Multispecific antibodies and their use

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to anti-CLEC5A (C-type lectin domain family 5 member A) antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, and their use. The present disclosure also relates to anti-CLEC5A multispecific antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof), and antibody-drug conjugates derived therefrom.

Background Art

[0002] Human C-type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-associated lectin-1 (MDL-1), is a type II transmembrane protein that is expressed only in myeloid lineages such as macrophages, monocytes, neutrophils, and dendritic cells (DCs). As a pattern recognition receptor, CLEC5A transmits signals to the cytoplasm via non-covalent binding to the adapter protein DAP12. Phosphorylation of DAP12 initiates a Syk kinase-based signaling cascade, activating macrophages and releasing chemokines and pro-inflammatory cytokines such as IL-6, TNF, CCL3, and CXCL8.

[0003] CLEC5A elicits immune responses related to bone marrow cells and is correlated with various infectious and inflammatory diseases. CLEC5A promotes the production of high levels of pro-inflammatory cytokines and chemokines in flavivirus infections, particularly dengue virus infection and Japanese encephalitis virus infection, and it has been suggested that CLEC5A is a promising therapeutic target for flavivirus infections because the progression of the disease can be reversed by anti-CLEC5A mAb or CLEC5A inhibitors. Furthermore, similar to several autoimmune diseases, elevated levels of CLEC5A have been found in rheumatoid arthritis, and CLEC5A activators increase the levels of pro-inflammatory cytokines. CLEC5A also plays an important role in cancer development and progression. Abnormally high expression of CLEC5A is significantly correlated with reduced overall survival in high-grade serous ovarian cancer (HGSOC), gastric cancer, and glioma.

[0004] Bispecific antibodies are artificial proteins that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual-targeting different disease mediators, and delivering payloads to target sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has been a major milestone in the development of bispecific antibodies.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Since bispecific antibodies have various applications, it is necessary to continue developing various therapeutic agents based on bispecific antibodies.

[0006] Considering the important role of CLEC5A in the immune system, there is a need to develop therapeutic agents targeting CLEC5A, particularly bispecific antibodies targeting CLEC5A.

[0007] This disclosure relates to anti-CLEC5A antibodies, their antigen-binding fragments, and their uses. This disclosure also relates to multispecific (e.g., bispecific) antibodies or their antigen-binding fragments, wherein the antibody or its antigen-binding fragment specifically binds to tumor-associated antigens (TAAs) and human C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the antibody or its antigen-binding fragment has an enhanced Fc. In some embodiments, the antibody or its antigen-binding fragment has increased binding affinity to FcγRIIa receptors and / or FcγRIIIa receptors. In some embodiments, the antibody or its antigen-binding fragment has a non-functional Fc.

[0008] The anti-CLEC5A antibodies described herein are not “typical agonists” compared to the reference antibody used in this disclosure (e.g., DX244). For example, the anti-CLEC5A antibodies described herein can mediate macrophage phagocytosis (similar to DX244), but with stronger potency and very low cytokine release levels. Furthermore, the TAA / CLEC5A bispecific antibodies described herein have been shown to induce killing of various TAA-expressing target cells by myeloid cells (e.g., monocytes and macrophages) with a very low E:T ratio, resulting in very low cytokine release. Without being constrained by theory, the anti-CLEC5A antibodies and TAA / CLEC5A bispecific antibodies described herein are considered to be usable to create potential myeloid cell engagers with high efficacy and a favorable safety profile.

[0009] In one embodiment, the Disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A (member A of the C-type lectin domain family 5), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region includes an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region includes an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region includes an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) including CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region includes an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region includes an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region includes a light chain variable region (VL) including CDRs 1, 2, and 3. With respect to an antibody or antigen-binding fragment thereof comprising a light chain variable region containing an amino acid sequence at least 80% identical to the CDR3 amino acid sequence, the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 3, 5, and 7, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 8 to 10, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 4, 6, and 7, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 8 to 10, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 18 to 20, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 14, 16, and 17, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 18 to 20, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 23, 25, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 28 to 30, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 24, 26, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 28 to 30, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 33, 35, and 37, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 38 to 40, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 34, 36, and 37, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 38 to 40, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 43, 45, and 47, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 48 to 50, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 44, 46, and 47, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 48 to 50, respectively; (11) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 53, 55, and 57, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 58 to 60, respectively; (12) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 54, 56, and 57, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 58 to 60, respectively; (13) The above-selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 63, 65, and 67, respectively, and the above-selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 68 to 70, respectively; (14) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 64, 66, and 67, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 68 to 70, respectively; (15) The amino acid sequences of the above-selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 73, 75, and 77, respectively, and the amino acid sequences of the above-selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 78 to 80, respectively; and (16) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 74, 76, and 77, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 78 to 80, respectively.

[0010] In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 3, 5, and 7, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 8 to 10, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13, 15, and 17, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 18 to 20, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 23, 25, and 27, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 33, 35, and 37, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 38 to 40, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 43, 45, and 47, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 48 to 50, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 53, 55, and 57, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 58 to 60, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 63, 65, and 67, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 68 to 70, respectively. In some embodiments, according to Kabat's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 73, 75, and 77, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 78 to 80, respectively.In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 6, and 7, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 8 to 10, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 14, 16, and 17, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 18 to 20, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 24, 26, and 27, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 34, 36, and 37, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 38 to 40, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 44, 46, and 47, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 48 to 50, respectively. In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 54, 56, and 57, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 58 to 60, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 64, 66, and 67, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 68 to 70, respectively. In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 74, 76, and 77, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 78 to 80, respectively.

[0011] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A, comprising a VH having an amino acid sequence at least 90% identical to a selected heavy chain variable region (VH) sequence, and a VL having an amino acid sequence at least 90% identical to a selected light chain variable region (VL) sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (2) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (3) The selected VH sequence is sequence number 31, and the selected VL sequence is sequence number 32; (4) The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 42; (5) The selected VH sequence is sequence number 51, and the selected VL sequence is sequence number 52; (6) The selected VH sequence is sequence number 61, and the selected VL sequence is sequence number 62; (7) The selected VH sequence is sequence number 71, and the selected VL sequence is sequence number 72; (8) The selected VH sequence is sequence number 81, and the selected VL sequence is sequence number 82; (9) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; (10) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86; (11) The selected VH sequence is sequence number 87, and the selected VL sequence is sequence number 88; (12) The selected VH sequence is sequence number 89, and the selected VL sequence is sequence number 90; and (13) The selected VH sequence is sequence number 91, and the selected VL sequence is sequence number 92.

[0012] In some embodiments, VH includes the sequence of sequence number 11, and VL includes the sequence of sequence number 12. In some embodiments, VH includes the sequence of sequence number 21, and VL includes the sequence of sequence number 22. In some embodiments, VH includes the sequence of sequence number 31, and VL includes the sequence of sequence number 32. In some embodiments, VH includes the sequence of sequence number 41, and VL includes the sequence of sequence number 42. In some embodiments, VH includes the sequence of sequence number 51, and VL includes the sequence of sequence number 52. In some embodiments, VH includes the sequence of sequence number 61, and VL includes the sequence of sequence number 62. In some embodiments, VH includes the sequence of sequence number 71, and VL includes the sequence of sequence number 72. In some embodiments, VH includes the sequence of sequence number 81, and VL includes the sequence of sequence number 82. In some embodiments, VH includes the sequence of sequence number 83, and VL includes the sequence of sequence number 84. In some embodiments, VH includes the sequence of sequence number 85, and VL includes the sequence of sequence number 86. In some embodiments, VH includes the sequence of sequence number 87, and VL includes the sequence of sequence number 88. In some embodiments, VH includes the sequence of sequence number 89, and VL includes the sequence of sequence number 90. In some embodiments, VH includes the sequence of sequence number 91, and VL includes the sequence of sequence number 92.

[0013] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human, mouse, or monkey CLEC5A.

[0014] In some embodiments, an antibody or its antigen-binding fragment can mediate macrophage phagocytosis (for example, having at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the target cell killing effect compared to a reference antibody (e.g., DX244)), and / or In some embodiments, an antibody or its antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release (e.g., less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to that induced by a reference antibody (e.g., DX244)).

[0015] In one embodiment, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLEC5A, and in some embodiments, the antibody or antigen-binding fragment thereof can mediate macrophage phagocytosis (e.g., the target cell killing effect is at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to a reference antibody (e.g., DX244)), and / or in some embodiments, the antibody or antigen-binding fragment thereof can induce low levels of cytokine (e.g., IL-6 or TNFα) release (e.g., less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to a reference antibody (e.g., DX244)).

[0016] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising: i) a first antigen-binding domain that specifically binds to a first antigen, wherein in some embodiments the first antigen is a tumor-associated antigen (TAA); and ii) a second antigen-binding domain that specifically binds to member A of the C-type lectin domain family 5 (CLEC5A).

[0017] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising: i) a first antigen-binding domain that specifically binds to a first antigen, wherein in some embodiments the first antigen is an autoimmune disease target; and ii) a second antigen-binding domain that specifically binds to CLEC5A.

[0018] In some embodiments, the antibody or its antigen-binding fragment has one or more of the following effects: i) Antibodies or their antigen-binding fragments can mediate target cell killing by bone marrow cells (e.g., monocytes and / or macrophages (e.g., M0, M1, and / or M2 macrophages)); ii) Antibodies or their antigen-binding fragments can mediate target cell killing 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 <1:10, <1:9, <1:8, <1:7, <1:6, <1:5, <1:4, <1:3, <1:2, <1:1, <2:1, <3:1, <4:1, <5:1, <6:1, <7:1, <8:1, <9:1, or <10:1); and iii) Antibodies or their antigen-binding fragments can induce low levels of cytokine (e.g., IL-6 or TNFα) release in bone marrow cells (e.g., monocytes and / or macrophages) (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 those induced by a reference antibody).

[0019] 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-strand variable fragment (scFv) domain, and in some embodiments, VH2 and VL2 are linked by a first linker. In some embodiments, the second antigen-binding domain is bound to the C-terminus of the light chain via a second linker. In some embodiments, the antibody or antigen-binding fragment described herein further comprises an Fc region. In some embodiments, the C-terminus of VH1 of the first antigen-binding domain is optionally bound to the Fc region via a CH1 domain. In some embodiments, the C-terminus of VH1 of the first antigen-binding domain is bound to the N-terminus of the Fc region, and the N-terminus of the second antigen-binding domain is bound to the C-terminus of the Fc region. In some embodiments, the antibody comprises a first heavy chain containing VH1 and a first light chain containing VL1, and a second heavy chain containing VH2 and a second light chain containing VL2. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations.

[0020] In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1. In some embodiments, the Fc region contains one or more of the following amino acid residues (all numbering follows 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), ranked 236th; (4) Alanine at position 236 (A), aspartic acid at position 293 (D), and glutamic acid at position 332 (E); (5) Aspartic acid (D) at position 293 and glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332; and (7) Alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329; The Fc region is selectively defucosylated.

[0021] In some embodiments, tumor-associated antigens (TAAs) include HER2, CD79b, EGFR, EpCAM, BCMA, CD38, GPRC5D, DLL3, CD70, GPC3, mesothelin, Fas ligand (FasL), CD1d, membrane glycolipids, globotriaosylceramide (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-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-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 extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scattering factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, insulin-like growth factor I receptor, integrin a5b1, integrin avb3, MSLN, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-Ra, TWEAK, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

[0022] In some embodiments, 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-18, IL-21, IL-22, IL-2 3, integrin, ITG-A4B1, ITG-A4B7, ITG-AVB6, TL1A, TNF-α, TNF-β, TNFSF13B, TSLP, TYK2, or VEGFR.

[0023] In some embodiments, TAA is HER2. In some embodiments, (1) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 100, 102, and 104, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 105 to 107, respectively, and the selected VH2 CDR1, 2, 3 amino acid sequences and the selected VL2 CDR1, 2, 3 amino acid sequences are selected from one of the sequences described herein, or (2) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 101, 103, and 104, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 105 to 107, respectively, and the selected VH2 CDR1, 2, 3 amino acid sequences and the selected VL2 CDR1, 2, 3 amino acid sequences are selected from one of the sequences described herein.

[0024] In some embodiments, the target of TAA or autoimmune disease is CD79b. In some embodiments, (1) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 200, 202, and 204, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 205 to 207, respectively, and the selected VH2 CDR1, 2, 3 amino acid sequences and the selected VL2 CDR1, 2, 3 amino acid sequences are selected from one of the sequences described herein; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 201, 203, and 204, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 205 to 207, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0025] In some embodiments, TAA is EGFR. In some embodiments, (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 126, 128, and 130, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 131 to 133, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 127, 129, and 130, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 131 to 133, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein; (3) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 136, 138, and 140, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 141 to 143, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein; or (4) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 137, 139, and 140, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 141 to 143, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein.

[0026] In some embodiments, TAA is EPCAM. In some embodiments, (1) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 150, 152, and 154, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 155 to 157, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein; or (2) The selected VH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 151, 153, and 154, respectively, and the selected VL1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs. 155 to 157, respectively, and the selected VH2 CDR1, 2, 3 amino acid sequences and the selected VL2 CDR1, 2, 3 amino acid sequences are selected from one of the sequences described herein; In some embodiments, the target of TAA or autoimmune disease is GPRC5D. In some embodiments, (1) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 164, 166, and 168, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 169 to 171, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 165, 167, and 168, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 169 to 171, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0027] In some embodiments, the target of TAA or autoimmune disease is BCMA. In some embodiments, (1) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 176, 178, and 180, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 181 to 183, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 177, 179, and 180, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 181 to 183, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0028] In some embodiments, the target of TAA or autoimmune disease is CD38. In some embodiments, (1) The selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 188, 190, and 192, respectively, and the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 193 to 195, respectively, and the selected VH2 CDR1,2,3 amino acid sequences and the selected VL2 CDR1,2,3 amino acid sequences are selected from one of the sequences described herein; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 189, 191, and 192, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 193 to 195, respectively, and the selected VH2 CDR1, 2, and 3 amino acid sequences and the selected VL2 CDR1, 2, and 3 amino acid sequences are selected from one of the sequences described herein.

[0029] In one embodiment, this disclosure relates to an antibody or an antigen-binding fragment that cross-competes with an antibody or an antigen-binding fragment described herein.

[0030] In one embodiment, this disclosure relates to nucleic acids comprising polynucleotides encoding antibodies or antigen-binding fragments thereof as described herein.

[0031] In one embodiment, this disclosure relates to a vector comprising a nucleic acid described herein. In one embodiment, this disclosure relates to a cell comprising a vector described herein.

[0032] In one aspect, the present disclosure relates to a method for reducing the rate of tumor growth, comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof as described herein.

[0033] In one aspect, the present disclosure relates to a method for killing tumor cells, comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof as described herein.

[0034] In one embodiment, the present disclosure relates to a method for 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 as described herein.

[0035] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0036] In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small 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 prelymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma. In some embodiments, the subject is further treated with an effective dose of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, and / or anti-PD-L1 antibody.

[0037] In one embodiment, the present disclosure relates to a method for treating a subject having an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's arteritis, cold agglutinin disease, warm autoimmune hemolytic anemia, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (e.g., granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) and microscopic polyangiitis (MPA)). In some embodiments, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, and / or rheumatoid arthritis.

[0038] In one embodiment, this disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.

[0039] As used herein, the term “antigen-binding domain” means one or more protein domains (for example, formed from amino acids derived from a single polypeptide, or formed from amino acids derived from two or more polypeptides (e.g., identical or different polypeptides) that are specifically capable of 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 specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. An example of an antigen-binding domain is an antigen-binding domain 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. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some examples, the antigen-binding domain can bind to a single antigen (e.g., one of effector antigens and tumor antigens).

[0040] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from an immunoglobulin light chain or any of three CDRs derived from an immunoglobulin heavy chain) and is capable of specifically binding to an antigen. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0041] As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0042] As used herein, the term “multispecific antibody” refers to an antibody comprising two or more different antigen-binding domains that collectively and specifically bind to two or more different epitopes. The two or more different epitopes may 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 embodiments, a multispecific antibody binds to two different epitopes (e.g., a “bispecific antibody”). In some embodiments, a multispecific antibody binds to three different epitopes (e.g., a “triplespecific antibody”). In some embodiments, a multispecific antibody binds to four different epitopes (e.g., a “quadrispecific antibody”). In some embodiments, a multispecific antibody binds to five different epitopes (e.g., a “quintuplespecific antibody”). Each binding specificity can exist at any appropriate valency. Non-limiting examples of multispecific antibodies are described herein.

[0043] As used herein, the term “bispecific antibody” means an antibody that binds to two different epitopes. Epitopes may be present on the same antigen or on different antigens.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0045] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0046] [Figure 1A] This paper presents a gating strategy for evaluating immune cell subsets in human PBMCs. [Figure 1B] This shows CLEC5A expression in a subset of immune cells in human PBMCs. [Figure 2A] This paper describes gating strategies used to evaluate immune cell subsets in human solid tumors. [Figure 2B-C] This shows CLEC5A expression in tumor-associated bone marrow cells. [Figure 3A] This paper describes gating strategies used to characterize non-neutrophil myeloid cells. [Figure 3B] This describes the characteristics of tumor-associated macrophages in human solid tumors. [Figure 3C-D] This shows CLEC5A expression in tumor-associated macrophages from human solid tumors. [Figure 4] This shows the binding affinity of the chimeric anti-CLEC5A antibody to human macrophages. [Figure 5] This shows TNFα release from M1 macrophages in plates coated with a chimeric anti-CLEC5A antibody. [Figure 6]This shows the binding affinity of the chimeric anti-CLEC5A antibody to mouse CLEC5A. [Figure 7A-B] This shows the results of BLI binding of a chimeric anti-CLEC5A antibody to human CLEC5A. [Figure 8A-E] This shows humanized anti-CLEC5A antibodies that bind to human CLEC5A, as measured by ELISA. [Figure 9A-F] A schematic diagram of an example of a TAA / CLEC5A bispecific antibody is shown. [Figure 10] This antibody exhibits bispecificity for HER2 / CLEC5A binding to human macrophages. [Figure 11] This antibody exhibits bispecificity of TAA / CLEC5A, which binds to human macrophages. [Figure 12] This antibody exhibits bispecificity for CD79b / CLEC5A binding to human macrophages. [Figure 13] This antibody exhibits bispecificity of TAA / CLEC5A binding to human CLEC5A. [Figure 14] This antibody exhibits bispecificity for HER2 / CLEC5A binding to human HER2. [Figure 15] This antibody exhibits bispecificity for EGFR / CLEC5A binding to human EGFR. [Figure 16] This antibody exhibits bispecificity of EpCAM / CLEC5A, which binds to DLD-1 cells. [Figure 17] This antibody exhibits bispecificity for CD79b / CLEC5A binding to Ramos cells. [Figure 18A-C] This shows SK-BR-3 cell killing by M0 macrophages mediated by a HER2 / CLEC5A bispecific antibody (E:T ratio: 5:1). [Figure 19] This shows SK-BR-3 cell elimination by M1 and M2 macrophages mediated by a HER2 / CLEC5A bispecific antibody (E:T ratio: 5:1). [Figure 20] This shows IFN-γ released in response to SK-BR-3 cell killing by M1 and M2 macrophages mediated by the HER2 / CLEC5A antibody (E:T ratio is 5:1). [Figure 21A-C]This shows SK-BR-3 cell elimination by M0 macrophages mediated by HER2 / CLEC5A bispecific antibodies in the presence of culture medium (Figure 21A), plasma (Figure 21B), or hIgG1 (Figure 21C). [Figure 22] This shows DLD-1 cell elimination by M0 macrophages mediated by an EGFR / CLEC5A bispecific antibody. [Figure 23] This shows DLD-1 cell elimination by M0 macrophages mediated by the EpCAM / CLEC5A bispecific antibody. [Figure 24] This shows the expression levels of EpCAM in various cancer cell lines. [Figure 25A-F] This demonstrates cancer cell killing by M0 macrophages mediated by the EpCAM / CLEC5A bispecific antibody. [Figure 26] This demonstrates multiple myeloma cancer cell killing by PBMCs mediated by various myeloid cell engagers. [Figure 27] This shows the expression levels of CD79b on B cells in malignant lymphoma cell lines and healthy PBMC donors. [Figure 28A-C] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by PBMCs (Study 1). [Figure 29A-B] CD79b / CLEC5A bispecific antibody-mediated Ramos cell killing by PBMCs was demonstrated (Study 2). [Figure 30A-C] CD79b / CLEC5A bispecific antibody-mediated Ramos cell killing by PBMCs was demonstrated (Study 3). [Figure 31A-B] This shows IL-6 levels from Ramos cell elimination mediated by the CD79b / CLEC5A bispecific antibody. [Figure 32] This shows TNFα levels from Ramos cell elimination mediated by the CD79b / CLEC5A bispecific antibody. [Figure 33A-C] CD79b / CLEC5A bispecific antibody-mediated endogenous B cell killing by PBMCs (Study 1). [Figure 34A-C]CD79b / CLEC5A bispecific antibody-mediated endogenous B cell killing by PBMCs (Study 2). [Figure 35A-B] CD79b / CLEC5A bispecific antibody-mediated endogenous B cell killing by PBMCs (Study 3). [Figure 36] This shows TNFα release from endogenous B cell killing by PBMCs mediated by the CD79b / CLEC5A bispecific antibody. [Figure 37A-F] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by M0 macrophages was demonstrated (Study 1). [Figure 38A-D] This study demonstrates cytokine release from Ramos cell killing by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Study 1). [Figure 39A-B] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by M0 macrophages at various E:T ratios (Study 2). [Figure 40A-B] This study demonstrates cytokine release from Ramos cell elimination by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Study 2). [Figure 41A-C] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by M0 macrophages was demonstrated (Study 3). [Figure 42] This study demonstrates cytokine release from Ramos cell killing by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody (Study 3). [Figure 43] This shows the survival rate of M0 macrophages in Ramos cell killing mediated by the CD79b / CLEC5A bispecific antibody (Study 3). [Figure 44A-B] The CD79b / CLEC5A bispecific antibody mediated Daudi cell killing by M0 macrophages at various E:T ratios (Study 1). [Figure 45A-B] The CD79b / CLEC5A bispecific antibody mediated Daudi cell killing by M0 macrophages at various E:T ratios (Study 2). [Figure 46A-D] This demonstrates cytokine release from Daudi cell elimination by M0 macrophages mediated by the CD79b / CLEC5A bispecific antibody. [Figure 47A-B] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by M1 macrophages was demonstrated (Study 1). [Figure 48A-B] This study demonstrates cytokine release from M1 macrophage-mediated killing of Ramos cells by the CD79b / CLEC5A bispecific antibody (Study 1). [Figure 49] CD79b / CLEC5A bispecific antibody-mediated M1 macrophage-mediated killing of Ramos cells was demonstrated (Study 2). [Figure 50] This study demonstrates cytokine release from M1 macrophage-mediated killing of Ramos cells by the CD79b / CLEC5A bispecific antibody (Study 2). [Figure 51] This shows the survival rate of M1 macrophages in Ramos cell killing mediated by the CD79b / CLEC5A bispecific antibody (Study 2). [Figure 52] This shows Daudi cell elimination by M1 macrophages mediated by the CD79b / CLEC5A bispecific antibody. [Figure 53] This shows cytokine release from Daudi cell killing by M1 macrophages mediated by the CD79b / CLEC5A bispecific antibody. [Figure 54A-D] The CD79b / CLEC5A bispecific antibody mediated Ramos cell killing by M2 macrophages was demonstrated (Study 1). [Figure 55A-D] This study demonstrates cytokine release from M2 macrophage-mediated killing of Ramos cells by the CD79b / CLEC5A bispecific antibody (Study 1). [Figure 56] CD79b / CLEC5A bispecific antibody-mediated Ramos cell killing by M2 macrophages was demonstrated (Study 2). [Figure 57]This study demonstrates cytokine release from M2 macrophage-mediated killing of Ramos cells by the CD79b / CLEC5A bispecific antibody (Study 2). [Figure 58] This shows the survival rate of M2 macrophages in Ramos cell killing mediated by the CD79b / CLEC5A bispecific antibody (Study 2). [Figure 59] This study demonstrates Daudi cell killing by M2 macrophages at various E:T ratios mediated by the CD79b / CLEC5A bispecific antibody. [Figure 60] This shows cytokine release from Daudi cell elimination by M2 macrophages mediated by the CD79b / CLEC5A bispecific antibody. [Figure 61A-B] This shows monocyte-mediated killing of Ramos cells by a CD79b / CLEC5A bispecific antibody. [Figure 62A-B] This shows cytokine release from monocyte-mediated monocyte-mediated killing of Ramos cells using a CD79b / CLEC5A bispecific antibody. [Figure 63A-B] This shows the monocyte survival rate in monocyte-mediated monocyte-mediated killing of Ramos cells using the CD79b / CLEC5A bispecific antibody. [Figure 64A-B] This shows B cell killing by monocytes mediated by the CD79b / CLEC5A bispecific antibody. [Figure 65A-B] This shows cytokine release from B cell killing by monocytes mediated by the CD79b / CLEC5A bispecific antibody. [Figure 66A-B] This shows the monocyte survival rate in B cell killing mediated by the CD79b / CLEC5A bispecific antibody. [Modes for carrying out the invention]

[0047] A multispecific antibody or its antigen-binding fragment is an artificial protein capable of simultaneously binding to two or more different epitopes (e.g., on two different antigens). In some embodiments, the multispecific antibody is a bispecific antibody. The bispecific antibody or its antigen-binding fragment may have two arms. Each arm has one heavy chain variable region and one light chain variable region, and can 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., to the C-terminus of the light or heavy chain).

[0048] This disclosure provides several anti-CLEC5A antibodies, their antigen-binding fragments, and methods for using these anti-CLEC5A antibodies and antigen-binding fragments to inhibit tumor growth, treat cancer, and treat autoimmune diseases.

[0049] This disclosure relates to a multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA) and a second antigen-binding domain that specifically binds to CLEC5A.

[0050] 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-related lectin 1 (MDL-1), is a type II transmembrane protein. C-type lectins are Ca 2+ It is characterized by a common C-type lectin domain (CTLD) that can bind to glycans and non-glycan ligands independently. 2+The CTLD, which binds to glycans in a dependent manner, is known as the "carbohydrate recognition domain" (CRD). The bone marrow type C lectin CLEC5A is a spleen tyrosine kinase (Syk)-bound type II membrane protein composed of a C-terminal CTLD and a short N-terminal cytoplasmic domain. Of the 15 groups of type C lectins, CLEC5A belongs to group V (NK cell receptor family), which includes CLEC7A (dectin-1), CLEC5A, CLEC2, CLEC1, NK receptors (NKG2D, NKRP1 family, NKG2 family, CD69, and CD94, etc.), mast cell-associated functional antigens (MAFAs), osteoclast inhibitory lectins (OCIL), and CD72. Similar to NKG2D, CLEC5A, when phosphorylated by Src upon activation, transmits signals via ITAM-containing DNAX-activated protein 12 (DAP12).

[0051] Human CLEC5A mRNA encodes a 165-residue polypeptide, with a signal peptide (amino acids 1-22) at the N-terminus, followed by a short intracellular cytoplasmic domain (amino acids 23-56), a transmembrane domain (amino acids 57-70), and an extracellular domain (amino acids 71-165). The transmembrane domain contains the positively charged amino acid Lys-58, which, upon activation, recruits DAP10 and DAP12 to associate with CLEC5A. CLEC5A is primarily expressed by myeloid cells such as monocytes, macrophages, neutrophils, and dendritic cells, and is further upregulated by interferon-gamma (IFN-γ). CLEC5A expression is also regulated by the PU.1 transcription factor, a key 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.

[0052] X-ray crystallography revealed that CLEC5A exists as a homodimeric protein when binding to dengue virus serotypes. Furthermore, crystallographic analysis of CLEC5A revealed its structural flexibility, suggesting that CLEC5A can adopt diverse structures within the body, and that its structure is ligand-dependent.

[0053] While NK receptors recognize stress-related autoantigens and are important in immune surveillance, group V spleen tyrosine kinase (Syk)-bound type C lectins in bone marrow cells recognize a variety of exogenous and endogenous antigens and are involved in host defense, sterile inflammation, platelet activation, and development. CLEC5A has been shown to interact with the glycan sites of dengue virus (DV), Japanese encephalitis virus (JEV), and influenza A virus (IAV). Furthermore, CLEC5A has been shown to interact with N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharides on Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus). Additionally, CLEC5A plays a crucial role in inflammatory responses associated with collagen-induced rheumatoid arthritis and concanavalin A-induced hepatitis. CLEC5A also interacts with exosomes released from activated platelets.

[0054] A detailed overview of CLEC5A and its function can be found in Sung, Pei-Shan, Wei-Chiao Chang, and Shie-Liang Hsieh, “CLEC5A: a promiscuous pattern recognition receptor to microbes and beyond,” Lectin in Host Defense Against Microbial Infections (2020): 57-73; Chen, Rui, et al., “A pan-cancer analysis reveals CLEC5A as a biomarker for cancer immunity and prognosis,” Frontiers in Immunology 13 (2022): 831-542; 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, the contents of which are incorporated herein by reference in their entirety.

[0055] Anti-CLEC5A antibody and antigen-binding fragment This disclosure provides antibodies and antigen-binding fragments 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 may act as agonists or antagonists to CLEC5A-mediated signaling. In some embodiments, the antibodies and antigen-binding fragments can bind to the extracellular domain of human CLEC5A.

[0056] This disclosure provides, for example, anti-CLEC5A antibodies 6A5, 6G9, 14A2, 5C7, 7G10, 3A7, 13E6, 9E11, their chimeric antibodies, and their humanized antibodies.

[0057] CDR sequences for 3A7 and antibodies derived from 3A7 include the heavy chain variable domain CDRs, SEQ ID NOs: 3, 5, and 7, and the light chain variable domain CDRs, SEQ ID NOs: 8-10, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 4, 6, and 7, and the light chain variable domain CDR sequences are shown in SEQ ID NOs: 8-10.

[0058] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18-20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 18-20.

[0059] CDR sequences for 6A5 and antibodies derived from 6A5 (e.g., humanized antibodies) include the heavy chain variable domain CDRs, SEQ ID NOs. 23, 25, and 27, and the light chain variable domain CDRs, SEQ ID NOs. 28-30, as defined by Kabat. CDRs can also be defined by Chothia. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 24, 26, and 27, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 28-30.

[0060] CDR sequences for 6G9 and antibodies derived from 6G9 include the heavy chain variable domain CDRs, SEQ ID NOs. 33, 35, and 37, and the light chain variable domain CDRs, SEQ ID NOs. 38-40, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 34, 36, and 37, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 38-40.

[0061] CDR sequences for 7G10 and antibodies derived from 7G10 include the heavy chain variable domain CDRs, SEQ ID NOs. 43, 45, and 47, and the light chain variable domain CDRs, SEQ ID NOs. 48-50, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 44, 46, and 47, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 48-50.

[0062] CDR sequences for 9E11 and antibodies derived from 9E11 include the heavy chain variable domain CDRs, SEQ ID NOs. 53, 55, and 57, and the light chain variable domain CDRs, SEQ ID NOs. 58-60, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 54, 56, and 57, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 58-60.

[0063] CDR sequences for 13E6 and antibodies derived from 13E6 include the heavy chain variable domain CDRs, SEQ ID NOs. 63, 65, and 67, and the light chain variable domain CDRs, SEQ ID NOs. 68-70, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 64, 66, and 67, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 68-70.

[0064] CDR sequences for 14A2 and antibodies derived from 14A2 include the heavy chain variable domain CDRs, SEQ ID NOs. 73, 75, and 77, and the light chain variable domain CDRs, SEQ ID NOs. 78-80, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 74, 76, and 77, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 78-80.

[0065] The amino acid sequence for the heavy chain variable region of the 3A7 antibody is shown in SEQ ID NO: 11. The amino acid sequence for the light chain variable region of the 3A7 antibody is shown in SEQ ID NO: 12.

[0066] The amino acid sequence for the heavy chain variable region of the 5C7 antibody is shown in SEQ ID NO: 21. The amino acid sequence for the light chain variable region of the 5C7 antibody is shown in SEQ ID NO: 22.

[0067] The amino acid sequence for the heavy chain variable region of the 6A5 antibody is shown in SEQ ID NO: 31. The amino acid sequence for the light chain variable region of the 6A5 antibody is shown in SEQ ID NO: 32.

[0068] The amino acid sequence for the heavy chain variable region of the 6G9 antibody is shown in SEQ ID NO: 41. The amino acid sequence for the light chain variable region of the 6G9 antibody is shown in SEQ ID NO: 42.

[0069] The amino acid sequence for the heavy chain variable region of the 7G10 antibody is shown in SEQ ID NO: 51. The amino acid sequence for the light chain variable region of the 7G10 antibody is shown in SEQ ID NO: 52.

[0070] The amino acid sequence for the heavy chain variable region of the 9E11 antibody is shown in SEQ ID NO: 61. The amino acid sequence for the light chain variable region of the 9E11 antibody is shown in SEQ ID NO: 62.

[0071] The amino acid sequence for the heavy chain variable region of the 13E6 antibody is shown in SEQ ID NO: 71. The amino acid sequence for the light chain variable region of the 13E6 antibody is shown in SEQ ID NO: 72.

[0072] The amino acid sequence for the heavy chain variable region of the 14A2 antibody is shown in SEQ ID NO: 81. The amino acid sequence for the light chain variable region of the 14A2 antibody is shown in SEQ ID NO: 82.

[0073] Humanization percentage refers to the percentage of identity of a heavy chain or light chain variable region sequence compared to a human antibody sequence in the International Immunogenetic Information System (IMGT) database. In some embodiments, the humanization percentage exceeds 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of methods for determining the humanization percentage and methods for determining top hits are well known in the art, for example, in Jones, et al. “The INNs and outs of antibody nonproprietary names.” MAbs. Vol.8. No.1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. Higher humanization percentages often have various advantages, such as being safer and more effective in humans, more likely to be accepted by human subjects, and / or less likely to have side effects. In some embodiments, the variable region is entirely human, derived from, for example, human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, human IGHD, and human IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and human IGKJ genes).

[0074] The amino acid sequence for the heavy chain variable region of the humanized 3A7 antibody is shown in SEQ ID NO: 83. The amino acid sequence for the light chain variable region of the humanized 3A7 antibody is shown in SEQ ID NO: 84.

[0075] The amino acid sequence for the heavy chain variable region of the humanized 5C7 antibody is shown in SEQ ID NO: 85. The amino acid sequence for the light chain variable region of the humanized 5C7 antibody is shown in SEQ ID NO: 86.

[0076] The amino acid sequence for the heavy chain variable region of the humanized 6A5 antibody is shown in SEQ ID NO: 87. The amino acid sequence for the light chain variable region of the humanized 6A5 antibody is shown in SEQ ID NO: 88.

[0077] The amino acid sequence for the heavy chain variable region of the humanized 7G10 antibody is shown in SEQ ID NO: 89. The amino acid sequence for the light chain variable region of the humanized 7G10 antibody is shown in SEQ ID NO: 90.

[0078] The amino acid sequence for the heavy chain variable region of the humanized 13E6 antibody is shown in SEQ ID NO: 91. The amino acid sequence for the light chain variable region of the humanized 13E6 antibody is shown in SEQ ID NO: 92.

[0079] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 83, 85, 87, 89, and 91. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 84, 86, 88, 90, and 92. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and both of these bind to CLEC5A.

[0080] Furthermore, in some embodiments, the antibody or antigen-binding fragments described herein are sequence numbers 3, 5, 7, 4, 6, 7, 13, 15, 17, 14, 16, 17, 23, 25, 27, 24, 26, 27, 33, 35, 37, 34, 36, 37, 43, 45, 47, 44, 46, 47, 53, 55, 57, 54, 56, 57, 63, 65, 67, sequence numbers One, two, or three heavy chain variable region CDRs selected from the group consisting of numbers 64, 66, 67, SEQ ID NOs. 73, 75, 77, and SEQ ID NOs. 74, 76, 77, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs. 8-10, SEQ ID NOs. 18-20, SEQ ID NOs. 28-30, SEQ ID NOs. 38-40, SEQ ID NOs. 48-50, SEQ ID NOs. 58-60, SEQ ID NOs. 68-70, and SEQ ID NOs. 78-80 may also be included.

[0081] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR1; the CDR2 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR2; and the CDR3 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR3. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are shown in Figure 22.

[0082] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0083] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0084] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0085] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 17, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0086] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0087] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 27, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0088] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0089] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 37, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0090] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0091] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 47, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0092] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 57, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0093] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 57, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0094] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 63, SEQ ID NO: 65, and SEQ ID NO: 67, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0095] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 67, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0096] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO: 77, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0097] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 77, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0098] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 9, and SEQ ID NO: 10, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0099] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 19, and SEQ ID NO: 20, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0100] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0101] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0102] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0103] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0104] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0105] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 79, and SEQ ID NO: 80, all of which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0106] Insertions, deletions, and substitutions may be located within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on Kabat's definition. In some embodiments, the CDR is determined based on Chothia's definition. In some embodiments, the CDR is determined based on a combination of Kabat's and Chothia's definitions.

[0107] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to CLEC5A. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising, or consisting thereof, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising, or consisting thereof, 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 sequence number 61 and the selected VL sequence is sequence number 62. In some embodiments, the selected VH sequence is sequence number 71 and the selected VL sequence is sequence number 72. In some embodiments, the selected VH sequence is sequence number 81 and the selected VL sequence is sequence number 82. In some embodiments, the selected VH sequence is sequence number 83 and the selected VL sequence is sequence number 84. In some embodiments, the selected VH sequence is sequence number 85 and the selected VL sequence is sequence number 86. In some embodiments, the selected VH sequence is sequence number 87 and the selected VL sequence is sequence number 88. In some embodiments, the selected VH sequence is sequence number 89 and the selected VL sequence is sequence number 90. In some embodiments, the selected VH sequence is sequence number 91 and the selected VL sequence is sequence number 92.

[0108] This disclosure also provides antibodies or antigen-binding fragments thereof that are competitive with the antibodies described herein. In some embodiments, the antibodies or antigen-binding fragments may bind to the same epitopes as the antibodies described herein.

[0109] This disclosure also provides an antibody or its antigen-binding fragment that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well 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 embodiment, this disclosure also provides an antibody or its antigen-binding fragment that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0110] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains contain or have the CDRs shown in Table 22. When a polypeptide forms a pair 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).

[0111] Anti-CLEC5A antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multimer, multispecific (e.g., bispecific), humanized antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0112] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to CLEC5A retains its ability to bind to CLEC5A. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site.

[0113] A single-chain Fv or scFv antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0114] The Fab fragment contains variable and constant domains of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment contains a pair of Fab fragments, generally commonally linked near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are well known in the art.

[0115] The antibodies and antibody fragments of this disclosure can be modified within the Fc region to confer a desired effector function or serum half-life. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0116] In some embodiments, the antibodies or antigen-binding fragments described herein recognize endogenous CLEC5A or recombinant CLEC5A. In some embodiments, the antibodies or antigen-binding fragments described herein recognize human CLEC5A (e.g., the extracellular region of human CLEC5A).

[0117] Multispecific antibodies or their antigen-binding fragments In one embodiment, the Specified

[0118] In some embodiments, a bispecific antibody or its antigen-binding fragment (e.g., anti-TAA / CLEC5A antibody) specifically binds to tumor-associated antigens (e.g., HER2) and CLEC5A, and such a bispecific antibody has a modified or enhanced Fc region (e.g., an Fc region including a GAALIE mutation, LALAPG mutation, S293D+I332E mutation, knob-into-hole (KIH) mutation, or non-fucosylated region).

[0119] In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain including a first heavy chain variable region (VH1) and a first light chain including a first light chain variable region (VL1); and a second heavy chain including a second heavy chain variable region (VH2) and a second light chain including a second light chain variable region (VL2).

[0120] 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.

[0121] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV) or VHH. In some embodiments, the second antigen-binding domain is an scFv or VHH.

[0122] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region containing the LALAPG mutation having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure containing the LALAPG mutation (L234A, L235A, and P329G mutations, according to EU numbering). In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329, according to EU numbering. In some embodiments, the Fc region contains 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.

[0123] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region containing the GAALIE mutation having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332, according to EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having 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 contains 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.

[0124] In some embodiments, the multispecific antibodies described herein are designed to have an Fc region containing aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure having the S239D+I332E mutation in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0125] In some embodiments, the multispecific antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the multispecific antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or a control multispecific antibody. In some embodiments, the antibody or its antigen-binding fragment has increased binding affinity to the FcγRIIa receptor and / or FcγRIIIa receptor.

[0126] In some embodiments, the multispecific antibodies described herein are designed to have a non-fucosylated Fc region. Non-fucosylated antibodies are designed so that the oligosaccharide in the antibody's Fc region does not contain a fucose sugar unit. Non-fucosylation of an antibody increases its antibody-dependent cytotoxic activity (ADCC).

[0127] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can take any preferred format. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain containing a light chain variable domain (VL) and a heavy chain variable domain (VH) linked by a first linker.

[0128] In some embodiments, a 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 linked to the CH1 domain and VL1 is linked to the CL domain. Schematic diagrams of this configuration are shown in Figures 9C and 9F.

[0129] This specification also provides an antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to member A of the C-type lectin domain family 5 (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the light chain of the antibody or the antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the C-terminus of the light chain of the antibody or the antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is linked to the light chain of the antibody or the antigen-binding fragment via a linker described herein. In some embodiments, the first antigen is TAA (e.g., HER2). A schematic diagram of this configuration is shown in Figure 9C.

[0130] This specification also provides an antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to member A of the C-type lectin domain family 5 (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the C-terminus of the heavy chain of the antibody or the antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the C-terminus of the Fc region of the antibody or the antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the C-terminus of the Fc region of the antibody or the antigen-binding fragment via a linker described herein. In some embodiments, the first antigen is TAA (e.g., HER2). A schematic diagram of this configuration is shown in Figure 9F.

[0131] This specification also provides an antibody or antigen-binding fragment thereof comprising a first chain comprising a first light chain and a first scFv or VHH, a second chain comprising a first heavy chain, a third chain comprising a second heavy chain, and a fourth chain comprising a second light chain and a second scFv or VHH, wherein the first light chain and the second light chain each comprise VL having the same sequence (VL1), and the first heavy chain and the second heavy chain each comprise 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, each of the first and second scFv or VHH comprises VH2 and / or VL2. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region. In some embodiments, the first and second heavy chains comprise an Fc region. 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. A schematic diagram of this configuration is shown in Figure 9C.

[0132] This specification also provides an antibody or antigen-binding fragment thereof comprising a first chain comprising a first light chain, a second chain comprising a first heavy chain and a first scFv or VHH, a third chain comprising a second heavy chain and a second scFv or VHH, and a fourth chain comprising a second light chain, wherein the first and second light chains each contain VL having the same sequence (VL1), and the first and second heavy chains each contain 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, each of the first and second scFv or VHH comprises VH2 and / or VL2. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region. In some embodiments, the first and second heavy chains comprise an Fc region. In some embodiments, a first scFv or VHH is ligated to the C-terminus of the Fc region of the first heavy chain. In some embodiments, a second scFv or VHH is ligated to the C-terminus of the Fc region of the second heavy chain. 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. A schematic diagram of this configuration is shown in Figure 9F.

[0133] This specification also provides 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 member A of the C-type lectin domain family 5 (CLEC5A). In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the heavy chain of the antibody or antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the Fc region of the antibody or antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the Fc region of the antibody or antigen-binding fragment via a linker described herein. In some embodiments, the first antigen is TAA (e.g., HER2). A schematic diagram of this configuration is shown in Figure 9D.

[0134] This specification also provides an antibody or antigen-binding fragment thereof comprising: a first chain comprising a first light chain comprising VL1; a second chain comprising a first heavy chain comprising VH1 and a first Fc region; and a third chain comprising scFv or VHH and a second Fc region. In some embodiments, scFv or VHH comprises VH2 and / or VL2. In some embodiments, the first Fc region comprises one or more knob mutations, and the second Fc region comprises one or more hole mutations. In some embodiments, the first Fc region comprises one or more hole mutations, and the second Fc region comprises one or more knob mutations. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. A schematic diagram of this configuration is shown in Figure 9D.

[0135] This specification also provides 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 the first antigen, and a third antigen-binding domain that specifically binds to member A of the C-type lectin domain family 5 (CLEC5A). In some embodiments, the third antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the heavy chain of the antibody or the antigen-binding fragment. In some embodiments, the third antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the Fc region of the antibody or the antigen-binding fragment. In some embodiments, the second antigen-binding domain that specifically binds to CLEC5A is ligated to the N-terminus of the Fc region of the antibody or the antigen-binding fragment via a linker as described herein. In some embodiments, the first antigen and / or the second antigen are TAAs (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. A schematic diagram of this configuration is shown in Figure 9B.

[0136] This specification also provides an antibody or an antigen-binding fragment thereof comprising: a first chain comprising a first light chain comprising VL1 and VL2; a second chain comprising a first heavy chain comprising VH1, VH2, and a first Fc region; and a third chain comprising scFv or VHH and a second Fc region. In some embodiments, scFv or VHH comprises VH3 and / or VL3. In some embodiments, the first Fc region comprises one or more 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. A schematic diagram of this configuration is shown in Figure 9B.

[0137] In some embodiments, the antibody or its antigen-binding fragment comprises a first heavy chain containing VH1 and a first light chain containing VL1, and a second heavy chain containing VH2 and a second light chain containing VL2. A schematic diagram of this configuration is shown in Figure 9A or Figure 9E. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations.

[0138] This specification also provides an antibody or antigen-binding fragment thereof comprising a first chain comprising a first light chain comprising VL1, a second chain comprising a first heavy chain comprising VH1, a third chain comprising a second heavy chain comprising VH2, and a fourth chain comprising a second light chain comprising VL2. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, VH1 and VL1 bind to a first antigen. In some embodiments, the first antigen is TAA. In some embodiments, VH2 and VL2 bind to a second antigen. In some embodiments, the second antigen is CLEC5A. In some embodiments, the first heavy chain 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.

[0139] This specification also provides an antibody or an antigen-binding fragment thereof comprising: a first chain comprising a first light chain comprising VL1; a second chain comprising a first heavy chain comprising VH1; a third chain comprising a second heavy chain comprising VH2 and scFv or VHH; and a fourth chain comprising a second light chain comprising VL2. In some embodiments, scFv or VHH comprises VH3 and / or VL3. A schematic diagram of this configuration is shown in Figure 9E. 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 contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations.

[0140] In some embodiments, the CrossMab substitution is introduced into 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.

[0141] In some embodiments, the CrossMab substitution is introduced into 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.

[0142] In some embodiments, the multispecific antibody has a heavy chain sequence containing the wild-type IgG1 Fc region (SEQ ID NO: 95). In some embodiments, the multispecific antibody has a heavy chain sequence containing an Fc region 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: 95.

[0143] In some embodiments, the multispecific antibody has a heavy chain sequence containing an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific antibody has a heavy chain sequence containing an Fc region 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: 96.

[0144] In some embodiments, the multispecific antibody has a heavy chain sequence containing an IgG1 Fc region (SEQ ID NO: 97) with an optimized mutation. In some embodiments, the multispecific antibody has a heavy chain sequence containing an Fc region 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: 97.

[0145] This disclosure provides modified antibodies, including multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies, such as chimeric antibodies, humanized antibodies, and human antibodies.

[0146] In some embodiments, a multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody, or an antibody fragment thereof, comprises a combination of an anti-HER2 antigen-binding domain and an anti-CLEC5A antigen-binding domain as shown in Table 22.

[0147] In some embodiments, anti-TAA (anti-HER2 / CLEC5A) antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0148] Any of the multispecific (bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies or their antigen-binding fragments described herein may be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or antigen-binding fragment, or sustain its biological activity, either in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0149] Multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies may be antibody variants (including derivatives and complexes) of multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies or antibody fragments. Additional 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 chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. A multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody or antigen-binding fragment may be an IgG (e.g., IgG1 Fc region shown in SEQ ID NO: 95) antibody or its antigen-binding fragment.

[0150] Fragments of multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies are suitable for use in the provided method, insofar as they retain the desired affinity and specificity for both TAA (e.g., HER2) and CLEC5A. Therefore, fragments of multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies retain the ability to bind to both TAA (e.g., HER2) and CLEC5A.

[0151] Tumor-associated antigens Tumor-associated antigens (TAAs) are antigenic substances produced by tumor cells. They can trigger an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells in diagnostic tests and may be candidates for use in cancer treatment. Numerous 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; each reference is incorporated herein in its entirety by reference).

[0152] In some embodiments, TAA is HER2, CD79b, BCMA, CD38, GPRC5D, DLL3, CD70, GPC3, Fas ligand (FasL), CD1d, membrane glycolipid, globotriaosylceramide (Gb3Cer / CD77), ganglioside (GD2, GD3, and GM2), GPRC5D, CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD1 9, WT-l, B7H3, TEM8, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-fetoprotein (AFP), BAFF, B lymphoma cells, CA242 antigen, CA-125, carbonic dehydration Enzyme 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, CD70, CD74, CD80, CEA, CNT0888, CTLA-4, DLL3, DR5, EGFR, EpCAM, CD3, FAP, Fibronectin Extradomain B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HGF, Human Scattering Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, Insulin-like Growth Factor I Receptor, Integrin a5b1, Integrin avb3, MSLN, MS4A1, MUC1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PD-1, PD-L1, PDGF-R a, TWEAK, Phosphatidylserine, Prostate Cancer Cells, RANKL, RON, SCH The group is selected from 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, OX40, and vimentin.

[0153] In some embodiments, a multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprises a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA) and a second antigen-binding domain that specifically binds to CLEC5A. In some embodiments, the first antigen-binding domain specifically binds to HER2. In some embodiments, the first antigen-binding domain specifically binds to a TAA selected from HER2, EGFR, EpCAM, CD79b, GPRC5D, BCMA, CD38, DLL3, CD70, GPC3, and mesothelin.

[0154] Anti-TAA / CLEC5A antibody and its antigen-binding fragment In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein have agonist activity for macrophage activation. In some embodiments, macrophage activation by 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 macrophage activation without contact with such antibody. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have a LALAPG mutation (SEQ ID NO: 96) in the Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein that bind to macrophages have mutations optimized for the Fc region (SEQ ID NO: 97).

[0155] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody described herein that binds to macrophages has a silent mutation (e.g., LALAPG mutation) in the Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibody described herein binds to target cells (e.g., HER2+ cancer cells) that have a silent mutation (e.g., LALAPG mutation) in the Fc region.

[0156] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., with the involvement of CLEC5A) and mediate 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) in the Fc region. In some embodiments, the anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein contain the wild-type human IgG1 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) in the Fc region.

[0157] In some embodiments, the multispecific (e.g., bispecific) anti-TAA / CLEC5A (e.g., anti-HER2 / CLEC5A) antibodies described herein activate macrophages (e.g., with the involvement of CLEC5A) and induce macrophage-mediated killing of target cells. In some embodiments, target cells (e.g., HER2+ cancer cells) are killed by macrophage-mediated killing. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the total number of target cells are killed by macrophage-mediated phagocytosis.

[0158] In some embodiments, the TAAs described herein are selected from HER2, EGFR, EpCAM, CD79b, GPRC5D, BCMA, CD38, DLL3, CD70, GPC3, and mesoserine.

[0159] Anti-HER2 / CLEC5A antibody and its antigen-binding fragment The receptor tyrosine protein kinase erbB-2 (HER2) is a protein encoded in humans by the ERBB2 gene. ERBB is an abbreviation for erythroblastic oncogene B, a gene originally isolated from the genome of birds. This human protein is also frequently referred to as HER2 (human epidermal growth factor receptor 2) or CD340 (differentiation antigen group 340).

[0160] HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. However, unlike other members of the ERBB family, HER2 does not directly bind to its ligand. HER2 activation is triggered by heterodimerization with other ERBB members, or homodimerization when HER2 concentrations are high, such as in cancer. Amplification or overexpression of this oncogene has been shown to play a crucial role in the development and progression of some aggressive breast cancers. In recent years, this protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0161] Detailed overviews of HER2 can be found, for example, on the NCBI website: “ERBB2 erb-b2 receptor tyrosine kinase 2 [Homo sapiens (human)]-Gene-NCBI”; “ERBB2” Genetics Home Reference; Barh D, Gunduz M (2015-01-22). Noninvasive Molecular Markers in Gynecologic Cancers. CRC Press. p.427. ISBN 9781466569393; and Hsu JL, Hung MC (2016). “The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer”. Cancer and Metastasis Reviews. 35(4):575-588. doi:10.1007 / s10555-016-9649-6. The entire contents of each are incorporated herein by reference.

[0162] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to HER2 / CLEC5A (e.g., human HER2 / CLEC5A). In one embodiment, this disclosure provides an anti-HER2 / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to HER2 and a second antigen-binding domain that specifically binds to CLEC5A.

[0163] 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).

[0164] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 100, 102, and 104, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 105, 106, and 107, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 101, 103, and 104, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 105, 106, and 107, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 3, 5, and 7, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 6, and 7, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0165] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 102, and 104, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 105, 106, and 107, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 3, 5, and 7, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively.

[0166] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 101, 103, and 104, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 105, 106, and 107, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 6, and 7, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively.

[0167] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 102, and 104, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 105, 106, and 107, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0168] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 101, 103, and 104, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 105, 106, and 107, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0169] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 108, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 109, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 11, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 12.

[0170] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 108, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 109, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 83, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 84.

[0171] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 108, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 109, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0172] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 108, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 109, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0173] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence is sequence number 108, and the selected VL sequence is sequence number 109.

[0174] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (2) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (3) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; and (4) The selected VH sequence is sequence number 86, and the selected VL sequence is sequence number 87.

[0175] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 108, and the selected VL sequence is sequence number 109.

[0176] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (2) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (3) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; and (4) The selected VH sequence is sequence number 86, and the selected VL sequence is sequence number 87.

[0177] In some embodiments, the first antigen-binding domain is the antigen-binding domain of trastuzumab, which is an anti-HER2 antibody. Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1 kappa monoclonal antibody that is a humanized format of a mouse anti-HER2 antibody (4D5) that selectively binds to the extracellular domain of HER2 with high affinity (Kd=5nM) in cell-based assays (e.g., U.S. Patent Nos. 5,677,171, 5,821,337, 6,054,297, 6,165,464, 6,339,142, 6,407,213, 6,639,055, 6,719,971, 6,800,738, 7,074,404, Coussens et al.). al(1985)Science230:1132-9;Slamon et al(1989)Science244:707-12;Slamon et al(2001)New Engl.J.Med.344:783-792).

[0178] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7 or 3A7, its chimeric antibody, and its humanized antibody.

[0179] CDR sequences for 3A7 and antibodies derived from 3A7 include the heavy chain variable domain CDRs, SEQ ID NOs: 3, 5, and 7, and the light chain variable domain CDRs, SEQ ID NOs: 8, 9, and 10, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 4, 6, and 7, and the light chain variable domain CDRs are shown in SEQ ID NOs: 8, 9, and 10.

[0180] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0181] In some embodiments, the first antigen-binding domain specifically binds to HER2 in humans, rabbits, mice, monkeys, or dogs, and / or the second antigen-binding domain specifically binds to CLEC5A in humans, rabbits, mice, monkeys, or dogs.

[0182] 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.

[0183] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0184] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0185] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the 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 alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0186] In some embodiments, the anti-HER2 / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0187] In some embodiments, the multispecific (or bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an Fc region containing aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody described herein is designed to have an IgG1 subtype structure having the S239D+I332E mutation in the EU numbering. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering.

[0188] In some embodiments, the multispecific (bispecific) anti-HER2 / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-HER2 / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0189] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0190] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains 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 to 9F.

[0191] 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 having the LALAPG mutation (SEQ ID NO: 96).

[0192] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 119.

[0193] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 3A7(2+2)Fc-LALAPG" or "HER2 / 3A7(2+2 A)Fc silencing", and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 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.

[0194] 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 having the LALAPG mutation (SEQ ID NO: 96).

[0195] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121.

[0196] 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+2 A)Fc silencing", and comprises a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 120, and a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121.

[0197] 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 having an optimized mutation (SEQ ID NO: 97).

[0198] In some embodiments, the multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody comprises a light chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 123.

[0199] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 5C7(2+2 A)Fc optimized" and contains 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.

[0200] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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. In some embodiments, the first heavy chain contains an IgG1 Fc region having a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain contains one or more hole mutations.

[0201] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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.

[0202] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains a second 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: 111. In some embodiments, the second heavy chain contains an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain contains one or more knob mutations.

[0203] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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.

[0204] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 3A7(1+1 A)Fc-LALAPG" or "HER2 / 3A7(1+1 A)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 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: 112. The light chain sequence comprises a second 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 sequence number 111, and 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 sequence number 113.

[0205] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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. In some embodiments, the first heavy chain contains an IgG1 Fc region having a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the first heavy chain contains one or more hole mutations.

[0206] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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.

[0207] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains a second 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: 115. In some embodiments, the second heavy chain contains an IgG1 Fc region having a LALAPG mutation (SEQ ID NO: 96). In some embodiments, the second heavy chain contains one or more knob mutations.

[0208] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody contains 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.

[0209] In some embodiments, a multispecific (e.g., bispecific) anti-HER2 / CLEC5A antibody is referred to as "HER2 / 5C7(1+1 A)Fc-LALAPG" or "HER2 / 5C7(1+1 A)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 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: 116. The light chain sequence comprises a second 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 sequence number 115, and 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 sequence number 117.

[0210] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0211] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0212] This disclosure also provides nucleic acids comprising polynucleotides encoding anti-HER2 / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-HER2 / CLEC5A antibody comprises the CDRs shown in Table 22. When a polypeptide pairs 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.

[0213] Anti-EGFR / CLEC5A antibody and its antigen-binding fragment Epidermal growth factor receptors (EGFR, ErbB-1, and HER1 in humans) are transmembrane proteins that are receptors for members of the epidermal growth factor family (EGF family), which are extracellular protein ligands. The epidermal growth factor receptors are members of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many cancer types, mutations affecting EGFR expression or activity may increase cancer progression. In humans, deficiencies in EGFR and other receptor tyrosine kinase signaling are associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a wide variety of tumors. Blocking EGFR signaling, for example by blocking the EGFR binding site in the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can suppress the growth of EGFR-expressing tumors and improve the patient's condition.

[0214] EGFR is a transmembrane protein that is activated by the binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFa). ErbB2 does not have a known direct activating ligand and may be constitutively activated or activated by heterodimerization with other family members such as EGFR. When EGFR is activated by a growth factor ligand, it transitions from an inactive monomer to an active homodimer.

[0215] Dimerization of EGFR stimulates its intrinsic intracellular protein tyrosine kinase activity. As a result, multiple tyrosine (Y) residues present in the C-terminal domain of EGFR are autophosphorylated. These include Y992, Y1045, Y1068, Y1148, and Y1173.

[0216] Autophosphorylation causes downstream activation and signal transduction by multiple other proteins that bind to the phosphorylated tyrosine via their phosphotyrosine-binding SH2 domains. These downstream signaling proteins mainly initiate multiple signaling cascades such as the MAPK pathway, Akt pathway, and JNK pathway, 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 co-associated receptors and can itself be activated in such a manner.

[0217] Mutations that cause overexpression (known as upregulation or amplification) of EGFR are associated with a number of cancers including lung adenocarcinoma (about 40% of cases), anal cancer, glioblastoma (about 50%), and epithelial tumors of the head and neck (about 80% - 100%). These somatic mutations involving EGFR cause its constitutive activation, leading to uncontrolled cell division. In glioblastoma, the specific EGFR mutation EGFRvIII is often observed. Mutations, amplifications, or dysregulations of EGFR or family members are involved in about 30% of all epithelial cancers. <0,

[0218] The present disclosure 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 disclosure provides an anti-EGFR / CLEC5A multispecific (e.g., bispecific) antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to CLEC5A.

[0219] 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).

[0220] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 126, 128, and 130, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 131, 132, and 133, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 127, 129, and 130, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 131, 132, and 133, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 136, 138, and 140, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 141, 142, and 143, respectively; and (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 137, 139, and 140, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 141, 142, and 143, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0221] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 126, 128, and 130, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 131, 132, and 133, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0222] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 127, 129, and 130, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 131, 132, and 133, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 14, 16, and 17, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0223] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 136, 138, and 140, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 141, 142, and 143, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0224] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 137, 139, and 140, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 141, 142, and 143, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0225] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 134, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 135, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0226] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 134, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 135, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0227] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 144, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 145, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0228] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 144, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 145, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0229] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 134, and the selected VL sequence is SEQ ID NO: 135. In some embodiments, the selected VH sequence is SEQ ID NO: 144, and the selected VL sequence is SEQ ID NO: 145.

[0230] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0231] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 134, and the selected VL sequence is sequence number 135. In some embodiments, the selected VH sequence is sequence number 144, and the selected VL sequence is sequence number 145.

[0232] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0233] In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody such as cetuximab, panitumumab, nesitumumab, or Eg-B4-VHH. In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody such as amivantamab. In some embodiments, the first antigen-binding domain is the antigen-binding domain of an anti-EGFR antibody such as nimotuzumab (EGFR2).

[0234] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0235] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0236] In some embodiments, the first antigen-binding domain specifically binds to EGFR in humans, rabbits, mice, monkeys, or dogs, and / or the second antigen-binding domain specifically binds to CLEC5A in humans, rabbits, mice, monkeys, or dogs.

[0237] 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.

[0238] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0239] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0240] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0241] In some embodiments, the anti-EGFR / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0242] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an Fc region containing aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the S239D+I332E mutation in the EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering.

[0243] In some embodiments, the multispecific (bispecific) anti-EGFR / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-EGFR / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0244] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0245] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies described herein may have the structure shown in any one of Figures 9A-9F.

[0246] In some embodiments, multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibodies contain 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 or 148. In some embodiments, the heavy chain contains an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97).

[0247] In some embodiments, a multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody contains 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.

[0248] In some embodiments, a multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody is referred to as "EGFR1 / 5C7(2+2 A)Fc optimized" and contains 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, a multispecific (e.g., bispecific) anti-EGFR / CLEC5A antibody is referred to as "EGFR2 / 5C7(2+2 A)Fc optimized" and contains 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.

[0249] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0250] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0251] The present invention also provides nucleic acids comprising polynucleotides encoding an anti-EGFR / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-EGFR / CLEC5A antibody comprises the CDRs shown in Table 22. When a 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.

[0252] Anti-EpCAM / CLEC5A antibody and its antigen-binding fragment Epithelial cell adhesion molecules (EpCAM), also known as tumor-associated calcium signaling transducer 1 (TACST-1), 17-1A, and CD326, are 40-kDa transmembrane glycoproteins that are highly expressed in epithelial cancers and at lower levels in normal monolayer epithelium. The structure and function of EpCAM are outlined, for example, in 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.

[0253] EpCAM is expressed in the basolateral membrane and plays a role in calcium-independent alloaffinity cell adhesion. The mature EpCAM molecule (after the removal of the 23-amino acid signal peptide) consists of a 242-amino acid extracellular domain at the N-terminus (including an epidermal growth factor-like repeat region, a human thyroglobulin (TY) repeat region, and a cysteine-depleted region), a 23-amino acid single transmembrane domain, and a 26-amino acid cytoplasmic domain at the C-terminus (including two α-actinin binding sites and an NPXY internalization motif). EpCAM is frequently overexpressed in epithelial-derived cancers and is also expressed in cancer stem cells, making it a molecule of great interest in treatment and diagnosis. Due to its high frequency and high levels of expression in carcinomas and their metastatic lesions, EpCAM functions as a prognostic marker, therapeutic target, and anchor molecule on ring tumor cells and disseminated tumor cells (CTCs / DTCs), which are considered major sources of metastatic cancer cells. The extracellular domain of EpCAM is 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 a nuclear complex, upregulating the expression of genes that promote cell proliferation. EpCAM may be involved in epithelial-mesenchymal transition (EMT) and contribute to the formation of large metastatic lesions.

[0254] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to EpCAM / CLEC5A (e.g., human EpCAM / CLEC5A). In one embodiment, this disclosure provides an anti-EpCAM / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to EpCAM and a second antigen-binding domain that specifically binds to CLEC5A.

[0255] 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).

[0256] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 150, 152, and 154, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 155, 156, and 157, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 151, 153, and 154, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 155, 156, and 157, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0257] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 150, 152, and 154, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 155, 156, and 157, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0258] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 151, 153, and 154, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 155, 156, and 157, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 14, 16, and 17, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0259] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 158, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 159, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0260] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 158, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 159, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0261] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 158, and the selected VL sequence is SEQ ID NO: 159.

[0262] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0263] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 158, and the selected VL sequence is sequence number 159.

[0264] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0265] In some embodiments, the first antigen-binding domain is the antigen-binding domain of the anti-EpCAM antibody solitomab.

[0266] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0267] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0268] In some embodiments, the first antigen-binding domain specifically binds to human, mouse, monkey, or dog EpCAM, and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0269] 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.

[0270] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0271] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0272] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0273] In some embodiments, the anti-EpCAM / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0274] In some embodiments, the multispecific (or bispecific) anti-EpCAM / CLEC5A antibody described herein is designed to have an Fc region containing aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody described herein is designed to have an IgG1 subtype structure having the S239D+I332E mutation in the EU numbering. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in the EU numbering.

[0275] In some embodiments, the multispecific (bispecific) anti-EpCAM / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-EpCAM / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0276] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0277] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibodies described herein may have the structure shown in any one of Figures 9A to 9F.

[0278] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody contains 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: 160 or 162. In some embodiments, the heavy chain contains an IgG1 Fc region with an optimized mutation (SEQ ID NO: 97).

[0279] In some embodiments, a multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody contains 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.

[0280] In some embodiments, a multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody is referred to as "EpCAM / 5C7(2+2 A)Fc optimized" and contains 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: 160, and contains 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. In some embodiments, the heavy 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: 162, and the 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: 163.

[0281] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0282] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0283] The present invention also provides nucleic acids comprising polynucleotides encoding an anti-EpCAM / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-EpCAM / CLEC5A antibody comprises the CDRs shown in Table 22. When a 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.

[0284] Anti-GPRC5D / CLEC5A antibody and its antigen-binding fragment G protein-coupled receptor family group C, member D (GPRC5D) is a protein encoded by the GPRC5D gene in humans. The protein encoded by this gene is a member of the G protein-coupled receptor family.

[0285] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to GPRC5D / CLEC5A (e.g., human GPRC5D / CLEC5A). In one embodiment, this disclosure provides an anti-GPRC5D / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment, comprising a first antigen-binding domain that specifically binds to GPRC5D and a second antigen-binding domain that specifically binds to CLEC5A.

[0286] 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).

[0287] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 164, 166, and 168, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 169, 170, and 171, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 165, 167, and 168, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 169, 170, and 171, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0288] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 164, 166, and 168, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 169, 170, and 171, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0289] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 165, 167, and 168, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 169, 170, and 171, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 14, 16, and 17, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 18, 19, and 20, respectively.

[0290] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 172, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 173, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0291] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 172, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 173, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0292] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 172, and the selected VL sequence is SEQ ID NO: 173.

[0293] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0294] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 172, and the selected VL sequence is sequence number 173.

[0295] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0296] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0297] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0298] In some embodiments, the first antigen-binding domain specifically binds to GPRC5D in humans, rabbits, mice, monkeys, or dogs, and / or the second antigen-binding domain specifically binds to CLEC5A in humans, rabbits, mice, monkeys, or dogs.

[0299] 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.

[0300] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0301] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0302] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having LALAPG mutations (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0303] In some embodiments, the anti-GPRC5D / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains 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.

[0304] In some embodiments, the multispecific (bispecific) anti-GPRC5D / CLEC5A antibody described herein is designed to have an Fc region containing 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-GPRC5D / CLEC5A antibody described herein is designed to have an IgG1 subtype structure having the S239D+I332E mutation in EU numbering. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0305] In some embodiments, the multispecific (bispecific) anti-GPRC5D / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-GPRC5D / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0306] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0307] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies described herein may have the structures shown in any one of Figures 9A to 9F.

[0308] In some embodiments, the multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody contains 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. In some embodiments, the heavy chain contains an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97).

[0309] In some embodiments, multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibodies contain 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.

[0310] In some embodiments, a multispecific (e.g., bispecific) anti-GPRC5D / CLEC5A antibody is referred to as "GPRC5D / 5C7(2+2 A)Fc optimized" and contains 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 contains 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.

[0311] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0312] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0313] This disclosure also provides nucleic acids comprising polynucleotides encoding anti-GPRC5D / CLEC5A antibodies. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-GPRC5D / CLEC5A antibody comprises the CDRs shown in Table 22. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to GPRC5D and / or CLEC5A.

[0314] Anti-BCMA / CLEC5A antibody and its antigen-binding fragment B-cell maturation antigen (BCMA or BCM), also known as tumor necrosis factor receptor superfamily 17 (TNFRSF17) in humans, is a protein encoded by the TNFRSF17 gene. BCMA is a cell surface receptor belonging to the TNF receptor superfamily that recognizes B-cell activator (BAFF). Serum B-cell maturation antigen (sBCMA) is a cleaved form of BCMA and is present at low levels in the serum of normal patients, but is generally elevated in patients with multiple myeloma (MM).

[0315] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to BCMA / CLEC5A (e.g., human BCMA / CLEC5A). In one embodiment, this disclosure provides an anti-BCMA / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to BCMA and a second antigen-binding domain that specifically binds to CLEC5A.

[0316] 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).

[0317] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 176, 178, and 180, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 181, 182, and 183, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 177, 179, and 180, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 181, 182, and 183, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0318] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 176, 178, and 180, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 181, 182, and 183, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0319] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 177, 179, and 180, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 181, 182, and 183, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 14, 16, and 17, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 18, 19, and 20, respectively.

[0320] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 184, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 185, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0321] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 184, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 185, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0322] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 184, and the selected VL sequence is SEQ ID NO: 185.

[0323] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0324] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 184, and the selected VL sequence is sequence number 185.

[0325] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0326] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0327] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0328] In some embodiments, the first antigen-binding domain specifically binds to BCMA in humans, rabbits, mice, monkeys, or dogs, and / or the second antigen-binding domain specifically binds to CLEC5A in humans, rabbits, mice, monkeys, or dogs.

[0329] 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.

[0330] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0331] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0332] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the LALAPG mutation (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0333] In some embodiments, the anti-BCMA / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0334] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein are designed to have an Fc region containing 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-BCMA / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the S239D+I332E mutation in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0335] In some embodiments, the multispecific (bispecific) anti-BCMA / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-BCMA / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0336] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0337] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains one or more knob mutations. In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies described herein may have the structures shown in any one of Figures 9A to 9F.

[0338] In some embodiments, multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibodies contain 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. In some embodiments, the heavy chain contains an IgG1 Fc region with an optimized mutation (SEQ ID NO: 97).

[0339] In some embodiments, a multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody contains 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.

[0340] In some embodiments, a multispecific (e.g., bispecific) anti-BCMA / CLEC5A antibody is referred to as "BCMA / 5C7(2+2 A)Fc optimized" and contains 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.

[0341] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0342] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0343] The present invention also provides nucleic acids comprising polynucleotides encoding an anti-BCMA / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-BCMA / CLEC5A antibody comprises the CDRs shown in Table 22. When a 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.

[0344] Anti-CD38 / CLEC5A antibody and its antigen-binding fragment CD38 (differentiation antigen group 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 functions in cell adhesion, signal transduction, and calcium signal transduction. In humans, the CD38 protein is encoded by the CD38 gene located on chromosome 4. CD38 is a homologous gene to CD157, located on human chromosome 4 (4p15).

[0345] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically binds to CD38 / CLEC5A (e.g., human CD38 / CLEC5A). In one embodiment, this disclosure provides an anti-CD38 / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to CD38 and a second antigen-binding domain that specifically binds to CLEC5A.

[0346] 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).

[0347] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The above-selected VH1 CDR1, 2, and 3 amino acid sequences and the above-selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 188, 190, and 192, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 193, 194, and 195, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 189, 191, and 192, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 193, 194, and 195, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 18, 19, and 20, respectively; and (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0348] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 188, 190, and 192, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 193, 194, and 195, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0349] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 189, 191, and 192, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 193, 194, and 195, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 14, 16, and 17, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0350] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 196, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 197, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0351] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 196, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 197, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0352] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 196, and the selected VL sequence is SEQ ID NO: 197.

[0353] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0354] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 196, and the selected VL sequence is sequence number 197.

[0355] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; and (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86.

[0356] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibody, its chimeric antibody, and its humanized antibody as described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibody 5C7, its chimeric antibody, and its humanized antibody.

[0357] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0358] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CD38, and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0359] 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.

[0360] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0361] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0362] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the LALAPG mutation (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 alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0363] In some embodiments, the anti-CD38 / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0364] In some embodiments, the multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies described herein are designed to have an Fc region containing 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 having the S239D+I332E mutation in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0365] In some embodiments, the multispecific (bispecific) anti-CD38 / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-CD38 / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0366] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0367] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains 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.

[0368] In some embodiments, multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibodies contain 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. In some embodiments, the heavy chain contains an IgG1 Fc region with an optimized mutation (SEQ ID NO: 97).

[0369] In some embodiments, a multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody contains 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.

[0370] In some embodiments, a multispecific (e.g., bispecific) anti-CD38 / CLEC5A antibody is referred to as "CD38 / 5C7(2+2 A)Fc optimized" and contains 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 contains 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.

[0371] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0372] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0373] This disclosure also provides nucleic acids comprising polynucleotides encoding an anti-CD38 / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-CD38 / CLEC5A antibody comprises the CDRs shown in Table 22. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to CD38 and / or CLEC5A.

[0374] Anti-CD79b / CLEC5A antibody and its antigen-binding fragment The CD79b molecule (also known as immunoglobulin-associated beta, or CD79B (differentiation antigen group 79B)) is a human gene. It is associated with agammaglobulinemia type 6. The B lymphocyte antigen receptor is a multimeric complex containing surface immunoglobulin (Ig), an antigen-specific component. Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta, which are necessary for the expression and function of the B cell antigen receptor. This gene encodes the Ig-beta protein of the B cell antigen component. Alternatively spliced ​​transcript variants encoding different isoforms have been described.

[0375] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to CD79b / CLEC5A (e.g., human CD79b / CLEC5A). In one embodiment, this disclosure provides an anti-CD79b / CLEC5A multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to CD79b and a second antigen-binding domain that specifically binds to CLEC5A.

[0376] 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).

[0377] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) comprises CDR1, 2, and 3, the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3, and the above selected VH1 CDR1, 2, and 3 amino acid sequences and the above selected VL1 CDR1, 2, and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 200, 202, and 204, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 205, 206, and 207, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 201, 203, and 204, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 205, 206, and 207, respectively; The second heavy chain variable region (VH2) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, and the VH2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3; The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 14, 16, and 17, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 3, 5, and 7, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively; (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 6, and 7, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively; (5) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 63, 65, and 67, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 68, 69, and 70, respectively; and (6) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 64, 66, and 67, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 68, 69, and 70, respectively.

[0378] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 200, 202, and 204, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 205, 206, and 207, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 15, and 17, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0379] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 201, 203, and 204, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 205, 206, and 207, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 14, 16, and 17, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 18, 19, and 20, respectively.

[0380] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 200, 202, and 204, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 205, 206, and 207, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 3, 5, and 7, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 8, 9, and 10, respectively.

[0381] In some embodiments, the selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 201, 203, and 204, respectively; the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 205, 206, and 207, respectively; the selected VH2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 4, 6, and 7, respectively; and the selected VL2 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 8, 9, and 10, respectively.

[0382] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 200, 202, and 204, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 205, 206, and 207, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 63, 65, and 67, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 68, 69, and 70, respectively.

[0383] In some embodiments, the selected VH1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 201, 203, and 204, respectively; the selected VL1 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 205, 206, and 207, respectively; the selected VH2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 64, 66, and 67, respectively; and the selected VL2 CDR1,2,3 amino acid sequences are shown in SEQ ID NOs. 68, 69, and 70, respectively.

[0384] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 21, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 22.

[0385] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 85, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 86.

[0386] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 11, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 12.

[0387] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 83, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 84.

[0388] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 71, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 72.

[0389] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 208, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 209, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 91, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 92.

[0390] In some embodiments, VH1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 208, and the selected VL sequence is SEQ ID NO: 209.

[0391] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86; (3) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (4) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; (5) The selected VH sequence is sequence number 71, and the selected VL sequence is sequence number 72; and (6) The selected VH sequence is sequence number 91, and the selected VL sequence is sequence number 92.

[0392] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3, which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3, which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence. In some embodiments, the selected VH sequence is sequence number 208, and the selected VL sequence is sequence number 209.

[0393] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (2) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86; (3) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (4) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; (5) The selected VH sequence is sequence number 71, and the selected VL sequence is sequence number 72; and (6) The selected VH sequence is sequence number 91, and the selected VL sequence is sequence number 92.

[0394] In some embodiments, the second antigen-binding domain is one of the antigen-binding domains of the anti-CLEC5A antibodies, their chimeric antibodies, and their humanized antibodies described herein. In some embodiments, the second antigen-binding domain is the antigen-binding domain of the anti-CLEC5A antibodies 5C7, 3A7, 13E6, their chimeric antibodies, and their humanized antibodies.

[0395] CDR sequences for 5C7 and antibodies derived from 5C7 include the heavy chain variable domain CDRs, SEQ ID NOs. 13, 15, and 17, and the light chain variable domain CDRs, SEQ ID NOs. 18, 19, and 20, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 14, 16, and 17, and the light chain variable domain CDRs are shown in SEQ ID NOs. 18, 19, and 20.

[0396] CDR sequences for 3A7 and antibodies derived from 3A7 include the heavy chain variable domain CDRs, SEQ ID NOs: 3, 5, and 7, and the light chain variable domain CDRs, SEQ ID NOs: 8, 9, and 10, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 4, 6, and 7, and the light chain variable domain CDRs are shown in SEQ ID NOs: 8, 9, and 10.

[0397] CDR sequences for 13E6 and antibodies derived from 13E6 include, as defined by Kabat, the CDRs of the heavy chain variable domain, SEQ ID NOs. 63, 65, and 67, and the CDRs of the light chain variable domain, SEQ ID NOs. 68, 69, and 70. Under Chothia's definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs. 64, 66, and 67, and the CDRs of the light chain variable domain are shown in SEQ ID NOs. 68, 69, and 70.

[0398] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CD79b, and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.

[0399] 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.

[0400] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFV), and / or the second antigen-binding domain is an scFv.

[0401] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region).

[0402] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies described herein are designed to have an IgG1 subtype structure having the LALAPG mutation (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 alanine (A) at position 234, alanine (A) at position 235, and glycine (G) at position 329 in EU numbering. In some embodiments, the Fc region contains 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.

[0403] In some embodiments, the anti-CD79b / CLEC5A antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to that of SEQ ID NO: 97.

[0404] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody described herein is designed to have an Fc region containing 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 antibody described herein is designed to have an IgG1 subtype structure having the S239D+I332E mutation in EU numbering. In some embodiments, the multispecific antibody described herein is designed to have an IgG1 Fc region having aspartic acid (D) at position 239 and glutamic acid (E) at position 332 in EU numbering.

[0405] In some embodiments, the multispecific (bispecific) anti-CD79b / CLEC5A antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-CD79b / CLEC5A antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0406] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0407] In some embodiments, a 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 linked to the CH1 domain, and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figure 9C. In some embodiments, the antibody or antigen-binding fragment described herein comprises a first heavy chain and a first light chain; as well as a second heavy chain and a second light chain. A schematic diagram of this configuration is shown in Figure 9A. In some embodiments, the Fc region contains a knob-into-hole (KIH) mutation. In some embodiments, the first heavy chain contains one or more knob mutations, and the second heavy chain contains one or more hole mutations. In some embodiments, the first heavy chain contains one or more hole mutations, and the second heavy chain contains 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.

[0408] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" 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: 210, and a second heavy chain sequence (indicated as "H2" 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: 211. In some embodiments, the first and / or second heavy chain includes an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first light chain sequence (indicated as "L1" 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: 212, and a second heavy chain sequence (indicated as "L2" 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: 213.

[0409] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies are referred to as "CD79b / 5C7(1+1 A)Fc silencing" and consist of 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: 210, and a second 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: 211. The sequence comprises a heavy chain sequence 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 sequence number 212, and 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 sequence number 213.

[0410] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" 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 sequence (indicated as "H2" 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: 228. In some embodiments, the first and / or second heavy chain includes an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97). In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first light chain sequence (indicated as "L1" 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: 229, and a second heavy chain sequence (indicated as "L2" 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: 230.

[0411] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1 A)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, and a second 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 sequence comprises a chain sequence 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 sequence number 229, and 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 sequence number 230.

[0412] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9B) 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, and a second heavy chain sequence (indicated as "H2" in Figure 9B) 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. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a light chain sequence (indicated as "L1" in Figure 9B) 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.

[0413] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is called "CD79b / 5C7(2+1 A)Fc silencing" and contains 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, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 9 It contains a second heavy chain sequence that is 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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 sequence number 219.

[0414] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9B) 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 sequence (indicated as "H2" in Figure 9B) 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 chains include an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody includes a light chain sequence (indicated as "L1" in Figure 9B) 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.

[0415] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1 A)Fc optimized" and contains 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, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, or 90% identical to SEQ ID NO: 235. It contains a second heavy chain sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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 sequence number 236.

[0416] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9C) 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. In some embodiments, the heavy chain contains an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain sequence (indicated as "L" in Figure 9C) 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.

[0417] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2 A)Fc silencing" and contains 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.

[0418] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9C) 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. In some embodiments, the heavy chain contains an IgG1 Fc region with an optimized mutation (SEQ ID NO: 97). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain (indicated 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.

[0419] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2 A)Fc optimized" and contains 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 contains 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.

[0420] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9D) 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, and a second heavy chain sequence (indicated as "H2" in Figure 9D) that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 215. In some embodiments, the first and / or second heavy chain comprises an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody comprises a light chain sequence (indicated as "L1" in Figure 9D) 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.

[0421] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is called "CD79b / 5C7(1+1 B)Fc silencing" and contains 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, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 9 It contains a second heavy chain sequence that is 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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.

[0422] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9D) 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 sequence (indicated as "H2" in Figure 9D) 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 chains include an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a light chain sequence (indicated as "L1" in Figure 9D) 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.

[0423] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(1+1 B)Fc optimized" and contains 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, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, or 90% identical to SEQ ID NO: 232. It contains a second heavy chain sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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 sequence number 233.

[0424] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9E) 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, and a second heavy chain sequence (indicated as "H2" in Figure 9E) 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. In some embodiments, the first and / or second heavy chains include an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a light chain sequence (indicated as "L1" in Figure 9E) 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.

[0425] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1 B)Fc silencing" and contains 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, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 9 It contains a second heavy chain sequence that is 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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 sequence number 222.

[0426] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a first heavy chain sequence (indicated as "H1" in Figure 9E) 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 sequence (indicated as "H2" in Figure 9E) 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 chains include an IgG1 Fc region having an optimized mutation (SEQ ID NO: 97). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody contains a light chain sequence (indicated as "L1" in Figure 9E) 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.

[0427] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+1 B)Fc optimized" and contains 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: 237, and approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, or 90% identical to SEQ ID NO: 238. It contains a second heavy chain sequence that is %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and contains 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 sequence number 239.

[0428] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9F) 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. In some embodiments, the heavy chain contains an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain sequence (indicated as "L" in Figure 9F) 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.

[0429] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2 B)Fc silencing" and contains 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 contains 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.

[0430] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9F) 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. In some embodiments, the heavy chain contains an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain sequence (indicated as "L" in Figure 9F) 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.

[0431] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 3A7(2+2 B)Fc silencing" and contains 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 contains 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.

[0432] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9F) 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. In some embodiments, the heavy chain contains an IgG1 Fc region having the LALAPG mutation (SEQ ID NO: 96). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain sequence (indicated as "L" in Figure 9F) 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.

[0433] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 13E6(2+2 B)Fc silencing" and contains 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 contains 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.

[0434] In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a heavy chain sequence (indicated as "H" in Figure 9F) 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. In some embodiments, the heavy chain contains an IgG1 Fc region with an optimized mutation (SEQ ID NO: 97). In some embodiments, multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibodies contain a light chain sequence (indicated as "L" in Figure 9F) 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.

[0435] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / CLEC5A antibody is referred to as "CD79b / 5C7(2+2 B)Fc optimized" and contains 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 contains 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.

[0436] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker may include a spacer array. Various spacer arrays are known in the art, including, but are not limited to, glycineserine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, and (GGGGS)n (SEQ ID NO: 99) (where n is an integer of at least 1). Those skilled in the art will be able to select a suitable spacer array.

[0437] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0438] This disclosure also provides nucleic acids comprising polynucleotides encoding an anti-CD79b / CLEC5A antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-CD79b / CLEC5A antibody comprises the CDRs shown in Table 22. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to CD79b and / or CLEC5A.

[0439] Antibody-drug conjugates (ADCs) The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated with therapeutic agents (drugs). The therapeutic agent can be covalently or noncovalently bound to the antibody or antigen-binding fragment, or to the antigen-binding protein construct (e.g., bispecific antibodies).

[0440] In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, meitansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as its analogues). Useful classes of cytotoxic, cell proliferation inhibitor, or immunomodulatory agents include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0441] In some embodiments, the therapeutic agent may be, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent, an immunotherapeutic agent, etc.), an antiviral agent, or an antibacterial agent. In some embodiments, the conjugateable therapeutic agent may be, but is not limited to, selected from MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0442] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of drastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed of auristatin E and a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO No. 02 / 088172, and U.S. Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5, These are listed in Nos. 521,284; Nos. 5,504,191; Nos. 5,410,024; Nos. 5,138,036; Nos. 5,076,973; Nos. 4,986,988; Nos. 4,978,744; Nos. 4,879,278; Nos. 4,816,444; and Nos. 4,486,414, each of which is incorporated herein by reference for all purposes.

[0443] Auristatin has been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and has been shown to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects in cancer cells. Numerous well-known assays exist in the art that can be used to measure whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects in desired cells.

[0444] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine; chlorambucil, chlornafadin, chlorophosphamide, estram Nitrogen mustards such as stine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimastine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; acrasinomycin, actinomycin, anthramycin, azaserine, bleomycin, kakutinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromo Antibiotics such as mycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; methotrexate and 5-fluorouracil (5- Antimetabolites such as FU; folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, furoxiuridine, and 5-FU; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone;Anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatrexate; defofamine; demecoltin; diazion; eflornithine; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; phena Met; Pirarubicin; Podophyllic acid; 2-Ethyl hydrazide; Procarbazine; PSK7; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2'-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxanes, e.g., Paclitaxel (TAXOL®, Bristol-Myers Examples include Squibb Oncology (Princeton, New Jersey) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition includes, for example, tamoxifen, raloxifen, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Furthermore, anti-hormonal agents that modulate or inhibit hormonal activity in tumors, such as anti-estrogen agents containing any of the pharmaceutically acceptable salts, acids, or derivatives mentioned above, are also included. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated in its entirety by reference.

[0445] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, e.g., an SPBD linker, or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via an incleavable linker, e.g., an MCC linker formed using SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be quickly made by a person skilled in the art who has knowledge of the art and takes into account relevant factors such as the binding site to the antigen-binding construct, any structural limitations of the drug, and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Numerous specific linker-toxin combinations have been described and can be used with the antigen-binding constructs described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide linkers with auristatin such as MMAE and MMAF, camptothecin such as SN-38, duocalmycin, and PBD dimer; incleavable MC linkers with auristatin MMAF and MMAE; acid-unstable hydrazone linkers with calicheamycin and doxorubicin; disulfide linkers with meitansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO) linkers with meitansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci.Rep.e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication No. 2015 / 0374847, and US20180193477A1, which are incorporated herein by reference in their entirety.

[0446] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method for coupling them to each other. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker complex, which still contains a reactive group for covalently conjugating the antibody. In some embodiments, a drug-maleimide complex (i.e., a maleimide-bound drug) can be used with the payload having the reactive group in this disclosure. Maleimide is the most common reactive group that can be bound to a thiol group in ADC preparation. Furthermore, organobromids and iodides are also frequently used.

[0447] ADCs can be prepared by one of several well-known routes in the art, using organic chemical reactions, conditions, and reagents familiar to those skilled in the art (see, for example, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) forming an antibody-linker intermediate Ab-L by covalent bonding, followed by a reaction with an activated drug site D, through the reaction of a nucleophilic or electrophilic group of the antibody with a divalent linker reagent; or (2) forming a drug-linker intermediate DL by covalent bonding, through the reaction of a nucleophilic or electrophilic group of the drug site with a linker reagent, followed by a reaction with a nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be used with various antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J.Org.Chem.67:1866-1872; Frisch et al., (1997) Bioconj.Chem.7:180-186; Bioconjugate Techniques (G.T. Humanson, 2013, Academic Press); US20210379193A1, and US20180193477A1, which are incorporated herein by reference in their entirety. Furthermore, a number of preformed drug-linkers suitable for reaction with selected antigen-binding constructs are also commercially available. For example, linker-toxins containing DM1, DM4, MMAE, MMAF, or duocalmycin SA are available from Creative BioLabs (Shirley, NY).

[0448] Several specific examples of methods for preparing ADCs are well known in the art and are described in U.S. Patent No. 8,624,003 (Pot Method), U.S. Patent No. 8,163,888 (One-Step Method), U.S. Patent No. 5,208,020 (Two-Step Method), and U.S. 20180193477A1, which are incorporated herein by reference in their entirety. Other methods are well known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0449] The drug load is expressed by the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, the drug load can be limited by the number of binding sites on the antibody. For example, when the binding is cysteinethiol, as in certain exemplary embodiments described herein, the drug load can range from 0 to 8 drug moieties per antibody. In certain embodiments, a high drug load, e.g., p ≥ 5, may cause aggregation, insolubility, toxicity, or cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug load for an antibody-drug conjugate is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In fact, it has been shown that for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody can be about 4. In some embodiments, the drug-to-antibody ratio (DAR) is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0450] Antibodies and their antigen-binding fragments In some embodiments, multispecific antibodies (e.g., bispecific antibodies) and their antigen-binding fragments can have various forms.

[0451] Generally, antibodies (also called immunoglobulins) can consist of two classes of polypeptide chains: a light chain and a heavy chain. The non-limiting anti-HER2 / CLEC5A antibodies of this disclosure may be intact four-unit immunoglobulin chain antibodies containing two heavy chains and two light chains. The heavy chains of the anti-HER2 / CLEC5A antibody may be any isotype including IgM, IgG, IgE, IgA, or IgD, or subisotypes including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains may be kappa light chains or lambda light chains.

[0452] The hypervariable region, known as the complementarity-determining region (CDR), forms a loop containing the antibody's principle antigen-binding surface. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form linked loops, sometimes even forming part of the β-sheet structure. The CDR of each chain is held in close proximity to the framework region and, together with the CDRs of other chains, contributes to the formation of the antigen-binding domain.

[0453] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422~439; Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et This is described in al., Nature 342(6252):877-83(Dec.1989); and Ponomarenko and Bourne, BMC Structural Biology 7:64(2007), and the entirety of each of these is incorporated herein by reference.

[0454] CDRs are important for recognizing the epitopes of antigens. As used herein, “epitope” refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0455] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, and are described, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5(2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference.

[0456] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, or camelids). The antigen-binding domain or antigen-binding fragment is any portion of the antibody that retains the specific binding activity of the intact antibody, i.e., any portion of the antibody that is specifically capable of binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide comprising a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0457] In some embodiments, the scFv of a multispecific (e.g., bispecific) antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFv has two antigen-binding regions (antigen-binding regions: A and B), the two antigen-binding regions capable of binding to their respective target antigens having different affinities.

[0458] In some embodiments, a multispecific (e.g., bispecific) antibody or its antigen-binding fragment may comprise one, two, or three heavy chain variable region CDRs selected from Table 22.

[0459] In some embodiments, the multispecific (e.g., bispecific) antibodies described herein may be conjugated to a therapeutic agent. The antibody-drug conjugate, comprising an antibody or its antigen-binding fragment, may be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mytoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as analogues). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, for example, a VC linker. Details of the linkers used in ADCs are described, for example, in Su, Z. et al. “Antibody-drug conjugates: Recent advances in linker chemistry.” Acta Pharmaceutica Sinica B (2021), which is incorporated in its entirety by reference.

[0460] Multiple specific (or bispecific) antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture media. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0461] Any of the multispecific (bispecific) antibodies or their antigen-binding fragments described herein can be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-HER2 / CLEC5A antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0462] Bispecific antibodies or their antigen-binding fragments can also exist in various forms. Many different formats of bispecific antibodies or their antigen-binding fragments are known in the art, for example, as described in Suurs, et al., “A review of bispecific antibodies and antibody constructs in oncology and clinical challenges,” Pharmacology & Therapeutics (2019), which is incorporated herein by reference in its entirety.

[0463] In some embodiments, the bispecific antibody is BiTE, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the bispecific antibodies are VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, Diabody, crossMab, DAF(2in1), DAF(4in1), DutaMab, DT-IgG, Knob-in-hole common light chain, Knob-in-hole assembly, Charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH These include IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Mini-antibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0464] In some embodiments, bispecific antibodies can be TrioMab. In TrioMab, the two heavy chains originate from different species, and their different sequences limit the formation of heavy-light chain pairs.

[0465] In some embodiments, the bispecific antibody has two different heavy chains and one common light chain. In some embodiments, the bispecific antibody has two different heavy chains and two different light chains. Heterodimerization of the heavy chains can be based on knob-into-hole or some other heavy chain pairing techniques.

[0466] In several embodiments, bispecific antibodies can be produced using CrossMAb technology. CrossMAb technology can enhance correct light chain association in bispecific heterodimeric IgG antibodies, enabling the production of various bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) based antibodies. These formats can be derived from any existing antibody pair using domain crossover without requiring recognition of a common light chain, post-translational processing / extracorporeal chemical assembly, or the introduction of a series of mutations to enhance correct light chain association. This method is described in Klein et al., “The use of CrossMAb technology for the generation of bi- and multispecific antibodies.” mAbs. Vol.8. No.6. Taylor & Francis, 2016, and is incorporated in its entirety by reference. In some embodiments, the CH1 in the heavy chain is exchanged with the CL domain in the light chain.

[0467] Bispecific antibodies may also be duobodies. The Fab exchange mechanism naturally present in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between heavy and light chains.

[0468] In the bivariable domain antibody (DVD-Ig), additional VH and variable light chain (VL) domains are added to each N-terminus for bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind individually to the corresponding N-terminus of each heavy chain, instead of the scFv.

[0469] In scFv-IgG, two scFv molecules are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two distinct divalent binding sites and is therefore also called tetravalent. There is no problem with heavy-light chain pairing in scFv-IgG.

[0470] In some embodiments, bispecific antibodies may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically bonding the C-terminuses of their heavy chains.

[0471] Bispecific antibodies may also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, a light chain, a heavy chain, and a third chain containing the Fc region and scFv are assembled. This ensures efficient preparation and purification.

[0472] In some embodiments, the bispecific antibody may be a TF. Three Fab fragments are linked by disulfide crosslinks. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format does not have an Fc region.

[0473] ADAPTIR has two scFvs bound to both sides of a certain Fc region. It discards the intact IgG that serves as the basis for its construct, but preserves the Fc region, extending its half-life and facilitating purification.

[0474] Biaffinity retargeting (DART) involves two peptide chains linking opposite fragments (i.e., VLA to VHB and VLB to VHA), and a sulfur bond fusing them together at the C-terminus. In DART, the sulfur bond can improve stability compared to BiTE.

[0475] In DART-Fc, the Fc region is bound to DART. This can be generated by assembling three chains (two via disulfide bonds, similar to DART). One chain contains half of the Fc region, which dimerizes with the third chain, expressing only the Fc region. The addition of the Fc region extends the half-life, resulting in a longer effective concentration and avoiding consecutive IV injections.

[0476] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are created by half of the Fc region and then dimerized. This format has divalent bonds to both targets, and therefore, it is a tetravalent molecule.

[0477] A tandem diabody (TandAb) contains two diabodies. Each diabody consists of a VHA and a VLB fragment, and another VHA and a VLB fragment, associated by covalent bonds. The two diabodies are linked by a peptide chain. This can improve stability compared to a diabody composed of two scFvs. It has two divalent bonding sites.

[0478] The scFv-scFv-toxin comprises a toxin and two scFvs containing a stabilizing linker. This can be used for the specific delivery of the payload.

[0479] In some embodiments, the bispecific antibodies in this disclosure are designed to be 1+1 (monovalent for each target) and have an IgG1 subtype structure. This allows for reduced avidity to cells with low expression levels of the first and second epitopes and increased avidity to cells co-expressing the first and second epitopes, thereby achieving improved targeting function.

[0480] In some embodiments, the bispecific antibody contains a KIH mutation. In some embodiments, the bispecific antibody contains a first arm containing a first antigen-binding domain that specifically binds to a first epitope, and a second arm containing a second antigen-binding domain that specifically binds to a second epitope. In some embodiments, the first antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations, and the second antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations. In some embodiments, the first ...

Claims

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

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 3, 5, and 7, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 8 to 10, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 13, 15, and 17, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 18 to 20, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 23, 25, and 27, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 28 to 30, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 33, 35, and 37, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 38 to 40, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 43, 45, and 47, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 48 to 50, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 53, 55, and 57, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 58 to 60, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 63, 65, and 67, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 68 to 70, respectively.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Kabat's definition, the VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 73, 75, and 77, respectively, and the VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 78 to 80, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 6, and 7, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 8 to 10, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 14, 16, and 17, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 18 to 20, respectively.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 24, 26, and 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 28 to 30, respectively.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 34, 36, and 37, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 38 to 40, respectively.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 44, 46, and 47, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 48 to 50, respectively.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 54, 56, and 57, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 58 to 60, respectively.

16. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 64, 66, and 67, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 68 to 70, respectively.

17. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 74, 76, and 77, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 78 to 80, respectively.

18. An antibody that binds to CLEC5A or an antigen-binding fragment thereof, The selected heavy chain variable region (VH) sequence comprises a VH having an amino acid sequence that is at least 90% identical to the selected heavy chain variable region (VL) sequence, and a VL having an amino acid sequence that is at least 90% identical to the selected light chain variable region (VL) sequence, wherein the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is sequence number 11, and the selected VL sequence is sequence number 12; (2) The selected VH sequence is sequence number 21, and the selected VL sequence is sequence number 22; (3) The selected VH sequence is sequence number 31, and the selected VL sequence is sequence number 32; (4) The selected VH sequence is sequence number 41, and the selected VL sequence is sequence number 42; (5) The selected VH sequence is sequence number 51, and the selected VL sequence is sequence number 52; (6) The selected VH sequence is sequence number 61, and the selected VL sequence is sequence number 62; (7) The selected VH sequence is sequence number 71, and the selected VL sequence is sequence number 72; (8) The selected VH sequence is sequence number 81, and the selected VL sequence is sequence number 82; (9) The selected VH sequence is sequence number 83, and the selected VL sequence is sequence number 84; (10) The selected VH sequence is sequence number 85, and the selected VL sequence is sequence number 86; (11) The selected VH sequence is sequence number 87, and the selected VL sequence is sequence number 88; (12) The selected VH sequence is sequence number 89 and the selected VL sequence is sequence number 90; and (13) The selected VH sequence is sequence number 91, and the selected VL sequence is sequence number 92, One of them is An antibody or its antigen-binding fragment.

19. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 11 and VL comprises the sequence of SEQ ID NO:

12.

20. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 21 and VL comprises the sequence of SEQ ID NO:

22.

21. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 31 and VL comprises the sequence of SEQ ID NO:

32.

22. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 41 and VL comprises the sequence of SEQ ID NO:

42.

23. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 51 and VL comprises the sequence of SEQ ID NO:

52.

24. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 61 and VL comprises the sequence of SEQ ID NO:

62.

25. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 71 and VL comprises the sequence of SEQ ID NO:

72.

26. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 81 and VL comprises the sequence of SEQ ID NO:

82.

27. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 83 and VL comprises the sequence of SEQ ID NO:

84.

28. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 85 and VL comprises the sequence of SEQ ID NO:

86.

29. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 87 and VL comprises the sequence of SEQ ID NO:

88.

30. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 89 and VL comprises the sequence of SEQ ID NO:

90.

31. The antibody or antigen-binding fragment thereof according to claim 18, wherein VH comprises the sequence of SEQ ID NO: 91 and VL comprises the sequence of SEQ ID NO:

92.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the antibody or antigen-binding fragment thereof specifically binds to CLEC5A of a human, mouse, or monkey.

33. The antibody or its antigen-binding fragment can mediate macrophage phagocytosis (for example, the target cell killing effect is at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to a reference antibody (e.g., DX244)), and / or, the antibody or its antigen-binding fragment can induce low levels of cytokine (e.g., IL-6 or TNFα) release (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% compared to that induced by a reference antibody (e.g., DX244)), the antibody or its antigen-binding fragment according to any one of claims 1 to 32.

34. An antibody or antigen-binding fragment thereof that binds to CLEC5A, wherein the antibody or antigen-binding fragment can mediate macrophage phagocytosis (for example, the target cell killing effect is at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to a reference antibody (e.g., DX244)), and / or, wherein the antibody or antigen-binding fragment thereof can induce low levels of cytokine (e.g., IL-6 or TNFα) release (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% compared to that induced by a reference antibody (e.g., DX244)).

35. An antibody or its antigen-binding fragment, i) A first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is a tumor-associated antigen (TAA), ii) comprising a second antigen-binding domain that specifically binds to member A of the C-type lectin domain family 5 (CLEC5A), or i) A first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is a target of an autoimmune disease, ii) comprising a second antigen-binding domain that specifically binds to CLEC5A, An antibody or its antigen-binding fragment.

36. The aforementioned antibody or its antigen-binding fragment has the following effects: i) The antibody or its antigen-binding fragment can mediate target cell killing by bone marrow cells (e.g., monocytes and / or macrophages (e.g., M0, M1, and / or M2 macrophages)); ii) The antibody or its antigen-binding fragment can mediate target cell killing by myeloid cells (e.g., monocytes and / or macrophages) at a low E:T (effector cell:target cell) ratio (optionally, where the low E:T ratio is less than 1:10, less than 1:9, less than 1:8, less than 1:7, less than 1:6, less than 1:5, less than 1:4, less than 1:3, less than 1:2, less than 1:1, less than 2:1, less than 3:1, less than 4:1, less than 5:1, less than 6:1, less than 7:1, less than 8:1, less than 9:1, or less than 10:1); and iii) The antibody or its antigen-binding fragment can induce low levels of cytokine release (e.g., IL-6 or TNFα) in bone marrow cells (e.g., monocytes and / or macrophages) (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 that induced by a reference antibody). Having one or more of the following: The antibody or antigen-binding fragment thereof according to claim 35.

37. The antibody or antigen-binding fragment thereof according to claim 35 or 36, 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).

38. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 37, wherein the second antigen-binding domain is a single-stranded variable fragment (scFv) domain, where VH2 and VL2 are linked by a first linker.

39. The antibody or antigen-binding fragment thereof according to claim 38, wherein the second antigen-binding domain is bound to the C-terminus of the light chain via a second linker.

40. An antibody or antigen-binding fragment according to any one of claims 35 to 39, further comprising an Fc region.

41. The antibody or antigen-binding fragment according to claim 40, wherein the C-terminus of VH1 of the first antigen-binding domain is optionally bound to the Fc region via the CH1 domain.

42. The antibody or antigen-binding fragment according to claim 40, wherein the C-terminus of VH1 of the first antigen-binding domain is bound to the N-terminus of the Fc region, and the N-terminus of the second antigen-binding domain is bound to the C-terminus of the Fc region.

43. The antibody according to claim 37 or the antigen-binding fragment thereof, wherein the antibody comprises a first heavy chain containing VH1 and a first light chain containing VL1, and a second heavy chain containing VH2 and a second light chain containing VL2.

44. The first heavy chain contains one or more knob mutations, and The second heavy chain contains one or more hole mutations. The antibody or antigen-binding fragment thereof according to claim 43.

45. The first heavy chain contains one or more hole mutations, and The second heavy chain contains one or more knob mutations. The antibody or antigen-binding fragment thereof according to claim 43.

46. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 45, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

47. The antibody or antigen-binding fragment thereof according to claim 46, wherein the Fc region is the Fc region of human IgG1.

48. The Fc region consists of one or more of the following amino acid residues (all numbering follows EU numbering): (1) Alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332; (2) Alanine at position 236 (A), aspartic acid at position 293 (D), leucine at position 330 (L), and glutamic acid at position 332 (E); (3) Alanine (A) ranked 236th; (4) Alanine at position 236 (A), aspartic acid at position 293 (D), and glutamic acid at position 332 (E); (5) Aspartic acid (D) at position 293 and glutamic acid (E) at position 332; (6) Aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332; and (7) Alanine (A) at 234th place, Alanine (A) at 235th place, and Glycine (G) at 329th place, Includes, The Fc region is optionally defucosylated. The antibody or antigen-binding fragment thereof according to claim 46 or 47.

49. The tumor-associated antigens (TAAs) mentioned above are HER2, CD79b, EGFR, EpCAM, BCMA, CD38, GPRC5D, DLL3, CD70, GPC3, Fas ligand (FasL), CD1d, membrane glycolipids, globotriaosylceramide (Gb3Cer / CD77), gangliosides (GD2, GD3, and GM2), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CLL1, CD105, CD71, SSC, MAGE, MUC16, and CD1 9, WT-l, B7H3, TEM8, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-fetoprotein (AFP), BAFF, B lymphoma cells, CA242 antigen, CA-125, carbonic anhydration Enzyme 9 (CA-IX), C-MET, CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, Fibronectin Extra Domain-B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HGF, Human Scattering Factor Receptor Kinase, IGF-I Receptor, IGF-I, IgGl, IL-5, IL-13, IL-6, IL-15, Insulin-like Growth Factor I Receptor, Integrin a5b1, Integrin avb3, MSLN, MS4A1, MUC1, Mucin CanAg, N-Glycolylneuraminic 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-beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF-I, VEGF-2, or vimentin. The autoimmune disease targets include CD79b, CD38, ACHE, BAFF, BTK, CCL2, CD19, CD20, CD25, CD40, CD 52, 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, I L-23, integrin, ITG-A4B1, ITG-A4B7, ITG-AVB6, TL1A, TNF-α, TNF-β, TNFSF13B, TSLP, TYK2, or VEGFR, An antibody or antigen-binding fragment thereof according to any one of claims 35 to 48.

50. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA is HER2.

51. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 100, 102, and 104, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 105 to 107, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 101, 103, and 104 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 105 to 107 respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 50.

52. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA or the autoimmune disease target is CD79b.

53. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 200, 202, and 204, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 205 to 207, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 201, 203, and 204 respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 205 to 207 respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 52.

54. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA is EGFR.

55. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 126, 128, and 130, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 131 to 133, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 127, 129, and 130, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 131 to 133, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; (3) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 136, 138, and 140, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 141 to 143, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (4) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 137, 139, and 140, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 141 to 143, respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 54.

56. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA is EpCAM.

57. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 150, 152, and 154, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 155 to 157, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 151, 153, and 154, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 155 to 157, respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 56.

58. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA or the autoimmune disease target is GPRC5D.

59. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 164, 166, and 168, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 169 to 171, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 165, 167, and 168, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 169 to 171, respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 58.

60. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA or the autoimmune disease target is BCMA.

61. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 176, 178, and 180, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 181 to 183, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 177, 179, and 180, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 181 to 183, respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 60.

62. The antibody or antigen-binding fragment thereof according to any one of claims 35 to 49, wherein the TAA or the autoimmune disease target is CD38.

63. (1) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 188, 190, and 192, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 193 to 195, respectively, and The selected VH2 CDR1,2,3 amino acid sequence and the selected VL2 CDR1,2,3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17; or (2) The selected VH1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 189, 191, and 192, respectively, and the selected VL1 CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 193 to 195, respectively, and The selected VH2 CDR1, 2, 3 amino acid sequence and the selected VL2 CDR1, 2, 3 amino acid sequence are selected from one of the sequences described in any one of claims 1 to 17. The antibody or antigen-binding fragment thereof according to claim 62.

64. An antibody or an antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described in any one of claims 1 to 63.

65. A nucleic acid comprising a polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 64.

66. A vector comprising the nucleic acid described in claim 65.

67. A cell comprising the vector according to claim 66.

68. A method for reducing the rate of tumor growth, The process involves contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 64. method.

69. A method for killing tumor cells, The process involves contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 64. method.

70. A method for increasing the immune response in a target, The method involves administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 64. method.

71. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition containing an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 64.

72. The method according to claim 71, wherein the cancer is a solid tumor or a hematological tumor.

73. The method according to claim 71 or 72, wherein the cancer is breast cancer, lung cancer, stomach 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.

74. The method according to claim 71 or 72, wherein the cancer is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prelymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, follicular lymphoma, and / or mantle cell lymphoma.

75. The method according to any one of claims 70 to 74, wherein the subject is further treated with an effective amount of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, and / or anti-PD-L1 antibody.

76. A method for treating a subject having an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a composition containing an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 64.

77. The method according to claim 76, 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 arteritis, 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).

78. The method according to claim 77, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, and / or rheumatoid arthritis.

79. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 64, and a pharmaceutically acceptable carrier.

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