the variable region of the heavy chain of the 9E11 antibody is listed in SEQ ID NO: 61. The amino acid sequence of the variable region of the light chain of the 9E11 antibody is listed in SEQ ID NO: 62.

[0144] The amino acid sequence of the variable region of the heavy chain of the 13E6 antibody is listed in SEQ ID NO: 71. The amino acid sequence of the variable region of the light chain of the 13E6 antibody is listed in SEQ ID NO: 72.

[0145] The amino acid sequence of the variable region of the heavy chain of the 14A2 antibody is listed in SEQ ID NO: 81. The amino acid sequence of the variable region of the light chain of the 14A2 antibody is listed in SEQ ID NO: 82.

[0146] The percentage of humanization refers to the percentage identity of the heavy or light chain variable region sequence to human antibody sequences in the International Immunogenetic Information System (IMGT) database. In some embodiments, the percentage of humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine popular matches are known in the art and are described, for example, in 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 percentages of humanization generally have several advantages, such as greater safety and efficacy in humans, greater tolerability in human subjects, and / or a lower likelihood of side effects. In some embodiments, the variable region is entirely human, for example derived from human heavy chain immunoglobulin locus sequences (e.g., recombinants of human IGHV, human IGHD, and human IGHJ genes) and / or human κ chain immunoglobulin locus sequences (e.g., recombinants of human IGKV and human IGKJ genes).

[0147] The amino acid sequence of the variable region of the heavy chain of the humanized 3A7 antibody is listed in SEQ ID NO: 83. The amino acid sequence of the variable region of the light chain of the humanized 3A7 antibody is listed in SEQ ID NO: 84.

[0148] The amino acid sequence of the variable region of the heavy chain of the humanized 5C7 antibody is listed in SEQ ID NO: 85. The amino acid sequence of the variable region of the light chain of the humanized 5C7 antibody is listed in SEQ ID NO: 86.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] The amino acid sequences of the heavy chain variable region and light chain variable region of the modified antibody are also provided. 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 may pair with the corresponding light chain variable region sequence to bind together to CLEC5A.

[0153] In addition, in some embodiments, the antibody or its antigen-binding fragment described herein may also contain 1, 2 or 3 heavy chain variable regions (CDRs) selected from the group consisting of: 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: 74, 76, 77; and / or contain 1, 2 or 3 CDRs selected from the group consisting of: The light chain variable regions CDRs are: 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.

[0154] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR3. In some embodiments, the antibody may have light chain variable regions (VLs) of CDRs 1, 2, and 3, wherein CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR1 amino acid sequence, CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR2 amino acid sequence, and CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs 1, 2, and 3 amino acid sequences are shown in Table 22.

[0155] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 3 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 7 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0156] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 4 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 7 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0157] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 13 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 17 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0158] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 14 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 16 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 17 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0159] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 23 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 25 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 27 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0160] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 24 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 27 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0161] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 33 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 37 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0162] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 34 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 37 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0163] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 43 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 47 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0164] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 44 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 47 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0165] In some embodiments, the antibody or antigen-binding fragment 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; and SEQ ID NO: 57 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0166] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 54 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 57 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0167] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 63 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 67 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0168] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 64 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 66 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 67 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0169] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 73 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 75 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 77 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0170] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 74 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 76 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 77 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0171] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 8 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 10 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0172] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 18 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 19 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 20 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0173] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 28 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 30 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0174] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 38 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 40 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0175] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 48 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 49 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 50 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0176] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 58 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 60 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0177] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 68 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 69 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 70 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0178] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO: 78 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 79 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 80 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0179] Insertion, deletion, and replacement 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 and Chothia definitions.

[0180] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VH CDR3.

[0181] 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.

[0182] The present invention also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In some aspects, the antibodies or antigen-binding fragments may bind to the same epitopes as the antibodies described herein.

[0183] This invention also provides antibodies or antigen-binding fragments thereof that cross-compete with any antibody or antigen-binding fragment described herein. Cross-compete 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 1 gp120 exterior envelope glycoprotein." Journal of virology 70.3 (1996): 1863-1872, the entire text of which is incorporated herein by reference. In one aspect, this 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, the entire text of which is incorporated herein by reference.

[0184] The present invention also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises a CDR or has a sequence as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to CLEC5A (e.g., human CLEC5A).

[0185] Anti-CLEC5A antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) or multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies described herein include polyclonal antibodies, monoclonal antibodies, polymeric antibodies, multispecific antibodies (e.g., bispecific), humanized antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly prepared antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments 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 antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0186] Antibody fragments are suitable for the provided methods as long as they retain the affinity and specificity required for a full-length antibody. Therefore, an antibody fragment binding to CLEC5A will retain its ability to bind to CLEC5A. Fv fragments are antibody fragments containing complete antigen recognition and binding sites. The region consists of a dimer composed of a heavy chain and a light chain variable domain tightly bound together, and its properties can be covalent, 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 the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-targeting CDRs) can have the ability to recognize and bind antigens, although its affinity is typically lower than that of the entire binding site.

[0187] Single-chain Fv or scFv antibody fragments contain the VH and VL domains (or regions) of the antibody, which are contained within a single polypeptide chain. Typically, scFv polypeptides also include a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0188] The Fab fragment contains a variable and a constant domain of the light chain and a variable and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked by a hinge cysteine ​​residue near its carboxyl terminus. Other chemical conjugations of antibody fragments are also known in the art.

[0189] The antibodies and antibody fragments of this invention can be modified in the Fc region to provide desired effector function or serum half-life. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).

[0190] In some embodiments, the antibodies described herein or their antigen-binding fragments recognize endogenous CLEC5A or recombinant CLEC5A. In some embodiments, the antibodies described herein or their antigen-binding fragments recognize human CLEC5A (e.g., the extracellular region of human CLEC5A).

[0191] Multispecific antibodies or their antigen-binding fragments In one aspect, this document provides an antibody or an 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 a member of the C-type lectin domain family 5 (CLEC5A). In some embodiments, the antibody or the antigen-binding fragment thereof includes a fragment crystallizable region (Fc region). In some embodiments, the antibody or the antigen-binding fragment thereof is a bispecific antibody.

[0192] In some embodiments, the bispecific antibody or its antigen-binding fragment (e.g., anti-TAA / CLEC5A antibody) specifically binds to tumor-associated antigens and CLEC5A, and such bispecific antibodies have modified or enhanced Fc regions (e.g., Fc regions with GAALIE mutation, LALAPG mutation, S293D+I332E mutation, kihool (KIH) mutation, or fucosylated Fc regions).

[0193] In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises: a first heavy chain having a first heavy chain variable region (VH1); a first light chain having a first light chain variable region (VL1); a second heavy chain having a second heavy chain variable region (VH2); and a second light chain having a second light chain variable region (VL2).

[0194] 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-stranded variable fragment (scFv) or a VHH. In some embodiments, the second antigen-binding domain is scFv or a VHH.

[0195] In some embodiments, the multispecific antibody described herein is designed to have an Fc region containing the LALAPG mutation: alanine (A) at position 234; alanine (A) at position 235; and glycine (G) at position 329, as per EU designation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 subtype structure with the LALAPG mutation (L234A, L235A, and P329G mutations in EU designation). In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region containing alanine (A) at position 234; alanine (A) at position 234; and glycine (G) at position 329, as per EU designation. In some embodiments, the Fc region contains about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0196] In some embodiments, the multispecific antibody described herein is designed to have an Fc region containing a GAALIE mutation: alanine at position 236 (A); leucine at position 330 (L); and glutamic acid at position 332 (E), as designated by EU. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region, wherein leucine at position 330 (L) and glutamic acid at position 332 (E), as designated by EU. In some embodiments, the Fc region contains an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0197] In some embodiments, the multispecific antibody described herein is designed with an Fc region having an aspartic acid (D) at position 239 and a glutamic acid (E) at position 332, as per EU designations. In some embodiments, the multispecific antibody described herein is designed with an IgG1 subtype structure having an S239D+I332E mutation, as per EU designations. In some embodiments, the multispecific antibody described herein is designed with an IgG1 Fc region having an aspartic acid (D) at position 239 and a glutamic acid (E) at position 332, as per EU designations.

[0198] In some embodiments, the multispecific antibodies described herein may be designed as IgG1 subtype structures with a club-and-mortar (KIH) mutation, which can promote heterodimerization and avoid mismatch 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 antibody or its antigen-binding fragment has increased binding affinity for FcγRIIa receptors and / or FcγRIIIa receptors.

[0199] In some embodiments, the multispecific antibodies described herein are designed to have a fucosylated Fc region. Fucosylated antibodies are designed such that the oligosaccharides in the antibody's Fc region do not contain any fucosylate units. When the antibody is fucosylated, antibody-dependent cytotoxicity (ADCC) increases.

[0200] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments 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.

[0201] 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. Figure 9C and Figure 9F A schematic diagram of the structure is shown.

[0202] This document also provides 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 a member of the C-type lectin domain family 5, member A (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to a light chain of the antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the C-terminus of the light chain of the antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the light chain of the antibody or antigen-binding fragment thereof via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Figure 9C A schematic diagram of this structure is shown.

[0203] This document also provides an antibody or an 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 a member of the C-type lectin domain family 5, member A (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the C-terminus of the heavy chain of the antibody or the antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the C-terminus of the Fc region of the antibody or the antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the C-terminus of the Fc region of the antibody or the antigen-binding fragment thereof via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Figure 9F A schematic diagram of this structure is shown.

[0204] This document also provides an antibody or 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 scFvs or VHHs comprise the same amino acid sequence. In some embodiments, the first and second scFvs or VHHs each comprise VH2 and / or VL2. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region. In some embodiments, the first and second heavy chains comprise Fc regions. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is a TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. Figure 9C A schematic diagram of the structure is shown.

[0205] This document also provides an antibody or 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 scFvs or VHHs comprise the same amino acid sequence. In some embodiments, the first and second scFvs or VHHs each comprise VH2 and / or VL2. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region. In some embodiments, the first and second heavy chains comprise Fc regions. In some embodiments, the first scFv or VHH is attached to the C-terminus of the Fc region of the first heavy chain. In some embodiments, the second scFv or VHH is attached to the C-terminus of the Fc region of the second heavy chain. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is a TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. Figure 9F A schematic diagram of this structure is shown in the image.

[0206] This document also provides 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 a member of the C-type lectin domain family 5, member A (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the N-terminus of the heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the N-terminus of the Fc region of the antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the N-terminus of the Fc region of the antibody or antigen-binding fragment thereof via a linker described herein. In some embodiments, the first antigen is a TAA (e.g., HER2). Figure 9D shows a schematic diagram of the structure.

[0207] This document also provides 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 scFv or VHH, and a second Fc region. In some embodiments, scFv or VHH comprises VH2 and / or VL2. In some embodiments, the first Fc region comprises one or more club-shaped mutations, and the second Fc region comprises one or more acetabular mutations. In some embodiments, the first Fc region comprises one or more acetabular mutations, and the second Fc region comprises one or more club-shaped mutations. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is a TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. Figure 9D A schematic diagram of the structure is shown.

[0208] This document also provides an antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain specifically binding to a first antigen; a second antigen-binding domain specifically binding to the first antigen; and a third antigen-binding domain specifically binding to a member of the C-type lectin domain family 5, member A (CLEC5A). In some embodiments, the third antigen-binding domain specifically binding to CLEC5A is linked to the N-terminus of the heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the third antigen-binding domain specifically binding to CLEC5A is linked to the N-terminus of the Fc region of the antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain specifically binding to CLEC5A is linked to the N-terminus of the Fc region of the antibody or antigen-binding fragment thereof 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. Figure 9B A schematic diagram of the structure is shown.

[0209] This document also provides an antibody or an 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 scFv or VHH and a second Fc region. In some embodiments, scFv or VHH comprises VH3 and / or VL3. In some embodiments, the first Fc region comprises one or more club-shaped mutations, and the second Fc region comprises one or more mortise-shaped mutations. In some embodiments, the first Fc region comprises one or more mortise-shaped mutations, and the second Fc region comprises one or more club-shaped 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 a TAA. In some embodiments, VH3 and VL3 bind to a third antigen. In some embodiments, the third antigen is CLEC5A. Figure 9B shows a schematic diagram of this structure.

[0210] In some embodiments, the antibody or its antigen-binding fragment comprises: a first heavy chain containing VH1 and a first light chain containing VL1; and a second heavy chain containing VH2 and a second light chain containing VL2. Figure 9A or Figure 9E shows a schematic diagram of this structure. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, while the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain contains one or more mortar mutations, while the second heavy chain contains one or more club mutations.

[0211] This document also provides an antibody or an 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, wherein the second heavy chain comprises VH2; and a fourth chain comprising a second light chain, wherein the second light chain comprises VL2. Figure 9A shows a schematic diagram of this structure. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is a 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 comprises one or more club-shaped mutations, and the second heavy chain comprises one or more mortis-shaped mutations. In some embodiments, the first heavy chain comprises one or more mortis-shaped mutations, and the second heavy chain comprises one or more club-shaped mutations.

[0212] This document also provides an antibody or an 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 the second heavy chain and scFv or VHH, wherein the second heavy chain comprises VH2; and a fourth chain comprising the second light chain, wherein the second light chain comprises VL2. In some embodiments, scFv or VHH comprises VH3 and / or VL3. Figure 9E shows a schematic diagram of the structure. 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 comprises one or more club-shaped mutations, and the second heavy chain comprises one or more mortise-shaped mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more pestle mutations.

[0213] In some embodiments, CrossMab substitution is 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.

[0214] In some embodiments, CrossMab substitution is introduced in both 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.

[0215] In some embodiments, the multispecific antibody has a heavy chain sequence comprising the wild-type IgG1 Fc region (SEQ ID NO: 95). In some embodiments, the multispecific antibody has a heavy chain sequence comprising about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the Fc region identical to that of SEQ ID NO: 95.

[0216] In some embodiments, the multispecific antibody has a heavy chain sequence comprising an IgG1 Fc region with a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific antibody has a heavy chain sequence comprising about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc region of SEQ ID NO: 96.

[0217] In some embodiments, the multispecific antibody has a heavy chain sequence comprising an IgG1 Fc region (SEQ ID NO: 97) with an optimized mutation. In some embodiments, the multispecific antibody has a heavy chain sequence comprising about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the Fc region identical to that of SEQ ID NO: 97.

[0218] The present invention provides multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies and modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies and human antibodies.

[0219] In some embodiments, a multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or an antibody fragment thereof comprises a combination of anti-HER2 and anti-CLEC5A antigen-binding domains as shown in Table 22.

[0220] In some embodiments, anti-TAA (e.g., anti-HER2 / CLEC5A) antibodies are bispecific antibodies. 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 cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In the method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains, a compensating "cavity" (e.g., alanine or threonine) of the same or similar size as the large side chain is formed at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers beyond other unwanted end products, such as homodimers. The method is described, for example, in WO96 / 27011, the entire text of which is incorporated herein by reference.

[0221] Any multispecific (e.g., bispecific) anti-TAA / CLEC5A antibody or its antigen-binding fragment described herein may be conjugated to a stable molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or solution). Non-limiting examples of stable molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). The conjugation of a stable molecule may increase the half-life or prolong the biological activity 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).

[0222] 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 described herein include polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly prepared antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. 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 (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or antigen-binding fragment is an IgG antibody (e.g., the IgG1 Fc region is shown in SEQ ID NO: 95) or an antigen-binding fragment thereof.

[0223] Multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody fragments are suitable for the provided methods, provided they maintain the required affinity and specificity for both TAA (e.g., HER2) and CLEC5A. Therefore, multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody fragments will retain the ability to bind to both TAA (e.g., HER2) and CLEC5A.

[0224] Tumor-associated antigens Tumor-associated antigens (TAAs) are antigenic substances produced by tumor cells. They can trigger an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells through diagnostic tests and are also potential candidates for cancer treatment. Many tumor-associated antigens are known in the field (see, for example, Yu et al., Cancers (Basel). 2023Apr; 15(8): 2323; and Tong et al., Mol Cancer, 2022 Nov 1;21(1):206, the full contents of each of which are incorporated herein by reference).

[0225] In some embodiments, TAA is selected from the group consisting of: HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, membrane glycoglucan, globulin-based 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, and 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 gangliosides, glycoprotein 75, GPNMB, HGF, human scattering factor receptor kinase, IGF-I receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1, MUC1, mucin Canag, N-glycoside neuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-RA, TWEAK, phosphatidylserine, prostate cancer cells, Rank1, 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.

[0226] In some embodiments, a multispecific (e.g., bispecific) antibody or its antigen-binding fragment 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.

[0227] Anti-TAA / CLEC5A antibody and its antigen-binding fragment In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein exhibit agonistic activity against macrophage activation. In some embodiments, macrophage activation is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% after exposure to the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein, compared to macrophage activation without exposure to such antibodies. In some embodiments, the macrophage-binding anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have a LALAPG mutation in their Fc region (SEQ ID NO: 96). In some embodiments, the macrophage-binding anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have an optimized mutation in their Fc region (SEQ ID NO: 97).

[0228] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have a silent mutation (e.g., LALAPG mutation) in their Fc region. 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 a silent mutation (e.g., LALAPG mutation) in their Fc region.

[0229] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., by binding to CLEC5A) to mediate the killing of target cells (e.g., HER2+ cancer cells). In some embodiments, the macrophage-activating anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have a silent mutation (e.g., LALAPG mutation, SEQ ID NO: 96) in their Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein comprise the wild-type human IgG1 Fc region. In some embodiments, the macrophage-binding anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have an optimized mutation (SEQ ID NO: 97) in their Fc region.

[0230] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., by binding to CLEC5A) and induce macrophage-mediated killing of target cells. In some embodiments, macrophage-mediated killing results in the killing of target cells (e.g., HER2+ cancer cells). In some embodiments, approximately 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 through macrophage-mediated phagocytosis.

[0231] In some embodiments, the TAAs described herein are selected from HER2, EGFR, EpCAM, CD79b, GPRC5D, BCMA, CD38, DLL3, CD70, GPC3, and mesothelin.

[0232] Anti-HER2 / CLEC5A antibody and its antigen-binding fragment Receptor tyrosine protein kinase erbB-2 (HER2) is a human protein encoded by the ERBB2 gene. ERBB is an abbreviation for erythrocyte oncogene B, which was originally isolated from the avian genome. This human protein is also frequently referred to as HER2 (human epidermal growth factor receptor 2) or CD340 (differentiation cluster 340).

[0233] HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. However, unlike other members of the ERBB family, HER2 does not directly bind to its ligand. At high HER2 concentrations, such as in cancer, HER2 activation is caused by heterodimerization or homodimerization with another ERBB member. Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of some aggressive breast cancers. In recent years, this protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0234] Detailed reviews and commentaries on HER2 can be found, for example, on the NCBI website: "ERBB2 erb-b2 receptortyrosine kinase 2 [Homo sapiens (human)] - Gene - NCBI"; "ERBB2". GeneticsHome 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 full content of each article is incorporated herein by reference.

[0235] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to HER2 / CLEC5A (e.g., human HER2 / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-HER2 / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to HER2; and a second antigen-binding domain that specifically binds to CLEC5A.

[0236] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0237] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) includes CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDR 1, 2, and 3 amino acid sequences, and the selected VL1 CDR 3 amino acid sequences are identical to the selected VH1 CDR1 amino acid sequence. The amino acid sequence of CDR 1, 2, and 3 is one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 100, 102, and 104, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 105, 106, and 107, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 101, 103, and 104, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 105, 106, and 107, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% amino acid sequence identical to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% amino acid sequence identical to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% amino acid sequence identical to the selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) includes CDRs 1, 2, and 3, wherein VL2 CDR1 contains at least 80% amino acid sequence identical to the selected VL2 CDR1 amino acid sequence, VL2 CDR2 contains at least 80% amino acid sequence identical to the selected VL2 CDR2 amino acid sequence, and VL2 CDR3 contains at least 80% amino acid sequence identical to the selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs The amino acid sequences of 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 3, 5, and 7, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 8, 9, and 10, respectively; (2) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 4, 6, and 7, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 8, 9, and 10, respectively; (3) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (4) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0238] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 100, 102, and 104, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 105, 106, and 107, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 3, 5, and 7, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8, 9, and 10, respectively.

[0239] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 101, 103, and 104, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 105, 106, and 107, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 4, 6, and 7, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8, 9, and 10, respectively.

[0240] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 100, 102, and 104, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 105, 106, and 107, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0241] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 101, 103, and 104, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 105, 106, and 107, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0242] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 108, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 109, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 11, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 12.

[0243] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 108, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 109, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 83, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 84.

[0244] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 108, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 109, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0245] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 108, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 109, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0246] In some embodiments, VH1 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL1 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence is SEQ ID NO:108, and the selected VL sequence is SEQ ID NO:109.

[0247] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence 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: 83, and the selected VL sequence is SEQ ID NO: 84; and (4) The selected VH sequence is SEQ ID NO: 86, and the selected VL sequence is SEQ ID NO: 87.

[0248] In some embodiments, VH1 comprises the same VH1 CDR1, VH CDR2, and VH CDR3 as the selected VH sequence; and VL1 comprises the same VL1 CDR1, VL1 CDR2, and VL1 CDR3 as the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 108, and the selected VL sequence is SEQ ID NO: 109.

[0249] In some embodiments, VH2 comprises the same VH2 CDR1, VH CDR2, and VH CDR3 as the selected VH sequence; and VL2 comprises the same VL2 CDR1, VL2 CDR2, and VL2 CDR3 as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence 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: 83, and the selected VL sequence is SEQ ID NO: 84; and (4) The selected VH sequence is SEQ ID NO: 86, and the selected VL sequence is SEQ ID NO: 87.

[0250] In some embodiments, the first antigen-binding domain is the antigen-binding domain of the anti-HER2 antibody trastuzumab. Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1 kappa monoclonal antibody. It is a humanized version of the mouse anti-HER2 antibody (4D5) and binds selectively with high affinity (Kd=5 nM) to the extracellular domain of HER2 in cell-based assays (see, e.g., US Pat. NO. 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).

[0251] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7 or 3A7, its chimeric antibody, and its humanized antibody.

[0252] The CDR sequences of 3A7 and 3A7-derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 3, 5, and 7, and CDRs of the light chain variable domain, SEQ ID NOs: 8, 9, and 10, as defined by Kabat. According to Chothia, 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.

[0253] The CDR sequences of 5C7 and 5C7-derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13, 15, and 17, and CDRs of the light chain variable domain, SEQ ID NOs: 18, 19, and 20, as defined by Kabat. According to Chothia, 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.

[0254] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog HER2; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0255] 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.

[0256] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0257] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0258] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU designations). In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an IgG1 Fc region, wherein alanine (A) at position 234; alanine (A) at position 234; and glycine (G) at position 329, as per EU designations. In some embodiments, the Fc region contains 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 anti-HER2 / CLEC5A antibody described herein may be designed to have an IgG1 Fc region, wherein alanine (A) at position 236; leucine (L) at position 330; and glutamic acid (E) at position 332, as designated by EU. In some embodiments, the Fc region comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0260] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an Fc region, wherein the aspartic acid (D) at position 239 and the glutamate (E) at position 332 are numbered according to EU. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an IgG1 isotype structure with the S239D+I332E mutation, numbered according to EU. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an IgG1 Fc region, wherein the aspartic acid (D) at position 239 and the glutamate (E) at position 332 are numbered according to EU.

[0261] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein can be designed as IgG1 isoform structures with a club-and-mortise mutation (KIH), which can promote heterodimerization and avoid mismatch between the two heavy chains. In some embodiments, the anti-HER2 / CLEC5A antibodies have a higher endocytosis rate than the corresponding monoclonal antibodies or control bispecific antibodies.

[0262] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can take 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.

[0263] 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. Figure 9C shows a schematic diagram of this structure. In some embodiments, the 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. Figure 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, while the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain contains one or more mortar mutations, while the second heavy chain contains one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein may have Figures 9A-9F Any one of the structures shown in the diagram.

[0264] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain sequence to SEQ ID NO: 118. In some embodiments, the heavy chain comprises an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96).

[0265] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 119.

[0266] 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+2 A) Fc-silence” 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 that of 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 that of SEQ ID NO: 119.

[0267] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain sequence to SEQ ID NO: 120. In some embodiments, the heavy chain comprises an IgG1 Fc region containing the LALAPG mutation (SEQ ID NO: 96).

[0268] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 121.

[0269] 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-silence” 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 that of 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 that of SEQ ID NO: 121.

[0270] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain sequence as SEQ ID NO: 122. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0271] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 123.

[0272] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as “HER2 / 5C7(2+2 A) Fc-optimized” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 122; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 123.

[0273] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical first heavy chain sequence to SEQ ID NO: 110. In some embodiments, the first heavy chain comprises an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain comprises one or more club-shaped mutations.

[0274] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first light chain sequence as SEQ ID NO: 112.

[0275] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second heavy chain sequence that is about 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 containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain comprises one or more acetabular mutations.

[0276] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same second light chain sequence as SEQ ID NO: 113.

[0277] 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-silence” 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 the first light chain sequence 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 the second heavy chain sequence is identical to SEQ ID NO: 111. 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; 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.

[0278] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical first heavy chain sequence to SEQ ID NO: 114. In some embodiments, the first heavy chain comprises an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain comprises one or more acetabular mutations.

[0279] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first light chain sequence as SEQ ID NO: 116.

[0280] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a second heavy chain sequence that is about 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 containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain comprises one or more club-shaped mutations.

[0281] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the second light chain sequence identical to SEQ ID NO: 117.

[0282] 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-silence”, 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 identical to SEQ ID NO: 115. 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; 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.

[0283] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer of at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0284] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0285] 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 CDRs as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to HER2 and / or CLEC5A.

[0286] Anti-EGFR / CLEC5A antibody and its antigen-binding fragment Epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein and a receptor for extracellular protein ligands of the epidermal growth factor family (EGF family). 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 affecting EGFR expression or activity can 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 various tumors. Blocking EGFR signaling, whether by blocking the EGFR binding site in the extracellular domain of the receptor or inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumors and improve patient conditions.

[0287] EGFR is a transmembrane protein that is activated by binding to its specific ligands, including epidermal growth factor and transforming growth factor a (TGFa). ErbB2 has no known direct activating ligands 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 transforms from an inactive monomeric form to an active homodimer. EGFR dimerization stimulates its intrinsic intracellular protein tyrosine kinase activity. Following 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 EGFR terminal domain. These include Y992, Y1045, Y1068, Y1148, and Y1173.

[0288] Autophosphorylation triggers downstream activation and signal transduction, which is carried out by several other proteins that bind phosphorylated tyrosine residues via their own phosphotyrosine-binding SH2 domain. These downstream signaling proteins initiate several signal transduction cascades, primarily the MAPK, Akt, and JNK pathways, leading to DNA synthesis and cell proliferation. These proteins regulate phenotypes such as cell migration, adhesion, and proliferation. The kinase domain of EGFR can also cross-phosphorylate tyrosine residues of other receptors it aggregates with, and can itself be activated in this manner.

[0289] Mutations that lead to EGFR overexpression (known as upregulation or amplification) are associated with a variety of cancers, including lung adenocarcinoma (40% of cases), anal cancer, glioblastoma (50%), and head and neck epithelial tumors (80-100%). These somatic mutations involving EGFR lead to its continuous activation, resulting in uncontrolled cell division. In glioblastoma, EGFR-specific mutations, known as EGFRvIII, are frequently observed. Approximately 30% of epithelial cancers are associated with mutations, amplifications, or dysregulation of EGFR or its family members.

[0290] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to EGFR / CLEC5A (e.g., human EGFR / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-EGFR / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to EGFR; and a second antigen-binding domain that specifically binds to CLEC5A.

[0291] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0292] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1CDR1 region contains at least 80% of the amino acid sequence identical to the selected VL1CDR1 amino acid sequence, the VL1CDR2 region contains at least 80% of the amino acid sequence identical to the selected VL1CDR2 amino acid sequence, and the VL1CDR3 region contains at least 80% of the amino acid sequence identical to the selected VL1CDR3 amino acid sequence. The selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 126, 128, and 130, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 131, 132, and 133, respectively; (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 127, 129, and 130, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 131, 132, and 133, respectively; (3) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 136, 138, and 140, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 141, 142, and 143, respectively; and (4) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 137, 139, and 140, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 141, 142, and 143, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence; Furthermore, the second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% identical amino acid sequences to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region contains at least 80% identical amino acid sequences to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region contains at least 80% identical amino acid sequences to the selected VL2 CDR3 amino acid sequence. The selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (2) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0293] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 126, 128, and 130, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 131, 132, and 133, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0294] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 127, 129, and 130, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 131, 132, and 133, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0295] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 136, 138, and 140, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 141, 142, and 143, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0296] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 137, 139, and 140, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 141, 142, and 143, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0297] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 134, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 135, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0298] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 134, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 135, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0299] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 144, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 145, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0300] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 144, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 145, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0301] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0302] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0303] 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.

[0304] In some embodiments, VH2 comprises the same VH2CDR1, VH CDR2, and VH CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2CDR1, VL2CDR2, and VL2CDR3 as the VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0305] In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody cetuximab, panitumumab, nexituzumab, or Eg-B4-VHH. In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody amivantatumab (EGFR1). In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody nimotuzumab (EGFR2).

[0306] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0307] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 13, 15, and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NOs: 18, 19, and 20. According to Chothia's 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.

[0308] 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.

[0309] 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-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0310] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0311] In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU designations). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 Fc region, wherein alanine (A) at position 234; alanine (A) at position 234; and glycine (G) at position 329, as per EU designations. In some embodiments, the Fc region contains about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0312] In some embodiments, the anti-EGFR / CLEC5A antibody described herein may 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, as designated by EU. In some embodiments, the Fc region contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0313] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an Fc region containing alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332, according to EU designations. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 isotype structure with the S239D+I332E mutation, according to EU designations. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region containing aspartic acid (D) at position 239, and glutamic acid (E) at position 332, according to EU designations.

[0314] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein can be designed as IgG1 subtype structures with a club-and-mortise mutation (KIH), which can promote heterodimerization and avoid mismatch between the two heavy chains. In some embodiments, the anti-EGFR / CLEC5A antibodies have a higher endocytosis rate than the corresponding monoclonal antibodies or control bispecific antibodies.

[0315] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can employ 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.

[0316] 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, while VL1 is connected to the CL domain. Figure 9C A schematic diagram of this structure is shown. In some embodiments, the 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. Figure 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, and the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody described herein may have the following characteristics: Figures 9A-9F Any of the structures shown in the diagram.

[0317] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 146 or 148. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0318] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 147 or 149.

[0319] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody is referred to as “EGFR1 / 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 that of 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 that of 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 includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 148; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 149.

[0320] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer of at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0321] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0322] The present invention also provides nucleic acids comprising polynucleotides encoding anti-EGFR / CLEC5A antibodies. The immunoglobulin heavy or light chain in the anti-EGFR / CLEC5A antibody comprises CDRs as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to EGFR and / or CLEC5A.

[0323] Anti-EpCAM / CLEC5A antibody and its antigen-binding fragment Epithelial cell adhesion molecule (EpCAM), also known as tumor-associated calcium signaling molecule 1 (TACST-1), 17-1A, and CD326, is a 40 kDa transmembrane glycoprotein that is highly expressed in epithelial cancers and at lower levels in normal monolayer epithelium. The structure and function of EpCAM have been described in the following literature: 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. EpCAM is expressed on the basolateral membrane and plays a role in calcium-independent allogeneic cell adhesion. The mature EpCAM molecule contains an N-terminus (with a 23-amino acid signal peptide removed after treatment), a 242-amino acid extracellular domain (containing an epidermal growth factor-like repeat region), a human thyroglobulin repeat region and a cysteine-deficient region, a single 23-amino acid transmembrane domain, and a C-terminus of a 26-amino acid cytoplasmic domain (containing two α-actin binding sites and an NPXY internalization motif). EpCAM is frequently overexpressed in epithelial-derived cancers and is expressed by cancer stem cells, making it a molecule of significant therapeutic and diagnostic value. Due to its frequent and high expression in cancer and its metastases, EpCAM can serve as a prognostic marker, a therapeutic target, and an anchoring molecule for circulating and disseminated tumor cells (CTCs / DTCs), which are considered the primary source of metastatic cancer cells. The extracellular domain of EpCAM can be cleaved to yield the soluble extracellular domain molecule EpEX and the intracellular molecule EPICD. EpICD has been shown to bind with other proteins to form nuclear complexes, thereby upregulating the expression of genes that promote cell proliferation. EpCAM may also be involved in the epithelial-to-mesenchymal transition (EMT) and may contribute to the formation of large metastatic foci.

[0324] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to EpCAM / CLEC5A (e.g., human EpCAM / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-EpCAM / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to EpCAM; and a second antigen-binding domain that specifically binds to CLEC5A.

[0325] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0326] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR3 amino acid sequence. The selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 150, 152, and 154, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 155, 156, and 157, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 151, 153, and 154, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 155, 156, and 157, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% identical amino acid sequences to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% identical amino acid sequences to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% identical amino acid sequences to the selected VH2 CDR3 amino acid sequence; and Furthermore, the second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR3 amino acid sequence. The selected amino acid sequences of VH2 CDR 1, 2, and 3, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (2) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0327] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 150, 152, and 154, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 155, 156, and 157, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0328] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 151, 153, and 154, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 155, 156, and 157, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0329] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 158, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 159, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0330] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 158, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 159, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0331] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0332] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0333] In some embodiments, VH1 includes VH1CDR1, VH1CDR2, and VH1CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1CDR1, VL1CDR2, and VL1CDR3, which are 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.

[0334] In some embodiments, VH2 comprises the same VH2CDR1, VH2CDR2, and VH2CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2CDR1, VL2CDR2, and VL2CDR3 as the VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0335] In some embodiments, the first antigen-binding domain is the antigen-binding domain of the anti-EpCAM antibody Solitomab.

[0336] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0337] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat, the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 13, 15, and 17, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 18, 19, and 20. According to Chothia, 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.

[0338] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog EpCAM; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0339] 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-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0340] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0341] In some embodiments, the multispecific (e.g., multispecific) anti-EpCAM / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU designations). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 Fc region, wherein alanine (A) at position 234; alanine (A) at position 234; and glycine (G) at position 329, as per EU designations. In some embodiments, the Fc region contains about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:96.

[0342] In some embodiments, the anti-EpCAM / CLEC5A antibody described herein may 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, as designated by EU. In some embodiments, the Fc region contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:97.

[0343] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody described herein is designed to have an Fc region comprising alanine (A) at position 236 (EU number); leucine (L) at position 330; and glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody described herein is designed to have an IgG1 isotype structure with the EU-designated S239D+I332E mutation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region comprising aspartic acid (D) at position 239 (EU number); and glutamic acid (E) at position 332.

[0344] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein can be designed with an IgG1 subtype structure having a club-and-mortise mutation (KIH), which can promote heterodimerization and avoid mismatch between the two heavy chains. In some embodiments, the anti-EpCAM / CLEC5A antibody has a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0345] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can employ 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.

[0346] 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, while VL1 is connected to the CL domain. Figure 9C A schematic diagram of this structure is shown. In some embodiments, the 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. Figure 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, and the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody described herein may have the following characteristics: Figures 9A-9F Any of the structures shown in the diagram.

[0347] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 160 or 162. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0348] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 161 or 163.

[0349] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody is referred to as “EpCAM / 5C7(2+2A)Fc-optimized” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 160; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 161.

[0350] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer of at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0351] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0352] The present invention also provides nucleic acids comprising polynucleotides encoding anti-EpCAM / CLEC5A antibodies. The immunoglobulin heavy or light chain in the anti-EpCAM / CLEC5A antibody comprises CDRs as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to EpCAM and / or CLEC5A.

[0353] Anti-GPRC5D / CLEC5A antibody and its antigen-binding fragment G protein-coupled receptor family C5 member D (GPRC5D) is a protein encoded by the GPRC5D gene in humans. The protein encoded by this gene is a member of the G protein-coupled receptor family.

[0354] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to GPRC5D / CLEC5A (e.g., human GPRC5D / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-GPRC5D / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to GPRC5D; and a second antigen-binding domain that specifically binds to CLEC5A.

[0355] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0356] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains at least 80% identical amino acid sequences to the selected VH1CDR1 amino acid sequence, the VH1CDR2 region contains at least 80% identical amino acid sequences to the selected VH1CDR2 amino acid sequence, and the VH1CDR3 region contains at least 80% identical amino acid sequences to the selected VH1CDR3 amino acid sequence; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR3 amino acid sequence. The selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 164, 166, and 168, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 169, 170, and 171, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 165, 167, and 168, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 169, 170, and 171, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% identical amino acid sequences to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% identical amino acid sequences to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% identical amino acid sequences to the selected VH2 CDR3 amino acid sequence; and Furthermore, the second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% identical amino acid sequences to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region contains at least 80% identical amino acid sequences to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region contains at least 80% identical amino acid sequences to the selected VL2 CDR3 amino acid sequence. The selected amino acid sequences of VH2 CDR 1, 2, and 3, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (2) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0357] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 164, 166, and 168, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 169, 170, and 171, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0358] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 165, 167, and 168, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 169, 170, and 171, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0359] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 172, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 173, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0360] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 172, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 173, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0361] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0362] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0363] In some embodiments, VH1 includes VH1CDR1, VH1CDR2, and VH1CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1CDR1, VL1CDR2, and VL1CDR3, which are 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.

[0364] In some embodiments, VH2 comprises the same VH2CDR1, VH2CDR2, and VH2CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2CDR1, VL2CDR2, and VL2CDR3 as the VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0365] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0366] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat, the CDR sequences of the heavy chain variable domain listed in SEQ ID NO: 13, 15, and 17, and the CDR sequences of the light chain variable domain listed in SEQ ID NO: 18, 19, and 20. According to Chothia, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 14, 16, and 17, and the CDR sequences of the light chain variable domain are listed in SEQ ID NO: 18, 19, and 20.

[0367] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog GPRC5D; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0368] 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-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0369] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0370] In some embodiments, the multispecific (e.g., multispecific) anti-GPRC5D / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU designation). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 Fc region comprising alanine (A) at position 234 according to EU designation; alanine (A) at position 234; and glycine (G) at position 329. In some embodiments, the Fc region comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0371] In some embodiments, the anti-GPRC5D / CLEC5A antibody described herein may 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, as designated by EU. In some embodiments, the Fc region contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0372] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody described herein is designed to have an Fc region comprising alanine (A) at position 236 (EU number); leucine (L) at position 330; and glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody described herein is designed to have an IgG1 isotype structure with the EU-designated S239D+I332E mutation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region comprising aspartic acid (D) at position 239 (EU number); and glutamic acid (E) at position 332.

[0373] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein can be designed as IgG1 subtype structures with a club-and-mortise mutation (KIH), which can promote heterodimerization and avoid mismatch between the two heavy chains. In some embodiments, the anti-GPRC5D / CLEC5A antibodies have a higher endocytosis rate than the corresponding monoclonal antibodies or control bispecific antibodies.

[0374] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can employ 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.

[0375] 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, while VL1 is connected to the CL domain. Figure 9C A schematic diagram of this structure is shown. In some embodiments, the 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. Figure 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, and the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody described herein may have the following characteristics: Figures 9A-9F Any of the structures shown in the diagram.

[0376] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 174. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0377] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 175.

[0378] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody is referred to as “GPRC5D / 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 that of 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 that of SEQ ID NO: 175.

[0379] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer of at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0380] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0381] The present invention also provides nucleic acids comprising polynucleotides encoding anti-GPRC5D / CLEC5A antibodies. The immunoglobulin heavy or light chain in the anti-GPRC5D / CLEC5A antibody comprises CDRs as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to GPRC5D and / or CLEC5A.

[0382] Anti-BCMA / CLEC5A antibody and its antigen-binding fragment 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 and recognizes B-cell activating factor (BAFF). Serum B-cell maturation antigen (sBCMA) is a cleaved form of BCMA, and its levels are low in the serum of normal patients but generally high in patients with multiple myeloma (MM).

[0383] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to BCMA / CLEC5A (e.g., human BCMA / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-BCMA / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to BCMA; and a second antigen-binding domain that specifically binds to CLEC5A.

[0384] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0385] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL1 CDR3 amino acid sequence. The selected VH1 CDR 1, 2, and 3 amino acid sequences and the selected VL1 CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 176, 178, and 180, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 181, 182, and 183, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 177, 179, and 180, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 181, 182, and 183, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% identical amino acid sequences to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% identical amino acid sequences to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% identical amino acid sequences to the selected VH2 CDR3 amino acid sequence; and Furthermore, the second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR3 amino acid sequence. The selected amino acid sequences of VH2 CDR 1, 2, and 3, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (2) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0386] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 176, 178, and 180, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 181, 182, and 183, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0387] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 177, 179, and 180, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 181, 182, and 183, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0388] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 184, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 185, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0389] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 184, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 185, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0390] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0391] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0392] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are 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.

[0393] In some embodiments, VH2 comprises the same VH2 CDR1, VH2 CDR2, and VH2 CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2 CDR1, VL2 CDR2, and VL2 CDR3 as the VL CDR1, VL CDR2, and VLCDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0394] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0395] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat, the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 13, 15, and 17, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 18, 19, and 20. According to Chothia, 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.

[0396] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog BCMA; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0397] 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-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0398] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0399] In some embodiments, the multispecific (e.g., multispecific) anti-BCMA / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with LALAPG mutations (L234A, L235A, and P329G mutations in EU designation). In some embodiments, the multispecific (e.g., multispecific) anti-EGFR / CLEC5A antibody described herein is designed to have an IgG1 Fc region comprising alanine (A) at position 234 in EU designation; alanine (A) at position 234; and glycine (G) at position 329. In some embodiments, the Fc region comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0400] In some embodiments, the anti-BCMA / CLEC5A antibody described herein may be designed to have an IgG1 Fc region comprising alanine (A) at position 236 (EU number); leucine (L) at position 330; and glutamic acid (E) at position 332. In some embodiments, the Fc region comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0401] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody described herein is designed to have an Fc region comprising alanine (A) at position 236 (EU number); leucine (L) at position 330; and glutamic acid (E) at position 332. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody described herein is designed to have an IgG1 isotype structure with the EU-designated S239D+I332E mutation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region comprising aspartic acid (D) at position 239 (EU number); and glutamic acid (E) at position 332.

[0402] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein can be designed as IgG1 isoform structures with a club-and-mortise mutation (KIH), which can promote heterodimerization and avoid mismatch between the two heavy chains. In some embodiments, the anti-BCMA / CLEC5A antibodies have a higher endocytosis rate than the corresponding monoclonal antibodies or control bispecific antibodies.

[0403] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can employ 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.

[0404] 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, while VL1 is connected to the CL domain. Figure 9C A schematic diagram of this structure is shown. In some embodiments, the 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. Figure 9A A schematic diagram of the structure is shown. In some embodiments, the Fc region contains a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain contains one or more club mutations, and the second heavy chain contains one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody described herein may have the following characteristics: Figures 9A-9F Any of the structures shown in the diagram.

[0405] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 174. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0406] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 187.

[0407] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody is referred to as “BCMA / 5C7(2+2 A)Fc-optimized” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 186; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 187.

[0408] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer of at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0409] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0410] The present invention also provides nucleic acids comprising polynucleotides encoding anti-BCMA / CLEC5A antibodies. The immunoglobulin heavy or light chain in the anti-BCMA / CLEC5A antibody comprises CDRs as shown in Table 22. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to BCMA and / or CLEC5A.

[0411] Anti-CD38 / CLEC5A antibody and its antigen-binding fragment CD38 (Cluster 38), also known as cyclic ADP-ribolytic enzyme, is a glycoprotein found 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).

[0412] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to CD38 / CLEC5A (e.g., human CD38 / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-CD38 / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to CD38; and a second antigen-binding domain that specifically binds to CLEC5A.

[0413] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0414] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains at least 80% identical amino acid sequences to the selected VH1 CDR1, the VH1 CDR2 region contains at least 80% identical amino acid sequences to the selected VH1 CDR2, and the VH1 CDR3 region contains at least 80% identical amino acid sequences to the selected VH1 CDR3; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains at least 80% identical amino acid sequences to the selected VL1 CDR1, the VL1 CDR2 region contains at least 80% identical amino acid sequences to the selected VL1 CDR2, and the VL1 CDR3 region contains at least 80% identical amino acid sequences to the selected VL1 CDR3, wherein the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 188, 190, and 192, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 193, 194, and 195, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 189, 191, and 192, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 193, 194, and 195, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% identical amino acid sequences to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% identical amino acid sequences to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% identical amino acid sequences to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR1, the VL2 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR2, and the VL2 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR3. The selected amino acid sequences of VH2 CDR 1, 2, and 3, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; and (2) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0415] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 188, 190, and 192, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 193, 194, and 195, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0416] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 189, 191, and 192, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 193, 194, and 195, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0417] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 196, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 197, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0418] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 196, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 197, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0419] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0420] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0421] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are 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.

[0422] In some embodiments, VH2 comprises the same VH2 CDR1, VH2 CDR2, and VH2 CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2 CDR1, VL2 CDR2, and VL2 CDR3 as the VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; and (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86.

[0423] In some embodiments, the second antigen-binding domain is any one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0424] The CDR sequences of 5C7 and 5C7-derived antibodies include, according to Kabat's definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NOs: 13, 15, and 17, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19, and 20, respectively. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16, and 17, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19, and 20, respectively.

[0425] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CD38; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0426] 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.

[0427] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0428] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0429] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody described herein is designed as an IgG1 subtype structure having LALAPG mutations (L234A, L235A, and P329G mutations in the EU designation). In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody described herein is designed to have an IgG1 Fc region having alanine (A) at position 234; alanine (A) at position 235; and glycine (G) at position 329, according to the EU designation. In some embodiments, the Fc region contains about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0430] In some embodiments, the anti-CD38 / CLEC5A antibody described herein may be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 (as per EU designation). In some embodiments, the Fc region contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0431] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody described herein is designed to have an Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU designation. In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with the S239D+I332E mutation in the EU designation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU designation.

[0432] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein may be designed with an IgG1 isoform structure containing a club-and-mortar (KIH) mutation, which promotes heterodimerization and avoids mismatch between the two heavy chains. In some embodiments, the anti-CD38 / CLEC5A antibody exhibits a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0433] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can be of 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.

[0434] 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. A schematic diagram of the structure is shown below. Figure 9C In some embodiments, the 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. A schematic diagram of the structure is shown below. Figure 9A In some embodiments, the Fc region includes a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain includes one or more club mutations, and the second heavy chain includes one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody described herein may have Figures 9A-9F Any one of the structures shown in the diagram.

[0435] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody comprises about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain sequence as SEQ ID NO: 198. In some embodiments, the heavy chain comprises an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97).

[0436] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain sequence as SEQ ID NO: 199.

[0437] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody is referred to as “CD38 / 5C7(2+2A)Fc optimized” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 198; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 199.

[0438] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0439] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0440] 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, wherein 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.

[0441] Anti-CD79b / CLEC5A antibody and its antigen-binding fragment The CD79b molecule, immunoglobulin-associated beta, also known as CD79B (differentiation cluster 79B), is a human gene. It is associated with agammaglobulinemia-6. The B-cell antigen receptor is a multimeric complex comprising the antigen-specific component surface immunoglobulin (Ig). Surface Ig binds nonvalently to two other proteins, Ig-alpha and Ig-beta, both essential for the expression and function of the B-cell antigen receptor. The gene encodes the Ig-beta protein, a component of the B-cell antigen. Alternative splicing transcriptomorphs encoding different isotypes have been described.

[0442] This invention provides multispecific (e.g., bispecific) antibodies that specifically bind to CD79b / CLEC5A (e.g., human CD79b / CLEC5A) and antigen-binding fragments thereof. In one aspect, this invention provides an anti-CD79b / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to CD79b; and a second antigen-binding domain that specifically binds to CLEC5A.

[0443] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0444] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1CDR1 region contains at least 80% identical amino acid sequences to the selected VH1 CDR1, the VH1 CDR2 region contains at least 80% identical amino acid sequences to the selected VH1 CDR2, and the VH1 CDR3 region contains at least 80% identical amino acid sequences to the selected VH1 CDR3; and The first light chain variable region (VL1) contains CDRs 1, 2, and 3, wherein the VL1 CDR1 region contains at least 80% identical amino acid sequences to the selected VL1 CDR1, the VL1 CDR2 region contains at least 80% identical amino acid sequences to the selected VL1 CDR2, and the VL1 CDR3 region contains at least 80% identical amino acid sequences to the selected VL1 CDR3, wherein the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following: (1) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 200, 202, and 204, respectively; and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205, 206, and 207, respectively; and (2) The selected amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 201, 203, and 204, respectively, and the selected amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205, 206, and 207, respectively; The second heavy chain variable region (VH2) contains complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH2 CDR1 contains at least 80% identical amino acid sequences to the selected VH2 CDR1 amino acid sequence, VH2 CDR2 contains at least 80% identical amino acid sequences to the selected VH2 CDR2 amino acid sequence, and VH2 CDR3 contains at least 80% identical amino acid sequences to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) contains CDRs 1, 2, and 3, wherein the VL2 CDR1 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR1, the VL2 CDR2 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR2, and the VL2 CDR3 region contains at least 80% of the same amino acid sequence as the selected VL2 CDR3. The selected amino acid sequences of VH2 CDR 1, 2, and 3, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are one of the following: (1) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; (2) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; (3) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 3, 5, and 7, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 8, 9, and 10, respectively; (4) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 4, 6, and 17, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18, 19, and 20, respectively; (5) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 63, 65, and 67, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 68, 69, and 70, respectively; (6) The selected amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 64, 66, and 67, respectively, and the selected amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 68, 69, and 70, respectively; In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 200, 202, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 13, 15, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0445] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 201, 203, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 14, 16, and 17, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0446] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 200, 202, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 3, 5, and 7, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8, 9, and 10, respectively.

[0447] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 201, 203, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 4, 6, and 7, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 8, 9, and 10, respectively.

[0448] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 200, 202, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 63, 65, and 67, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 68, 69, and 70, respectively.

[0449] In some embodiments, the selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 201, 203, and 204, respectively; the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 205, 206, and 207, respectively; the selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 64, 66, and 67, respectively; and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NO: 68, 69, and 70, respectively.

[0450] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 21, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 22.

[0451] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 85, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 86.

[0452] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 11, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 12.

[0453] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 83, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 84.

[0454] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 71, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 72.

[0455] In some embodiments, the first heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 208, the first light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 209, the second heavy chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 91, and the second light chain variable region contains at least 80%, 85%, 90%, 95%, 99%, or 100% of the sequence identical to SEQ ID NO: 92.

[0456] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to 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.

[0457] In some embodiments, VH2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VH sequence, and VL2 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86; (3) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (4) The selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; (5) The selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72; (6) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92; In some embodiments, VH1 includes VH1CDR1, VH1CDR2, and VH1CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; VL1 includes VL1CDR1, VL1CDR2, and VL1CDR3, which are 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.

[0458] In some embodiments, VH2 comprises the same VH2 CDR1, VH2 CDR2, and VH2 CDR3 as the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises the same VL2 CDR1, VL2 CDR2, and VL2 CDR3 as the VL CDR1, VL CDR2, and VLCDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (2) The selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86; (3) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (4) The selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; (5) The selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72; (6) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92; In some embodiments, the second antigen-binding domain is the antigen-binding domain of any one of the anti-CLEC5A antibodies, their chimeric antibodies, and their humanized antibodies described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibodies 5C7, 3A7, 13E6, their chimeric antibodies, and their humanized antibodies.

[0459] The CDR sequences of 5C7 and 5C7-derived antibodies include: according to Kabat's definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NO: 13, 15, and 17, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 18, 19, and 20, respectively. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 14, 16, and 17, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 18, 19, and 20, respectively.

[0460] The CDR sequences of 3A7 and 3A7-derived antibodies include: according to Kabat's definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NO: 3, 5, and 7, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 8, 9, and 10, respectively. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 4, 6, and 7, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 8, 9, and 10, respectively.

[0461] The CDR sequences of 13E6 and 13E6-derived antibodies include: according to Kabat's definition, the CDRs of the heavy chain variable domain are listed in SEQ ID NO: 63, 65, and 67, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 68, 69, and 70, respectively. According to Chothia's definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NO: 64, 66, and 67, respectively, and the CDRs of the light chain variable domain are listed in SEQ ID NO: 68, 69, and 70, respectively.

[0462] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CD79b; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0463] 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.

[0464] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv); and / or the second antigen-binding domain is scFv.

[0465] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0466] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein is designed as an IgG1 subtype structure having LALAPG mutations (L234A, L235A, and P329G mutations in the EU designation). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein is designed to have an IgG1 Fc region having alanine (A) at position 234; alanine (A) at position 235; and glycine (G) at position 329, according to the EU designation. In some embodiments, the Fc region contains about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 96.

[0467] In some embodiments, the anti-CD79b / CLEC5A antibody described herein may be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 (as per EU designation). In some embodiments, the Fc region contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 97.

[0468] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein is designed to have an Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU designation. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein is designed to have an IgG1 subtype structure with the S239D+I332E mutation in the EU designation. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region comprising aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU designation.

[0469] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein may be designed with an IgG1 isoform structure containing a club-and-mortar (KIH) mutation, which promotes heterodimerization and avoids mismatch between the two heavy chains. In some embodiments, the anti-CD79b / CLEC5A antibody has a higher endocytosis rate than the corresponding monoclonal antibody or control bispecific antibody.

[0470] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragments described herein can be of 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.

[0471] 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. A schematic diagram of the structure is shown below. Figure 9C In some embodiments, the 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. A schematic diagram of the structure is shown below. Figure 9A In some embodiments, the Fc region includes a club-and-mortar (KIH) mutation. In some embodiments, the first heavy chain includes one or more club mutations, and the second heavy chain includes one or more mortar mutations. In some embodiments, the first heavy chain includes one or more mortar mutations, and the second heavy chain includes one or more club mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein may have Figures 9A-9F Any one of the structures shown in the diagram.

[0472] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 210. Figure 9A The sequence shown as "H1" in the image, and a second heavy chain 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: 211 (in... Figure 9AThe sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes a first 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 that in SEQ ID NO: 212. Figure 9A The sequence is shown as "L1"), and a second light chain 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: 213 (in Figure 9A The sequence is displayed as "L2" in the image.

[0473] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (1+1A)Fc silencing” and includes 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; and a first light chain sequence that is identical to SEQ ID NO: 210. SEQ ID 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.

[0474] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 227. Figure 9AThe sequence is shown as "H1" in the image, and a second heavy chain 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: 228. Figure 9A The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes a first 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 that in SEQ ID NO: 229. Figure 9A The sequence is shown as "L1"), and a second light chain 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: 230. Figure 9A The sequence is displayed as "L2" in the image.

[0475] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (1+1A) Fc Optimized” and includes 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; and a first light chain sequence that is identical to SEQ ID NO: 228. 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 to SEQ ID NO: 230; 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.

[0476] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 217. Figure 9B The sequence shown as "H1" in the image, and a second heavy chain 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: 218. Figure 9B The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region containing 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 that in SEQ ID NO: 219. Figure 9B The sequence is displayed as "L1".

[0477] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (2+1A) Fc silencing” and comprises 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 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 identical to SEQ ID NO: 218. NO:219 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequence.

[0478] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 234. Figure 9B The sequence shown as "H1" in the image, and a second heavy chain 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: 235 (in... Figure 9B The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain as SEQ ID NO: 236 (in the diagram). Figure 9B The sequence is displayed as "L1".

[0479] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (2+1A) Fc Optimized” and includes 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 that of 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 that of 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 that of SEQ ID NO: 236. Approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequences.

[0480] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 223. Figure 9C The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain (in the diagram) identical to that in SEQ ID NO: 224. Figure 9C The sequence is displayed as "L".

[0481] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7(2+2A)Fc silencing” 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 that of 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 that of SEQ ID NO: 224.

[0482] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 240. Figure 9C The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain as SEQ ID NO: 241. Figure 9CThe sequence is displayed as "L".

[0483] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 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 that of 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 that of SEQ ID NO: 241.

[0484] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 214. Figure 9D The sequence shown as "H1" in the image, and a second heavy chain 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: 215 (in... Figure 9D The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain as SEQ ID NO: 216 (in the diagram). Figure 9D The sequence is displayed as "L1".

[0485] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (1+1B) Fc silencing” and comprises 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 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 identical to SEQ ID NO: 216. Approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequences.

[0486] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical first heavy chain (in SEQ ID NO: 231) to SEQ ID NO: 231. Figure 9D The sequence shown as "H1" in the image, and a second heavy chain 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 that of SEQ ID NO: 232. Figure 9D The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes 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 that in SEQ ID NO: 233. Figure 9D The sequence is displayed as "L1".

[0487] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (1+1B) Fc Optimized” and includes 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 that of 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 that of 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 that of SEQ ID NO: 233. Approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequences.

[0488] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical first heavy chain (in SEQ ID NO: 220) to the first heavy chain of SEQ ID NO: 220. Figure 9E The sequence shown as "H1" in the image, and a second heavy chain 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 the second heavy chain of SEQ ID NO: 221. Figure 9E The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains comprise an IgG1 Fc region containing a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain as SEQ ID NO: 222 (in the diagram). Figure 9E The sequence is displayed as "L1".

[0489] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (2+1B) Fc silencing” and comprises 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 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 identical to SEQ ID NO: 221. NO:222 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequence.

[0490] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same first heavy chain as SEQ ID NO: 237. Figure 9E The sequence shown as "H1" in the image, and a second heavy chain 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: 238. Figure 9E The sequence is shown as "H2" in the diagram. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes 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 that in SEQ ID NO: 239. Figure 9E The sequence is displayed as "L1".

[0491] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7 (2+1B) Fc Optimized” and includes 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: 238; and a second heavy chain sequence that is identical to SEQ ID NO: 238. NO:239 is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain sequence.

[0492] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 225. Figure 9F The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain (in the diagram) identical to that in SEQ ID NO: 226. Figure 9F The sequence is displayed as "L".

[0493] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7(2+2B)Fc silencing” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 225; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 226.

[0494] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 244. Figure 9F The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain (in the diagram) identical to that in SEQ ID NO: 245. Figure 9F The sequence is displayed as "L".

[0495] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 3A7 (2+2B) Fc silencing” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 244; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 245.

[0496] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 246. Figure 9F The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain (in the diagram) identical to that in SEQ ID NO: 247. Figure 9F The sequence is displayed as "L".

[0497] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 13E6 (2+2B) Fc silencing” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 246; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 247.

[0498] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain as SEQ ID NO: 242. Figure 9F The sequence is shown as "H" in the diagram. In some embodiments, the heavy chain includes an IgG1 Fc region containing an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain as SEQ ID NO: 243 (in the diagram). Figure 9FThe sequence is displayed as "L".

[0499] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as “CD79b / 5C7(2+2B)Fc optimized” and includes about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the heavy chain sequence identical to that of SEQ ID NO: 242; and about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the light chain sequence identical to that of SEQ ID NO: 243.

[0500] The linkers described herein can 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 known as GS linkers), such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99), where n represents an integer at least 1. Those skilled in the art will be able to select an appropriate spacer sequence.

[0501] In some embodiments, a club-and-mortar mutation is introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the chance of mispairing between the two heavy chains.

[0502] 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, wherein 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.

[0503] Example The following embodiments will further describe the invention, but these embodiments do not limit the scope of the invention as described in the claims.

[0504] Example 1: CLEC5A expression of immune cell subsets in human PBMCs Using gating strategies to assess immune cell subsets in human PBMCs Figure 1AFrozen PBMCs from healthy donors (Stanford Blood Center) were thawed, stained with a fixable viability dye (Zombie Aqua™, BioLegend), and then labeled with a set of antibodies specific to immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), 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 containing 2% heat-inactivated FBS (fetal bovine serum) and 0.05% BSA (bovine serum albumin)), and then analyzed by flow cytometry (Attune™ CytPik™, Invitrogen). 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.

[0505] Example 2: CLEC5A expression in tumor-associated myeloid cells of human solid tumors CLEC5A expression was assessed in tumor-associated myeloid cells of human solid tumors. Frozen human tumor cells isolated from six solid tumor indications (renal cancer, lung cancer, ovarian cancer, colorectal cancer, cholangiocarcinoma, and pancreatic cancer) were thawed (Discovery LifeSciences), stained with a fixable viability dye (Zombie Aqua™, BioLegend), and then labeled with a set of antibodies targeting immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), and 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 minutes at room temperature, washed, resuspended in FACS buffer (PBS containing 2% heat-inactivated FBS and 0.05% BSA), and then analyzed by flow cytometry (Attune™ CytPik™, Invitrogen). Gated strategies were used to assess immune cell subsets in human solid tumors ( Figure 2A ).like Figure 2B-2C As shown, in human tumors, the majority of neutrophils (80–98%, represented as live / CD45+ / CD19- / CD3- / CD56- / CD15+ / CD16+) and non-neutrophilic myeloid cells (>43%, represented as live / CD45+ / CD19- / CD3- / CD56- / CD15-) are positive for CLEC5A. The highest expression of CLEC5A was observed in non-neutrophilic 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%) of a mixture of B cells, T cells, and NK cells (live / CD45+ / CD19+ / CD3+ / CD56+).

[0506] Example 3: CLEC5A expression in tumor-associated macrophages (TAMs) in human solid tumors CLEC5A expression was evaluated in tumor-associated macrophages (TAMs) of human solid tumors. Specifically, frozen human tumor cell isolates from six solid tumor indications (renal cancer, lung cancer, ovarian cancer, colorectal cancer, cholangiocarcinoma, and pancreatic cancer) were thawed (Discovery Life Sciences), stained with a fixable viability dye (Zombie Aqua™, BioLegend), and then labeled with a set of antibodies targeting immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), and 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 minutes at room temperature, washed, resuspended in FACS buffer (PBS containing 2% heat-inactivated FBS and 0.05% BSA), and analyzed by flow cytometry (Attune™ CytPik™, Invitrogen). A gating strategy was used to characterize non-neutrophilic myeloid cells (live / CD45+ / CD19- / CD3- / CD56- / CD15-) isolated from human tumor cells. Figure 3A ).like Figure 3B As shown, the majority (~30-60%) of non-neutrophilic myeloid cells are identified as TAMs due to their dual expression of CD11b and TREM2. Only a subset of TAMs express CD206 (>45% of CD11b+ cells). Figure 3C-3D As shown, CLEC5A is highly expressed in most (>70%) TREM2+TAM and CD206+TAM.

[0507] Example 4: Production of CLEC5A antibody Immunization To generate monoclonal antibodies against CLEC5A, two New Zealand white rabbits (Acro Biosystems, Cat#: CLA-H5243) were immunized with human CLEC5A His-tagged protein according to a 63-day rabbit immunization protocol. During the first injection period, 400 μg of CFA emulsion antigen was injected subcutaneously on day 1, followed by three subcutaneous injections (200 μg of IFA emulsion antigen) on days 7, 21, and 42, with a final intravenous booster injection on day 63.

[0508] antibody titer On days 28 and 49, 5 mL of serum was collected from each rabbit for titer determination. Antibody titers were determined post-immunization by binding serially diluted serum to a human CLEC5A ELISA. Briefly, 96-well plates were coated overnight at 4°C with human CLEC5A (1 µg / mL). The plates were blocked for 1 hour at room temperature with PBS containing 1% BSA (pH 7.4, Fisher Scientific, Cat#: 21-040-CM). Samples (serum diluted 1:1000 and titrated 3-fold) were added to the plates and incubated for 1 hour at room temperature, followed by washing with PBS. HRP donkey anti-rabbit IgG secondary antibody (BioLegend, Cat#: 406401) was then added to the plates and incubated for 1 hour at room temperature, followed by washing with PBS. The substrate was added to the plates and reacted for 5 minutes. OD readings at 450 nm were measured using a microplate reader (CLARIOstar®).

[0509] Rabbit spleen cell isolation and B cell sorting Rabbit spleens were harvested five days after the final booster injection. Splenic cells were prepared in a sterile cell filter placed at the bottom of a 100 mm sterile culture dish containing 20 mL RPMI + 1% penicillin / streptomycin (P / S). The spleen tissue was transferred to the cell filter using sterile forceps. Specifically, the spleen was held with forceps, cut into small pieces, and then pressed through the mesh of the cell filter. Tissue fragments were washed with 10 mL RPMI + 1% P / S. Splenic cells 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 of ACK buffer (Gibco Cat#: A1049201) was added to resuspend the cells. The resuspended 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 min and aspirate the supernatant. Resuspend the pellet in 15 mL RPMI + 10% FBS + 1% P / S. Transfer the cells through a 100µm cell filter into a 50 mL conical tube to remove cell clumps. Then, arrange the spleen cells at the desired density (e.g., 4×10⁻⁶). 7 (cells / mL) were seeded into appropriate culture medium for sorting. The remaining spleen cells were then seeded at 6 × 10⁻⁶ cells / mL. 7 Cells / vial (~1.8 mL) were frozen overnight at -80 °C in 90% serum + 10% DMSO. The frozen cells were then transferred to a liquid nitrogen tank for long-term storage.

[0510] For B cell sorting, freshly isolated or thawed spleen cells (~2×10⁻⁶) are sorted. 8Spleen cells were incubated overnight in B cell medium (RPMI-1640, 15% FBS, 1×HEPES, 1×2-ME (2-mercaptoethanol), 1% penicillin / streptomycin) and then sorted. The appropriate 96-well B cell culture plates were prepared the day before sorting. On the day of sorting, suspended and loosely attached spleen cells were collected by gently pipetting the medium onto the culture surface of the flask. The cells were then transferred to conical tubes and centrifuged at 400×g for 3 min. The cell pellet was washed twice with fluorescence-activated cell sorting (FACS) buffer (1×PBS + 0.5% BSA). Biotinylated antigen was added at 5 µg / mL (final concentration). The mixture was incubated at room temperature (RT) for 20 min. The staining mixture was then centrifuged at 400×g for 3 min, and the cells were resuspended in FACS buffer. The cells were transferred to 1.5 mL amber Eppendorf™ tubes. The staining antibody mixture was then added to the cells. Incubate the staining mixture at 4°C for 15–30 minutes, then centrifuge at 400×g for 3 minutes. Wash the cell pellet twice with FACS buffer. After washing, incubate the cell pellet in 1×PBS + 1% FBS at a concentration of ~10⁻⁶. 7 Resuspend at a concentration of cells / mL. Using FACS, antigen-specific individual B cells were sorted into 96-well plates (20 plates per rabbit). The 96-well B cell culture plates containing the sorted B cells were incubated at 37°C and 5% CO2 for 12 days.

[0511] ELISA screening of single B cell cultures, LEM supernatant, and purified antibodies On day 8 post-sorting, 15 μL of B cell culture supernatant was collected from each well for antigen-specific ELISA detection. In short, the B cell culture supernatant was transferred to a human CLEC5A-coated ELISA plate (a 384-well plate coated with CLEC5A antibody and blocked with BSA). Cells were incubated at room temperature for 1 hour and washed three times with PBS containing 0.05% Tween-20. Antibody detection was performed using goat anti-rabbit IgGHRP + TMB substrate (VWR, Cat#: 5120-007). B cell supernatant meeting the OD450 cutoff value (>0.5 or 3 times higher than pre-immunization serum) was then selected.

[0512] Human CLEC5A-specific antibodies were initially screened and sorted from 1920 supernatants derived from 40-well (96-well) plates using ELISA. Approximately 200 B cell clones that were bound to the human CLEC5A antigen (ELISA OD cutoff value 0.9) were identified.

[0513] On day 12 post-sorting, B cell culture plates were centrifuged at 400×g for 3 minutes. The supernatant from positive clones (OD greater than the selected cutoff value of 0.9 for antigen-specific ELISA) was collected, and the cell pellet was stored in 100 μL of DNA / RNA blocking buffer (Zymo, Cat#: R1100-250) in a 250 μL PCR tube. Additional assays were performed on the collected supernatant to confirm ELISA binding to the cell surface.

[0514] Heavy and light chain genes from positive clones were cloned into a linear expression module (LEM). The LEM was then transfected into production cells, and the culture supernatant was detected by ELISA as described above.

[0515] Screening for CLEC5A-specific antibodies on cell surface CLEC5A is primarily expressed in bone marrow 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 vendor's instructions. Approximately 50,000 monocytes were incubated on ice for 60 min each in FACS buffer (PBS + 0.5% BSA) with undiluted B cell or LEM supernatant or serially diluted purified antibody. 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) for 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 the LEM. After testing, eight clones (6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6, and 9E11) that showed binding to human CLEC5A on the cell surface were selected. Finally, recombinant CLEC5A antibodies were expressed for further functional assays.

[0516] Example 5: Chimeric anti-CLEC5A antibody and molecular structure To express the chimeric rabbit / human IgG1 antibody, the human IgG1 heavy chain constant region (hIgG1-Hc-LALAPG variant) and the human light chain Kappa constant region (CL-kappa) were synthesized and cloned into pcDNA3.4 (GeneScript). The pcDNA3.4 containing the human IgG1-hIgG1-Hc-LALAPG variant (pcDNA3.4-huIgG1-Hc-LALAPG) was further digested with EcoRI / NheI to clone the VH sequence. Digestion with EcoRI / BsiWI yielded the VL sequence of the vector pcDNA3.4 (pcDNA3.4-huKappa-Lc) expressing human CL-κ. The VH and VL sequences of eight selected 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 via IDT. The designed sequences overlapped at the 5' and 3' ends, allowing for annealing and assembly with the corresponding vector ends (NEB NEBuilder® HiFi DNA Assembly). The assembled plasmids were transformed into competent *E. coli* cells (NEB® 5-alpha), and the correct sequences were cloned based on sequencing results (ElimBiopharm). The cells were further cultured in LB broth containing carbenicillin (100 μg / mL) for plasmid purification (QIAGEN Plasmid Plus Kits). The plasmids were eluted with nuclease-free water (Sigma) and stored at -80°C.

[0517] Example 6: Transfection and purification of chimeric anti-CLEC5A antibody All antibody light and heavy chain coding sequences were generated via 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 × 10⁶ cells / mL with fresh cell culture medium at transfection. 6 cells / mL. Per 4 × 10 6 Each cell was transfected with 1 μg of plasmid DNA via liposome transfection. The transfected cells were cultured in a shaker at 32°C, 5% CO2, 70% humidity, and 125 rpm.

[0518] Protein expression titers were measured every 5 days. Cell supernatants were collected by centrifugation 10–14 days post-transfection and filtered through a 0.22 μm PES filter. The expressed antibody was purified by protein A chromatography (TOYOPEARL® AF-r Protein A HC-650 Mresin, Tosoh) and aggregates were removed by anion exchange resin (TOYOPEARL NH2-750F, Tosoh).

[0519] The purified antibodies were analyzed by HPLC (Dionex UltiMate® 3000-RSUHPLC, ThermoFisher) controlled by Chromeleon® software. Antibody purity and aggregation were resolved 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 and were sterilized by filtration through a 0.22 μm PES filter before binding and functional assays.

[0520] Example 7: Binding of chimeric anti-CLEC5A antibody to human macrophages The binding affinity of anti-CLEC5A antibody (an antibody with silenced Fc) to human macrophages was evaluated. CD14+ monocytes (purified from human PBMC donor #651 using the EasySep™ Human Monocyte Enrichment Kit (CD16 not removed, StemCell Technologies, Cat#: 19058) were differentiated into M0 macrophages after treatment with 50 ng / mL macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. Approximately 50,000 M0 macrophages were incubated on ice for 30 min with serially diluted antibody in FACS buffer (PBS + 0.5% BSA). After incubation, the cells were washed twice and detected using anti-human IgGPE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). To perform the analysis, the mean fluorescence intensity (MFI) of each antibody was determined, and graphs were plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Figure 4 As shown in the table below, the eight selected anti-CLEC5A antibodies can bind to M0 macrophages (CLEC5A+) cells with different affinities.

[0521] Table 1: Binding of anti-CLEC5A antibody to human macrophages Example 8: TNFα release from chimeric anti-CLEC5A antibody TNFα release from anti-CLEC5A antibody was assessed. CD14+ monocytes (purified from human PBMC donor #651 using the EasySep™ Human Monocyte Enrichment Kit (CD16 not removed, StemCell Technologies, Cat#: 19058)) were differentiated into M1 cells for 6 days with 50 ng / mL macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) and then treated for 24 hours with 50 ng / mL M-CSF and 50 ng / mL IFN-… (StemCell Technologies, Cat#: 78020). Anti-CLEC5A antibody was serially diluted and coated onto 96-well plates at 4°C for 24 hours. After incubation, the plates were washed with PBS. Approximately 100,000 M1 macrophages were added to wells coated with anti-CLEC5A antibody 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 GraphPadPrism software (Version 9.4.1, GraphPad Software Inc.). Figure 5 As shown, all eight anti-CLEC5A antibodies exhibited moderate levels of TNFα release, lower than the baseline DX244, suggesting that anti-CLEC5A antibodies may be safe.

[0522] Example 9: Binding of chimeric anti-CLEC5A antibody to mouse CLEC5A ELISA was used to assess whether antibodies with silenced Fc in CLEC5A could cross-react with recombinant mouse CLEC5A protein. Corning high-binding flat-bottom 96-well plates (Cat#: 3361) were coated with 1 µg / mL mouse CLEC5A (R&D Systems, Cat#: 8438-CL-050) and incubated at 4°C for 48 hours. The protein antigen was aspirated, and the wells were washed three times with 400 μL of wash buffer (PBS containing 0.05% Tween® 20) using a plate washer. The antibody with silenced Fc in CLEC5A was prepared as a 5 µg / mL primary antibody and serially diluted 3-fold to 11 spots (dilution concentration) with reagent buffer (PBS + 0.05% BSA). 100 μL of the primary antibody was added to the corresponding wells and incubated at room temperature for 1 hour. The primary antibody was aspirated, and the wells were washed three times with 400 μL of wash buffer (PBS containing 0.05% Tween® 20) using a plate washer. The secondary antibody BioLegend donkey anti-human HRP (Cat#: 410902) was diluted 1:10,000 with reagent buffer (PBS + 0.5% BSA), and 100 μL was added to each well. The plate was incubated at room temperature for 30 minutes. The secondary antibody was then aspirated, and the wells were washed three times with 400 μL of washing buffer (PBS containing 0.05% Tween® 20). 100 μL of Thermo 1-step TMB Turbo (Cat#: 34022) was added to each well, and the plate was incubated at room temperature in the dark for 10 minutes. 100 μL of Fisher 1N Sulfuric Acid (Cat#: SA212-2) stop solution was added to each well, and the data were read from the plate at 450 nm using a microplate reader. The OD450 of each antibody was plotted and analyzed using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Figure 6 As shown in the table below, anti-CLEC5A antibodies 6A5, 14A2, and 5C7 exhibit cross-reactivity with mouse CLEC5A.

[0523] Table 2: Binding of anti-CLEC5A antibody to mouse CLEC5A For BLI (Biolayer Interferometry) binding, 10 μg / mL of anti-CLEC5A antibody was captured on an anti-human IgG Fc probe (Gator Bio, Cat#: 160024). Binding and dissociation with recombinant human CLEC5A and HisTag (ACRO Biosystems, Cat#: NC1-H52H4) were then measured at a single concentration of 11 nM, dissolved in PBS + 0.05% Tween® 20. The absolute KD was determined using a 1:1 binding model. BLI binding assay results are shown below. Figures 7A-7B As shown in the figure. The results indicate that anti-CLEC5A antibodies 3A7 and 5C7 can bind to human CLEC5A with high affinity.

[0524] Example 10: Humanization and ELISA binding of chimeric anti-CLEC5A antibody By transplanting the CDR of the major antibody into the closest conjugate to IgBLAST ( www.ncbi.nlm.nih.gov / igblast / ) and / or IMGT / DomainGapAlig ( www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi Within the selected human germline frameworks of the identified rabbit framework, four rabbit anti-human CLEC5A monoclonal antibody clones, 3A7, 5C7, 6A5, 7G10, and 13E6, were humanized. Based on sequence similarity within the framework and CDR, human germline IGHV, IGKV, IGHJ, and IGKJ were selected. Certain human germline framework residues were reverted to their corresponding rabbit residues to maintain the typical loop structure and light / heavy chain interface. (Padlan E.A., Mol. Immunol., 1994, 31:169; Foote J. and Winter G., JMB, 1992, 224:487). Through humanization, humanized antibodies against h3A7, h5C7, h6A5, h7G10, and h13E6 were generated.

[0525] ELISA was performed to assess whether the humanized anti-CLEC5A antibody could bind to recombinant human CLEC5A protein compared to the corresponding chimeric parent clone. Corning high-binding flat-bottom 96-well plates (Cat#: 3361) were coated with 1 µg / mL human CLEC5A (ACROBiosystems, Cat#: CLA-H5243) and incubated at 4°C for 48 hours. The protein antigen was aspirated, and the wells were washed three times with 400 μL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. The anti-CLEC5A antibody was prepared as a 5 µg / mL primary antibody and serially diluted 3-fold to 11 spots (dilution concentration) with reagent buffer (PBS + 0.05% BSA). 100 μL of the primary antibody was added to the corresponding wells and incubated at room temperature for 1 hour. The primary antibody was aspirated, and the wells were washed three times with 400 μL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. The secondary antibody BioLegend donkey anti-human HRP (Cat#: 410902) was diluted 1:10,000 in reagent buffer (PBS + 0.5% BSA), and 100 μL was added to each well. The plate was incubated at room temperature for 30 minutes. The secondary antibody was aspirated, and the wells were washed three times with 400 μL of wash buffer (PBS containing 0.05% Tween® 20) using a plate washer. 100 μL of Thermo 1-step TMB Turbo (Cat#: 34022) was added to each well, and the plate was incubated at room temperature in the dark for 10 minutes. 100 μL of Fisher 1N Sulfuric Acid (Cat#: SA212-2) stop solution was added to each well, and the readings were taken at 450 nm using a microplate reader. The OD450 of each antibody was plotted and analyzed using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). Figures 8A-8E As shown in the table below, all humanized anti-CLEC5A antibodies exhibited similar affinity for the human CLEC5A protein compared to the corresponding chimeric parent clones, indicating that the humanization of the above clones was successful.

[0526] Table 3: Binding of humanized anti-CLEC5A antibody to human CLEC5A (ELISA) Example 11: Molecular construction of TAA / CLEC5A bispecific antibody like Figures 9A-9FAs shown, six different forms of TAA / CLEC5A bispecific antibodies were generated. Based on sequencing results (Elim Biopharm), competent *E. coli* (NEB® 5-alpha) cells were transformed with assembled plasmids encoding clones with the correct sequences. Transformed cells were further cultured in LB broth containing carbenicillin (100 μg / mL) to purify the plasmids (QIAGEN® Plasmid Plus Kits). The plasmids were eluted in nuclease-free water (Sigma) and stored at -80°C.

[0527] Antibodies were expressed in CHO cells (ExpiCHO™ expression system, Gibco) by transfection with pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc containing paired VH and VL sequences. ExpiCHO™ cells were cultured in ExpiCHO™ expression medium and maintained at 0.3 to 6 × 10⁻⁶ cells / mL according to the supplier's instructions at 37°C, 125 rpm, 5% CO₂, and 80% humidity. 6 Be...

Claims

1. An antibody or antigen-binding fragment, comprising: (1) A first antigen-binding domain, wherein the domain specifically binds to a first antigen, wherein the first antigen is differentiation cluster 79b (CD79b); and (2) A second antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A).

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 first heavy chain variable region (VH1) comprising CDR1, 2, and 3, and the first light chain variable region (VL1) comprising CDR1, 2, and 3, wherein: (1) The amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 200, 202, and 204, respectively, and the amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205-207, respectively; or (2) The amino acid sequences of VH1 CDR 1, 2, and 3 are listed in SEQ ID NO: 201, 203, and 204, respectively, and the amino acid sequences of VL1 CDR 1, 2, and 3 are listed in SEQ ID NO: 205-207, respectively.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the VH1 comprises at least 90% of the same amino acid sequence as SEQ ID NO: 208, and the VL1 comprises at least 90% of the same amino acid sequence as SEQ ID NO:

209.

4. The antibody or antigen-binding fragment according to any one of claims 1-3, wherein the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), the second heavy chain variable region (VH2) comprising CDR1, 2, and 3, and the second light chain variable region (VL2) comprising CDR1, 2, and 3, wherein: (1) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 3, 5, and 7, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 8-10, respectively; (2) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 4, 6, and 7, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 8-10, respectively; (3) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 13, 15, and 17, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18-20, respectively; (4) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 14, 16, and 17, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 18-20, respectively; (5) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 23, 25, and 27, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 28-30, respectively; (6) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 24, 26, and 27, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 28-30, respectively; (7) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 33, 35, and 37, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 38-40, respectively; (8) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 34, 36, and 37, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 38-40, respectively; (9) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 43, 45, and 47, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 48-50, respectively; (10) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 44, 46, and 47, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 48-50, respectively; (11) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 53, 55, and 57, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 58-60, respectively; (12) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 54, 56, and 57, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 58-60, respectively; (13) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 63, 65, and 67, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 68-70, respectively; (14) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 64, 66, and 67, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 68-70, respectively; (15) The amino acid sequences of VH2 CDR 1, 2, and 3 are listed in SEQ ID NO: 73, 75, and 77, respectively, and the amino acid sequences of VL2 CDR 1, 2, and 3 are listed in SEQ ID NO: 78-80, respectively; or (16) The amino acid sequences of VH2 CDR 1, 2 and 3 are listed in SEQ ID NO: 74, 76 and 77 respectively, and the amino acid sequences of VL2 CDR 1, 2 and 3 are listed in SEQ ID NO: 78-80 respectively.

5. The antibody or antigen-binding fragment according to any one of claims 1-4, wherein the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein the VH2 comprises an amino acid sequence that is at least 90% identical to the selected VH2 sequence, and the VL2 comprises an amino acid sequence that is at least 90% identical to the selected VL2 sequence, wherein the selected VH2 sequence and the selected VL2 sequence are one of the following: (1) The selected VH2 sequence is SEQ ID NO: 11, and the selected VL2 sequence is SEQ ID NO: 12; (2) The selected VH2 sequence is SEQ ID NO: 21, and the selected VL2 sequence is SEQ ID NO: 22; (3) The selected VH2 sequence is SEQ ID NO: 31, and the selected VL2 sequence is SEQ ID NO: 32; (4) The selected VH2 sequence is SEQ ID NO: 41, and the selected VL2 sequence is SEQ ID NO: 42; (5) The selected VH2 sequence is SEQ ID NO: 51, and the selected VL2 sequence is SEQ ID NO: 52; (6) The selected VH2 sequence is SEQ ID NO: 61, and the selected VL2 sequence is SEQ ID NO: 62; (7) The selected VH2 sequence is SEQ ID NO: 71, and the selected VL2 sequence is SEQ ID NO: 72; (8) The selected VH2 sequence is SEQ ID NO: 81, and the selected VL2 sequence is SEQ ID NO: 82; (9) The selected VH2 sequence is SEQ ID NO: 83, and the selected VL2 sequence is SEQ ID NO: 84; (10) The selected VH2 sequence is SEQ ID NO: 85, and the selected VL2 sequence is SEQ ID NO: 86; (11) The selected VH2 sequence is SEQ ID NO: 87, and the selected VL2 sequence is SEQ ID NO: 88; (12) The selected VH2 sequence is SEQ ID NO: 89, and the selected VL2 sequence is SEQ ID NO: 90; and (13) The selected VH2 sequence is SEQ ID NO: 91, and the selected VL2 sequence is SEQ ID NO:

92.

6. The antibody or antigen-binding fragment according to any one of claims 1-5, wherein: The first antigen-binding domain includes a first heavy chain variable region (VH1) and a first light chain variable region (VL1). The second antigen-binding domain includes a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

7. The antibody or antigen-binding fragment according to any one of claims 6, wherein the second antigen-binding domain is a single-chain variable fragment (scFv) domain; VH2 and VL2 are linked by a first linker peptide; optionally, the second antigen-binding domain is linked to the C-terminus of the light chain by a second linker peptide.

8. The antibody or antigen-binding fragment according to any one of claims 6-7, further comprising an Fc region, wherein: i) The C-terminus of the VH1 domain of the first antigen-binding domain is connected to the Fc region, optionally via the CH1 domain; or ii) 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.

9. The antibody or antigen-binding fragment thereof according to any one of claims 6-8, wherein the antibody comprises a first heavy chain composed of VH1 and a first light chain composed of VL1; and comprises a second heavy chain composed of VH2 and a second light chain composed of VL2.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein: (1) The first heavy chain contains one or more pestle mutations; the second heavy chain contains one or more mortar mutations; or (2) The first heavy chain contains one or more mortar mutations; the second heavy chain contains one or more pestle mutations.

11. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; optionally, the Fc region is the Fc region of human IgG1.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the Fc region comprises one or more of the following groups of amino acid residues (all numbered according to the EU numbering system): (1) Position 236 is alanine (A); position 330 is leucine (L); position 332 is glutamic acid (E); (2) Position 236 is alanine (A); position 293 is aspartic acid (D); position 330 is leucine (L); position 332 is glutamic acid (E); (3) Position 236 is alanine (A); (4) Position 236 is alanine (A); position 293 is aspartic acid (D); position 332 is glutamic acid (E); (5) Position 293 is aspartic acid (D) and position 332 is glutamic acid (E); (6) Position 293 is aspartic acid (D); position 330 is leucine (L); position 332 is glutamic acid (E); and (7) Position 234 is alanine (A); position 235 is alanine (A), position 329 is glycine (G); Optionally, the Fc region is afucosylated.

13. A nucleic acid comprising a polynucleotide encoding an antibody or an antigen-binding fragment thereof of any one of claims 1-12.

14. Use of a composition comprising an antibody or an antigen-binding fragment thereof of any one of claims 1-12 for the preparation of a pharmaceutical agent for the treatment of cancer or an autoimmune disease, the method comprising administering a therapeutically effective amount of the pharmaceutical agent to a subject.

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