WO2025011639A1 - Multispecific antibody and use thereof - Google Patents

Abstract

Provided are an anti-CLEC5A (C-type lectin domain family 5 member A) antibody and an antigen-binding fragment thereof, comprising a first antigen-binding domain (TAA) that specifically binds to a tumor-associated antigen and a second antigen-binding domain and Fc region that specifically bind to CLEC5A; or comprising a second antigen-binding domain and Fc region that specifically bind to an autoimmune disease target and specifically bind to the CLEC5A.

Description

Multispecific antibodies and their uses

CROSS-REFERENCE TO RELATED APPLICATIONS

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

Technical Field

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

Background Art

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

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

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

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

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

Summary of the invention

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(3) Alanine (A) at position 236;

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

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

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

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

Optionally, the Fc region is aglycosylated.

In some embodiments, the tumor-associated antigen (TAA) is HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycocoll, globulinyl-calcium amide (GB3Cer/CD77), gangliosides (GD2, GD3 and GM2), B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD19, WT-L, B7H3, TEM8, CD22, LI-CAM, ROR-I, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cells, CA242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-I receptor, IGF-I, IgGI, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A 1. MUC1, mucin Canag, N-glycosylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R A, TWEAK, phosphatidylserine, prostate cancer cells, Rankl, Ron, SCH 900105, SDC1, SLAMF7, TAG-72, Tenascin C, TGF B Beta 2, TGF-B, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or Vimentin.

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

In some embodiments, the TAA is HER2.

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

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

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

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

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

In some embodiments, TAA is EGFR. In some embodiments,

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

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

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

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

In some embodiments, the TAA is EpCAM. In some embodiments,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the gating strategy used to assess immune cell subsets in human PBMCs.

FIG. 1B shows CLEC5A expression by immune cell subsets in human PBMCs.

FIG2A shows the gating strategy used to assess immune cell subsets in human solid tumors.

Figures 2B-2C show CLEC5A expression in tumor-associated myeloid cells.

Figure 3A shows the gating strategy used to characterize non-neutrophil myeloid cells.

FIG. 3B shows the characteristics of tumor-associated macrophages in human solid tumors.

3C-3D show the expression of CLEC5A in tumor-associated macrophages in human solid tumors.

FIG4 shows the binding affinity of chimeric anti-CLEC5A antibodies to human macrophages.

FIG5 shows M1 macrophage-mediated TNFα release in chimeric anti-CLEC5A antibody-coated plates.

FIG6 shows the binding affinity of chimeric anti-CLEC5A antibodies to mouse CLEC5A.

7A-7B show the BLI binding results of chimeric anti-CLEC5A antibodies to human CLEC5A.

8A-8E show the binding of humanized anti-CLEC5A antibodies to human CLEC5A as determined by ELISA.

9A-9F show schematic diagrams of examples of TAA/CLEC5A bispecific antibodies.

FIG. 10 shows the binding of HER2/CLEC5A bispecific antibodies to human macrophages.

FIG. 11 shows the binding of TAA/CLEC5A bispecific antibodies to human macrophages.

FIG. 12 shows the binding of CD79b/CLEC5A bispecific antibodies to human macrophages.

FIG. 13 shows the binding of TAA/CLEC5A bispecific antibodies to human CLEC5A.

FIG. 14 shows the binding of HER2/CLEC5A bispecific antibodies to human HER2.

FIG. 15 shows the binding of EGFR/CLEC5A bispecific antibodies to human EGFR.

FIG. 16 shows the binding of EpCAM/CLEC5A bispecific antibody to DLD-1 cells.

FIG. 17 shows the binding of CD79b/CLEC5A bispecific antibody to Ramos cells.

Figures 18A-18C show that the HER2/CLEC5A bispecific antibody mediates the killing of SK-BR-3 cells by M0 macrophages (E:T ratio of 5:1).

FIG. 19 shows that the HER2/CLEC5AFc bispecific antibody mediates the killing of SK-BR-3 cells by M1 and M2 macrophages (E:T ratio of 5:1).

FIG. 20 shows the release of INF-γ when HER2/CLEC5A antibody mediates the killing of SK-BR-3 cells by M1 and M2 macrophages (E:T ratio of 5:1).

Figures 21A-21C show that the HER2/CLEC5A bispecific antibody mediates killing of SK-BR-3 cells by M0 macrophages in the presence of culture medium (Figure 21A), plasma (Figure 21B), or hIgG1 (Figure 21C).

FIG. 22 shows that the EGFR/CLEC5A bispecific antibody mediates the killing of DLD-1 cells by M0 macrophages.

FIG. 23 shows that EpCAM/CLEC5A bispecific antibody mediates the killing of DLD-1 cells by M0 macrophages.

FIG. 24 shows the expression levels of EpCAM in different tumor cell lines.

Figures 25A-25F show that EpCAM/CLEC5A bispecific antibody mediates the killing of cancer cells by M0 macrophages.

FIG. 26 shows that different myeloid cell engagers mediate PBMC killing of multiple myeloma cancer cells.

FIG. 27 shows CD79b expression levels in malignant lymphoma cell lines and B cells from healthy PBMC donors.

Figures 28A-28C show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by PBMCs (experiment 1).

29A-29B show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by PBMCs (experiment 2).

Figures 30A-30C show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by PBMCs (experiment 3).

Figures 31A-31B show IL-6 levels when CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells.

FIG. 32 shows TNFα levels when CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells.

Figures 33A-33C show that the CD79b/CLEC5A bispecific antibody mediates killing of endogenous B cells by PBMCs (experiment 1).

Figures 34A-34D show that the CD79b/CLEC5A bispecific antibody mediates killing of endogenous B cells by PBMCs (Experiment 2).

Figures 35A-35B show that the CD79b/CLEC5A bispecific antibody mediates killing of endogenous B cells by PBMCs (experiment 3).

FIG. 36 shows the release of TNFα when CD79b/CLEC5A bispecific antibody mediates PBMC killing of endogenous B cells.

Figures 37A-37F show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M0 macrophages (experiment 1).

Figures 38A-38D show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M0 macrophages (experiment 1).

39A-39B show that the CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M0 macrophages at different E:T ratios (Experiment 2).

Figures 40A-40B show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Romas cells by M0 macrophages (experiment 2).

Figures 41A-41C show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M0 macrophages (experiment 3).

FIG. 42 shows the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M0 macrophages (experiment 3).

FIG. 43 shows the survival rate of M0 macrophages when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M0 macrophages (experiment 3).

44A-44B show that the CD79b/CLEC5A bispecific antibody mediates killing of Daudi cells by M0 macrophages at different E:T ratios (Experiment 1).

Figures 45A-45B show that the CD79b/CLEC5A bispecific antibody mediates killing of Daudi cells by M0 macrophages at different E:T ratios (Experiment 2).

Figures 46A-46D show that the CD79b/CLEC5A bispecific antibody mediates cytokine release when M0 macrophages kill Daudi cells.

Figures 47A-47B show that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M1 macrophages (Experiment 1).

Figures 48A-48B show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M1 macrophages (experiment 1).

FIG. 49 shows that CD79b/CLEC5A bispecific antibody mediates M1 macrophage killing of Ramos cells (experiment 2).

FIG. 50 shows the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M1 macrophages (experiment 2).

FIG. 51 shows the survival rate of M1 macrophages when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M1 macrophages (experiment 2).

FIG. 52 shows that the CD79b/CLEC5A bispecific antibody mediates the killing of Daudi cells by M1 macrophages.

FIG. 53 shows the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Daudi cells by M1 macrophages.

Figures 54A-54D show that the CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M2 macrophages (Experiment 1).

Figures 55A-55D show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M2 macrophages (experiment 1).

FIG. 56 shows that CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M2 macrophages (experiment 2).

FIG. 57 shows the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M2 macrophages (experiment 2).

FIG. 58 shows the survival rate of M2 macrophages when CD79b/CLEC5A bispecific antibody mediates the killing of Ramos cells by M2 macrophages (experiment 2).

FIG. 59 shows that the CD79b/CLEC5A bispecific antibody mediates the killing of Daudi cells by M2 macrophages at different E:T ratios.

FIG. 60 shows the release of cytokines when CD79b/CLEC5A bispecific antibody mediates the killing of Daudi cells by M2 macrophages.

Figures 61A-61B show that CD79b/CLEC5A bispecific antibody mediates monocyte killing of Ramos cells.

Figures 62A-62B show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates monocyte killing of Ramos cells.

Figures 63A-63B show the monocyte survival rate when CD79b/CLEC5A bispecific antibody mediates monocyte killing of Ramos cells.

Figures 64A-64B show that CD79b/CLEC5A bispecific antibody mediates monocyte killing of B cells.

Figures 65A-65B show the release of cytokines when CD79b/CLEC5A bispecific antibody mediates monocyte killing of B cells.

Figures 66A-66B show monocyte survival rate when CD79b/CLEC5A bispecific antibody mediates monocyte killing of B cells.

Detailed Description

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

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

The present disclosure relates to multispecific (eg, bispecific) antibodies or antigen-binding fragments thereof, comprising a first antigen-binding domain that specifically binds to a tumor-associated antigen and a second antigen-binding domain that specifically binds to CLEC5A.

Human C-type lectin domain family 5 member A (CLEC5A)

Human C-type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-binding lectin-1 (MDL-1), is a type II transmembrane protein. C-type lectins are characterized by a common structural C-type lectin domain that can bind glycans and non-glycan ligands in a Ca2 ⁺ -independent manner. The CTLD that binds glycans in a Ca2 ⁺ -dependent manner is called the "carbohydrate recognition domain" (CRD). The myeloid C-type lectin CLEC5A is a spleen tyrosine kinase (Syk)-coupled type II membrane protein that contains a C-terminal CTLD and a short N-terminal cytoplasmic domain. Among the 15 groups of C-type lectins, CLEC5A belongs to group V (NK cell receptor family), which includes CLEC7A, CLEC5A, CLEC2, CLEC1, NK receptors (such as NKG2D, NKRP1 family, NKG2 family, CD69 and CD94), mast cell-associated functional antigen (MAFA), osteoclast inhibitory lectin, and CD72. Similar to NKG2D, CLEC5A signals through ITAM-containing DNAX-activated protein 12 (DAP12) when phosphorylated by Src after activation.

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

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

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

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

Anti-CLEC5A antibodies and antigen-binding fragments

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

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

The CDR sequences of 3A7 and 3A7-derived antibodies include the CDR sequences of the heavy chain variable domain listed in SEQ ID NOs: 3, 5, 7, and the CDR sequences of the light chain variable domain listed in SEQ ID NOs: 8-10 according to the Kabat definition. Definitions: 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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.

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

In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain 1, 2 or 3 heavy chain variable region CDRs selected from the following groups: SEQ ID NO: 3, 5, 7, SEQ ID NO: 4, 6, 7, SEQ ID NO: 13, 15, 17, SEQ ID NO: 14, 16, 17, SEQ ID NO: 23, 25, 27, SEQ ID NO: 24, 26, 27, SEQ ID NO: 33, 35, 37, SEQ ID NO: 34, 36, 37, SEQ ID NO: 43, 45, 47, SEQ ID NO: 44, 46, 47, SEQ ID NO: 53, 55, 56, 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 contains 1, 2 or 3 light chain variable region CDRs selected from the following groups: SEQ ID NO: 8-10, SEQ ID NO: 18-20, SEQ ID NO: 28-30, SEQ ID NO: 38-40, SEQ ID NO: 48-50, SEQ ID NO: 58-60, SEQ ID NO: 68-70 and SEQ ID NO: 78-80.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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) that contains or consists 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) that contains or consists 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.

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

The present invention also provides antibodies or antigen-binding fragments thereof that cross-compete with any antibody or antigen-binding fragment described herein. Cross-competition assays are known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein." Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope sorting assays are known in the art and are described, for example, in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, the entire text of which is incorporated herein by reference.

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

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

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

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

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

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

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

Multispecific antibodies or antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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. Figures 9C and 9F show schematic diagrams of the structures.

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

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

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

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

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

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

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

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

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

Also provided herein are antibodies or antigen-binding fragments thereof comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1; a third chain comprising a second heavy chain, wherein the second heavy chain comprises VH2; and a fourth chain comprising a second light chain, wherein the second light chain comprises VL2. FIG9A shows a schematic diagram of this structure. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. In some embodiments, the first heavy chain comprises one or more knob mutations and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations and the second heavy chain comprises one or more knob mutations.

Also provided herein are antibodies or antigen-binding fragments thereof comprising: a first chain comprising a first light chain, wherein the first light chain comprises VL1; a second chain comprising a first heavy chain, wherein the first heavy chain comprises VH1; a third chain comprising a second heavy chain and an scFv or VHH, wherein the second heavy chain comprises VH2; and a fourth chain comprising a second light chain, wherein the second light chain comprises VL2. In some embodiments, the scFv or VHH comprises VH3 and/or VL3. 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 knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations, and the second heavy chain comprises one or more knob mutations.

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

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

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

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

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

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

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

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 culture. For example, the interface can include at least a portion of the CH3 domain of the antibody constant domain. In the method, one or more small amino acid side chains on the interface of the first antibody molecule are replaced by larger side chains (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 on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products, such as homodimers. The method is described, for example, in WO 96/27011, which is incorporated herein by reference in its entirety.

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

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

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

Tumor-associated antigens

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

In some embodiments, the TAA is selected from the group consisting of HER2, CD79b, EGFR, EpCAM, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycocoll, globulinyl-calcium amide (GB3Cer/CD77), gangliosides (GD2, GD3 and GM2), B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD 19, WT-L, B7H3, TEM8, CD22, LI-CAM, ROR-I, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cells, CA242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD 40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-I receptor, IGF-I, IgGI, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1 , MUC1, mucin Canag, N-glycosylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R A, TWEAK, phosphatidylserine, prostate cancer cells, Rankl, Ron, SCH 900105, SDC1, SLAMF7, TAG-72, Tenascin C, TGF B Beta 2, TGF-B, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or Vimentin.

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

Anti-TAA/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

Anti-HER2/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

a second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and a second light chain variable region (VL2) comprising CDRs 1, 2 and 3. 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDR 1, 2, 3 amino acid sequence, and the selected VL2 CDR 1, 2, 3 amino acid sequence are one of the following:

(1) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, and 7, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8, 9, and 10, respectively;

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

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

(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.

In some embodiments, the first antigen binding domain is the antigen binding domain of the anti-HER2 antibody trastuzumab. Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1 kappa monoclonal antibody that is a humanized version of the murine anti-HER2 antibody (4D5) that selectively binds to the extracellular domain of HER2 with high affinity (Kd = 5 nM) in cell-based assays (see, e.g., US Pat. 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).

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

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

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, according to the Kabat definition. According to the Chothia definition, the CDR sequences of the heavy chain variable domain are listed in SEQ ID NOs: 14, 16 and 17, and the CDRs of the light chain variable domain are listed in SEQ ID NOs: 18, 19 and 20.

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

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

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

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

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

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

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

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

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

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 antibodies or antigen-binding fragments thereof described herein comprise a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Figure 9A shows a schematic diagram of the structure. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations, and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-HER2/CLEC5A antibodies described herein may have the structure shown in any one of Figures 9A-9F.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, a 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, In some embodiments, the second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain comprises one or more knob mutations.

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

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

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

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

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

Anti-EGFR/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

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

(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.

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

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

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

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.

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

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

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

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

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

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

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

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 antibodies or antigen-binding fragments thereof described herein comprise a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Figure 9A shows a schematic diagram of the structure. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations, and the second heavy chain includes one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR/CLEC5A antibodies described herein may have a structure as shown in any one of Figures 9A-9F.

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

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

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

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

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

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

Anti-EpCAM/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

In some embodiments, VH2 comprises a VH2 CDR1, VH2 CDR2, and VH2 CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and VL2 comprises a VL CDR1, VL CDR2, and VH2 CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. wherein the VL2 CDR1, VL2 CDR2 and VL2 CDR3 are identical, 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.

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

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

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

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

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

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

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

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

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

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

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

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 antibodies or antigen binding fragments thereof described herein comprise a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Figure 9A shows a schematic diagram of the structure. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations, and the second heavy chain includes one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM/CLEC5A antibodies described herein may have a structure as shown in any one of Figures 9A-9F.

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

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

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

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

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

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

Anti-GPRC5D/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

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

(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.

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

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

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

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

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

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

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

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

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

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

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 antibodies or antigen binding fragments thereof described herein comprise a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Figure 9A shows a schematic diagram of the structure. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations, and the second heavy chain includes one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D/CLEC5A antibodies described herein may have a structure as shown in any one of Figures 9A-9F.

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

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

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 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: 174; and a light chain sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 175.

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

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

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

Anti-BCMA/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

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

(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.

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

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

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

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

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

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

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

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

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

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

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. Figure. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a first heavy chain and a first light chain; and a second heavy chain and a second light chain. Figure 9A shows a schematic diagram of the structure. In some embodiments, the Fc region comprises a knob-hole (KIH) mutation. In some embodiments, the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations. In some embodiments, the first heavy chain comprises one or more hole mutations, and the second heavy chain comprises one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA/CLEC5A antibodies described herein may have a structure as shown in any one of Figures 9A-9F.

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

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

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

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

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

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

Anti-CD38/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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.

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

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

(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.

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the second antigen binding domain is linked 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 linked to the Fc region. In some embodiments, VH1 is connected to the CH1 domain and VL1 is connected to the CL domain. The structural schematic diagram is shown in Figure 9C. In some embodiments, the antibodies or antigen-binding fragments thereof described herein include a first heavy chain and a first light chain; and a second heavy chain and a second light chain. The structural schematic diagram is shown in Figure 9A. In some embodiments, the Fc region includes a knob-hole (KIH) mutation. In some embodiments, the first heavy chain includes one or more knob mutations and the second heavy chain includes one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations and the second heavy chain includes one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD38/CLEC5A antibodies described herein may have the structure shown in any one of Figures 9A-9F.

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

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

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

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

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

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

Anti-CD79b/CLEC5A antibodies and antigen-binding fragments thereof

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

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

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

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

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

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

(2) The selected VH1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 201, 203, and 204, respectively, and the selected VL1 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 205, 206, and 207, respectively;

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

the second light chain variable region (VL2) comprises CDR 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3,

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

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

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

(3) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, and 7, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8, 9, and 10, respectively;

(4) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, and 17, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 18, 19, and 20, respectively;

(5) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, and 67, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68, 69, and 70, respectively;

(6) The selected VH2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL2 CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68, 69, and 70, respectively;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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. The structural schematic diagram is shown in Figure 9C. In some embodiments, the antibodies or antigen-binding fragments thereof described herein include a first heavy chain and a first light chain; and a second heavy chain and a second light chain. The structural schematic diagram is shown in Figure 9A. In some embodiments, the Fc region includes a knob-hole (KIH) mutation. In some embodiments, the first heavy chain includes one or more knob mutations and the second heavy chain includes one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations and the second heavy chain includes one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibodies described herein may have the structure shown in any one of Figures 9A-9F.

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first heavy chain (shown as "H1" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 210, and a second heavy chain (shown as "H2" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 211. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first light chain (shown as "L1" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 212, and a second light chain (shown as "L2" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 213.

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

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first heavy chain (shown as "H1" sequence in Figure 9A) 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: 227, and a second heavy chain (shown as "H2" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 228. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region containing optimized mutations (SEQ ID NO: 97). In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first light chain (shown as "L1" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 229, and a second light chain (shown as "L2" in Figure 9A) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 230.

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

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

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

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first heavy chain (shown as "H1" in Figure 9B) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 234, and a second heavy chain (shown as "H2" in Figure 9B) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 235. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a light chain (shown as "L1" in FIG. 9B ) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 236.

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

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises an antibody that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, In some embodiments, the multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a light chain (shown as "L" in FIG. 9C ) sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 224.

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

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

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

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first heavy chain (shown as “H1” in FIG. 9D ) sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 214, and a second heavy chain (shown as “H2” in FIG. 9D ) sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 215. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, a multispecific (eg, bispecific) anti-CD79b/CLEC5A antibody comprises A light chain (shown as "L1" in Figure 9D) sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:216.

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

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

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

In some embodiments, a 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first heavy chain (shown in FIG. 9E as "H1") sequence, and a second heavy chain (shown in FIG. 9E as "H2") sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 221. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region comprising a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a light chain (shown as "L1" in Figure 9E) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 222.

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

In some embodiments, a multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a first heavy chain (shown as "H1" in Figure 9E) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 237, and a second heavy chain (shown as "H2" in Figure 9E) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 238. In some embodiments, the first and/or second heavy chain comprises an IgG1 Fc region comprising an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b/CLEC5A antibody comprises a light chain (shown as "L1" in Figure 9E) sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 239.

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

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

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

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

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

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

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

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

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

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

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

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, when When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CD79b and/or CLEC5A.

Example

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

Example 1: CLEC5A expression in immune cell subsets in human PBMCs

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

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

Expression of CLEC5A was evaluated in tumor-associated myeloid cells of human solid tumors. Frozen human isolated tumor cells (Discovery Life Sciences) from 6 solid tumor indications (renal, lung, ovarian, colorectal, cholangiocarcinoma, and pancreatic) were thawed, stained with a fixable viability dye (Zombie Aqua ^™ , BioLegend), and then labeled with a panel of antibodies against immune cell subsets: CD45 (H130, Bio-Legend), CD3 (UCHT1, BioLegend), CD19 (HIB19, BioLegend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), TREM2 (237920, R&D Biosciences), CD11b (IRFCF44, Invitrogen), CD11c (IRFCF44, Invitrogen), CD11d ... 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). A gating strategy was used to evaluate immune cell subsets in human solid tumors (Figure 2A). As shown in Figures 2B-2C, in human tumors, most neutrophils (80-98%, expressed as live/CD45+/CD19-/CD3-/CD56-/CD15+/CD16+) and non-neutrophil myeloid cells (>43%, expressed as live/CD45+/CD19- /CD3-/CD56-/CD15-) were positive for CLEC5A. The highest expression of CLEC5A was observed in non-neutrophil myeloid cells. Little or no CLEC5A expression was observed in non-immune cells (live/CD45-), while some expression was observed in a subset of cells (<14%) in a cell mixture of B cells, T cells, and NK cells (live/CD45+/CD19+/CD3+/CD56+).

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

The expression of CLEC5A was evaluated in tumor-associated macrophages (TAMs) of human solid tumors. Specifically, frozen human isolated tumor cells (Discovery Life Sciences) from 6 solid tumor indications (renal, lung, ovarian, colorectal, cholangiocarcinoma, and pancreatic) were thawed, stained with a fixable viability dye (Zombie Aqua ^™ , BioLegend), and then labeled with a panel of antibodies against immune cell subsets: CD45 (H130, BioLegend), CD3 (UCHT1, BioLegend), CD19 (HIB19, Bio-Legend), CD56 (5.1H11, BioLegend), CD14 (M5E2, BD Biosciences), CD16 (3G8, BioLegend), CD15 (HI98, BD Biosciences), CD11b (IRFCF44, Invitrogen), TREM2 (237920, R&D Biosciences), CD11b (IRFCF44, Invitrogen), CD11c (IRFCF44, Invitrogen), CD11d ... Systems), CD206 (15-2, BioLegend) and CLEC5A (283834, R&D Systems). After staining, the 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). A gating strategy was used to characterize non-neutrophil myeloid cells (live/CD45+/CD19-/CD3-/CD56-/CD15-) in human isolated tumor cells (Figure 3A). As shown in Figure 3B, most (~30-60%) non-neutrophil myeloid cells were found to be TAMs due to their dual expression of CD11b and TREM2. Only a portion of TAMs express CD206 (>45% of CD11b+ cells). As shown in Figures 3C-3D, CLEC5A was highly expressed in the majority (>70%) of TREM2+TAMs and CD206+TAMs.

Example 4: Generation of CLEC5A Antibodies

Immunization

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

Antibody titer

On days 28 and 49, 5 mL of serum was collected from each rabbit for titer testing. Antibody titers after immunization were determined by combining serially diluted serum with human CLEC5A ELISA. Briefly, 96-well plates were coated with human CLEC5A (1 μg/mL) overnight at 4°C. The plates were blocked with PBS containing 1% BSA (pH 7.4, Fisher Scientific, Cat#: 21-040-CM) for 1 hour at room temperature. Samples (serum diluted 1:1000 and titrated 3 times) were added to the plates and Incubate at room temperature for 1 hour and then wash with PBS. Then add HRP donkey anti-rabbit IgG secondary antibody (BioLegend, Cat#: 406401) to the plate and incubate at room temperature for 1 hour and then wash with PBS. Add substrate to the plate and react for 5 minutes. Use a microplate reader ( ) Measure the OD reading at 450 nm.

Rabbit splenocyte isolation and B cell sorting

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

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

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

On day 8 after sorting, 15 μL of B cell culture supernatant was collected from each well for antigen-specific ELISA detection. Briefly, B cell culture supernatant was transferred to human CLEC5A-coated ELISA plates (384-well plates coated with CLEC5A antibody and blocked with BSA). The cells were incubated at room temperature for 1 hour and washed 3 times with PBS plus 0.05% Tween-20. Antibodies were detected using goat anti-rabbit IgG HRP+TMB substrate (VWR, Cat#: 5120-007). B cell supernatants that met the OD450 cutoff (>0.5 or 3 times higher than pre-immune serum) were then selected.

A preliminary ELISA screening of 1920 supernatants from 40 well plates (96-well plates) for human CLEC5A-specific antibodies in sorted B cells was performed. Approximately more than 200 human CLEC5A antigen-binding (ELISA OD cutoff 0.9) B cell clones were identified.

On day 12 after sorting, the B cell culture plate was centrifuged at 400 × g for 3 minutes. Supernatants of positive clones (OD greater than the selected cutoff of 0.9 for antigen-specific ELISA) were collected and cell pellets were stored in 100 μL DNA/RNA blocking solution (Zymo, Cat#: R1100-250) in a 250 μL PCR tube. Additional assays were performed on the collected supernatants to confirm ELISA and cell surface binding.

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

Screening of cell surface CLEC5A specific antibodies

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

Example 5: Chimeric anti-CLEC5A antibodies and molecular structures

To express chimeric rabbit/human IgG1 antibodies, 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 of hIgG1-Hc-LALAPG of IgG1 (pcDNA3.4-huIgG1-Hc-LALAPG) was further digested with EcoRI/NheI to clone the VH sequence. The VL sequence of the vector pcDNA3.4 (pcDNA3.4-huKappa-Lc) expressing human CL-κ was obtained by digestion with EcoRI/BsiWI. The VH and VL sequences of the selected 8 rabbit anti-CLEC5A antibodies 6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6 and 9E11 and the VH and VL sequences of the reference antibody DX244 were synthesized by IDT, and the sequences were designed to overlap at the 5' and 3' ends so that they could be annealed and assembled with the ends of the corresponding vectors (NEB). HiFi DNA Assembly). The assembled plasmid was transformed into competent E. coli cells ( 5-alpha), and the correct sequence was cloned according to the sequencing results (Elim Biopharm), and further cultured with LB containing carbenicillin (100 μg/mL) for plasmid purification (QIAGEN Plasmid Plus Kits). The plasmid was eluted with nuclease-free water (Sigma) and stored at -80°C.

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

All antibody light chain and heavy chain coding sequences were generated by direct DNA synthesis and cloned into mammalian expression vectors. The cloned sequences were verified by sanger sequencing. Chinese hamster ovary (CHO) cells were grown in CHOgro medium (Mirus) supplemented with 4mM L-glutamine and 0.33% Poloxamer-188 and diluted to 4×10 ⁶ cells/mL with fresh cell culture medium at the time of transfection. Every 4×10 ⁶ cells were transfected with 1 μg of plasmid DNA by liposome transfection. The transfected cells were cultured in a shaking incubator at 32°C, 5% CO ₂ and 70% humidity and 125rpm.

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

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

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

The ability of anti-CLEC5A antibodies (antibodies with silent Fc) to bind 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 7 days of action of 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). Approximately 50,000 M0 macrophages were incubated with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) on ice for 30 minutes. After incubation, cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and analyzed using GraphPad Prism software. (Version 9.4.1; GraphPad Software Inc.) was used to draw the graphs. As shown in FIG4 and the table below, the selected eight anti-CLEC5A antibodies can bind to M0 macrophages (CLEC5A+) cells with different affinities.

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

Example 8: TNFα Release by Chimeric Anti-CLEC5A Antibodies

Evaluation of TNFα release by anti-CLEC5A antibodies. CD14+ monocytes (purified from human PBMC donor #651 using EasySep ^™ Human Monocyte Enrichment Kit (CD16 not removed, StemCell Technologies, Cat#: 19058)) were differentiated with 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 6 days and then differentiated into M1 with 50 ng/mL M-CSF and 50 ng/mL IFN-… (StemCell Technologies, Cat#: 78020) for 24 hours. Anti-CLEC5A antibodies were serially diluted and coated on 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 the anti-CLEC5A antibody-coated wells in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After 24 hours, the supernatant was collected and TNFα was measured using a human TNFα ELISA kit (R&D systems, Cat#: DY210-05) according to the supplier's instructions. For analysis, TNFα levels (in pg/mL) were determined and plotted using GraphPadPrism software (Version 9.4.1, GraphPad Software Inc.). As shown in Figure 5, all eight anti-CLEC5A antibodies showed moderate levels of TNFα release, which was lower than the benchmark DX244, indicating that the anti-CLEC5A antibodies may be safe.

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

ELISA binding was used to evaluate whether the CLEC5A antibody with a silent Fc could cross-react with the recombinant mouse CLEC5A protein. Corning high-binding flat-bottom 96-well culture plates (Cat#: 3361) were coated with 1 μg/mL mouse CLEC5A (R&D Systems, Cat#: 8438-CL-050) and placed at 4°C for 48 hours. The protein antigen was aspirated and washed with 400 μL of wash buffer (PBS containing 0.05% 20) Wash the wells 3 times. Prepare the CLEC5A Fc-silent antibody as 5 μg/mL primary antibody and dilute it 3 times to 11 points (dilution concentration) with reagent buffer (PBS + 0.05% BSA). Add 100 μL of primary antibody to the corresponding wells and incubate at room temperature. 1 hour. Aspirate the primary antibody and wash with 400 μL washing buffer (PBS containing 0.05% 20) Wash the wells 3 times. Dilute the secondary antibody BioLegend Donkey Anti-Human HRP (Cat#: 410902) with reagent buffer (PBS + 0.5% BSA) at 1:10,000, add 100 μL to each well, and incubate at room temperature for 30 minutes. Aspirate the secondary antibody and wash with 400 μL washing buffer (PBS containing 0.05% 20) Wash the wells 3 times. Add 100 μL Thermo1-step TMB Turbo (Cat#: 34022) to each well and incubate in the dark at room temperature for 10 minutes. Add 100 μL Fisher 1NSulfuric Acid (Cat#: SA212-2) stop solution to each well and read the data in the plate at 450 nm using an ELISA reader. Use GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) to plot the OD450 of each antibody for analysis. As shown in Figure 6 and the table below, anti-CLEC5A antibodies 6A5, 14A2, and 5C7 show cross-reactivity with mouse CLEC5A.

Table 2: Binding of anti-CLEC5A antibodies to mouse CLEC5A

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

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

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

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

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

Example 11: Molecular construction of TAA/CLEC5A bispecific antibody

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

Antibodies were expressed using CHO cells (ExpiCHO ^™ Expression System, Gibco) by transfection of pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc containing paired VH and VL sequences. ExpiCHO ^™ cells were cultured with ExpiCHO ^™ Expression Medium and maintained between 0.3 and 6×10 ⁶ /mL at 37°C, 125rpm, 5% CO ₂ and 80% humidity according to the supplier's instructions. 25mL of fresh ExpiCHO ^™ cells (6×10 ⁶ /mL, viability>95%) were prepared from a 1-day culture inoculated at 3×10 ⁶ /mL in a 125mL baffled flask. 1mL of serum-free medium (OptiPRO ^™ SFM, Gibco) containing pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc (12μg of each plasmid) was added to the culture. Mix thoroughly with 1 mL OptiPRO ^TM SFM containing 80 μL of transfection reagent (ExpiFectamine ^TM CHO reagent, Gibco). Add the transfection mixture to 25 mL ExpiCHO ^TM cells and culture at 37°C. The next day, the transfected culture was added with 150 μL ExpiFectamine ^TM CHO Enhancer, 6 mL ExpiCHO ^TM Feed and 1× penicillin/streptomycin (Gibco) and transferred to a 32°C incubator. Monitor the cell density and viability of the transfected culture, and use BLI technology combined with Protein A biosensor (Gator Bio) to determine the IgG1 antibody titer in the culture medium. After about 5 days, the culture medium containing secreted IgG1 antibodies was collected (centrifuged at 2000 × g for 10 minutes), filtered (Thermo Scientific ^TM Nalgene ^TM Rapid-Flow ^TM sterile disposable filter), and further purified using a gravity flow column (Bio-Rad) filled with Protein A resin (TOYOPEARL AF-rProtein A Hc-650F). The IgG1 antibodies were eluted with 3.5 mL of glycine-HCl (100 mM, pH 2.7), immediately neutralized with 1 M Tris-HCl (pH 8.5), dialyzed with ThermoScientific ^TM Slide-A-Lyzer ^TM G2 dialysis card (20K MWCO) in 1 × PBS buffer (pH 7.2), and stored at 4°C. The concentration of the purified IgG1 antibody was determined using a NanoDrop ^™ One/OneC microvolume UV-Vis spectrophotometer (Thermo Scientific ^™ ), and the quality of the IgG1 antibody was checked using SDS-PAGE gels under denaturing and native conditions.

Example 12: Transfection and purification of TAA/CLEC5A bispecific antibodies

All antibody light and heavy chain coding sequences were generated by direct DNA synthesis and cloned into mammalian expression vectors. The cloned sequences were verified by Sanger sequencing.

Chinese hamster ovary (CHO) cells were grown in CHOgro ^TM medium (Mirus) supplemented with 4 mM L-glutamine and 0.33% Poloxamer-188 and diluted to 4 × 10 ⁶ cells/mL with fresh cell culture medium for transfection. 1 μg of plasmid DNA was used per 4 × 10 ⁶ cells by lipofectamine transfection. The transfected cells were kept in an incubator at 32°C and shaken at 125 rpm in an environment of 5% CO ₂ and 70% humidity.

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

The purified antibodies were analyzed by HPLC (Dionex 3000-RS UHPLC, Thermo Fisher) was used for analysis. Software control. A size exclusion column TSKgel UP-SW3000, 2 μm, 4.6 mm ID×15 cm (Tosoh) was used to determine the purity and aggregation of the antibody.

All purified antibodies had a monomer purity of 97% or higher and were sterilized through a 0.22 μm PES membrane filter for binding and functional assays.

Example 13: Binding of HER2/CLEC5A bispecific antibody to human macrophages

The binding ability of HER2/CLEC5A bispecific antibodies to human macrophages was evaluated. CD14+ monocytes (purified from human PBMC donor #441 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 depletion) (StemCell Technologies, Cat#: 19058)) were cultured for 7 days under the action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) to differentiate into M0 macrophages. About 50,000 M0 macrophages were incubated on ice for 30 minutes with serial dilutions of antibodies in FACS buffer (PBS+0.5% BSA). After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.).

As shown in Figure 10 and the table below, the binding affinity of the HER2/CLEC5A antibody in the 2+2A format and the 1+1A format to M0 macrophage (CLEC5A+) cells is different. However, the binding affinity of the HER2/CLEC5A antibody in the 2+2 format is lower than that of the antibody in the 1+1 format. The sequences of the rabbit-human chimeric antibodies 3A7 and 5C7 were used.

Table 4: Binding of HER2/CLEC5A bispecific antibodies to human macrophages

Example 14: Binding of TAA/CLEC5A bispecific antibodies to human macrophages

The binding ability of different TAA/CLEC5A bispecific antibodies to human macrophages was evaluated. CD14+ monocytes (purified from human PBMC donor #900 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were cultured for 7 days under the action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) and differentiated into M0 macrophages. Approximately 50,000 M0 macrophages were incubated with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) on ice for 30 minutes. After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) As shown in Figures 11-12 and the table below, different TAA/CLEC5A bispecific antibodies can bind to M0 macrophages (CLEC5A+) cells.

Table 5: Binding of TAA/CLEC5A bispecific antibodies to human macrophages

Example 15: Binding of TAA/CLEC5A bispecific antibodies to human CLEC5A

ELISA binding assay evaluates the affinity of different TAA/CLEC5A bispecific antibodies to recombinant human CLEC5A protein. 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 washed with 400 μL of wash buffer (0.05% 20) Wash the wells 3 times with a plate washer. Prepare the TAA/CLEC5A bispecific antibody as a primary antibody at 5 μg/mL and serially dilute 3-fold to 11 points (dilution concentration) with reagent buffer (PBS + 0.05% BSA). Add 100 μL of primary antibody to the appropriate wells and incubate at room temperature for 1 hour. Remove the primary antibody and wash with 400 μL of washing buffer (containing 0.05% The wells were washed three times with 20% PBS, and the secondary antibody BioLegend donkey anti-human HRP (Cat#: 410902) was diluted 1:10,000 with reagent buffer (PBS + 0.5% BSA), 100 μL was added to each well, and incubated at room temperature for 30 minutes. The secondary antibody was aspirated and 400 μL washing buffer (containing 0.05% 20% PBS) was used to wash the wells 3 times. 100 μL Thermo 1-step TMB Turbo (Cat#: 34022) was added to each well and incubated for 10 minutes in the dark at room temperature. 100 μL Fisher 1N Sulfuric Acid (Cat#: SA212-2) stop solution was added to each well and the OD value was read at 450 nm using a microplate reader. For analysis, GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) was used to plot the OD450 of each antibody. As shown in Figure 13 and the table below, all TAA/CLEC5A bispecific antibodies have similar affinities for human CLEC5A.

Table 6: Binding of TAA/CLEC5A bispecific antibodies to human CLEC5A

Example 16: Binding of HER2/CLEC5A bispecific antibody to human HER2

The affinity of HER2/CLEC5A bispecific antibody to recombinant human HER2 protein was detected by ELISA binding assay. Corning high binding flat-bottom 96-well plates (Cat#: 3361) were coated with 1 μg/mL human HER2 (ACROBiosystems, Cat#: HE2-H52R8) and incubated at 4°C for 48 hours. The protein antigen was removed by aspiration and washed with 400 μL of washing buffer (0.05% 20) Wash the wells 3 times with a plate washer. HER2/CLEC5A (2+2A) antibody with optimized Fc was prepared as primary antibody at 5 μg/mL and serially diluted 3-fold to 11 points (dilution concentration) with reagent buffer (PBS+0.05% BSA). 100 μL of primary antibody was added to the appropriate wells and incubated at room temperature for 1 hour. The primary antibody was aspirated and washed with 400 μL of washing buffer (0.05% 20inPBS) were washed 3 times and the plates were washed with reagent buffer (PBS + 0.5% BSA) Dilute the secondary antibody BioLegend Donkey Anti-Human HRP (Cat#: 410902) at 1:10,000, add 100 μL to each well, and incubate at room temperature for 30 minutes. Aspirate the secondary antibody and wash with 400 μL washing buffer (0.05% BSA in PBS) through a plate washer. 20) Wash the wells 3 times. Add 100 μL Thermo 1-step TMB Turbo (Cat#: 34022) to each well and incubate at room temperature in the dark for 10 minutes. Add 100 μL Fisher 1N Sulfuric Acid (Cat#: SA212-2) stop solution to each well and read the OD value at 450nm using a plate reader. For analysis, the OD450 of each antibody was plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in Figure 14 and the table below, the HER2/CLEC5A bispecific antibody binds to human HER2 with high affinity.

Table 7: Binding of HER2/CLEC5A bispecific antibodies to human HER2

Example 17: Binding of EGFR/CLEC5A bispecific antibody to human EGFR

ELISA binding was performed to determine the affinity of different anti-EGFR hIgG1 (Cetuximab, Panitumumab, Necitumumab, Eg-B4-VHH) and EGFR/CLEC5A (2+2A) antibodies with optimized Fc to the EGFR portion of Amivantamab (EGFR1) and Nimotuzumab (EGFR2) for recombinant human EGFR protein. Specifically, Corning high-binding flat-bottom 96-well plates (Cat#: 3361) were coated with 1 μg/mL human EGFR (ACROBiosystems, Cat#: EGR-H5222) at 4°C for 48 hours. The protein antigen was aspirated and washed with 400 μL of washing buffer (0.05% in PBS) by a plate washer. 20) Wash the wells 3 times. EGFR hIgG1 and EGFR/CLEC5A (2+2A) antibodies with optimized Fc were prepared as primary antibodies at 5 μg/mL and serially diluted 3-fold to 11 points (dilution concentration) with reagent buffer (PBS+0.05% BSA). 100 μL of primary antibody was added to the appropriate wells and incubated at room temperature for 1 hour. The primary antibody was aspirated and washed with 400 μL of washing buffer (0.05% BSA in PBS) through a plate washer. 20) Wash the wells 3 times. Dilute the secondary antibody BioLegend Donkey Anti-Human HRP (Cat#: 410902) in reagent buffer (PBS + 0.5% BSA) at 1:10,000, add 100 μL to each well, and then incubate at room temperature for 30 minutes. Aspirate the secondary antibody and wash with 400 μL washing buffer (0.05% BSA in PBS) using a plate washer. 20) Wash the wells 3 times. Add 100 μL Thermo 1-step TMB Turbo (Cat#: 34022) to each well and incubate at room temperature in the dark for 10 minutes. Add 100 μL Fisher1N Sulfuric Acid (Cat#: SA212-2) stop solution to each well and read the OD value at 450 nm using a plate reader. For analysis, GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.) was used to plot the OD450 of each antibody. As shown in Figure 15 and the table below, the EGFR/CLEC5A bispecific antibody has a lower binding affinity to human EGFR than Cetuximab and Necitumumab.

Table 8: Binding of EGFR/CLEC5A bispecific antibodies to human EGFR

Example 18: Binding of EpCAM/CLEC5A bispecific antibody to DLD-1

The binding ability of anti-EpCAM antibodies (Solitomab EpCAM-binding partial IgG format) and EpCAM/CLEC5A (2+2A) antibodies with optimized Fc to DLD-1 cells (EPCAM+) was evaluated. Approximately 50,000 DLD-1 cells (ATCC, CCL-221) were incubated with serial dilutions of antibodies in FACS buffer (PBS+0.5% BSA) on ice for 30 minutes. After incubation, cells were washed twice and probed with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in Figure 16 and the table below, the anti-EpCAM antibodies showed higher binding affinity to DLD-1 cells compared to the EpCAM/CLEC5A bispecific antibodies.

Table 9: Binding of EpCAM/CLEC5A bispecific antibodies to DLD-1

Example 19: Binding of CD79b/CLEC5A bispecific antibody to Ramos cells

The binding ability of CD79b/CLEC5A bispecific antibodies to Ramos cells (CD79b+) was evaluated. Approximately 50,000 Ramos cells (ATCC, CRL-1596) were incubated with serially diluted antibodies in FACS buffer (PBS+0.5% BSA) on ice for 30 minutes. After incubation, the cells were washed twice and detected with anti-human IgG PE secondary antibody (Jackson Immuno Research, Cat#: 109-116-170). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in Figure 17, the results indicate that the 2+2A and 2+2B forms of CD79b/CLEC5A bispecific antibodies have similar affinities for Ramos cells.

Example 20: HER2/CLEC5A bispecific antibody mediates killing of SK-BR-3 cells by M0 macrophages

The killing ability of the HER2/CLEC5A bispecific antibody on the target cancer SK-BR-3 cells (HER2+) was evaluated by effector macrophages (CLEC5A+) M0 cells through phagocytosis and engulfment mechanisms. Specifically, CD14+ monocytes (purified from human PBMC donor #031 using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 depletion) (StemCell Technologies, Cat#: 19058)) were cultured for 7 days in the presence of 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) and differentiated into M0 macrophages. SK-BR-3 cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+SK-BR-3 (E:T ratio of 5:1) in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were suspended in 100 μL of FACS buffer containing SYTOX ^TM Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and used Flow cytometry was used for analysis. SK-BR-3 cells were divided into CFSE+ by FACS, and the absolute cell counts of CFSE+ cells were obtained by collecting a fixed volume under all treatment conditions. The target cell killing percentage was calculated as follows: (absolute number of CFSE+SK-BR-3 cells in the non-treatment group - absolute number of CFSE+SYTOX-SK-BR-3 cells in the treatment group) / absolute number of CFSE+SK-BR-3 cells in the non-treatment group × 100.

As shown in Figures 18A-18C and the table below, the HER2/CLEC5A bispecific antibody effectively kills the HER2+ cancer cells SK-BR-3, indicating that the HER2/CLEC5A bispecific antibody can kill tumor cells expressing the target antigen HER2 by recruiting macrophages.

Table 10: Killing of target cancer cells by HER2/CLEC5A bispecific antibodies

Example 21: HER2/CLEC5A bispecific antibody mediates killing of SK-BR-3 cells by M1 and M2 macrophages

The killing ability of effector macrophages (CLEC5A+) differentiated into M1 or M2 states by HER2/CLEC5A bispecific antibody was evaluated against target cancer SK-BR-3 cells (HER2+) through phagocytic and engulfing mechanisms. CD14+ monocytes (purified from human PBMC donor #900 using EasySep ^TM Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 6 days of treatment with 50 μg/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were differentiated into M1 with 50 μg/mL IFN-γ (StemCell Technologies, Cat#: 78020) or into M2 with 25 μg/mL IL-10 (StemCell Technologies, Cat#: 78024) for another 24 hours (day 7). SK-BR-3 cells were labeled with CFSE (ThermoFisher, Cat#: C34554). Approximately 100 μg/mL of antibodies were added to complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) and then incubated for 24 hours. 100,000 macrophages were incubated with 20,000 CFSE+SK-BR-3 (E:T ratio of 5:1) at 37°C for 24 hours. After incubation, the cells were suspended in 100 μL FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry was used for analysis. SK-BR-3 cells were selected as CFSE+ by FACS, and the absolute cell counts of CFSE+ cells were obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as follows: (absolute number of CFSE+SK-BR-3 cells in the non-treatment group-absolute number of CFSE+SYTOX-SK-BR-3 cells in the treatment group)/absolute number of CFSE+SK-BR-3 cells in the non-treatment group × 100. As shown in Figure 19 and the table below, surprisingly, the HER2/CLEC5A bispecific antibody can effectively kill HER2+ cancer cells SK-BR-3 by recruiting M1 and M2 macrophages.

Table 11: HER2/CLEC5A bispecific antibody mediates killing of target cancer cells by M1 and M2 macrophages

Example 22: Cytokine production by HER2/CLEC5A bispecific antibody

The ability of the HER2/CLEC5A bispecific antibody to activate M1 and M2 polarized macrophages in the presence or absence of target SK-BR-3 cells (HER2+) was evaluated. CD14+ monocytes (purified from human PBMC donor #900 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages for 6 days in the presence of 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were polarized to M1 with 50 ng/mL IFN-γ (StemCell Technologies, Cat#: 78020) or to M2 with 25 ng/mL IL-10 (StemCell Technologies, Cat#: 78024) for another 24 hours (day 7). Approximately 100,000 macrophages were incubated with 20,000 SK-BR-3 (E:T ratio of 5:1) at 37°C for 24 hours in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After incubation, the supernatant was collected for cytokine analysis. The cytokine IFN-γ was measured using the V-PLEX Plus pro-inflammatory Panel 1 Human Kit (MesoScale Discovery, Cat#: K15049G-1). As shown in Figure 20, the HER2/CLEC5A bispecific antibody activated M1 and M2 macrophages only in the presence of SK-BR-3, with low levels of cytokine release.

Example 23: Effects of plasma and hIgG1 on target cell killing mediated by HER2/CLEC5A bispecific antibody

The effects of human plasma and purified hIgG1 antibodies on HER2/CLEC5A antibody-mediated killing of cancer SK-BR-3 cells (HER2+) by effector macrophages (CLEC5A+) were evaluated. CD14+ monocytes (purified from human PBMC donor #308 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 depletion) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages in the presence of 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. SK-BR-3 cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+SK-BR-3 (E:T ratio of 5:1) in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours with the addition of medium (control), 50% plasma (heparin) or hIgG1 (BioLegend, Cat#: 403502). After incubation, the cells were suspended in 100 μL of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and analyzed using Flow cytometry was used for analysis. SK-BR-3 cells were set as CFSE+ by FACS, and the absolute cell counts of CFSE+ cells were obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as follows: (absolute number of CFSE+SK-BR-3 cells in the non-treatment group-absolute number of CFSE+SYTOX-SK-BR-3 cells in the treatment group)/absolute number of CFSE+SK-BR-3 cells in the non-treatment group × 100. As shown in Figures 21A-21C and the table below, the activity of different HER2/CLEC5A bispecific antibodies was not significantly reduced even in the presence of plasma or hIgG1.

Table 12: HER2/CLEC5A bispecific antibody-mediated target cell killing in the presence of culture medium, plasma or hIgG1

Example 24: EGFR/CLEC5A bispecific antibody mediates macrophage killing of DLD-1 cells

Different EGFR hIgG1 (Cetuximab, Panitumumab, Necitumumab, Eg-B4-VHH) and EGFR/CLEC5A (2+2A) Fc optimized antibodies were evaluated with the EGFR portion of Amivantamab (EGFR1) and Nimotuzumab (EGFR2) antibodies to mediate the killing effect of macrophages (CLEC5A+) on targeted cancer DLD-1 cells (EGFR+) through phagocytic and engulfing mechanisms. Specifically, CD14+ monocytes (purified from human PBMC donor #900 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages in the presence of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. DLD-1 cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+DLD-1 (E:T ratio of 5:1) at 37°C for 24 hours in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After incubation, the cells were suspended in 100 μL of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry was used for analysis. DLD-1 cells were set as CFSE+ by FACS, and the absolute cell counts of CFSE+ cells were obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+DLD-1 cells in the non-treatment group-absolute number of CFSE+SYTOX-DLD-1 cells in the treatment group)/absolute number of CFSE+DLD-1 cells in the non-treatment group × 100. As shown in Figure 22 and the table below, the EGFR/CLEC5A bispecific antibody has a higher killing effect on EGFR+ target cells.

Table 13: EGFR/CLEC5A bispecific antibody mediates macrophage killing of DLD-1 cells

Example 25: EpCAM/CLEC5A bispecific antibody mediates macrophage killing of DLD-1 cells

The anti-EpCAM antibody (EpCAM binding portion of Solitomab in IgG format) and EpCAM/CLEC5A (2+2A) Fc optimized antibody were evaluated for their ability to mediate macrophage (CLEC5A+) killing of targeted cancer DLD-1 cells (EpCAM+) through phagocytic and engulfing mechanisms. CD14+ monocytes (purified from human PBMC donor #900 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 7 days in the presence of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). DLD-1 cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+DLD-1 (E:T ratio of 5:1) serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were suspended in 100 μL of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry was used for analysis. DLD-1 cells were divided into CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting fixed volumes under all treatment conditions. The target cell killing percentage was calculated as: (absolute number of CFSE+DLD-1 cells in the non-treatment group-absolute number of CFSE+SYTOX-DLD-1 cells in the treatment group)/absolute number of CFSE+DLD-1 cells in the non-treatment group × 100. As shown in Figure 23 and the table below, the EpCAM/CLEC5A bispecific antibody has a higher killing effect on EpCAM+ target cells.

Table 14: EpCAM/CLEC5A bispecific antibody mediates macrophage killing of DLD-1 cells

Example 26. EpCAM/CLEC5A bispecific antibody mediates macrophage killing of various cancer cells

hEpCAM expression levels in various cancer cell lines were evaluated. Various cancer cell lines A549, DLD-1, MCF-7, SKPR3, SKOV3 and T47D were used. 50,000 cancer cells were stained with 0.5 nM anti-EpCAM antibody-AF488 (1:200 dilution, BioLegend, Cat#: 302208) on ice for 30 minutes. The cells were washed twice with PBS and then stained with DAPI (1:5000 dilution, 1 mg/ml, Invitrogen, Cat#: D1306) for 5 minutes at room temperature and then analyzed by flow cytometry ( Northern Lights TM) were tested. The raw data were analyzed by FlowJo TM Portal10 and graphed using GraphPad Prism 10. As shown in Figure 24, these cell lines exhibited different EpCAM densities. The killing effect of EpCAM/CLEC5A bispecific antibody-mediated M0 macrophages on target cell lines with different EpCAM densities was evaluated. Various cancer cell lines A549, DLD-1, MCF-7, SKPR3, SKOV3 and T47D were used. CD14+ monocytes (purified from human PBMCs using EasySep ^TM Human Monocyte Enrichment Kit (CD16 not removed) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 7 days of action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). Tumor cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+ tumor cells (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were resuspended in 100 μl FACS buffer with SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with The cells were analyzed by cytometer. FACS gated tumor cells as CFSE+, and the absolute cell counts of CFSE+ cells were obtained by collecting a fixed volume under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+ tumor cells in the non-treatment group - absolute number of CFSE+SYTOX- tumor cells in the treatment group) / absolute number of CFSE+ tumor cells in the non-treatment group × 100. As shown in Figures 25A-25F, the EpCAM/CLEC5A bispecific antibody had a higher killing effect on target cells with different EpCAM densities.

Example 27: Different myeloid cell engagers mediate PBMC killing of multiple myeloma cancer cells

The killing effect of myeloid cell binders on multiple myeloma cancer cells (NCI-H929) in healthy human PBMCs was evaluated. Specifically, myeloid cell binders (e.g., GPRC5D/5C7(2+2A)Fc-optimized, BCMA/5C7(2+2A)Fc-optimized, CD38/5C7(2+2A)Fc-optimized) were serially diluted. NCI-H929 cells (as target cells) were first stained with CFSE and then mixed with healthy human PBMCs (E:T=10:1) in a 96-well round-bottom plate. The cells were then incubated with serially diluted bispecific antibodies (1- to 3-fold dilutions from 100 nM) for 48 hours. After incubation, the plates were spun down and the cells were stained with live/dead staining dye. The stained cells were processed using a CytoFLEX LX flow cytometer, and the cell killing rate was calculated as follows: (number of live tumor cells in the non-treated group - number of live tumor cells in the treated group) / number of live tumor cells in the non-treated group × 100.

As shown in Figure 26, the maximum killing rate of BCMA/5C7 (2+2A) Fc-optimized and CD38/5C7 (2+2A) Fc-optimized bispecific antibodies against NCI-H929 was about 99%; the maximum killing rate of GPRC5D/5C7 (2+2A) Fc-optimized bispecific antibody was about 92%. The results show that myeloid cell engagers can effectively kill multiple myeloid cancer cells with different targets.

Example 28: CD79b expression levels in malignant lymphoma cell lines and B cells from healthy PBMC donors

hCD79b expression levels on malignant lymphoma cell lines and B cells in healthy PBMC donors were evaluated. Malignant lymphoma cell lines (Ramos and Daudi) were from ATCC, and frozen PBMCs were from healthy donors (Stanford Blood Center). 50,000 Ramos or Daudi cells and 20,0000 PBMCs were stained with 0.5nM anti-CD79b antibody-AF488 and anti-CD19 antibody-PE (1:200 dilution, BioLegend, Cat#: 302208) on ice for 30 minutes. The cells were washed twice with PBS and then stained with DAPI (1:5000 dilution, 1mg/mL, Invitrogen, Cat#: D1306) for 5 minutes at room temperature and then analyzed by flow cytometry ( The raw data were analyzed by FlowJo™ Portal 10 and plotted using GraphPad Prism. As shown in Figure 27, the expression level of hCD79b in Ramos and Daudi cells was about 4-10 times higher than that in healthy B cells in PBMCs.

Example 29: PBMC immunophenotyping

To understand the proportion of various cell populations in PBMCs, immunophenotyping of cells was performed. Healthy human blood was obtained from the Stanford Blood Center. PBMCs were isolated using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058). PBMCs were washed twice with FACS buffer (1×PBS+2% BSA+2mM EDTA). 1×10 ⁵ PBMCs were seeded in each well of a V-bottom 96-well plate. Then 50 μL of antibody mixture (BV421CD19 (BD Biosciences, Cat#: 562440), BV605 CD11b (BioLegend, Cat#: 101237), BV711 CD11 (BD Biosciences, Cat#: 563130, FITC CD56 (BD Biosciences, Cat#: 340410), PE CD8 (BioLegend, Cat#: 344706), Percpcy5.5 CD14 (BD Biosciences, Cat#: 550787), PEcy7 CD16 (BD Biosciences, Cat#: 557744), APC CD3 (BD Biosciences, Cat#: 555335), AF700 CD4 (BD Biosciences, Cat#: 566318), AF700 HLA-DR (BD Biosciences, Cat#: 566319), Biosciences, Cat#: 560743)) was added to each well. The mixture was incubated on ice for 30 minutes; then the cells were washed twice with FACS buffer (after centrifugation at 1200 rpm for 5 minutes), and the cells were suspended in 100 μL SYTOX ^TM Blue Dead Cell Stain (1:1000) buffer and incubated on ice in the dark for 20 minutes. Analyze cells by flow cytometry. The proportions of various cell populations were calculated based on the number of surface marker-positive cells (CD19+B cells, CD56+NK cells, CD14+ monocytes, CD8+T cells, CD4+T cells, CD11c+/HLA-DR+DC cells and CD11b+/CD16+neutrophils).

Table 15: PBMC immunophenotypes from different donors

Example 30: CD79b/CLEC5A bispecific antibody mediates PBMC killing of Ramos cells

To detect the tumor killing effect of fresh human PBMCs and Ramos cells mediated by CD79b/CLEC5A bispecific antibody. Fresh human PBMCs were isolated from healthy human blood donated by SBC (Stanford Blood Center) and cultured using U-bottom 96-well plates. Ramos cells were stained with CFSE staining buffer (Invitrogen). 2×10 ⁴ CFSE-stained Ramos cells (dissolved in 50 μL culture medium) were added to each designated well. 2×10 ⁵ PBMCs (dissolved in 130 μL culture medium) were added to each designated well. 20 μL of the antibody to be tested serially diluted in complete culture medium was added to each designated well, and the final total volume of each well was 200 μL. The culture plate with mixed cells was incubated at 37°C and 5% CO _2. Samples were collected after 24 hours of incubation. The cell pellet was suspended in FACS buffer and washed twice. The cells were stained with SYTOX ^TM Blue Dead Cell Stain and PE-conjugated CD19. Use Cells were analyzed by cell analyzer (live Ramos cell gating: CFSE+SYTOX- or CD19+CFSE+SYTOX-). The killing efficiency was calculated as follows: (Killing rate % = (control live Ramos cell count - treatment live Ramos cell count) / control live Ramos cell count) × 100. As shown in Figures 28A-28C, all CD79b/CLEC5A bispecific antibodies showed a superior Ramos cell killing effect than commercial Mosunetuzumab at 24 hours, and CD79b/CLEC5A bispecific antibodies showed higher endogenous B killing than Mosunetuzumab in PBMCs of all three donors.

The effect of CD79b/CLEC5A bispecific antibody-mediated Ramos killing on malignant B cell depletion was evaluated. Specifically, fresh PBMCs were isolated from healthy blood samples provided by the Stanford Blood Center using Ficoll-paque gradient centrifugation. Ramos cells (target cells) were first stained with CFSE and then mixed with PBMCs (effector cells) at a ratio of E:T = 10:1. The cells were then placed in a round-bottom 96-well plate with titrated antibodies and incubated for 24 hours at 37°C and 5% _CO2 . The true E:T ratio calculated using monocytes: B (endogenous B cells + Ramos cells) is 0.5:1, and if NK cells are included as effector cells, it is 1.5:1. After 24 hours, the cells were spun down. For flow cytometry analysis, the supernatant was collected for ELISA analysis of cytokine release. The cell samples were washed once with PBS and then stained with Zombie Aqua ^TM 1:1000 dilution at 4°C for 20 minutes. After staining, the cells were washed once with PBS, resuspended in flow buffer, and (Northern light ^TM ) flow cytometer was used for analysis. Ramos cell killing rate % = (non-treatment group Ramos cells - treatment group Ramos cells) / non-treatment group Ramos cells × 100. As shown in Figures 29A-29B and the following table, for donor 234, compared with clinically used Mosunetuzumab (IC50 is about 0.25nM), CD79b/CLEC5A bispecific antibodies with different structures all showed higher Ramos killing effects (IC50 is about 60% to 80% higher). For example, the IC50 range of CD79b/CLEC5A bispecific antibodies ranges from 0.0047nM for CD79b/5C7(2+1A)Fc-optimized to 0.338nM for CD79b/5C7(1+1A)Fc-optimized. For donor 431, if calculated as monocytes: B cells, the true E:T <1:1, if NK cells are included as effector cells, E:T = 2:1. The results of Ramos intervention PBMC killing showed that compared with Mosunetuzumab (IC50 of 0.0069nM), CD79b/CLEC5A bispecific antibodies with different structures all showed higher Ramos killing effects (IC50 was about 80% to 100% higher). For example, the IC50 of CD79b/CLEC5A bispecific antibodies ranged from 0.0062nM for CD79b/5C7(2+2B)Fc-silence to 0.0329nM for CD79b/5C7(1+1B)Fc-silence.

Table 16: CD79b/CLEC5A bispecific antibody mediates PBMC killing of Ramos cells (experiment 2)

In order to detect the killing effect of CD79b/CLEC5A bispecific antibody on Ramos cells, a co-culture system of human PBMCs and Ramos cells was established. PBMCs were isolated from healthy human blood using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 depletion) (StemCell Technologies, Cat#: 19058). Ramos cells were stained with CFSE (Invitrogen). 20 μl of serially diluted bispecific antibody was added to each well of a U-bottom 96-well plate. 2×10 ⁵ PBMC and 2×10 ⁴ Ramos cells (E:T ratio = 10:1) in 180 μl complete medium (RPMI1640 + 10% FBS) were added to each well. The mixture was incubated at 37°C and 5% CO ₂ for 24 hours. The cells were centrifuged at 1500 rpm for 5 minutes. The cell pellet was washed and suspended in FACS buffer, stained and The cells were analyzed by a cell analyzer. The staining group included SYTOX ^TM Blue Dead Cell Stain and Brilliant Violet 421 ^TM anti-human CD19 antibody (BV421 CD19). Live CFSE+ cells were classified as viable tumor cells. The killing effect calculation formula is: killing = (control tumor cell number - treatment tumor cell number) / control tumor cell number. As shown in Figures 30A-30C and the table below, all CD79b/CLEC5A bispecific antibodies with different structures showed high Ramos killing effects at very low E:T ratios.

Table 17: CD79b/CLEC5A bispecific antibody mediates PBMC killing of Ramos cells (experiment 3)

The ability of the CD79b/CLEC5A bispecific antibody to stimulate IL-6 secretion in the presence of targeted Ramos cells (CD79b+) was evaluated. Approximately 200,000 PBMCs were incubated with 20,000 Ramos cells (E:T ratio of 10:1) at 37°C for 24 hours in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After incubation, the supernatant was collected for IL-6 analysis. As shown in Figures 31A-31B, in the presence of targeted Ramos cells, IL-6 levels were not significantly increased by the CD79b/CLEC5A bispecific antibody. However, IL-6 levels were increased in a dose-dependent manner by commercialized mosunetuzumab. The results showed that the CD79b/CLEC5A bispecific antibody had a better safety profile than mosunetuzumab.

To detect cytokine release in the Ramos interventional assay, supernatants from the Ramos killing assay were collected at 24 hours and the TNFα concentration in the supernatant was measured using an ELISA kit (TNF alpha Human Uncoated ELISA Kit, Invitrogen, Cat#: 88-7346-88). 25 μL of sample was tested with 25 μL of reagent diluent (2-fold dilution) in a 96-well high-binding culture plate coated with capture antibody for 24 hours. As shown in Figure 32, the concentration of TNFα released was high in the mosunetuzumab treatment group, but was low or at background level in the CD79b/CLEC5A bispecific antibody treatment group.

Example 31: CD79b/CLEC5A bispecific antibody mediates PBMC killing of endogenous B cells

The killing effect of CD79b/CLEC5A bispecific antibody on endogenous B cells was detected. Specifically, fresh human PBMCs were isolated from healthy human blood donated by SBC (Stanford blood Center) using U-bottom 96-well plates. 2×10 ⁵ PBMCs in 130 μl of culture medium were added to each designated well. Serially diluted test antibodies in 20 μl of complete culture medium were added to each designated well, and the final total volume of each well was 200 μl. The culture plate with mixed cells was incubated at 37°C and 5% CO _2. Samples were collected after 24 hours of incubation. The cell pellet was suspended in FACS buffer and washed twice. The cells were stained with SYTOX ^TM blue dead cell stain and PE-conjugated anti-CD19 antibody. Cells were analyzed by cell analyzer (live B cell gating: CFSE+SYTOX- or CD19+CFSE+SYTOX-). Endogenous B cell killing rate % = (endogenous B cells in non-treatment group - endogenous B cells in treatment group) / endogenous B cells in non-treatment group × 100. As shown in Figures 33A-33C, the CD79b/CLEC5A bispecific antibody showed a higher endogenous B cell killing effect than Mosunetuzumab in PBMCs of all three donors.

To test the efficacy of CD79b/CLEC5A bispecific antibody to deplete endogenous B cells, we used healthy human PBMCs. Fresh PBMCs were isolated from healthy blood samples provided by the Stanford Blood Center using Ficoll-paque gradient centrifugation. 2×10 ⁵ /well PBMCs were plated with titrated antibodies in a round-bottom 96-well plate and incubated for 24 hours at 37°C and 5% CO _2. The true E:T ratio, which varied from donor to donor, was calculated using monocytes to endogenous B cells or monocytes plus NK cells to endogenous B cells. After 24 hours, cells were centrifuged for flow cytometry analysis and supernatants were collected for cytokine release analysis by ELISA. Cell samples were washed once with PBS and then stained with Zombie Aqua ^TM 1:1000 dilution for 20 minutes at 4°C. After staining, cells were washed once with PBS and then stained with CD19-BV421 (BioLegend, Cat#: 302234) for 30 minutes at 4°C. The stained cells were washed once with PBS, resuspended in flow buffer, and then Flow cytometry (Northern light ^TM ) was used for analysis. Endogenous B cell killing rate % = (endogenous B cells in non-treatment group - endogenous B cells in treatment group) / non-treatment Group endogenous B cells × 100. As shown in Figures 34A-34D and the table below, for donor 023, the E:T of monocytes: endogenous B cells is 2:1, and if NK cells are included as effector cells, the number is 7:1. Mosunetuzumab used clinically has only about 65% effect at 10nM, while CD79b/CLEC5A bispecific antibodies with different structures all show higher endogenous B cell killing effects, about 80% to 96%. For donor 234, monocytes: endogenous B cells (E:T) <1:1, if NK cells are included as effector cells, E:T = 3:1. Compared with Mosunetuzumab used clinically (IC50 is 0.086nM), CD79b/CLEC5A bispecific antibodies with different structures all show higher endogenous B cell killing effects, about 90%. For donor 431, the ratio of monocytes to endogenous B cells (E:T) was <1:1. If calculated by (monocytes + NK): endogenous B, then E:T = 2:1. CD79b/CLEC5A bispecific antibodies with different structures all showed higher endogenous B cell killing effects. Similar results were observed in donor 367.

Table 18. Cytotoxicity of PBMCs to endogenous B cells mediated by different myeloid cell engagers

The tumor killing effect of endogenous B cells mediated by CD79b/CLEC5A bispecific antibody was tested. Specifically, fresh human PBMCs were isolated from healthy human blood donated by SBC (Stanford Blood Center), and 2×105/well PBMCs were incubated with titrated antibodies in round-bottom 96-well plates at 37°C and 5% CO2 for 24 hours. Samples were collected after 24 hours of incubation. The cell pellet was resuspended in FACS buffer and rinsed twice. The cells were stained with SYTOX ^TM blue dead cell stain and PE-coupled anti-CD19 antibodies. Endogenous B cell killing rate % = (endogenous B cells in the non-treatment group-endogenous B cells in the treatment group)/endogenous B cells in the non-treatment group × 100. The results are shown in Figures 35A-35B.

To detect cytokine release in the endogenous B cell killing assay, the supernatant of the endogenous B cell killing assay was collected at 24 hours and the TNFα concentration in the supernatant was measured using an ELISA kit (TNF alpha human uncoated ELISA kit, Invitrogen, Cat#: 88-7346-88). 25 μL of sample was tested in 25 μL of reagent diluent (2-fold dilution) in a 96-well high binding culture plate coated with the capture antibody for 24 hours. As shown in Figure 36, in the CD79b/CLEC5A bispecific antibody treatment group, the TNFα concentration was at a low or background level, indicating that the myeloid cell engager is safer than the benchmark antibody Mosunetuzumab.

Example 32: CD79b/CLEC5A bispecific antibody mediates M0 macrophage killing of Ramos cells

The killing effect of different structural CD79b/CLEC5A bispecific antibodies on target cancer Ramos cells (CD79b+) by mediating macrophages (CLEC5A+) was evaluated. CD14+ monocytes (purified from human PBMC donors #100, #233, #022, #431, #159 and #161 using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 7 days of action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). Ramos cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were resuspended in 100 μl FACS buffer with SYTOX ^™ Blue Dead Cell Stain (ThermoFisher, Cat#: S34857) and stained with Cytometer was used for analysis. Ramos cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figures 37A-37F and the table below, all CD79b/CLEC5A myeloid cell conjugates can effectively kill CD79b+ cancerous Ramos cells.

Table 19: Different myeloid cell engagers mediate the killing of Ramos cells by M0 macrophages

Cytokine release of CD79b/CLEC5A bispecific antibodies of different structures in the presence or absence of target Ramos cells was evaluated. CD14+ monocytes (purified from human PBMC donors #100, donor #233, donor #022, and donor #431 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages under the action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. Approximately 100,000 macrophages were incubated with 20,000 Ramos cells (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the supernatant was collected for cytokine analysis. TNFα and IL-6 levels in the supernatant were measured using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the supplier's instructions. As shown in Figures 38A-38D, IL-6 and TNFα were only expressed in the target Ramos cells. In the presence of CD79b and CD79b/CLEC5A antibodies, CLEC5A was released from macrophages in a dose-dependent manner and at very low levels, indicating that myeloid cell engagers can be safely used clinically.

To evaluate the killing effect of M0 macrophages on cancerous B cells (Ramos) mediated by different E:T ratios, human monocytes were isolated from PBMCs (donors #22461 and #22657) using the EasySep ^™ Human Monocyte Enrichment Kit (StemCell Technologies, Cat#: 19058) without CD16 depletion. The isolated monocytes were induced with 50ng/mL M-CSF (StemCell Technologies, Cat#: 78057). After 7 days, monocytic differentiated macrophages (M0) were obtained. M0 macrophages and Ramos cells at different E:T ratios were incubated with myeloid cell engagers to test their function in M0 macrophages. The myeloid cell engager CD79b/CLEC5A bispecific antibody was serially diluted. Ramos cells were first stained with CFSE, then added to M0 macrophages (E:T = 5:1, E:T = 2:1 or E:T = 1:1) in a 96-well round-bottom plate (fixed Ramos cell number: 2×104/well), and then incubated with serially diluted antibodies (1 to 3 dilutions starting from 10 nM). After incubation for 24 hours, the plate was centrifuged and the supernatant was stored at -80°C. The cells were stained with live/dead staining dye and then processed on a CytoFLEXLX flow cytometer. The cell killing rate was calculated as: (number of live tumor cells in the non-treatment group - number of live tumor cells in the treatment group) / number of live tumor cells in the non-treatment group × 100. As shown in Figures 39A-39B, the myeloid cell binder exhibited a potent Ramos cell killing effect at different E:T ratios, even at low E:T ratios (e.g., E:T = 1:1). At 24 h, the maximum specific tumor cell killing rate of Ramos was approximately 99% (E:T=5:1), 95% (E:T=2:1), and 80% (E:T=1:1), indicating that myeloid cell engagers can effectively promote target cell killing at a low E:T ratio of M0 macrophages to target cells.

To evaluate the release of cytokines that mediate the killing of cancerous B cells (Ramos) by M0 macrophages at different E:T ratios, human monocytes were isolated from PBMCs (donors #22461 and #22657) using the EasySep ^™ Human Monocyte Enrichment Kit (StemCell Technologies, Cat#: 19058) without CD16 removal. The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). After 7 days, monocytic differentiated macrophages (M0) were obtained. M0 macrophages and Ramos cells at different E:T ratios were incubated with myeloid cell engagers to detect their function in M0 macrophages. Myeloid cell engager CD79b/CLEC5A bispecific antibody was serially diluted. Ramos cells were first stained with CFSE, then added to M0 macrophages (E:T=5:1, E:T=2:1 or E:T=1:1) in a 96-well round-bottom plate (fixed Ramos count: 2×10 ⁴ /well), and then incubated with serially diluted CD79b/CLEC5A bispecific antibodies (1-3 times dilution starting from 10 nM). After incubation for 24 hours, the plate was centrifuged and the supernatant was recovered for ELISA analysis. ELISA was performed according to the instructions of the ELISAMAX ^TM Deluxe Set Human TNF-α (BioLegend, Cat#: 430204) and ELISAMAX ^TM Deluxe Set Human IL-6 (BioLegend, Cat#: 430504) kits. Use Absorbance Reader (MOLECULARDEVICEs, S/N3052431867) measured the absorbance at 450 nm within 15 minutes. In the absence of target cells (Ramos) incorporation, the myeloid cell binder did not mediate TNFα or IL-6 release in vitro. As shown in Figures 40A-40B, in the presence of target cells (Ramos), the myeloid binder showed weak TNFα and IL-6 release at different E:T ratios in vitro. The maximum release of TNFα at 24 hours was less than 150pg/mL, while the maximum release of IL-6 was less than 200pg/mL, indicating that the myeloid cell binder can effectively promote target cell killing, but induces M0 macrophages to release very weak cytokines to target cells.

The killing effect of macrophages (CLEC5A+) on target cancer Ramos cells (CD79b+) mediated by CD79b/CLEC5A bispecific antibodies with different structures was evaluated. CD14+ monocytes (purified from human PBMC donor #022 and donor 0112 using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were induced to differentiate for 7 days with 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) and differentiated into M0 macrophages. Ramos cells were labeled with CFSE (ThermoFisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. Approximately 80,000 macrophages were incubated with 40,000 CFSE+Ramos (E:T ratio of 2:1) serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. Approximately 60,000 macrophages were incubated with 60,000 CFSE+Ramos (E:T ratio of 1:1) serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were suspended in 100 μl of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and analyzed using Flow cytometry was used for analysis. Ramos cells were divided into CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figures 41A-41C, all myeloid cell conjugates can effectively kill CD79b+ cancerous Ramos cells.

The cytokine release of the CD79b/CLEC5A bispecific antibodies of different structures was evaluated. After incubation, the supernatant was collected for cytokine analysis. TNFα and IL-6 levels in the supernatant were measured using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the supplier's instructions. As shown in Figure 42, both IL-6 and TNFα were at very low levels, indicating that the myeloid cell engager can be safely used in clinical practice.

In order to understand whether the activated M0 macrophages further induce suicide and reduce their number, we analyzed the number of M0 macrophages to detect their status. Ramos cells were stained with 0.25uM CSFE after dead cells were removed. The number of M0 macrophages is shown in Figure 43, and there is no obvious change in the number of macrophages.

Example 33: CD79b/CLEC5A bispecific antibody mediates killing of Daudi cells by M0 macrophages

To evaluate the killing effect of M0 macrophages on cancerous B cells (Daudi) at different E:T ratios, human monocytes were isolated from PBMC (donors #21232, #22657 and #22461) using the EasySep ^TM Human Monocyte Enrichment Kit (StemCell Technologies, Cat#: 19058) without CD16 removal. The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). After 7 days, monocytic differentiated macrophages (M0) were obtained. M0 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engagers to detect their function in M0 macrophages. The myeloid cell engager CD79b/CLEC5A bispecific antibody was serially diluted. Daudi cells were first stained with CFSE and then added to M0 macrophages (E:T=5:1, E:T=2:1, E:T=1:1, or E:T=1:2) in 96-well round-bottom plates (fixed Daudi count: 2×10 ⁴ /well) and incubated with serially diluted antibodies (1 to 3 times dilution starting from 10 nM). After 24 hours of incubation, the culture plates were centrifuged and the supernatants were stored at -80°C. Cells were stained with live/dead staining dye and then processed on a CytoFLEX LX flow cytometer. The cell killing percentage was calculated as follows: (number of live tumor cells in the non-treatment group - number of live tumor cells in the treatment group)/number of live tumor cells in the non-treatment group × 100. As shown in Figures 44A-44B, the myeloid cell binder exhibited potent Daudi cell killing effects on M0 macrophages at different E:T ratios in vitro. For Daudi cells, the maximum specific tumor cell killing rate was about 97%-99% at E:T=5:1 after 24 hours, 90%-96% at E:T=2:1, and 60%-89% at E:T=1:1, indicating that myeloid cell binders can effectively promote target cell killing at low E:T ratios of M0 macrophages to target cells. As shown in Figures 45A-45B, myeloid cell binders showed potent and comparable Daudi cell killing effects in M0 macrophages in vitro at different E:T ratios. For Daudi cells, the maximum specific tumor cell killing rate at 24 hours was about 99% (E:T=5:1), 96% (E:T=2:1), 92% (E:T=1:1), and 90% (E:T=1:2), indicating that myeloid cell binders can promote effective target cell killing at relatively low E:T ratios of M0 macrophages to target cells.

To evaluate the release of cytokines upon killing of cancerous B cells (Daudi) by myeloid cell engager CD79b/CLEC5A bispecific antibody in M0 macrophages at different E:T ratios, human monocytes (StemCell Technologies, Cat#: 19058) were isolated from PBMCs (donors #21232, #22657, and #22461) using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 depletion). The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). On day 7, M0 macrophages were obtained. M0 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engager CD79b/CLEC5A bispecific antibody to test their effects on M0 macrophages. Function in cells. Myeloid cell engagers (CD79b/CLEC5A bispecific antibodies) were serially diluted, and Daudi cells were first stained with CFSE, then added to M0 macrophages in 96-well round-bottom plates (E:T=5:1, E:T=2:1, E:T=1:1 or E:T=1:2) (fixed Daudi count: 2×10 ⁴ /well), and then incubated with serially diluted CD79b/CLEC5A bispecific antibodies (1 to 3 times dilution starting from 10nM). After 24 hours of incubation, the plates were centrifuged and the supernatant was recovered for ELISA analysis. ELISA was performed according to the instructions of the ELISAMAX ^TM Deluxe Set Human TNF-α (BioLegend, Cat#: 430204) and ELISAMAX ^TM Deluxe Set Human IL-6 (BioLegend, Cat#: 430504) kits. Use The absorbance at 450 nm was measured by an absorbance reader (MOLECULARDEVICEs, S/N 3052431867) within 15 minutes. Without the addition of target cells (Daudi), the myeloid cell engager (CD79b/CLEC5A bispecific antibody) did not show TNFα or IL-6 release in vitro. As shown in Figures 46A-46D, when target cells (Daudi) were added, the myeloid cell engager showed less TNFα and IL-6 release in vitro. After 24 hours, the maximum release of TNFα was less than 150 pg/mL, while the maximum release of IL-6 was less than 200 pg/mL, indicating that the myeloid cell engager can effectively promote target cell killing and induce M0 macrophages to release very weak cytokines to target cells.

Example 34: CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M1 macrophages

The killing effect of different CD79b/CLEC5A bispecific antibodies on target cancer Ramos cells (CD79b+) by mediating macrophages (CLEC5A+) was evaluated. CD14+ monocytes (purified from human PBMC donor #100 and donor 431 using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 6 days of treatment with 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, 50ng/mL IFN-γ (StemCell Technologies, Cat#: 78020) was used for another 24 hours to polarize M0 macrophages to M1 macrophages (day 7). Ramos cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were resuspended in 100 μl FACS buffer with SYTOX ^™ Blue Dead Cell Stain (ThermoFisher, Cat#: S34857) and stained with Cytometer was used for analysis. Ramos cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figures 47A-47B and the table below, all myeloid cell binders can effectively kill CD79b+ cancerous Ramos cells.

Table 20: CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M1 macrophages

Cytokine release of CD79b/CLEC5A bispecific antibodies of different structures in the presence or absence of target Ramos cells was evaluated. CD14+ monocytes (purified from human PBMC donor #100 and donor #431 using EasySep ^TM human monocyte enrichment kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 6 days of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were differentiated into M1 macrophages after 24 hours of 50ng/mL IFN-γ (StemCell Technologies, Cat#: 78020). Approximately 100,000 macrophages were incubated with 20,000 Ramos cells (E:T ratio of 5:1) at 37°C in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) for 24 hours. After incubation, the supernatant was collected for cytokine analysis. TNFα and IL-6 levels in the supernatant were measured using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the supplier's instructions. As shown in Figures 48A-48B, IL-6 and TNFα were both released from macrophages in a dose-dependent manner and at low levels only in the presence of the target Ramos and CD79b/CLEC5A bispecific antibodies, indicating that myeloid cell engagers can be safely used in the clinic.

The killing effect of macrophages (CLEC5A+) on target cancer Ramos cells (CD79b+) mediated by CD79b/CLEC5A bispecific antibodies with different structures was evaluated. CD14+ monocytes (purified from human PBMC donor #022 using the EasySep ^TM Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 6 days of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were differentiated into M1 macrophages (day 7) after 24 hours of 50ng/mL IFN-γ (StemCell Technologies, Cat#: 78020). Ramos cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) at 37°C for 24 hours in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After incubation, the cells were suspended in 100 μl of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry The Ramos cells were set as CFSE+ by FACS, and the absolute cell counts of CFSE+ cells were obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figure 49, all myeloid cell binders can effectively kill CD79b+ cancerous Ramos cells.

The cytokine release of CD79b/CLEC5A bispecific antibodies with different structures was evaluated. After incubation, the supernatant was collected for cytokine analysis, and the TNFα and IL-6 levels in the supernatant were determined using ELISA kits (R&D Systems, Cat#: DY206 and DY210) according to the instructions. The results are shown in Figure 50.

In order to understand whether the activated M1 macrophages further lead to self-killing and reduce their number, we analyzed the number of M1 macrophages to detect their status. Ramos cells were stained with 0.25uM CSFE after dead cells were removed. The number of macrophages is shown in Figure 51, and there is no obvious change in the number of M1 macrophages.

Example 35: CD79b/CLEC5A bispecific antibody mediates M1 macrophage killing of Daudi cells

To evaluate the killing effect of myeloid cell engager CD79b/CLEC5A bispecific antibody on M1 macrophages against cancerous B cells (Daudi) at different E:T ratios, human monocytes were isolated from PBMCs using the EasySep ^™ Human Monocyte Enrichment Kit (StemCell Technologies, Cat#: 19058) without CD16 removal. The isolated monocytes were induced with 50ng/mL M-CSF (StemCell, Cat#: 78057). On day 6, complete medium containing 50ng/mL M-CSF and 50ng/mL IFN-… (StemCell, Cat#: 78020) was added. On day 7, M1 macrophages were obtained. M1 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engagers to test their function in M1 macrophages. Myeloid cell engagers (CD79b/CLEC5A bispecific antibody) were serially diluted, Daudi cells were first stained with CFSE, and then added to M1 macrophages in 96-well round-bottom plates (E:T=5:1 or E:T=2:1) (fixed Daudi count: 2×10 ⁴ /well), and then incubated with serially diluted CD79b/hCLEC5A bispecific antibodies (1 to 3 times dilution starting from 10 nM). After 24 hours of incubation, the culture plates were centrifuged and the supernatants were stored at -80°C. Cells were stained with live/dead staining dye and then processed on a CytoFLEX LX flow cytometer. The cell killing rate was calculated as follows: (number of live tumor cells in the non-treated group - number of live tumor cells in the treated group)/number of live tumor cells in the non-treated group × 100. As shown in Figure 52, myeloid cells showed strong Daudi cell killing effects at different E:T ratios, and myeloid cell binders showed considerable Daudi cell killing efficacy on M1 macrophages in vitro. The maximum specific tumor cell killing rate of Daudi cells at 24 hours was about 96% (E:T = 5:1) and 85% (E:T = 2:1), indicating that myeloid cell binders can effectively promote target cell killing at different E:T ratios of M1 macrophages to target cells.

To evaluate the release of cytokines upon killing of cancerous B cells (Daudi) by myeloid cell engager CD79b/CLEC5A bispecific antibody in M1 at different E:T ratios, human monocytes (StemCell Technologies, Cat#: 19058) were isolated from PBMC using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal). The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). On day 6, complete medium containing 50 ng/mL M-CSF and 50 ng/mL IFN-… (StemCell Technologies, Cat#: 78020) was used. On day 7, M1 macrophages were obtained. M1 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engagers to test their function in M1 macrophages. Myeloid cell engagers (CD79b/CLEC5A bispecific antibodies) were serially diluted. Daudi cells were first stained with CFSE, then added to M1 macrophages (E:T=5:1 or E:T=2:1) in a 96-well round-bottom plate (fixed Daudi count: 2×10 ⁴ /well), and then incubated with gradient dilutions of CD79b/CLEC5A bispecific antibody (1 to 3 times dilution starting from 10 nM). After incubation for 24 hours, the plate was centrifuged and the supernatant was recovered for ELISA analysis. ELISA was performed according to the instructions of the ELISAMAX ^TM Deluxe Set Human TNF-α (BioLegend, Cat#: 430204) and ELISAMAX ^TM Deluxe Set Human IL-6 (BioLegend, Cat#: 430504) kits. Use The absorbance at 450 nm was measured by an absorbance reader (MOLECULAR DEVICEs, S/N 3052431867) within 15 minutes. Without the addition of target cells (Daudi), the myeloid cell binder showed no TNFα and IL-6 release in vitro. As shown in FIG53 , after the addition of target cells (Daudi), the myeloid cell binder showed comparable TNFα and IL-6 release in vitro. The maximum release of TNFα at 24 hours was less than 1000 pg/mL, while the maximum release of IL-6 was less than 1600 pg/mL, indicating that the myeloid cell binder can effectively promote target cell killing and induce M1 macrophages to release cytokines to target cells.

Example 36: CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M2 macrophages

The myeloid cell engager CD79b/CLEC5A antibody was evaluated for its ability to mediate the killing of target cancer Ramos cells (CD79b+) by M2 macrophages (CLEC5A+). CD14+ monocytes (purified from human PBMC donors #100, 233, 022, and 431 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal) (StemCell Technologies, Cat#: 19058)) were treated with 50 ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 6 days and then differentiated into M0 macrophages. On day 6, 25 ng/mL IL-10 (StemCell Technologies, Cat#: 78024) was used for another 24 hours to differentiate from M0 macrophages into M2 macrophages (day 7). Ramos cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the cells were resuspended in SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857). Resuspend in 100 μl FACS buffer and Cytometer was used for analysis. Ramos cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figures 54A-54D and the table below, all myeloid cell binders with different structures can effectively kill CD79b+ cancerous Ramos cells.

Table 21: CD79b/CLEC5A bispecific antibody mediates killing of Ramos cells by M2 macrophages

Cytokine release of CD79b/CLEC5A bispecific antibodies of different structures in the presence or absence of target Ramos cells was evaluated. CD14+ monocytes (purified from human PBMC donors #100, 233, 022, and 431 using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages for 6 days under the action of 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were further differentiated into M2 macrophages for 24 hours (day 7) under the action of 25ng/mL IL-10 (StemCell Technologies, Cat#: 78024). Approximately 100,000 macrophages were incubated with 20,000 Ramos cells (E:T ratio of 5:1) in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours. After incubation, the supernatant was collected for cytokine analysis. TNFα and IL-6 levels in the supernatant were measured using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the supplier's instructions. As shown in the figure As shown in Figures 55A-55D, only in the presence of target Ramos cells and CD79b/CLEC5A bispecific antibody, M2 macrophages released a small amount of IL-6 in a dose-dependent manner. TNFα levels did not change regardless of the presence or absence of target Ramos cells, indicating that myeloid cell engagers can be safely used in the clinic.

The killing effect of targeted cancer Ramos cells (CD79b+) mediated by M2 macrophages (CLEC5A+) of myeloid cell engager CD79b/CLEC5A antibody was evaluated. CD14+ monocytes (purified from human PBMC donor 022 using EasySep ^™ Human Monocyte Enrichment Kit (without CD16 depletion) (StemCell Technologies, Cat#: 19058)) were differentiated into M0 macrophages after 6 days with 50ng/mL macrophage colony stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On day 6, M0 macrophages were differentiated into M2 macrophages (day 7) after 24 hours with 25ng/mL IL-10 (StemCell Technologies, Cat#: 78024). Ramos cells were labeled with CFSE (Thermo Fisher, Cat#: C34554). Approximately 100,000 macrophages were incubated with 20,000 CFSE+Ramos (E:T ratio of 5:1) at 37°C for 24 hours in the presence of serially diluted antibodies in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin). After incubation, the cells were suspended in 100 μl of FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry was used for analysis. Ramos cells were set as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+Ramos cells in the non-treatment group-absolute number of CFSE+SYTOX-Ramos cells in the treatment group)/absolute number of CFSE+Ramos cells in the non-treatment group × 100. As shown in Figure 56, all myeloid cell conjugates can effectively kill CD79b+ cancerous Ramos cells.

The cytokine release of CD79b/CLEC5A bispecific antibodies with different structures was evaluated. After incubation, the supernatant was collected for cytokine analysis, and the TNFα and IL-6 levels in the supernatant were determined using ELISA kits (R&D Systems, Cat#: DY206 and DY210) according to the instructions. The results are shown in Figure 57.

In order to determine whether the activated M2 macrophages further lead to self-killing and reduce their number, we analyzed the number of M2 macrophages to detect their status. Ramos cells were stained with 0.25uM CSFE after removing dead cells. The number of macrophages is shown in Figure 58, and there is no significant change in the number of M2 macrophages.

Example 37: CD79b/CLEC5A bispecific antibody mediates killing of Daudi cells by M2 macrophages

To evaluate the killing effect of M2 macrophages on cancerous B cells (Daudi) mediated by myeloid cell engager CD79b/CLEC5A bispecific antibody at different E:T ratios, human monocytes were isolated from PBMCs using EasySep ^™ Human Monocyte Enrichment Kit (StemCell Technologies, Cat#: 19058) without CD16 depletion. The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). On day 6, monocytes were added with Complete medium containing 50 ng/mL M-CSF and 25 ng/mL IL-10 (StemCell Technologies, Cat#: 78024). On day 7, M2 macrophages were obtained. M2 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engagers to test their functions in M2 macrophages. Myeloid cell engagers (CD79b/CLEC5A bispecific antibodies) were serially diluted. Daudi cells were first stained with CFSE and then added to M2 macrophages (E:T=2:1, E:T=1:1 or E:T=1:2) in 96-well round-bottom plates (fixed Daudi count: 2×10 ⁴ /well), which were then incubated with serially diluted CD79b/CLEC5A bispecific antibodies (1 to 3-fold dilutions starting from 10 nM). After incubation for 24 hours, the plates were centrifuged and the supernatants were stored at -80°C. The cells were stained with live/dead staining dye and then processed on a CytoFLEX LX flow cytometer. The cell killing percentage was calculated as: (number of live tumor cells in the non-treatment group - number of live tumor cells in the treatment group) / number of live tumor cells in the non-treatment group × 100. As shown in Figure 59, the myeloid cell binder showed a strong Daudi cell killing effect at different E:T ratios, and the CD79b/CLEC5A bispecific antibody showed a comparable Daudi cell killing effect in M2 macrophages in vitro. The maximum specific tumor cell killing rate of Daudi cells at 24 hours was approximately 97-99% (E:T = 2:1), 88-96% (E:T = 1:1) and 67-82% (E:T = 1:2), indicating that the myeloid cell binder can effectively promote target cell killing at different E:T ratios of M2 macrophages to target cells.

To evaluate the release of cytokines when myeloid cell engager CD79b/CLEC5A bispecific antibody mediates M2 macrophage killing of cancerous B cells (Daudi) at different E:T ratios, human monocytes (StemCell Technologies, Cat#: 19058) were isolated from PBMC using the EasySep ^™ Human Monocyte Enrichment Kit (without CD16 removal). The isolated monocytes were induced with 50 ng/mL M-CSF (StemCell Technologies, Cat#: 78057). On day 6, complete medium containing 50 ng/mL M-CSF and 25 ng/mL IL-10 (StemCell Technologies, Cat#: 78024) was added. On day 7, M2 macrophages were obtained. M2 macrophages and Daudi cells at different E:T ratios were incubated with myeloid cell engagers (CD79b/CLEC5A bispecific antibodies) to test their function in M2 macrophages. The myeloid cell binder was graded diluted. Daudi cells were first stained with CFSE, and then added to M2 macrophages (E:T=2:1, E:T=1:1 or E:T=1:2) in a 96-well round-bottom plate (fixed Daudi count: 2×10 ⁴ /well), and then further incubated with graded diluted CD79b/CLEC5A bispecific antibody (1 to 3-fold dilution starting from 10nM). After incubation for 24 hours, the culture plate was centrifuged and the supernatant was recovered for ELISA analysis. ELISA was performed according to the instructions of the ELISAMAX ^TM Deluxe Set Human TNF-α (BioLegend, Cat#: 430204) and ELISAMAX ^TM Deluxe Set Human IL-6 (BioLegend, Cat#: 430504) kits. Use The absorbance at 450 nm was measured by an absorbance reader (MOLECULAR DEVICEs, S/N 3052431867) for 15 minutes. As shown in FIG60 , after the addition of target cells (Daudi), the myeloid cell engagers showed comparable TNFα and IL-6 release in vitro. The maximum release of TNFα at 24 hours was less than 150 pg/mL, while the maximum release of IL-6 was less than 150 pg/mL. At 200 pg/mL, it was shown that the myeloid cell engager could effectively promote the killing of target cells and induce very low cytokine release of M2 macrophages to target cells.

Example 38: CD79b/CLEC5A bispecific antibody mediates monocyte killing of B cells

The killing effect of monocytes (CLEC5A+) on target cancer B cells (CD79b+) mediated by myeloid cell engager CD79b/CLEC5A antibodies was evaluated. Fresh monocyte donors #131 and #445 were isolated from PBMCs using the StemCell Human Monocyte Enrichment Kit (without CD16 depletion) and allowed to rest for 1 hour before assay. Approximately 100,000 monocytes were incubated with 20,000 CFSE+ B cells (E:T ratio of 5:1) in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours in the presence of serially diluted antibodies. After incubation, the cells were suspended in 100 μl FACS buffer containing SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Flow cytometry was used for analysis. B cells were divided into CFSE+ by FACS, and the absolute cell number of CFSE+ cells was obtained by collecting fixed volumes under all treatment conditions. The percentage of target cell killing was calculated as: (absolute number of CFSE+B cells in the non-treatment group-absolute number of CFSE+SYTOX-B cells in the treatment group)/absolute number of CFSE+B cells in the non-treatment group × 100. As shown in Figures 61A-61B, all myeloid cell conjugates can effectively kill CD79b+ cancerous Ramos cells.

The cytokine release of CD79b/CLEC5A bispecific antibodies with different structures was evaluated. After incubation, the supernatant was collected for cytokine analysis, and the levels of TNFα and IL-6 in the supernatant were determined using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the instructions. The results are shown in Figures 62A-62B.

To determine whether activated monocytes further lead to self-killing and reduce their number, we analyzed the number of monocytes to test their status. B cells were stained with 0.25uM CSFE after dead cell removal. The number of monocytes is shown in Figures 63A-63B, and there is no obvious change in the number of monocytes.

Example 39: CD79b/CLEC5A bispecific antibody mediates monocyte killing of B cells

The killing effect of monocytes (CLEC5A+) on target cancer B cells (CD79b+) mediated by myeloid cell engager CD79b/CLEC5A antibodies was evaluated. Fresh monocytes were isolated from PBMCs donors #131 and #445 using the StemCell Human Monocyte Enrichment Kit (without CD16 removal) and allowed to stand for 1 hour before being measured. Approximately 100,000 monocytes were incubated with 20,000 CFSE+ B cells (E:T ratio of 5:1) in complete RPMI medium (containing 10% heat-inactivated FBS and 5% penicillin/streptomycin) at 37°C for 24 hours in the presence of serially diluted antibodies. After incubation, the cells were suspended in 100 μl FACS buffer containing SYTOX ^TM Blue Dead Cell Stain (Thermo Fisher, Cat#: S34857) and stained with Analysis was performed by flow cytometry. B cells were divided as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume under all treatment conditions. The ratio was calculated as: (absolute number of CFSE+B cells in the non-treated group - absolute number of CFSE+SYTOX-B cells in the treated group)/absolute number of CFSE+B cells in the non-treated group × 100. The results are shown in Figures 64A-64B.

The cytokine release of CD79b/CLEC5A bispecific antibodies with different structures was evaluated. After incubation, the supernatant was collected for cytokine analysis, and the levels of TNFα and IL-6 in the supernatant were determined using ELISA kits (R&D Systems, Cat# DY206 and DY210, respectively) according to the instructions. The results are shown in Figures 65A-65B.

To determine whether activated monocytes further lead to self-killing and reduce their number, we analyzed the number of monocytes to test their status. B cells were stained with 0.25uM CSFE after dead cell removal. The number of monocytes is shown in Figures 66A-66B, and there is no obvious change in the number of monocytes.

The sequences discussed in this context are listed in the table below.

Table 22: Some amino acid sequences described in the present invention

Other embodiments

It should be understood that although the present invention has been described in conjunction with its detailed description, the above description is intended to illustrate rather than limit the scope of the present invention, and the scope of the present invention is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims (29)

An antibody or antigen-binding fragment comprising: (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is a tumor-associated antigen (TAA); and (ii) a second antigen binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A); or (i) a first antigen binding domain that specifically binds to a first antigen, wherein the first antigen is an autoimmune disease target; and (ii) a second antigen binding domain that specifically binds to CLEC5A; The antibody or antigen-binding fragment has one or more of the following effects: (1) The antibody or antigen-binding fragment can mediate the killing of target cells by myeloid cells (e.g., monocytes and/or macrophages (e.g., M0, M1, M1 and/or M2 macrophages)); (2) the antibody or antigen-binding fragment can mediate killing of target cells by myeloid cells (e.g., monocytes and/or macrophages) having a low E:T ratio (effector cell: target cell), optionally wherein the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1 or less than 10:1; and (3) The antibody or antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release from myeloid cells (e.g., monocytes and/or macrophages), for example, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% cytokine release compared to a control antibody. The antibody or antigen-binding fragment according to claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2). The antibody or antigen-binding fragment of claim 2, wherein the second antigen-binding domain is a single-chain fragment variable region (scFv) in which VH2 and VL2 are connected by a first linker. The antibody or antigen-binding fragment of claim 3, wherein the second antigen-binding domain is connected to the C-terminus of the light chain via a second linker. The antibody or antigen-binding fragment according to claim 2, further comprising an Fc region. The antibody or antigen-binding fragment according to claim 5, wherein the C-terminus of the VH1 of the first antigen-binding domain is connected to the Fc region via the CH1 domain. The antibody or antigen-binding fragment according to claim 5, wherein 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. The antibody or antigen-binding fragment according to claim 2, wherein the antibody comprises a first heavy chain consisting of VH1 and a first light chain consisting of VL1; and comprises a second heavy chain consisting of VH2 and a second light chain consisting of VL2. The antibody or antigen-binding fragment according to claim 8, wherein: (1) the first heavy chain comprises one or more knob mutations, and the second heavy chain comprises one or more hole mutations; or (2) The first heavy chain comprises one or more hole mutations, and the second heavy chain comprises one or more knob mutations. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment according to claim 10, wherein the Fc region is the Fc region of human IgG1. The antibody or antigen-binding fragment according to claim 10 or 11, wherein the Fc region comprises one or more amino acid residues (all numbering is according to EU numbering): (1) Alanine (A) at position 236; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (2) Alanine (A) at position 236; Aspartic acid (D) at position 293; Leucine (L) at position 330 and Glutamic acid (E) at position 332; (3) Alanine (A) at position 236; (4) Alanine (A) at position 236; Aspartic acid (D) at position 293; and Glutamic acid (E) at position 332; (5) Aspartic acid (D) at position 293 and Glutamic acid (E) at position 332; (6) aspartic acid (D) at position 293; leucine (L) at position 330 and glutamic acid (E) at position 332; and (7) Alanine (A) at position 234; Alanine (A) at position 235 and Glycine (G) at position 329; Optionally, the Fc region is aglycosylated. The antibody or antigen-binding fragment according to any one of claims 1 to 12, wherein the tumor-associated antigen (TAA) is HER2, CD79b, EGFR, EpCAM, BCMA, CD38, GPRC5D, DLL3, CD70, GPC3, FAS ligand (FASL), CD1d, glycocoll, globulin-based-calcium amide (GB3Cer/CD77), gangliosides (GD2, GD3 and GM2), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CLL1, CD105, CD71 , SSC, MAGE, MUC16, CD19, WT-L, B7H3, TEM8, CD22, LI-CAM, ROR-I, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cells, CA242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8) 、CD33、CD4、CD40、CD44 v6、CD51、CD52、CD56、CD74、CD80、CEA、CNT0888、CTLA-4、DR5、CD3、FAP、fibronectin extra domain B、folate receptor 1、GD2、GD3 ganglioside acid、glycoprotein 75、GPNMB、HGF、human scatter factor receptor kinase、IGF-I receptor、IGF-I、IgGI、IL-5、IL-13、IL-6、IL-15、insulin-like growth factor I receptor、integrin a5b1、integrin avb3、MSLN , MS4A1, MUC1, mucin Canag, N-glycosylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R A, TWEAK, phosphatidylserine, prostate cancer cells, Rankl, Ron, SCH 900105, SDC1, SLAMF7, TAG-72, Tenascin C, TGF B Beta 2, TGF-B, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or Vimentin; The autoimmune disease target is CD79b, CD38, ACHE, BAFF, BTK, CCL2, CD19, CD20, CD25, CD40, CD52, CD80, CD86, ETAR, ETBR, FCGRT, GM-CSF, JAK1, IFNAR, IFNB1, IFNG, IgE, IgG Fc, IL1A, IL1B, IL-2, IL-4, IL-5, IL-6, IL6R, IL7, IL-12, IL-13, IL-17, IL-17R, IL-18, IL-21, IL-22, IL-23, integrin, ITG-A4B1, ITG-A4B7, ITG-AVB6, TL1A, TNF-α, TNF-β, TNFSF13B, TSLP, TYK2 or VEGFR. The antibody or antigen-binding fragment according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment that binds to CLEC5A comprises: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3; and a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3, wherein the selected VH CDR 1, 2 and 3 amino acid sequence and the selected VL CDR 1, 2 and 3 amino acid sequence are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 3, 5, and 7, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (2) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 4, 6, 7, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 8-10, respectively; (3) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 13, 15, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (4) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 14, 16, 17, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 18-20, respectively; (5) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 23, 25, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (6) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 24, 26, 27, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 28-30, respectively; (7) the selected VH CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 33, 35, 37, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (8) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 34, 36, and 37, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 38-40, respectively; (9) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 43, 45, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (10) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 44, 46, and 47, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 48-50, respectively; (11) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 53, 55, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (12) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 56, and 57, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 58-60, respectively; (13) the selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 63, 65, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (14) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 66, and 67, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 68-70, respectively; (15) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 73, 75, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively; (16) The selected VH CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 74, 76, and 77, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 78-80, respectively. The antibody or antigen-binding fragment of claim 14, wherein an antibody or antigen-binding fragment that binds to CLEC5A comprises: The heavy chain variable region (VH) comprises an amino acid sequence at least 90% identical to a selected VH, and the light chain variable region (VL) comprises an amino acid sequence at least 90% identical to a selected VL, wherein the selected VH and selected VL sequences are one of the following: (1) The selected VH sequence is SEQ ID NO: 11, and the selected VL sequence is SEQ ID NO: 12; (2) the selected VH sequence is SEQ ID NO: 21, and the selected VL sequence is SEQ ID NO: 22; (3) the selected VH sequence is SEQ ID NO: 31, and the selected VL sequence is SEQ ID NO: 32; (4) the selected VH sequence is SEQ ID NO: 41, and the selected VL sequence is SEQ ID NO: 42; (5) the selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52; (6) The selected VH sequence is SEQ ID NO: 61, and the selected VL sequence is SEQ ID NO: 62; (7) the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72; (8) The selected VH sequence is SEQ ID NO: 81, and the selected VL sequence is SEQ ID NO: 82; (9) the selected VH sequence is SEQ ID NO: 83, and the selected VL sequence is SEQ ID NO: 84; (10) the selected VH sequence is SEQ ID NO: 85, and the selected VL sequence is SEQ ID NO: 86; (11) the selected VH sequence is SEQ ID NO: 87, and the selected VL sequence is SEQ ID NO: 88; (12) the selected VH sequence is SEQ ID NO: 89, and the selected VL sequence is SEQ ID NO: 90; (13) The selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92. The antigen or antigen-binding fragment cross-competes with the antigen or antigen-binding fragment of any one of claims 1-15. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. A vector comprising the nucleic acid of claim 17. A cell comprising the vector according to claim 18. A method for increasing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15. The method of claim 21, wherein the cancer is a solid tumor or a hematological tumor. The method according to claim 21 or 22, wherein the cancer is breast cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer and/or liver cancer. The method of claim 21 or 22, wherein the cancer is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, leukemia, splenic villous lymphocytic lymphoma, hairy cell leukemia, follicular lymphoma, and/or mantle cell lymphoma. The method according to any one of claims 20-24, wherein the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody and/or an anti-PD-L1 antibody. A method for treating a subject suffering from an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15. The method of claim 26, wherein 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, Takayasu's disease, cold agglutinin disease, warm autoimmune hemolytic anemia, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, including, for example, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) and microscopic polyangiitis (MPA). The method of claim 27, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus and/or rheumatoid arthritis. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier.