Multispecific antibodies targeting epcam and CLEC5a and the uses thereof

Abstract

This disclosure relates to an antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to EpCAM and a second antigen-binding domain that specifically binds to CLEC5A.

Description

[0001] Atorney Docket No.: 55679-0015WO3

[0002] MULTISPECIFIC ANTIBODIES TARGETING EPCAM AND CLEC5A AND THE USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is an International Application which claims priority to International Application No. PCT / CN2025 / 071655, filed January 10, 2025, and International Application No. PCT / CN2025 / 136539, filed November 20, 2025. The disclosure of the foregoing applications are hereby incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “55679-0015WO3.XML.” The XML file, created on January 9, 2026, is 116,799 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This disclosure relates to antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, and antibody drug conjugates (ADCs) derived therefrom.

[0008] BACKGROUND

[0009] A bispecific antibody is an artificial protein 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 of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become a major milestone in the development of bispecific antibodies. As bispecific antibodies have various applications, there is a need to continue developing various therapeutics based on bispecific antibodies.

[0010] SUMMARY

[0011] In one aspect, this disclosure relates to antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to epithelial cell adhesion molecule (EpCAM) and / or human C-type lectin domain family 5 member A (CLEC5A). In some embodiments, the antibodies or antigen-binding fragments thereof have an enhanced Fc. In some embodiments, theAtorney Docket No.: 55679-0015WO3

[0012] antibodies or antigen-binding fragments thereof have an increased binding affinity to the FcyRIIa receptor and / or the FcyRIIIa receptor. In some embodiments, the antibodies or antigen-binding fragments thereof have a non-functional Fc.

[0013] In one aspect, this disclosure relates to antibodies or antigen-binding fragments thereof, comprising: i) a first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is EpCAM; and ii) a second antigen-binding domain that specifically binds to CLEC5A.

[0014] In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is epithelial cell adhesion molecule (EpCAM); and a second antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A).

[0015] 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).

[0016] In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain, wherein the VH2 and VL2 are linked by a first linker.

[0017] In some embodiments, the second antigen-binding domain is connected to the C-terminus of a light chain through a second linker.

[0018] In some embodiments, the second antigen-binding domain is connected to the C-terminus of a heavy chain through a second linker.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc region.

[0021] In some embodiments, the C-terminus of the VH1 of the first antigen-binding domain is connected to the Fc region, optionally through a CHI domain.

[0022] In some embodiments, the C-terminus of the VH1 of the first antigen-binding domain is connected to the N-terminus of a Fc region, and the N-terminus of the second antigen-binding domain is connected to the C-terminus of the Fc region.Atorney Docket No.: 55679-0015WO3

[0023] In some embodiments, the antibody comprises a first heavy chain comprising the VH1 and a first light chain comprising the VL1, and a second heavy chain comprising the second antigen-binding domain.

[0024] In some embodiments, the C-terminus of the second antigen-binding domain is connected to the Fc region, optionally through a CHI domain.

[0025] In some embodiments, the N-terminus of the second antigen-binding domain is connected to the Fc region, optionally through a second linker.

[0026] In some embodiments, the first heavy chain comprises one or more knob mutations; and the second heavy chain comprises one or more hole mutations.

[0027] In some embodiments, the first heavy chain comprises one or more hole mutations; and the second heavy chain comprises one or more knob mutations.

[0028] In some embodiments, the Fc region is an Fc region of human IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is an Fc region of human IgGl.

[0029] In some embodiments, the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering): an Alanine (A) at position 236; a Leucine (L) at position 330; and a Glutamic acid (E) at position 332.

[0030] In some embodiments, the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering): an Alanine (A) at position 234; an Alanine (A) at position 235; and a Glycine (G) at position 329.

[0031] In some embodiments, the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering): an Aspartic acid (D) at position 239; and an glutamic acid (E) at position 332.

[0032] In some embodiments, the Fc region is afucosylated.

[0033] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; andAtorney Docket No.: 55679-0015WO3

[0034] the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence,

[0035] wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0036] (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;

[0037] (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;

[0038] (3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0039] (4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0040] (5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; and

[0041] (6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively.

[0042] In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2Atorney Docket No.: 55679-0015WO3

[0043] CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0044] the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0045] (1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0046] (2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0047] (3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; and

[0048] (4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively.

[0049] In some embodiments, (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0050] (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ IDAtorney Docket No.: 55679-0015WO3

[0051] NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0052] (3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0053] (4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0054] (5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0055] (6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0056] (7) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;

[0057] (8) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ IDAtorney Docket No.: 55679-0015WO3

[0058] NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;

[0059] (9) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;

[0060] (10) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;

[0061] (11) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; or

[0062] (12) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively.

[0063] In some embodiments, the Fc region has increased complement-dependent cytotoxicity (CDC), increased antibody-dependent cellular cytotoxicity (ADCC) or increased half-life.

[0064] In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.

[0065] In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein to the subject.

[0066] In some embodiments, the cancer is a solid tumor or a blood tumor.Atorney Docket No.: 55679-0015WO3

[0067] In some embodiments, 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, cancer of the small intestine, pancreatic cancer and / or liver cancer.

[0068] In some embodiments, the subject is further treated with an effective amount of an anti -4- IBB antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, or an anti-PD-Ll antibody.

[0069] In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein.

[0070] In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein.

[0071] In one aspect, the disclosure is related to a method of increasing immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein.

[0072] In one aspect, the disclosure is related to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier.

[0073] As used herein, the term “antigen-binding domain” refers to one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s) (e.g., an effector antigen or tumor antigen). In some examples, an 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. One example of an antigen-binding domain is an antigen-binding domain formed by VH-VL. In some embodiments, an antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-bindingAtorney Docket No.: 55679-0015WO3

[0074] domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a tumor antigen).

[0075] As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, singlechain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

[0076] As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.

[0077] As used herein, the term “multispecific antibody” is an antibody that includes two or more different antigen-binding domains that collectively specifically bind 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 on different antigens (e.g., different proteins present on the surface of the same cell or present on the surface of different cells). In some aspects, a multispecific antibody binds two different epitopes (i.e., a “bispecific antibody”). In some aspects, a multispecific antibody binds three different epitopes (i.e., a “trispecific antibody”). In some aspects, a multispecific antibody binds four different epitopes (i.e., a “quadspecific antibody”). In some aspects, a multispecific antibody binds five different epitopes (i.e., a “quintspecific antibody”). Each binding specificity may be present in any suitable valency. Non-limiting examples of multispecific antibodies are described herein.

[0078] As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative onlyAtorney Docket No.: 55679-0015WO3

[0080] and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0081] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0082] DESCRIPTION OF THE DRAWINGS FIGs. 1A-1D: schematic illustrations of structures of EpCAM / CLEC5A bispecific antibodies.

[0083] FIG 2: Quantification of tumor cell surface EpCAM antigen densities.

[0084] FIGs.3A-3B: ELISA Binding of EpCAM / CLEC5 A bispecific antibodies to Human CLEC5 A.

[0085] FIGs. 4A-4B: ELISA Binding of EpCAM / CLEC5Abispecific antibodies to Human EpCAM.

[0086] FIGs. 5A-5E: Binding of EpCAM / CLEC5Abispecific antibodies to U937 Cells.

[0087] FIG. 6: Binding of EpCAM / CLEC5 A bispecific antibodies to Human Monocytes.

[0088] FIG. 7: Binding of EpCAM / CLEC5Abispecific antibodies to Human MO macrophages. FIG. 8: Binding of EpCAM / CLEC5 A bispecific antibodies to Human M2 macrophages. FIGs. 9A-9E: Binding of EpCAM / CLEC5Abispecific antibodies to DLD-1.

[0089] FIG. 10: Binding of EpCAM / CLEC5Abispecific antibodies to MCF7.

[0090] FIG. 11: Binding of EpCAM / CLEC5Abispecific antibodies to HCT116.

[0091] FIG. 12: Binding of EpCAM / CLEC5Abispecific antibodies to SKBR3

[0092] FIG. 13: Binding of EpCAM / CLEC5Abispecific antibodies to T47D.

[0093] FIGs. 14A-14B: Binding of EpCAM / CLEC5Abispecific antibodies to SKOV3.

[0094] FIG. 15: Binding of EpCAM / CLEC5Abispecific antibodies to A549.

[0095] FIGs. 16A-16C: EpCAM / CLEC5A bispecific antibodies mediated DLD-1 cell killing by PBMC.

[0096] FIGs. 17A-17H: EpCAM / CLEC5Abispecific antibodies mediated DLD-1 cell killing by MO Macrophages.

[0097] FIG. 18: EpCAM / CLEC5A bispecific antibodies mediated DLD-1 cell killing by Ml Macrophages.

[0098] FIG. 19: EpCAM / CLEC5A bispecific antibodies mediated DLD-1 cell killing by M2 Macrophages.

[0099] FIGs. 20A-20G: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by MO Macrophages.

[0100] FIGs. 21A-21B: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by M2 Macrophages.Atorney Docket No.: 55679-0015WO3

[0101] FIGs. 22A-22B: EpCAM / CLEC5A bispecific antibodies mediated MCF7 cell killing by MO Macrophages.

[0102] FIGs.23A-23B: EpCAM / CLEC5 A bispecific antibodies mediated HCT116 cell killing by MO Macrophages.

[0103] FIGs. 24A-24C: EpCAM / CLEC5A bispecific antibodies mediated T47D cell killing by MO Macrophages.

[0104] FIG. 25: EpCAM / CLEC5A bispecific antibodies mediated LNCaP cell killing by MO Macrophages.

[0105] FIG. 26: shows the mean tumor volume curve in anti -turn or Study 1 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the mean tumor volume of each treatment group.

[0106] FIGs. 27A-27E: show the individual tumor volume curve in anti -turn or Study 1 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the tumor volume of an individual mouse.

[0107] FIG. 28: shows the mean tumor volume curve in rechallenge testing using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the mean tumor volume of each treatment group.

[0108] FIG. 29: shows the mean tumor volume curve in anti -turn or Study 2 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the mean tumor volume of each treatment group.

[0109] FIGs. 30A-30D: show the individual tumor volume curve in anti -turn or Study 2 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the tumor volume of an individual mouse.

[0110] FIG. 31: shows the mean tumor volume curve in anti -tumor Study 3 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the mean tumor volume of each treatment group.

[0111] FIGs. 32A-32D: show the individual tumor volume curve in anti -turn or Study 3 using B-hEpCAM MC38 colon carcinoma implantation model. Each curve represents the tumor volume of an individual mouse.

[0112] FIG. 33: shows the mean body weight of mice in in anti-tumor Study 3.

[0113] DETAILED DESCRIPTION

[0114] This disclosure relates to antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom. InAtorney Docket No.: 55679-0015WO3

[0115] some embodiments, the antibodies or antigen-binding fragments thereof comprise a first antigenbinding domain that specifically binds to EpCAM and a second antigen-binding domain that specifically binds to CLEC5A.

[0116] Anti-EpCAM Antibodies and Antigen-binding Fragments Thereof

[0117] EpCAM (epithelial cell adhesion molecule), also known as CD326, is a conserved type I transmembrane glycoprotein of 35 kDa expressed on epithelial cells of higher eukaryotes. It was initially discovered as a carcinoma cell surface antigen and has since then gained immense importance as a carcinoma marker with applications in diagnosis, prognosis, and therapeutic intervention. Soon after EpCAM’ s discovery it was proposed that it functions as a homophilic cell-cell adhesion molecule, hence its name — epithelial cell adhesion molecule. Owing to its frequent and high expression on carcinomas and their metastases, EpCAM serves as a prognostic marker, a therapeutic target, and an anchor molecule on circulating and disseminated tumor cells (CTCs / DTCs), which are considered the major source for metastatic cancer cells. Today, EpCAM is reckoned as a multifunctional transmembrane protein involved in the regulation of cell adhesion, proliferation, migration, sternness, and epithelial-to-mesenchymal transition (EMT) of carcinoma cells. To fulfill these functions, EpCAM is instrumental in intra- and intercellular signaling as a full-length molecule and following regulated intramembrane proteolysis, generating functionally active extra- and intracellular fragments. EpCAM represents a marker for the epithelial status of primary and systemic tumor cells and emerges as a measure for the metastatic capacity of CTCs. Consequentially, EpCAM has reclaimed potential as a prognostic marker and target on primary and systemic tumor cells.

[0118] Detailed reviews of EpCAM can be found, for example, at Keller, Laura, Stefan Werner, and Klaus Pantel. "Biology and clinical relevance of EpCAM." Cell stress 3.6 (2019): 165; Eyvazi, Shirin, et al. "Antibody based EpCAM targeted therapy of cancer, review and update." Current Cancer Drug Targets 18.9 (2018): 857-868; Gires, Olivier, et al. "Expression and function of epithelial cell adhesion molecule EpCAM: where are we after 40 years?" Cancer and Metastasis Reviews 39.3 (2020): 969-987; Gaber, Aljaz, Brigita Lenarcic, and Miha Pavsic. "Current view on EpCAM structural biology." Cells 9.6 (2020): 1361, the entire content of each of which is incorporated herein by reference.

[0119] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can specifically bind to EpCAM (e.g., human EpCAM).

[0120] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and a light chain variable region (VL).Atorney Docket No.: 55679-0015WO3

[0121] In some embodiments, the heavy chain variable region (VH) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and

[0122] the light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence,

[0123] wherein the selected VH CDRs 1, 2, and 3 amino acid sequences, and the selected VL CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0124] (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;

[0125] (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;

[0126] (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0127] (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0128] (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; and

[0129] (6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively.

[0130] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL comprises an amino acidAtorney Docket No.: 55679-0015WO3

[0131] sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0132] (1) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10;

[0133] (2) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; and

[0134] (3) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30.

[0135] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0136] (1) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10;

[0137] (2) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; and

[0138] (3) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30.

[0139] In some embodiments, the antibodies comprise an antigen-binding domain of the anti-EpCAM antibody EpCAMl, EpCAM2 or EpCAM3, the chimeric antibodies thereof, or the humanized antibodies thereof.

[0140] The CDR sequences for EpCAMl, and EpCAMl derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 3, and 5, and CDRs of the light chain variable domain, SEQ ID NOs: 6, 7, and 8, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 2, 4, and 5, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 6, 7, and 8.

[0141] The CDR sequences for EpCAM2, and EpCAM2 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 11, 13, and 15, and CDRs of the light chain variable domain, SEQ ID NOs: 16, 17, and 18, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 12, 14, and 15, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 16, 17, and 18.

[0142] The CDR sequences for EpCAM3, and EpCAM3 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 21, 23, and 25, and CDRs of the light chain variableAtorney Docket No.: 55679-0015WO3

[0143] domain, SEQ ID NOs: 26, 27, and 28, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 22, 24, and 25, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 26, 27, and 28.

[0144] Anti-CLEC5A Antibodies and Antigen-binding Fragments Thereof

[0145] Human C-type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-associating lectin-1 (MDL-1), is a Il-type transmembrane protein and only expresses on the myeloid lineages including macrophages, monocytes, neutrophiles, and dendritic cells (DCs). As a pattern recognition receptor, CLEC5A transmits signals into cytoplasm through non-covalent binding to the adaptor protein DAP12. Phosphorylation of DAP12 then initiates a Syk kinase-based signaling cascade resulting in macrophages activation and release of chemokines and proinflammation cytokines, including IL-6, TNF, CCL3, and CXCL8. CLEC5A can trigger myeloid cell-related immune response and correlates with diverse infection and inflammatory diseases. CLEC5A promotes the production of high levels of pro-inflammatory cytokines and chemokines in flavivirus infections, especially dengue and Japanese encephalitis virus infections, and anti-CLEC5A mAb or CLEC5A inhibitors can reverse disease progression, suggesting that CLEC5A is a promising therapeutic target for flavivirus infections. In addition, similar in several autoimmune diseases, a high level of CLEC5A is found in active rheumatoid arthritis and CLEC5A activator increases proinflammation cytokines level. CLEC5A is also a pivotal contributor to cancer development and progression. Aberrant high CLEC5A expression significantly correlates with decreased overall survival in high-grade serious ovarian cancer (HGSOC), gastric cancer, and glioma.

[0146] The human CLEC5A mRNA encodes a 165-residue polypeptide with an N-terminal signal peptide (a.a. 1-22), followed by a short intracellular cytoplasmic domain (a.a. 23-56), a transmembrane domain (a.a. 57-70) and an extracellular domain (a.a. 71-165). The transmembrane domain contains a positively charged amino acid, Lys-58, which recruits DAP 10 and DAP 12 to associate with CLEC5A after activation. CLEC5A is mainly expressed by myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells, and is further upregulated by interferongamma (IFN-y). In addition, CLEC5 A expression is under the control of the PU.1 transcription factor, which is a central regulator of myeloid cell differentiation. CLEC5A expression is upregulated by the nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting CLEC5 A is regulated by oxidative stress. An X-ray crystal structure has revealed that CLEC5 A is a homodimeric protein when it binds to dengue virus serotype. Moreover, the CLEC5A crystal structure revealed conformational flexibility,Atorney Docket No.: 55679-0015WO3

[0147] suggesting that CLEC5A can adopt various conformations in vivo and that its conformation is liganddependent.

[0148] A detailed review of CLEC5A and its functions 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 pancancer analysis reveals CLEC5A as a biomarker for cancer immunity and prognosis” Frontiers in Immunology 13 (2022): 831542; and Wang, Quhui, et al., “CLEC5A promotes the proliferation of gastric cancer cells by activating the PI3K / AKT / mTOR pathway” Biochemical and Biophysical Research Communications 524.3 (2020): 656-662; each of which is incorporated by reference in its entirety.

[0149] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can specifically bind to CLEC5A (e.g., human CLEC5A).

[0150] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and a light chain variable region (VL).

[0151] In some embodiments, the heavy chain variable region (VH) comprises CDRs 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and

[0152] the light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence,

[0153] wherein the selected VH CDRs 1, 2, and 3 amino acid sequences, and the selected VL CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0154] (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;Atorney Docket No.: 55679-0015WO3

[0155] (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0156] (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; and

[0157] (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively.

[0158] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0159] (1) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; and

[0160] (2) the selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50.

[0161] In some embodiments, the VH comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL comprising VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0162] (1) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; and

[0163] (2) the selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50.

[0164] In some embodiments, the antibodies comprise an antigen-binding domain of the anti-CLEC5 A antibody 5C7 or 13E6, the chimeric antibodies thereof, or the humanized antibodies thereof.

[0165] The CDR sequences for 5C7, and 5C7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 31, 33, and 35, and CDRs of the light chain variable domain, SEQ ID NOs: 36, 37, and 38, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 32, 34, and 35, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 36, 37, and 38.Atorney Docket No.: 55679-0015WO3

[0166] The CDR sequences for 13E6, and 13E6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 41, 43, and 45, and CDRs of the light chain variable domain, SEQ ID NOs: 46, 47, and 48, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 42, 44, and 45, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 46, 47, and 48.

[0167] Anti-EpCAM / CLEC5A Antibodies and Antigen-binding Fragments Thereof

[0168] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can specifically bind to EpCAM (e.g., human EpCAM) and / or CLEC5A (e.g., human CLEC5A).In one aspect, the disclosure provides antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to EpCAM; and a second antigen-binding domain that specifically binds to CLEC5A.

[0169] In some embodiments, the antigen-binding domain that binds to EpCAM comprises a heavy chain variable region (VH1) and a light chain variable region (VL1).

[0170] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; and

[0171] the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence,

[0172] wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0173] (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;Atorney Docket No.: 55679-0015WO3

[0174] (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;

[0175] (3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0176] (4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;

[0177] (5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; and

[0178] (6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively.

[0179] In some embodiments, the antigen-binding domain that binds to EpCAM comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0180] In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; and

[0181] the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:Atorney Docket No.: 55679-0015WO3

[0182] (1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0183] (2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0184] (3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; and

[0185] (4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively.

[0186] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0187] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0188] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0189] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acidAtorney Docket No.: 55679-0015WO3

[0190] sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0191] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;

[0192] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;

[0193] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;

[0194] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;

[0195] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;

[0196] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acidAtorney Docket No.: 55679-0015WO3

[0197] sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;

[0198] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; or

[0199] In some embodiments, the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively.

[0200] In some embodiments, the VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0201] (1) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10;

[0202] (2) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; and

[0203] (3) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30.

[0204] In some embodiments, the VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0205] (1) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; and

[0206] (2) the selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50.Atorney Docket No.: 55679-0015WO3

[0207] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 39, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 40.

[0208] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 50.

[0209] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 20, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 39, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 40.

[0210] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 20, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 50.

[0211] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 30, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 39, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 40.Atorney Docket No.: 55679-0015WO3

[0212] In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 29, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 30, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 50.

[0213] In some embodiments, the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL1 comprising VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0214] (1) the selected VH sequence is SEQ ID NO: 9, and the selected VL sequence is SEQ ID NO: 10;

[0215] (2) the selected VH sequence is SEQ ID NO: 19, and the selected VL sequence is SEQ ID NO: 20; and

[0216] (3) the selected VH sequence is SEQ ID NO: 29, and the selected VL sequence is SEQ ID NO: 30.

[0217] In some embodiments, the VH2 comprises VH2 CDR1, VH2CDR2, and VH2 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0218] (1) the selected VH sequence is SEQ ID NO: 39, and the selected VL sequence is SEQ ID NO: 40; and

[0219] (2) the selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50.

[0220] In some embodiments, the first antigen-binding domain is the antigen-binding domain of anti- EpCAM antibody EpCAMl, EpCAM2 or EpCAM3, the chimeric antibodies thereof, or the humanized antibodies thereof.Atorney Docket No.: 55679-0015WO3

[0221] The CDR sequences for EpCAMl, and EpCAMl derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 3, and 5, and CDRs of the light chain variable domain, SEQ ID NOs: 6, 7, and 8, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 2, 4, and 5, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 6, 7, and 8.

[0222] The CDR sequences for EpCAM2, and EpCAM2 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 11, 13, and 15, and CDRs of the light chain variable domain, SEQ ID NOs: 16, 17, and 18, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 12, 14, and 15, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 16, 17, and 18.

[0223] The CDR sequences for EpCAM3, and EpCAM3 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 21, 23, and 25, and CDRs of the light chain variable domain, SEQ ID NOs: 26, 27, and 28, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 22, 24, and 25, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 26, 27, and 28.

[0224] In some embodiments, the second antigen-binding domain is the antigen-binding domain of anti-CLEC5A antibody 5C7 or 13E6, the chimeric antibodies thereof, or the humanized antibodies thereof.

[0225] The CDR sequences for 5C7, and 5C7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 31, 33, and 35, and CDRs of the light chain variable domain, SEQ ID NOs: 36, 37, and 38, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 32, 34, and 35, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 36, 37, and 38.

[0226] The CDR sequences for 13E6, and 13E6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 41, 43, and 45, and CDRs of the light chain variable domain, SEQ ID NOs: 46, 47, and 48, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 42, 44, and 45, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 46, 47, and 48.

[0227] In some embodiments, the first antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog EpCAM; and / or the second antigen-binding domain specifically binds to human, rabbit, mouse, monkey, or dog CLEC5A.Atorney Docket No.: 55679-0015WO3

[0228] In some embodiments, the first antigen-binding domain is a human or humanized antigenbinding domain; and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0229] In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv); and / or the second antigen-binding domain is a scFv.

[0230] In some embodiments, the antibodies or antigen-binding fragments thereof comprise a fragment crystallizable region (Fc region).

[0231] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an IgGl subtype structure with the LALAPG mutations (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an IgGl Fc region having an Alanine (A) at position 234; an Alanine (A) at position 235; and a Glycine (G) at position 329 in EU numbering. In some embodiments, The Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 52.

[0232] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an IgGl subtype structure with the GAALIE mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein can be designed to have an IgGl Fc region having an Alanine (A) at position 236, a Leucine (L) at position 330, and a Glutamic acid (E) at position 332 in EU numbering. In some embodiments, The Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 53.

[0233] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an Fc region that comprises an Aspartic acid (D) at position 239 and a glutamic acid (E) at position 332 in EU numbering. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an IgGl subtype structure with the S239D+I332E mutations in EU numbering. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof have an IgGl Fc region having an Aspartic acid (D) at position 239 and an glutamic acid (E) at position 332 in EU numbering.Atorney Docket No.: 55679-0015WO3

[0234] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragments thereof have an IgGl subtype structure with knobs-into-holes (KIH) mutations, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the anti-EpCAM / CLEC5A antibody has a higher endocytosis rate than the corresponding monoclonal antibodies or the control bispecific antibodies.

[0235] The first antigen-binding fragment and the second antigen-binding fragment of the bi-specific antibodies or antigen-binding fragments described herein can be in any suitable configurations. In some embodiments, the second antigen-binding domain is a single-chain fragment variable (scFv) domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) linked by a first linker.

[0236] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain through a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to a Fc region. In some embodiments, the VH1 is linked to a CHI domain, and the VL1 is linked to a CL domain. A schematic illustration of this configuration is shown in FIG. ID.

[0237] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the heavy chain of the first antigen-binding domain through a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to a Fc region. In some embodiments, the VH1 is linked to a CHI domain, and the VL1 is linked to a CL domain. A schematic illustration of this configuration is shown in FIG. IB or FIG. 1C.

[0238] In some embodiments, the second antigen-binding domain is linked to a Fc region, optionally through a hinge domain. In some embodiments, the heavy chain variable domain of the first antigenbinding domain is linked to the Fc region. In some embodiments, the VH1 is linked to a CHI domain, and the VL1 is linked to a CL domain. A schematic illustration of this configuration is shown in FIG.

[0239] 1A

[0240] In some embodiments, the Fc region comprises knobs-into-holes (KIH) mutations. In some embodiments, the first heavy chain includes one or more knob mutations, and the second heavy chain includes one or more hole mutations. In some embodiments, the first heavy chain includes one or more hole mutations, and the second heavy chain includes one or more knob mutations.

[0241] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein or antigen-binding fragment thereof includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Atorney Docket No.: 55679-0015WO3

[0242] 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0243] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 56.

[0244] In some embodiments, the antibody is referred to as “EpCAMl / 5C7 1+1 Fc-S” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55; 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: 56.

[0245] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58. In some embodiments, the heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0246] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 59.Atorney Docket No.: 55679-0015WO3

[0247] In some embodiments, the antibody is referred to as “EpCAMl / 5C72+1 Fc-S” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58; 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: 59.

[0248] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 60. In some embodiments, the heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52).

[0249] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 61.

[0250] In some embodiments, the antibody is referred to as “EpCAMl / 5C72+2A Fc-S” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60; 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: 61.

[0251] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 62. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0252] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a light chain sequence thatAtorney Docket No.: 55679-0015WO3

[0253] 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: 64.

[0254] In some embodiments, the antibody is referred to as “EpCAMl / 5C7 1+1 Fc-E” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 62; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63; 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: 64.

[0255] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0256] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 67.

[0257] In some embodiments, the antibody is referred to as “EpCAMl / 5C72+1 Fc-E” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66; 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: 67.

[0258] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,Atorney Docket No.: 55679-0015WO3

[0259] 97%, 98%, 99% or 100% identical to SEQ ID NO: 68. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0260] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 69.

[0261] In some embodiments, the antibody is referred to as “EpCAMl / 5C72+2A Fc-E” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68; 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: 69.

[0262] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 70. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0263] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 71.

[0264] In some embodiments, the antibody is referred to as “EpCAM2 / 5C72+2A Fc-E” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 70; 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: 71.

[0265] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 72. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0266] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 73.Atorney Docket No.: 55679-0015WO3

[0267] In some embodiments, the antibody is referred to as “EpCAM2 / 5C72+2B Fc-E” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72; 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: 73.

[0268] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0269] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 76.

[0270] In some embodiments, the antibody is referred to as “EpCAM3 / 5C7 1+1 Fc-S” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75; 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: 76.

[0271] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 77. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical toAtorney Docket No.: 55679-0015WO3

[0272] SEQ ID NO: 78. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0273] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 79.

[0274] In some embodiments, the antibody is referred to as “EpCAM3 / 5C72+1 Fc-S” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 77; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 78; 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: 79.

[0275] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 80. In some embodiments, the heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52).

[0276] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 81.

[0277] In some embodiments, the antibody is referred to as “EpCAM3 / 5C7 2+2A Fc-S (G4S)i” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80; 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: 81.

[0278] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 82. In some embodiments, the heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52).Atorney Docket No.: 55679-0015WO3

[0279] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 83.

[0280] In some embodiments, the antibody is referred to as “EpCAM3 / 5C72+2A Fc-S” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82; 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: 83.

[0281] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 84. In some embodiments, the heavy chain includes an IgGl Fc region comprising the LALAPG mutations (SEQ ID NO: 52).

[0282] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 85.

[0283] In some embodiments, the antibody is referred to as “EpCAM3 / 13E62+2AFc-S” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 84; 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: 85.

[0284] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 86. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 87. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.Atorney Docket No.: 55679-0015WO3

[0285] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 88.

[0286] In some embodiments, the antibody is referred to as “EpCAM3 / 5C7 1+1 Fc-E” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 86; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 87; 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: 88.

[0287] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 89. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigenbinding fragments thereof include a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90. In some embodiments, the first and / or second heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53). In some embodiments, the first heavy chain includes one or more hole mutations. In some embodiments, the second heavy chain includes one or more knob mutations.

[0288] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 91.

[0289] In some embodiments, the antibody is referred to as “EpCAM3 / 5C72+1 Fc-E” and includes a first heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 89; a second heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90; 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: 91.Atorney Docket No.: 55679-0015WO3

[0290] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 92. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0291] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 93.

[0292] In some embodiments, the antibody is referred to as “EpCAM3 / 5C72+2B Fc-E” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 92; 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: 93.

[0293] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 94. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0294] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 95.

[0295] In some embodiments, the antibody is referred to as “EpCAM3 / 5C7 2+2A Fc-E (G4S)i” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94; 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: 95.

[0296] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 96. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).Atorney Docket No.: 55679-0015WO3

[0297] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 97.

[0298] In some embodiments, the antibody is referred to as “EpCAM3 / 5C7 2+2A Fc-E (048)2” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96; 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: 97.

[0299] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 98. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0300] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 99.

[0301] In some embodiments, the antibody is referred to as “EpCAM3 / 5C72+2A Fc-E” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98; 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: 99.

[0302] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 100. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0303] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 101.

[0304] In some embodiments, the antibody is referred to as “EpCAM3 / 13E62+2A Fc-E (G4S)I” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,Atorney Docket No.: 55679-0015WO3

[0305] 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 100; 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: 101.

[0306] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 102. In some embodiments, the heavy chain includes an IgGl Fc region comprising the GAALIE mutations (SEQ ID NO: 53).

[0307] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof include 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: 103.

[0308] In some embodiments, the antibody is referred to as “EpCAM3 / 13E6 2+2A Fc-E L” and includes a heavy chain sequence that is about or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 102; 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: 103.

[0309] The linkers described herein can be any suitable linkers known in the art. In some embodiments, the linker can comprise a spacer sequence. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, (GSGGS)n (SEQ ID NO: 104), (GGGS)n (SEQ ID NO: 105), (GGGGS)n (SEQ ID NO: 106) where n represents an integer of at least 1. Those of skill in the art would be able to select the appropriate spacer sequence.

[0310] In some embodiments, knobs-into-holes mutations were introduced in the Fc regions of the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom to reduce the chance of wrong pairing between the two heavy chains.

[0311] The immunoglobulin heavy chain or immunoglobulin light chain in the anti-EpCAM / CLEC5 A antibody comprises CDRs as shown in FIG.24. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to EpCAM and / or CLEC5A.

[0312] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) and antigen-binding fragments thereof have an agonistic activity on theAtorney Docket No.: 55679-0015WO3

[0313] activation of macrophages. In some embodiments, the activation of macrophages 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% upon contact with the antibodies described herein, compared to the activation of macrophages without contact with such antibody.

[0314] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein bind to macrophages. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein bind to target cells (e.g., EpCAM+ cancer cells).

[0315] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein activate macrophages (e.g., through the engagement of CLEC5A), which mediate the killing of target cells (e.g., EpCAM+ cancer cells). In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein include a wild-type human IgGl Fc region. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) described herein include a mutated human IgGl Fc region (e.g., enhanced Fc or silenced Fc). In some embodiments, the mutated human IgGl Fc region (e.g., silenced Fc) comprises the LALAPG mutations (L234A, L235A, and P329G mutations in EU numbering). In some embodiments, the mutated human IgGl Fc region (e.g., silenced Fc) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 52. In some embodiments, the mutated human IgGl Fc region (e.g., enhanced Fc) comprises the GAALIE mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the mutated human IgGl Fc region (e.g., enhanced Fc) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 53.

[0316] In some embodiments, the antibodies, multispecific (e.g., bispecific) anti-EpCAM / CLEC5A antibody described herein activates macrophages (e.g., through the engagement of CLEC5A) and induces macrophage-mediated target cell killing. In some embodiments, the macrophage-mediated killing results in the killing of target cells (e.g., EpCAM+ cancer cells). In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total number of the target cells were killed through macrophage-mediated phagocytosis.

[0317] Antibodies and Antigen Binding Fragments thereof

[0318] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) and antigen-binding fragments thereof can have various forms.Atorney Docket No.: 55679-0015WO3

[0319] In general, antibodies (also called immunoglobulins) can be made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody (e.g., a multispecific antibody, such as a bispecific anti-EpCAM / CLEC5A antibody) of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.

[0320] The hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigenbinding domain.

[0321] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (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 al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0322] The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

[0323] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., inAtorney Docket No.: 55679-0015WO3

[0324] 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; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0325] The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, or camelid). The antigen-binding domain or antigen binding fragment is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen binding fragment thereof can comprise e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigenbinding domains include, e.g., the heavy chain and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

[0326] In some embodiments, the scFv in 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), and the two antigen binding regions can bind to the respective target antigens with different affinities.

[0327] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can comprise one, two, or three heavy chain variable region CDRs selected from Table 25.

[0328] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5 A antibodies) or antigen-binding fragments thereof can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibodies or antigen-binding fragments thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, di one, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, andAtorney Docket No.: 55679-0015WO3

[0329] cyclophosphamide and analogs). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of the linkers used for ADCs can be found “e.g., in Su, Z. et al. "Antibody-drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), which is incorporated herein by reference in its entirety.

[0330] Multispecific (e.g., bispecific) antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody 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). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.

[0331] Any of the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibodies or antigen-binding fragments thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an anti-EpCAM / CLEC5A antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).

[0332] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) are bispecific antibodies. The antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., 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 the entirety.

[0333] In some embodiments, the antibody is a BiTe, a (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, aHSAbody, scDiabody-HAS, or a tandem-scFv. In some embodiments, the antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, aAtorney Docket No.: 55679-0015WO3

[0334] SEEDbody, a LUZ-Y, a Fcab, a Kk-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a 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 IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFvl-PEG-scFv2.

[0335] In some embodiments, the antibody can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.

[0336] In some embodiments, the antibody can have two different heavy chains and one common light chain. In some embodiments, the antibody has two different heavy chains and two different light chains. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.

[0337] In some embodiments, CrossMAb technique can be used produce bispecific antibodies. CrossMAb technique can be used enforce correct light chain association in bispecific heterodimeric IgG antibodies, this technique allows the generation of various bispecific antibody formats, including bi-(l+l), tri-(2+l) and tetra-(2+2) valent bispecific antibodies, as well as non-Fc tandem antigenbinding fragment (Fab)-based antibodies. These formats can be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing / in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association. The 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, which is incorporated by reference in its entirety. In some embodiments, the CHI in the heavy chain and the CL domain in the light chain are swapped.

[0338] The antibody can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgGl antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy-light chains.

[0339] In Dual-variable-domain antibody (DVD-Ig), additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.

[0340] In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.Atorney Docket No.: 55679-0015WO3

[0341] In some embodiments, the antibody can have a IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.

[0342] The antibody can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.

[0343] In some embodiments, the antibody can be a TF. Three Fab fragments are linked by disulfide bridges. Two fragments target the tumor associated antigen (e.g., EpCAM) and one fragment targets a hapten. The TF format does not have an Fc region.

[0344] ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.

[0345] Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs.

[0346] In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

[0347] In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets; thus it is a tetravalent molecule.

[0348] Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.

[0349] The ScFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.

[0350] In some embodiments, the antibody in the present disclosure is designed to be 1+1 (monovalent for each target). In some embodiments, the antibody in the present disclosure is designed to be 2+1 (bivalent for a first target and monovalent for a second target). In some embodiments, the antibody in the present disclosure is designed to be 2+2 (bivalent for each target). In some embodiments, the antibody in the present disclosure has an IgGl subtype structure. In some embodiments, the antibody in the present disclosure can reduce the avidity to cells with low expression levels of the first and second epitope, and increase the avidity to cells that co-express the first and second epitope, to achieve enhanced targeting function.Atorney Docket No.: 55679-0015WO3

[0351] In some embodiments, the antibodies include KIH mutations. In some embodiments, the antibody includes a first arm comprising a first antigen-binding domain that specifically binds to the first epitope, and a second arm comprising a second antigen-binding domain that specifically binds to the second epitope. In some embodiments, the first antigen-binding domain includes a heavy chain that including one or more knob mutations (a knob heavy chain), and the second antigen-binding domain includes a heavy chain including one or more hole mutations (a hole heavy chain). In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (a hole heavy chain), and the second antigen-binding domain that includes a heavy chain including one or more knob mutations (a knob heavy chain).

[0352] Antibody Characteristics

[0353] In some embodiments, the antibodies, or antigen-binding fragments thereof can initiate macrophage-mediated target cell (tumor cell) killing, CDC or ADCC.

[0354] The disclosure provides antibodies and antigen-binding fragments thereof that can specifically bind to a EpCAM (e.g., human EpCAM) and / or CLEC5A (e.g., human CLEC5A). The antibodies or antigen-binding fragments thereof described herein can bind to antigens on target cells (e.g., EpCAM-expressing cells such as EpCAMT cells) and recruit effector cells such as macrophages for the killing of the target cells.

[0355] General techniques can be used to measure the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some implementations, the antibodies or antigenbinding fragments thereof can bind to the EpCAM with a dissociation rate (koff) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.

[0356] In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / MS, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 105 / Ms, less than 1 x 106 / Ms, or less than 1 x 107 / MS.

[0357] In some embodiments, the antibodies or antigen-binding fragments thereof can bind to the EpCAM with a KD of less than 1 x 10’6M, less than 1 x 10’7M, less than 1 x 10’8M, less than 1 x 10’9M, or less than 1 x 10’10M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10’7M, greater than 1 x 10’8M, greater than 1 x 10’9M, or greater than 1 x 10’10M.Atorney Docket No.: 55679-0015WO3

[0358] The antibodies or antigen-binding fragments thereof can also include an antigen-binding domain that can specifically bind to CLEC5 A. In some embodiments, the antibodies or antigen-binding fragments thereof described herein activate macrophages and enhances macrophage-mediated cell killing (e.g., tumor cell killing). In some embodiments, the antibody can reduce tumor volume in an animal. In some embodiments, the antibody can reduce or slow down the progression of an autoimmune disease.

[0359] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have an agonistic activity on macrophage activation. In some embodiments, the activation of macrophages 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%, 96%, 97%, 98%, 99% or 100%, compared to the activation of macrophages without the contact with the antibodies or antigen-binding fragments thereof described herein.

[0360] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can bind to the CLEC5A with a dissociation rate (koff) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.

[0361] In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / MS, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 105 / Ms, less than 1 x 106 / Ms, or less than 1 x 107 / MS.

[0362] Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, KD is less than 1 x 10’6M, less than 1 x 10’7M, less than 1 x 10’8M, less than 1 x 10’9M, or less than 1 x 10’10M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10’7M, greater than 1 x 10’8M, greater than 1 x 10’9M, or greater than 1 x 10’10M.

[0363] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A), as measured by ELISA (see Example 4). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, lessAtorney Docket No.: 55679-0015WO3

[0364] than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A) with an EC50 of 0.5-1 nM, 0.4-1 nM, or 0.3-1 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A) with higher affinities compared to a control antibody.

[0365] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM), as measured by ELISA (see Example 5). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM) with an EC50 of 0.5-5 nM, 1-10 nM, or 0.2-5 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM) with higher affinities compared to a control antibody.

[0366] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to CLEC5A (e.g., human CLEC5A), as measured by SPR (see Example 6). In some embodiments, the antibodies or antigen-binding fragments thereof can bind to CLEC5A (e.g., human CLEC5A) with a KD of less than 1 x 10'6M, less than 1 x 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, less than 1 x 10'10M, less than 1 x 10'11M, or less than 1 x 10'12M. InAtorney Docket No.: 55679-0015WO3

[0367] some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10'7M, greater than 1 x 10'8M, greater than 1 x 10'9M, greater than 1 x 10'10M, greater than 1 x 10'11M, or greater than 1 x IO’12M. In some embodiments, the KD is between 1 x 10'12M and 1x10'11M.

[0368] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM (e.g., human EpCAM), as measured by SPR (see Example 7). In some embodiments, the antibodies or antigen-binding fragments thereof can bind to EpCAM (e.g., human EpCAM) with a KD of less than 1 x 10'6M, less than 1 x 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, or less than 1 x 10'10M. In some embodiments, the KD is less than 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10'6M, 1 x 10'7M, greater than 1 x 10'8M, greater than 1 x 10'9M, or greater than 1 x 10'10M. In some embodiments, the KD is between 1 x 10'7M and 1x10'6M.

[0369] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM (e.g., rehsus EpCAM). In some embodiments, the antibodies or antigen-binding fragments thereof can bind to EpCAM (e.g., rehsus EpCAM) with a KD of less than 1 x 10'6M, less than 1 x 10'7M, less than 1 x w8M, less than 1 x w9M, or less than 1 x 10'10M. In some embodiments, the KD is less than 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10'6M, 1 x 10'7M, greater than 1 x 10"8M, greater than 1 x 10'9M, or greater than 1 x 10'10M. In some embodiments, the KD is between 1 x IO’9M and 1 x 10’8M.

[0370] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to CLEC5A positive tumor cells (e.g., U937 cells) (see Example 8). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A positive tumor cells (e.g., U937 cells) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADCAtorney Docket No.: 55679-0015WO3

[0371] derived therefrom can bind to CLEC5A positive tumor cells (e.g., U937 cells) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A positive tumor cells (e.g., U937 cells) with an EC50 of 0.1-50 nM, 1-20 nM, or 1-10 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to CLEC5A positive tumor cells (e.g., U937 cells) with higher affinities compared to a control antibody.

[0372] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to human monocytes (see Example 9). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human monocytes with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human monocytes with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human monocytes with an EC50 of 0.1-50 nM, 1-20 nM, or 1-10 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human monocytes with higher affinities compared to a control antibody.

[0373] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to human macrophages (e.g., M0 macrophages and / or M2 macrophages) (see Examples 10-11). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human macrophages with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human macrophages with an EC50 of higher than 200 nM,Atorney Docket No.: 55679-0015WO3

[0374] higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human macrophages with an EC50 of 1-100 nM, 4-50 nM, or 3-60 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to human macrophages with higher affinities compared to a control antibody.

[0375] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., DLD-1 cells) (see Example 12). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., DLD-1 cells) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., DLD-1 cells) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., DLD-1 cells) with an EC50 of 0.1-50 nM, 0.1-20 nM, or 0.1-10 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., DLD-1 cells) with higher affinities compared to a control antibody.

[0376] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., MCF7 cells) (see Example 13). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., MCF7 cells) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., MCF7 cells) with an EC50 ofAtorney Docket No.: 55679-0015WO3

[0377] higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., MCF7 cells) with an EC50 of 0.1-50 nM, 0.5-20 nM, or 1-10 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., MCF7 cells) with higher affinities compared to a control antibody.

[0378] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., HCT116 cells) (see Example 14). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., HCT116 cells) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., HCT116 cells) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., HCT116 cells) with an EC50 of 0.1-100 nM, 0.5-50 nM, or 0.5-1 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., HCT116 cells) with higher affinities compared to a control antibody.

[0379] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., SKBR3 cells, T47D cells, SKOV3 cells, A549 cells) (see Examples 15-18). In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., SKBR3 cells, T47D cells, SKOV3 cells, A549 cells) with an EC50 of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.2 nM, or less than 0.15 nM. InAtorney Docket No.: 55679-0015WO3

[0380] some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., SKBR3 cells, T47D cells, SKOV3 cells, A549 cells) with an EC50 of higher than 200 nM, higher than 100 nM, higher than 50 nM, higher than 10 nM, higher than 5 nM, higher than 1 nM, higher than 0.75 nM, higher than 0.5 nM, higher than 0.25 nM, higher than 0.2 nM, or higher than 0.15 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., SKBR3 cells, T47D cells, SKOV3 cells, A549 cells) with an EC50 of 0.02-100 nM, 0.1-50 nM, or 0.5-10 nM. In some embodiments, the antibodies or antigen binding fragments as described herein or ADC derived therefrom can bind to EpCAM positive tumor cells (e.g., SKBR3 cells, T47D cells, SKOV3 cells, A549 cells) with higher affinities compared to a control antibody.

[0381] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can initiate macrophage-mediated target cell (tumor cell) killing, CDC or ADCC. In some embodiments, the antibodies or antigen-binding fragments thereof described herein activate macrophages and enhances macrophage-mediated cell killing (e.g., tumor cell killing). In some embodiments, the antibody can reduce tumor volume in an animal. In some embodiments, the antibody can reduce or slow down the progression of an autoimmune disease.

[0382] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can initiate PBMC-mediated target cell (e.g., DLD-1 cell) killing (see Example 19). In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a PBMC mediated cell killing ECso of less than 5 pM, less than 10 pM, less than 50 pM, less than 0.1 nM, less than 0.2 nM, less than 0.5 nM, less than 0.8 nM, less than 1 nM, less than 2 nM, less than 5 nM, less than 8 nM, less than 10 nM, less than 20 nM, less than 50 nM, less than 100 nM, less than 200 nM, less than 500 nM, or less than 800 nM, as determined using the method described herein. In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a PBMC mediated cell killing ECso of more than 5 pM, more than 10 pM, more than 50 pM, more than 0.1 nM, more than 0.2 nM, more than 0.5 nM, more than 0.8 nM, more than 1 nM, more than 2 nM, more than 5 nM, more than 8 nM, more than 10 nM,Atorney Docket No.: 55679-0015WO3

[0383] more than 20 nM, more than 50 nM, more than 100 nM, more than 200 nM, more than 500 nM, or more than 800 nM, as determined using the method described herein. In some embodiments, the antibodies, antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a PBMC mediated cell killing ECso value that is 1 pM-50 pM, 0.1-1 nM, 0.2-2 nM, 0.5-5 nM, 0.8-8 nM, 1-10 nM, 2-20 nM, 5-50 nM, 8-80 nM, 10-100 nM, 20-200 nM, 50-500 nM, or 80-800 nM. In some embodiments, the antibodies, antigen binding fragments thereof, or the antigenbinding protein constructs described herein have a PBMC mediated cell killing ECso value that is lpM-800nM. In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a PBMC mediated cell killing ECso value that is less than or about 50%, less than or about 30%, less than or about 20%, less than or about 10%, less than or about 5%, less than or about 1% as compared to that of a control antibody.

[0384] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies) or antigen-binding fragments thereof can initiate macrophage (e.g., M0 macrophage or M2 macrophage) mediated target cell (e.g., DLD-1 cell, SKBR3 cell, MCF7 cell, HCT116 cell, T47D cell, LNCaP cell) killing (see Examples 20-26). In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a macrophage mediated cell killing ECso of less than 5 pM, less than 10 pM, less than 50 pM, less than 0.1 nM, less than 0.2 nM, less than 0.5 nM, less than 0.8 nM, less than 1 nM, less than 2 nM, less than 5 nM, less than 8 nM, less than 10 nM, less than 20 nM, less than 50 nM, less than 100 nM, less than 200 nM, less than 500 nM, or less than 800 nM, as determined using the method described herein. In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a macrophage mediated cell killing ECso of more than 5 pM, more than 10 pM, more than 50 pM, more than 0.1 nM, more than 0.2 nM, more than 0.5 nM, more than 0.8 nM, more than 1 nM, more than 2 nM, more than 5 nM, more than 8 nM, more than 10 nM, more than 20 nM, more than 50 nM, more than 100 nM, more than 200 nM, more than 500 nM, or more than 800 nM, as determined using the method described herein. In some embodiments, the antibodies, antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a macrophage mediated cell killing ECso value that is 1 pM-50 pM, 0.1-1 nM, 0.2-2 nM, 0.5-5 nM, 0.8-8 nM, 1-10 nM, 2-20 nM, 5-50 nM, 8-80 nM, 10-100 nM, 20-200 nM, 50-500 nM, or 80-800 nM. In some embodiments, the antibodies, antigen binding fragmentsAtorney Docket No.: 55679-0015WO3

[0385] thereof, or the antigen-binding protein constructs described herein have a macrophage mediated cell killing EC50 value that is lpM-800nM. In some embodiments, the antibodies, the antigen binding fragments thereof, or the antigen-binding protein constructs described herein have a macrophage mediated cell killing EC50 value that is less than or about 50%, less than or about 30%, less than or about 20%, less than or about 10%, less than or about 5%, less than or about 1% as compared to that of a control antibody.

[0386] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom have a tumor growth inhibition rate or percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibodies, antigen-binding fragments thereof, or ADCs derived therefrom have a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. The TGI (%) can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the treatment starts. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:

[0387] TGI (%) = [l-(Ti-T0) / (Vi-V0)]xl00%

[0388] Ti is the average tumor volume in the treatment group on day i. TO is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

[0389] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibodydependent cell-mediated cytotoxicity (ADCC). In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgGl, human IgG2, human IgG3, or human IgG4.

[0390] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom do not have a functional Fc region. For example, the antibodies or antigen-binding fragments thereof are Fab, Fab’, F(ab’)2, and Fv fragments. In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom have an Fc region without effector function. In some embodiments, the Fc is aAtorney Docket No.: 55679-0015WO3

[0391] human IgGl Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALAPG mutations (L234A, L235A, P329G mutations in EU numbering). The amino acid sequence of the LALAPG mutant of human IgGl is shown in SEQ ID NO: 52.

[0392] In some embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom comprise a fragment crystallizable region (Fc region). In some embodiments, the antibody described herein can be designed to have an IgGl subtype structure with the GAALIE mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the antibody described herein can be designed to have an IgGl Fc region having an Alanine (A) at position 236, a Leucine (L) at position 330, and a Glutamic acid (E) at position 332 in EU numbering. In some embodiments, The Fc region comprises an amino acid sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 53.

[0393] Some other modifications to the Fc region can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and / or in vivo.

[0394] In some embodiments, multispecific (e.g., bispecific) antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).

[0395] Recombinant Vectors

[0396] The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosedAtorney Docket No.: 55679-0015WO3

[0397] herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and / or a vector comprising the polynucleotide), and the production of antibodies, multispecific (e.g., bispecific) antibody polypeptides or fragments thereof by recombinant techniques.

[0398] As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

[0399] A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0400] In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.Atorney Docket No.: 55679-0015WO3

[0401] For expression, the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the S V40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0402] As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

[0403] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

[0404] Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0405] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods EnzymoL, 153: 516-544 (1997).Atorney Docket No.: 55679-0015WO3

[0406] Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0407] Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0408] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

[0409] The polypeptide can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

[0410] The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein.

[0411] The disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any nucleotideAtorney Docket No.: 55679-0015WO3

[0412] sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any amino acid sequence as described herein.

[0413] In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0414] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

[0415] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

[0416] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0417] The disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a polypeptide of a heavy chain as described herein. In some embodiments, the nucleic acid includes aAtorney Docket No.: 55679-0015WO3

[0418] polynucleotide encoding a polypeptide of a light chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.

[0419] In some embodiments, the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to EpCAM (e.g., human EpCAM) and / or CLEC5 A (e.g., human CLEC5 A). In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to the EpCAM (e.g., human EpCAM) and / or CLEC5A (e.g., human CLEC5A).

[0420] Preparation of antibodies

[0421] The antibodies can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, a DNA molecule encoding the antibodies can be obtained through chemical synthesis or PCR amplification, the obtained DNA molecules can be inserted into an expression vector, and then the host cells can be transfected. Then, the transfected host cells can be cultured under specific conditions to express the antibodies. The antigen-binding fragments can be obtained by hydrolyzing intact antibody molecules.

[0422] In another aspect, the disclosure provides isolated nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof.

[0423] In another aspect, the disclosure provides a vector (for example, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule described herein. In certain embodiments, vectors are, for example, plasmids, cosmids, phages, and the like.

[0424] In another aspect, the disclosure provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0425] On the other hand, a method for preparing the antibodies or antigen-binding fragments thereof is provided, comprising the following steps: the host cells as described above are cultured under conditions that allow protein expression, and the antibodies or antigen-binding fragments thereof are recovered from the cultured host cell culture.

[0426] Conjugate

[0427] In another aspect, the disclosure also provides conjugates comprising the antibodies or antigenbinding fragments thereof, and a conjugation moiety.Atorney Docket No.: 55679-0015WO3

[0428] In certain embodiments, the antibodies or antigen-binding fragments thereof are optionally conjugated to the coupling moiety via a linker.

[0429] In certain embodiments, the coupling moiety is selected from protein tags. Such protein tags are well known in the art, examples of which include but are not limited to His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification tags, detection tags or tracer tags). In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof have a purification tag attached to their C-terminus.

[0430] In certain embodiments, the coupling moiety is selected from a detectable label such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H, 1251, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g. Cy7, Alexa 750)), luminescent substances (e.g. chemiluminescent substances such as acridinium esters), magnetic beads (e.g. Dynabeads®), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avidin (e.g., streptavidin) modified to bind the above labels, and biotin). In certain embodiments, such labels can be adapted for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, detectable labels as described above can be linked to bispecific antibodies via linkers of varying lengths to reduce potential steric hindrance.

[0431] In certain embodiments, the coupling moiety is selected from therapeutic agents, such as anti-neoplastic drugs.

[0432] In certain embodiments, the coupling moiety is selected from another biologically active polypeptide.

[0433] Methods of Treatment

[0434] The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies),Atorney Docket No.: 55679-0015WO3

[0435] antigen-binding fragments thereof, or ADCs derived therefrom, to a subject who is in need of, or who has been determined to be in need of, such treatment.

[0436] As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent described herein for the treatment of a condition associated with cancer will result in decreased number of cancer cells and / or alleviated symptoms.

[0437] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0438] In some embodiments, the cancer is a chemotherapy resistant cancer.

[0439] In one aspect, the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibitAtorney Docket No.: 55679-0015WO3

[0440] progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and / or duration of one or more symptoms of the cancer in a subject.

[0441] In one aspect, the disclosure features methods that include administering a therapeutically effective amount of the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., B cell lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, nonsmall cell lung cancer (NSCLC), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or renal cancer. In some embodiments, the cancer is a solid tumor such as 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, cancer of the small intestine, pancreatic cancer and / or liver cancer.

[0442] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods is provided. Veterinary and non-veterinary applications are contemplated. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0443] In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

[0444] As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subj ect to which the antibody, antigen binding fragment, antibody-drug conjugates, antibody-encoding polynucleotide, vector comprising the polynucleotide, and / or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

[0445] An effective amount can be administered in one or more administrations. By way of example, an effective amount of the antibody, or antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and / or delayAtorney Docket No.: 55679-0015WO3

[0446] proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro.

[0447] Effective amounts and schedules for administering the antibody, antigen binding fragment, antibody-drug conjugates, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibody, antigen binding fragment, antibody-drug conjugates, and / or compositions disclosed herein, the route of administration, the particular type of the agent or compositions disclosed herein used and other drugs being administered to the mammal.

[0448] A typical daily dosage of an effective amount of the antibody, antigen binding fragment, antibody-drug conjugates (e.g., such as anti-EpCAM / CLEC5A antibody, anti-EpCAM / CLEC5A antigen binding fragment, anti-EpCAM / CLEC5A antibody-drug conjugates) is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage can be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0449] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be an inhibitor of EpCAM. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EpCAM inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of CLEC5 A, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K / mT0R inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and / or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-l) (IDO1) (e.g., epacadostat).

[0450] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of EpCAM, an inhibitor of CLEC5A, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.Atorney Docket No.: 55679-0015WO3

[0451] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0452] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL- 10 antagonist, an IL-4 antagonist, an IL- 13 antagonist, an IL- 17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

[0453] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0454] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-lBB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.

[0455] Pharmaceutical Compositions and Routes of Administration

[0456] Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies (e.g., multispecific antibodies, such as bispecific anti-EpCAM / CLEC5A antibodies), antigen-binding fragments thereof, or ADCs derived therefrom. The pharmaceutical compositions may be formulated in any manner known in the art.

[0457] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceuticallyAtorney Docket No.: 55679-0015WO3

[0458] acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant.

[0459] Compositions containing one or more of any of the antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-EpCAM / CLEC5A antibodies, anti-EpCAM / CLEC5A antigenbinding fragments, or anti-EpCAM / CLEC5A antibody-drug conjugates) described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).

[0460] Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0461] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of the antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-EpCAM / CLEC5A antibodies, anti-EpCAM / CLEC5A antigen-binding fragments, or anti-EpCAM / CLEC5A antibody-drug conjugates) will be an amount that treats the disease (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer), or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured), decreases the severity, frequency, and / or duration of one or more symptoms of a disease in a subject (e.g., a human). The effectiveness and dosing of any of the antibodies or antigenbinding fragments, or antibody-drug conjugates (e.g., anti-EpCAM / CLEC5A antibodies, anti-EpCAM / CLEC5A antigen-binding fragments, or anti-EpCAM / CLEC5A antibody-drug conjugates) described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).Atorney Docket No.: 55679-0015WO3

[0462] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigenbinding fragments, or antibody-drug conjugates (e.g., anti-EpCAM / CLEC5A antibodies, anti-EpCAM / CLEC5A antigen-binding fragments, or anti-EpCAM / CLEC5A antibody-drug conjugates) described herein per kilogram of the subject’s weight (e.g., about 1 pg / kg to about 500 mg / kg; about 100 pg / kg to about 500 mg / kg; about 100 pg / kg to about 50 mg / kg; about 10 pg / kg to about 5 mg / kg; about 10 pg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the therapeutic agent in vivo.

[0463] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen-binding fragments, or antibody-drug conjugates (e.g., anti-EpCAM / CLEC5A antibodies, anti-EpCAM / CLEC5A antigen-binding fragments, or anti-EpCAM / CLEC5A antibodydrug conjugates) for various uses as described herein.

[0464] EXAMPLES

[0465] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0466] Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of protection claimed in the application. The experimental methods in the examples are all conventional methods unless otherwise specified. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.Atorney Docket No.: 55679-0015WO3

[0467] Example 1. Molecular construction

[0468] Different formats of EpCAM / CLEC5 A bi specific antibodies were generated as shown in FIGs.

[0469] 1A-1D. The EpCAM binding part was selected fromEpCAMl, EpCAM2 andEpCAM3. The CLEC5A binding part was selected from 5C7 and 13E6. Fc-E represents enhanced Fc. Fc-S represents silenced Fc.

[0470] Competent E. coli (NEB® 5-alpha) cells were transformed with the assembled plasmid encoding the clone with correct sequences, based on sequencing results (Elim Biopharm). The transformed cells were further cultured with LB containing carbenicillin (100 pg / ml) for plasmid purification (QIAGEN® Plasmid Plus Kits). Plasmids were eluted in nuclease-free water (Sigma) and stored at -80°C.

[0471] Example 2. Transfection and purification of EpCAM / CLEC5A bispecific antibodies Antibodies were expressed with CHO cells (ExpiCHO™ Expression System, Gibco) by transfection of both pcDNA3.4-huIgGl-Hc (containing VH sequence) and pcDNA3.4-huKappa-Lc (containing VL sequence) vectors. Specifically, ExpiCHO™ cells were cultured with the ExpiCHO™ expression medium and maintained at 0.3 to 6 * 106cells / ml according to manufacturer’s recommendation at 37°C, 125 rpm, 5% CO2 and 80% humidity. In a 125 ml baffled flask, 25 ml of fresh ExpiCHO™ cells (6 x 106cells / ml and viability > 95%) were prepared from 1 day-long culture seeded at 3 * 106 / ml. 1 ml serum-free medium (OptiPRO™ SFM, Gibco) containing both pcDNA3.4-huIgGl-Hc and pcDNA3 ,4-huKappa-Lc (12 pg of each plasmid) was mixed well (by pipetting) with 1 ml OptiPRO™ SFM containing 80 pl transfection reagent (ExpiFectamine™ CHO Reagent, Gibco). The transfection mixture was then added to the 25 ml ExpiCHO™ cells and cultured at 37°C. On the next day, the transfection culture was transferred to a 32°C incubator after adding 150 pl ExpiFectamine™ CHO Enhancer, 6 ml ExpiCHO™ Feed and 1 x penicillin / streptomycin (Gibco). The cell density and viability of the transfection culture was monitored and the IgGl antibody titer in the medium was determined using Biolayer interferometry (BLI) with Protein A biosensor (Gator Bio). After about 5 days, the culture medium containing secreted IgGl antibodies were collected (by centrifugation at 2000xg, 10 minutes), filtered (Thermo Scientific™ Nalgene™ Rapid-Flow™ Sterile Disposable Filter) and further purified using Protein A resin (TOYOPEARL AF-rProtein A Hc-650F)-Atorney Docket No.: 55679-0015WO3

[0472] packed gravity -flow column (Bio-Rad). IgGl antibodies were eluted with 3.5 ml Glycine-HCl (100 mM, pH 2.7), immediately neutralized with 1 M Tris-HCl (pH 8.5), dialyzed with Thermo Scientific™ Slide-A-Lyzer™ G2 Dialysis Cassettes (20K MWCO) in lx PBS buffer (pH 7.2) and stored at 4°C. The concentration of the purified IgGl antibody was determined with a NanoDrop™ One / OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific™) and the quality of IgGl antibody was checked by SDS-PAGE gel under both denaturing and non-denaturing conditions.

[0473] Purified antibodies were analyzed by HPLC (Dionex UltiMate® 3000-RS UHPLC, Thermo Fisher) controlled by Chromeleon® software. A size exclusion column TSKgel UP-SW3000, 2 pm, 4.6 mm IDxl5 cm (Tosoh) was used to resolve antibodies purity and aggregation.

[0474] All purified antibodies had 97% or greater monomer purity, which were sterilized through a 0.22 pm PES membrane filter, and used for binding and functional assays.

[0475] Example 3. Quantification of tumor cell surface EpCAM antigen densities

[0476] To estimate EpCAM antigen expression levels on malignant tumor cell surface, cell lines (DLD-1, MCF7, HCT116, SKBR3, T47D, SKOV3, A549) were stained at a saturating concentration of anti-EpCAM antibody on ice for 30min. After being washed with PBS, the cells were stained with a secondary Phycoerythrin (PE)-labeled anti-human IgG (Invitrogen, Cat: 12-4998-82) for 30 min on ice. Cells were then washed with PBS and resuspended in FACS buffer for flow cytometry analysis using Cytek northern lights. PE mean fluorescent intensity (MFI) of EpCAM staining was then calculated using FlowJo™ (Version 10.9.0). The EpCAM MFI was used to calculate PE molecules per cell (indicating EpCAM receptor density) using a PE fluorescence quantitation kit (BD Quantibrite™ Beads, Cat: 340495). Results were analyzed and graphed using GraphPad prism software (Version 9.4.1; GraphPad Software Inc.). As shown in FIG.2, different cell lines have different surface antigen densities. DLD-1, HCT116 and MCF7 have high EpCAM antigen density, and H226, A549 and SKOV3 have low EpCAM antigen density.

[0477] Example 4. ELISA Binding of EpCAM / CLEC5A Bispecific Antibodies to Human CLEC5A

[0478] ELISA analysis was performed to determine affinities of different EpCAM / CLEC5 Abispecific antibodies. Specifically, Corning high binding flat bottom 96-well plates (Cat#: 3361) were coatedAttorney Docket No.: 55679-0015WO3

[0479] with 1 pg / mL of human CLEC5A (Aero, CLA-H5243) for 24 hours at 4°C. Protein antigen was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. EpCAM / CLEC5A bispecific antibodies were used as the primary antibody starting at 4.2 pg / mL and serially diluted 4-fold through 7 points (diluted concentrations) with reagent buffer (PBS + 0.05% BSA). 50 pL of primary antibody was added to appropriate wells and incubated for 1 hour at room temperature. Primary antibody was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. Secondary antibody BioLegend donkey anti -human HRP (Cat#: 410902) was diluted 1:10,000 in reagent buffer (PBS + 0.5% BSA), and 50 pL was added per well, followed by an incubation for 30 minutes at room temperature. Secondary antibody was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. 50 pL of Thermo 1-step TMB Turbo (Cat#: 34022) was added to each well and incubated for 4 minutes at room temperature in the dark. 50 pL of Fisher IN Sulfuric Acid (Cat#: SA212-2) stop solution was added to each well and the plate was read in a plate reader at 450 nm. For analysis, OD450 of each antibody was plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 1 and FIGs. 3A-3B, all the EpCAM / CLEC5Abispecific antibodies were found to bind human CLEC5A.

[0480] Table 1 : ELISA Binding of EpCAM / CLEC5A Bispecific Antibodies to Human CLEC5A

[0481]

[0482] Atorney Docket No.: 55679-0015WO3

[0483] Example 5. ELISA Binding of EpCAM / CLEC5A Bispecific Antibodies to Human EpCAM

[0484] ELISA analysis was performed to determine affinities of different EpCAM / CLEC5A bispecific antibodies. Specifically, Corning high binding flat bottom 96-well plates (Cat#: 3361) were coated with 1 pg / mL of human EpCAM (ACROBiosystems, Cat#: EGR-H5223) for 24 hours at 4°C. Protein antigen was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. EpCAM / CLEC5A bispecific antibodies were used as the primary antibody starting at 4.2 pg / mL and serially diluted 4-fold through 7 points (diluted concentrations) with reagent buffer (PBS + 0.05% BSA). 50 pL of primary antibody was added to appropriate wells and incubated for 1 hour at room temperature. Primary antibody was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. Secondary antibody BioLegend donkey anti -human HRP (Cat#: 410902) was diluted 1:10,000 in reagent buffer (PBS + 0.5% BSA), and 50 pL was added per well, followed by an incubation for 30 minutes at room temperature. Secondary antibody was aspirated, and wells were washed 3 times with 200 pL of wash buffer (0.05% Tween® 20 in PBS) using a plate washer. 50 pL of Thermo 1-step TMB Turbo (Cat#: 34022) was added to each well and incubated for 4 minutes at room temperature in the dark. 50 pL of Fisher IN Sulfuric Acid (Cat#: SA212-2) stop solution was added to each well and the plate was read in a plate reader at 450 nm. For analysis, OD450 of each antibody was plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 2 and FIGs. 4A-4B, all the EpCAM / CLEC5 A bispecific antibodies were found to bind human EpCAM.

[0485] Table 2: ELISA Binding of EpCAM / CLEC5 A Bispecific Antibodies to Human EpCAM

[0486]

[0487] Attorney Docket No.: 55679-0015WO3

[0488]

[0489] Example 6. SPR Binding of EpCAM / CLEC5A Bispecific Antibodies to Human CLEC5A SPR analysis was used to evaluate if EpCAM / CLEC5 A bispecific antibodies can cross-react with human CLEC5 A protein. SPR was performed by Biacore 8000+. 5pg / mL of antibody was immobilized on Series S Sensor Chip CM5 by amine coupling. The analyte hCLEC5A (Aero, CLA-H5243) with different concentrations (0, 1.23, 3.70, 11.11, 33.33, 100, 300nM) were injected respectively for an association phase of 180 s, followed by 600 s dissociation. As shown in the table 3, EpCAM / CLEC5A bispecific antibodies were found to bind human CLEC5A with pMol affinity.

[0490] Table 3: SPR binding of EpCAM / CLEC5Abispecific antibodies to human CLEC5A

[0491]

[0492] Attorney Docket No.: 55679-0015WO3

[0493] Example 7. SPR Binding of EpCAM / CLEC5A Bispecific Antibody to Human EpCAM SPR analysis was used to evaluate if EpCAM / CLEC5A bispecific antibodies can cross-react with human EpCAM protein. SPR was performed by Biacore 8000+. 5pg / mL of antibody was immobilized on Series S Sensor Chip CM5 by amine coupling. The analyte hEpCAM (Aero, EPM-H5223) with different concentrations (0, 46.88, 93.75, 187.5, 375, 750, 1500nM) were injected respectively for an association phase of 120 s, followed by 300 s dissociation. As shown in the table 4, EpCAM / CLEC5A bispecific antibodies were found to bind human EpCAM with around E-07 M affinity.

[0494] Table 4: SPR binding of EpCAM / CLEC5Abispecific antibody to human EpCAM

[0495]

[0496] Example 8. Binding of EpCAM / CLEC5A bispecific antibodies to U937 Cells

[0497] Cell binding of EpCAM / CLEC5A bispecific antibodies to human monocytic cell line U937 (AddexBio, Cat# C0003005) was assessed. About 50,000 U937 cells were incubated with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson, Co.). Graphs were generated using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in theAtorney Docket No.: 55679-0015WO3

[0498] table 5 and FIGs. 5A-5E, all EpCAM / CLEC5A bispecific antibodies can bind to U937 (CLEC5A+) cells with various affinities.

[0499] Table 5: EpCAM / CLEC5 A bispecific antibodies binding to U937 (CLEC5A+) cells

[0500]

[0501] Example 9. Binding of EpCAM / CLEC5A bispecific antibodies to Human Monocytes Cell binding of AF488-conjugated EpCAM / CLEC5A bispecific antibodies to CD14+ monocytes was assessed. About 100,000 PBMCs (Donor D2343) were stained with fixable live / dead Zombia Aqua (Biolegend, Cat 77143,) for 20 min, and washed. Non-specific binding to Fc was blocked with human TruStain FcX (Biolegend, Cat 422304) for 30 min. The cells were incubated with PercpCy5.5-labeled anti-human CD14 (BD Biosciences, Cat 550787) combined with serially diluted AF488-conjugated EpCAM / CLEC5A bispecific antibodies in FACS buffer (PBS + 10% HI FBS + 0.5% BSA) for 30 minutes on ice. For analysis, the mean fluorescence intensity (MFI of AF488) of each antibody on CD14+ monocytes was determined using FlowJo software (Version 10.9.1, Becton Dickinson Co.) and plotted using GraphPad Prism software (Version 9.4.1, GraphPad Software Inc.). As shown in FIG. 6, all tested EpCAM / CLEC5A bispecific antibodies can bind to U937 (CLEC5A+) cells with various affinities.

[0502] Example 10. Binding of EpCAM / CLEC5A bispecific antibodies to Human M0 macrophages

[0503] Cell binding of EpCAM / CLEC5A bispecific antibodies to human monocyte-derived macrophages (MDMs) was assessed. Monocytes were isolated using the human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058) and differentiated to macrophages (MDMs) with 50 ng / mL of macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) in RPMI with 10% HI-FBS for 7 days. After 7 days, macrophages were harvested and 50,000 cells were plated per well in 96 well plates to analyze cell binding. Cells were stained withAtorney Docket No.: 55679-0015WO3

[0504] fixable live / dead Zombia Aqua (Biolegend, Cat 77143,) for 20 min, and washed. Non-specific binding to Fc was blocked with 10% goat serum (Southern Biotech, Cat 0060-01) for 30 min. The cells were stained with serially diluted EpCAM / CLEC5A bispecific antibodies in FACS buffer (PBS + 10% HI FBS + 0.5% BSA) for 30 minutes on ice. After staining with primary antibodies, cells were washed twice and incubated with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170) for 30 min. Flow cytometry analysis was performed on the Attune CytPix. Mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson Co.), and results were plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in FIG. 7, all tested EpCAM / CLEC5A bispecific antibodies can bind to M0 (CLEC5A+) cells with various affinities.

[0505] Example 11. Binding of EpCAM / CLEC5A bispecific antibodies to Human M2 macrophages

[0506] Binding abilities of different EpCAM / CLEC5A bispecific antibodies to M2 macrophages were evaluated. CD14+ monocytes (purified from human PBMC donor #DC233432 with EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058)) were differentiated to macrophages with 50 ng / mL of macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057). On Day 6, 25ng / ml IL- 10 (StemCell Technologies, Cat# 78024) was added to polarize the cell. Then, next day, M2 macrophages were obtained. In order to exclude the influence of M2 macrophage’s Fc receptors on the experiment, the antibodies were labeled with AF488 using Alexa Fluor™ 488 Antibody Labeling Kit (Invitrogen, Cat#: A20181). About 100,000 M2 macrophages were incubated with serially diluted EpCAM / CLEC5A bispecific antibodies (1 to 3 dilutions from 100 nM) in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice. For analysis, the mean fluorescence intensity (MFI of AF488) of each antibody was determined and plotted using GraphPad Prism software (Version 7.00; GraphPad Software Inc.). As shown in FIG. 8, different EpCAM / CLEC5A bispecific antibodies can bind M2 macrophages (CLEC5A+) cells.Atorney Docket No.: 55679-0015WO3

[0507] Example 12. Binding of EpCAM / CLEC5A bispecific antibodies to DLD-1

[0508] Binding abilities of different EpCAM / CLEC5 A bispecific antibodies to DLD-1 cells (EpCAM+) were evaluated. About 100,000 DLD-1 cells (ATCC, CCL-221) were incubated with serially diluted EpCAM / CLEC5A bispecific antibodies (1 to 3 dilutions from 10 nM) in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice and detected with anti -human IgG Fc PE secondary antibody (Invitrogen, Cat#: 12-4998-82). For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined and plotted using GraphPad Prism software (Version 7.00; GraphPad Software Inc.). As shown in FIG. 9A and the table 6A, the tested EpCAM / CLEC5Abispecific antibodies all had high binding affinity to DLD-1 cells.

[0509] Table 6A: EpCAM / CLEC5Abispecific antibodies binding to DLD-1

[0510]

[0511] Cell binding of EpCAM / CLEC5A bispecific antibodies to DLD1 cells (ATCC, Cat CCL-221) was assessed. About 50,000 DLD1 cells were stained with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with antihuman IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109- 116-170), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 6B and FIGs. 9B-9D, all EpCAM / CLEC5A bispecific antibodies can bind to DLD1 (EpCAM+) cells with various affinities depending on antibody clone and antibody format (2+2, 2+1, 1+1).

[0512] Table 6B: EpCAM / CLEC5Abispecific antibodies binding to DLD-1Attorney Docket No.: 55679-0015WO3

[0513]

[0514] Cell binding of EpCAM / CLEC5A bispecific antibodies to DLD1 cells (ATCC, Cat CCL-221) was assessed. About 50,000 DLD1 cells were stained with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with antihuman IgG PE secondary antibody (Invitrogen, Cat 12-4998-82), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using Flow Jo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 6C and FIG. 9E, all tested EpCAM / CLEC5A bispecific antibodies bind to DLD1 (EpCAM+) cells with similar affinity.

[0515] Table 6C: EpCAM / CLEC5Abispecific antibodies binding to DLD-1

[0516]

[0517] Atorney Docket No.: 55679-0015WO3

[0518] Example 13. Binding of EpCAM / CLEC5A bispecific antibodies to MCF7

[0519] Cell binding of EpCAM / CLEC5A bispecific antibodies to MCF7 cells (ATCC, Cat HTB-22) was assessed. About 50,000 MCF7 cells were stained with serially diluted antibodies in FACS buffer (PBS +2%BSA+2mM EDTA) for 45 minutes on ice. After incubation, cells were washed twice, stained with anti -human IgG PE secondary antibody (Invitrogen, Cat 12-4998-82), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using Flow Jo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 7 and FIG. 10, all tested EpCAM / CLEC5Abispecific antibodies bind to MCF7 (EpCAM+) cells with different affinities depending on anti-EpCAM antibody clone.

[0520] Table 7: EpCAM / CLEC5Abispecific antibodies binding to MCF7

[0521]

[0522] Example 14. Binding of EpCAM / CLEC5A bispecific antibodies to HCT116

[0523] Cell binding of EpCAM / CLEC5A bispecific antibodies to HCT116 cells (ATCC, Cat CCL-247) was assessed. About 50,000 HCT116 cells were stained with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with anti-human IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 8 and FIG. 11, all EpCAM / CLEC5A bispecific antibodies bind to HCT116 (EpCAM+) cells with different affinities.

[0524] Table 8: EpCAM / CLEC5Abispecific antibodies binding to HCT116Attorney Docket No.: 55679-0015WO3

[0525]

[0526] Example 15. Binding of EpCAM / CLEC5A bispecific antibodies to SKBR3

[0527] Cell binding of EpCAM / CLEC5A bispecific antibodies to SKBR3 cells (ATCC, Cat HTB-30) was assessed. About 50,000 SKBR3 cells were stained with serially diluted antibodies in FACS buffer (PBS +2%BSA+2mM EDTA) for 45 minutes on ice. After incubation, cells were washed twice, stained with anti-human IgG PE secondary antibody (Invitrogen, Cat 12-4998-82), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using Flow Jo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 9 and FIG. 12, all EpCAM / CLEC5A bispecific antibodies bind to SKBR3 (EpCAM+) cells with variable affinities.

[0528] Table 9: EpCAM / CLEC5Abispecific antibodies binding to SKBR3

[0529]

[0530] Example 16. Binding of EpCAM / CLEC5A bispecific antibodies to T47D

[0531] Cell binding of EpCAM / CLEC5A bispecific antibodies to T47D cells (ATCC, Cat HTB-133) was assessed. About 50,000 T47D cells were stained with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with antihuman IgG PE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170), and washed again.Atorney Docket No.: 55679-0015WO3

[0532] Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 10 and FIG. 13, all EpCAM / CLEC5A bispecific antibodies bind to T47D (EpCAM+) cells with variable affinities.

[0533] Table 10: EpCAM / CLEC5 A bispecific antibodies binding to T47D

[0534]

[0535] Example 17. Binding of EpCAM / CLEC5A bispecific antibodies to SKOV3

[0536] Cell binding of EpCAM / CLEC5A bispecific antibodies to SKOV3 cells (ATCC, CatHTB-77) was assessed. About 50,000 SKOV3 cells were stained with serially diluted antibodies in FACS buffer (PBS +2%BSA+2mM EDTA) for 45 minutes on ice. After incubation, cells were washed twice, stained with anti -human IgG PE secondary antibody (Invitrogen, Cat 12-4998-82), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using Flow Jo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 11 and FIGs. 14A-14B, all EpCAM / CLEC5A bispecific antibodies bind to SKOV3 (EpCAM+) cells with different affinities.

[0537] Table 11: EpCAM / CLEC5 A bispecific antibodies binding to SKOV3

[0538]

[0539] Atorney Docket No.: 55679-0015WO3

[0540]

[0541] Example 18. Binding of EpCAM / CLEC5A bispecific antibodies to A549

[0542] Cell binding of EpCAM / CLEC5A bispecific antibodies to A549 cells (ATCC, Cat CCL-185) was assessed. About 50,000 A549 cells were stained with serially diluted antibodies in FACS buffer (PBS + 0.5% BSA) for 30 minutes on ice. After incubation, cells were washed twice, stained with antihuman IgGPE secondary antibody (Jackson ImmunoResearch, Cat#: 109-116-170), and washed again. Flow cytometric analysis was performed on a Cytek Northern Lights flow cytometer. For analysis, the mean fluorescence intensity (MFI of PE) of each antibody was determined using FlowJo software (Version 10.9.1, Becton Dickinson, Co.) and then plotted using GraphPad Prism software (Version 9.4.1; GraphPad Software Inc.). As shown in the table 12 and FIG. 15, high affinity EpCAM / CLEC5A bispecific antibodies bind to A549 (EpCAM+) cells, while low affinity EpCAM / CLEC5A bispecific antibodies do not.

[0543] Table 12: EpCAM / CLEC5Abispecific antibodies binding to A549

[0544]

[0545] Example 19. EpCAM / CLEC5A bispecific antibodies mediated DLD-1 cell killing by PBMC

[0546] To screen for antibodies clones and compare the function of different formats and the impact of linker amount on function, EpCAM / CLEC5A bispecific antibody-mediated tumor killing was tested with a coculture system of fresh human PBMCs and DLD1 cells. Fresh human PBMCs were isolated from donated health human blood from SBC (Stanford Blood Center) and U-bottom 96 well plates are used. DLD1 cells were stained with CFSE staining buffer (Invitrogen). 2 x 104CFSE-stained DLD1 cells in 50 pl medium were added into each designated well. 2 x io5PBMCs in 130 pl medium were added into each designated well. 20 pl of serially diluted testing antibodies in complete culture mediaAttorney Docket No.: 55679-0015WO3

[0547] were added into each designated well, and total final volume for each well was 200 pl. The plate with well-mixed cells was incubated at 37°C with 5% CO2. Samples were collected 24 hours after the incubation. Cells were washed one time with PBS-calcium / magnesium, then 100 pl 0.25% trypsin was added into each well and the plate was incubated at 37°C for 15 minutes. 100 pl complete culture medium was added to stop the trypsin digestion. The cells suspension was aspirated up and down to dissociate DLD1 cells completely. Then, the cells suspensions were transferred to a V-bottom 96 well plate. The cells were washed one more time with PBS. Cells were stained with NIR live and dead dye for 15 minutes on ice. Cells were analyzed with a Cytek® cytometer (live DLD1 cells gating: CFSE+ NIR-). Killing efficacy was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table 13 and FIGs. 16A-16C., EpCAM / CLEC5A bispecific antibodies showed approximately 50%~80% max killing.

[0548] Table 13: EpCAM / CLEC5Abispecific antibodies mediated DLD-1 cell killing by PBMC

[0549]

[0550] Atorney Docket No.: 55679-0015WO3

[0551] Example 20. EpCAM / CLEC5A Bispecific Antibodies Mediated DLD-1 Cell Killing by MO Macrophages

[0552] The effects of EpCAM / CLEC5A bispecific antibodies with different structures to the cell killing of target cancer DLD1 cells (EpCAM+) by effector macrophages (CLEC5A+) were evaluated. CD14+ monocytes (purified from human PBMC donors #120, #654, #120, #838, #033, #234, #1148 and #3199 with Easy Sep™ Human Monocyte Enrichment Kit without CD 16 Depletion (StemCell Technologies, Cat#: 19058)) were differentiated to M0 macrophages with 50 ng / mL of macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. DLD1 cells were labeled with CFSE (ThermoFisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+ DLD1 (E:T ratio of 5:1) (E:T ratio of 3:1 for doner #3199) in the presence of serially diluted antibodies in complete RPMI media (with 10% heat-inactivated FBS and 5% penicillin / streptomycin) for 24 hours at 37°C. After incubation, the cells were washed with PBS -Calcium / magnesium and treated with 0.25% trypsin for 15 minutes on ice. The cells were collected and washed with PBS for one time, then cells were stained with NIR live and dead dye for 15 minutes on ice and were resuspended in 100 pl of FACS buffer and analyzed with a Cytek® cytometer. DLD1 cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume for all treatment conditions. Percentage of target cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIGs.17A-17H, all the tested EpCAM / CLEC5Abispecific antibodies with different EpCAM arms showed comparable efficacy of DLD1 killing. No significant difference in DLD1 killing was observed between Fc-enhanced format and Fc-silenced format.

[0553] Table 14: EpCAM / CLEC5Abispecific antibodies mediated DLD-1 cell killing by M0 macrophages

[0554]

[0555] Atorney Docket No.: 55679-0015WO3

[0556]

[0557] Atorney Docket No.: 55679-0015WO3

[0558]

[0559] After incubation, the supernatant was collected for cytokine analysis. TNFa and IL-6 levels in supernatants were very low.

[0560] Example 21. EpCAM / CLEC5A Bispecific Antibodies Mediated DLD-1 Cell Killing by Ml Macrophages

[0561] EpCAM / CLEC5A bispecific antibodies were evaluated in polarized monocyte derived Ml macrophage killing of target cancer cell line DLD-1. Monocytes were isolated from human PBMC donor 3199 using StemCell’s EasySep human monocyte isolation kit (cat # 19059) following the

[0562] manufacturer’s protocol. Monocytes were seeded at 1 x 106per mL to tissue culture (TC) treated plates in RPMI with 10% HI-FBS, IX glutamax & 50 ng / mL of human macrophage colony stimulating factor (M-CSF, StemCell cat # 78057) and cultured at 37°C in a 5% CO2 humidified incubator for 7 days for differentiation. On day 6, 50 ng / mL of human IFN-gamma (StemCell cat # 78020) was used to induce polarization into Ml macrophages. Target cells (cancer cell line DLD-1) were harvested and stained with 0.25 uM CFSE (Invitrogen cat # C34554) following the manufacturer’s protocol. Target DLD-1 cells were resuspended in RPMI with 10% HI-FBS & IX glutamax and 20,000 target cells were added to 96-well u-bottom TC treated plates containing Ml macrophage at an E:T ratio of 3:1. Following overnight incubation, cells were harvested with 0.05% trypsin, stained with L / D NIR (Invitrogen cat # L34975A, diluted 1 :2000 in PBS) on ice in the dark for 15 minutes, resuspended in 100 uL of FACS buffer (PBS with 2% HI-FBS & 0.5% BSA) and analysed on Attune Cytpix flow cytometer (60 pL was analysed). Total killing was calculated from live CFSE+ target DLD-1 cell counts inAtorney Docket No.: 55679-0015WO3

[0563] EpCAM / CLEC5 A treated wells compared to untreated wells in Flow Jo using the following formula: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group x 100. As shown in the table below and FIG. 18, all the tested EpCAM / CLEC5A bispecific antibodies with different G4S linkers showed comparable efficacy of DLD1 killing. No significant difference in DLD1 killing was observed between Fc-enhanced format and Fc-silenced format.

[0564] Table 15: EpCAM / CLEC5Abispecific antibodies mediated DLD-1 cell killing by Ml macrophages

[0565]

[0566] After incubation, the supernatant was collected for cytokine analysis. TNFa and IL-6 levels in supernatants were very low.

[0567] Example 22. EpCAM / CLEC5A Bispecific Antibodies Mediated DLD-1 Cell Killing by M2 Macrophages

[0568] EpCAM / CLEC5A bispecific antibodies were evaluated in polarized monocyte derived Ml macrophage killing of target cancer cell line DLD-1. Monocytes were isolated from human PBMC donor 3199 using StemCell’s EasySep human monocyte isolation kit (cat # 19059) following the

[0569] manufacturer’s protocol. Monocytes were seeded at 1 x 106per mL to tissue culture (TC) treated plates in RPMI with 10% HI-FBS, IX glutamax & 50 ng / mL of human macrophage colony stimulating factor (M-CSF, StemCell cat # 78057) and cultured at 37°C in a 5% CO2 humidified incubator for 7 days for differentiation. On day 6, 25 ng / mL of IL-10 (StemCell cat # 78024) was used to induce polarization into M2 macrophages. Target cells (cancer cell line DLD-1) were harvested and stained with 0.25 uM CFSE (Invitrogen cat # C34554) following the manufacturer’s protocol. Target DLD-1 cells wereAtorney Docket No.: 55679-0015WO3

[0570] resuspended in RPMI with 10% HI-FBS & IX glutamax and 20,000 target cells were added to 96-well u-bottom TC treated plates containing Ml macrophage at an E:T ratio of 3:1. Following overnight incubation, cells were harvested with 0.05% trypsin, stained with L / D NIR (Invitrogen cat # L34975A, diluted 1 :2000 in PBS) on ice in the dark for 15 minutes, resuspended in 100 uL of FACS buffer (PBS with 2% HI-FBS & 0.5% BSA) and analysed on Attune Cytpix flow cytometer (60 pL was analysed). Total killing was calculated from live CFSE+ target DLD-1 cell counts in EpCAM / CLEC5A treated wells compared to untreated wells in FlowJo using the following formula: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in nontreatment group x 100. As shown in the table below and FIG. 19, all the tested EpCAM / CLEC5A bispecific antibodies with different G4S linkers showed comparable efficacy of DLD1 killing. No significant difference in DLD1 killing was observed between Fc-enhanced format and Fc-silenced format.

[0571] Table 16: EpCAM / CLEC5Abispecific antibodies mediated DLD-1 cell killing by M2 macrophages

[0572]

[0573] After incubation, the supernatant was collected for cytokine analysis. TNFa and IL-6 levels in supernatants were very low.

[0574] Example 23. EpCAM / CLEC5A Bispecific Antibodies Mediated SKBR3 Cell Killing by M0 Macrophages

[0575] EpCAM / CLEC5 Abispecific antibodies with different EpCAM antibody clones were evaluated for their effects in cell killing of target cancer cell line SK-BR-3 by M0 monocyte derived macrophage (MDM). Monocytes were isolated from human PBMC donor 0112 using StemCell’s EasySep monocyte isolation kit without CD16 depletion (cat # 19058) following the manufacturer’s protocol. Monocytes were seeded at HIO6per mL to tissue culture (TC) treated plates in RPMI with 10% HI-Atorney Docket No.: 55679-0015WO3

[0576] FBS, Ixglutamax and 50 ng / mL of human macrophage colony stimulating factor (M-CSF, StemCell cat # 78057) and cultured at 37°Cin a 5% CO2 humidified incubator for 7 days for differentiation. On day 7, MDMs were harvested with enzyme free dissociation buffer (Millipore cat # S-014-M), resuspended in RPMI with 10% HI-FBS and Ixglutamax. 100,000 MDMs were added to 96-well u-bottom TC treated plates and incubated at 37 degree C in a 5% CO2 humidified incubator for one hour. Target cells (cancer cell line SK-BR-3) were harvested and stained with 0.25 pM CFSE (Invitrogen cat # C34554) following the manufacturer’s protocol. Target cells were resuspended in RPMI with 10% HI-FBS and Ixglutamax and about 20,000 target cells were added to 96-well u-bottom TC treated plates containing MDMs at an E:T ratio of 5:1. A 3 -fold 11 -point serial dilution of EpCAM / CLEC5A antibodies starting at 10 nM in RPMI with 10% HI-FBS and Ixglutamax were added to 96-well u-bottom TC treated plates containing MDMs and target cancer cells and incubated at 37°C in a 5% CO2 humidified incubator overnight. Following overnight incubation, cells were harvested, stained with L / D NIR (Invitrogen cat # L34975A, diluted 1:2000 in PBS) on ice in the dark for 15 minutes, resuspended in 100 pL of FACS buffer (PBS with 2% HI-FBS and 0.5% BSA) and analysed on Attune Cytpix flow cytometer (60 pL was analysed). Total killing was calculated from live CFSE+ target SK-BR-3 cell counts in EpCAM / CLEC5 A treated wells compared to untreated wells in FlowJo using the following formula: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIGs.

[0577] 20A-20B, EC50s ranged from 14-27 pM with max killing ranging from 71-96%. EpCAM3 / 5C72+2A Fc-E and EpCAM2 / 5C72+2A Fc-E had the highest max killing at 95.9% and 93.4%, respectively. Table 17A: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by M0 macrophages

[0578]

[0579] Atorney Docket No.: 55679-0015WO3

[0580]

[0581] To assess the abilities of EpCAM / CLEC5A bispecific antibodies with different EpCAM affinity to kill low level EpCAM tumor cells, SKBR killing by effector macrophages (CLEC5A+) were evaluated. CD14+ monocytes (purified from human PBMC donors #859, #1148 with EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058)) were differentiated to MO macrophages with 50 ng / mL macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. SKBR3 cells were labeled with CFSE (ThermoFisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+ SKBR3 (E:T ratio of 3:1) in the presence of serially diluted antibodies in complete RPMI media (with 10% heat-inactivated FBS and 5% penicillin / streptomycin) for 24 hours at 37°C. After incubation, the cells were washed with PBS -Calcium / magnesium and treated with 0.25% trypsin for 15 minutes on ice. The cells were collected and washed with PBS for one time, then cells were stained with NIR live and dead dye for 15 minutes on ice and were resuspended in 100 pl of FACS buffer and analyzed with a Cytek® cytometer. SKBR3 cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume for all treatment conditions. Percentage of target cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIGs. 20C-20E, all the EpCAM / CLEC5A bispecific antibodies displayed potent SKBR3 killing.

[0582] Table 17B: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by M0 macrophages

[0583]

[0584] Attorney Docket No.: 55679-0015WO3

[0585]

[0586] To estimate breast cancer cell (SK-BR-3) killing efficacy of different myeloid cell engager EpCAM / CLEC5 A bispecific antibodies in MO macrophages at different E:T ratios, human monocytes were isolated from PBMCs (donors #DC233432) by EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058). Isolated monocytes were induced by 50 ng / mLM-CSF (StemCell Technologies, Cat#: 78057). On Day 7, MO macrophages were obtained. Different E:T ratios of MO macrophages and SK-BR-3 cells were incubated with the myeloid cell engagers to test their functions in MO macrophages. The myeloid cell engagers (EpCAM / CLEC5A bispecific antibodies) were serially diluted. SK-BR-3 cells were stained with CFSE first, then spiked into M0 macrophages (E:T=3 : 1, or E:T=1 : 1) in a 96-well-round-bottom plate (Fixed SK-BR-3 counts: 2 x 104cells / well), which were further incubated with serially diluted EpCAM / CLEC5A bispecific antibodies (1 to 3 dilutions from 10 nM). After 24 hours of incubation, the plate was spun down. Cells were harvested by enzyme free cell dissociation buffer (Sigma, Cat#: S-014-B). Then, cells were stained with live / dead staining dye, and then analyzed on a CytoFLEX LX flow cytometer. Percentage of cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIGs. 20F-20G, all the myeloid engagers showed potent SK-BR-3 cell killing effects in M0 macrophages in vitro at different E:T ratios. The maximum specific tumor cell killing was aroundAttorney Docket No.: 55679-0015WO3

[0587] 95%-98% at E:T=3:1, 75%-89% at E:T=1:1 for SK-BR-3 cells at 24 hours, indicating that myeloid cell engagers can promote target cell killing efficiently at low E: T ratios of MO macrophages to target cells.

[0588] Table 17C: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by MO macrophages

[0589]

[0590] Example 24. EpCAM / CLEC5A Bispecific Antibodies Mediated SKBR3 Cell Killing by M2 Macrophages

[0591] Breast cancer cell (SK-BR-3) killing efficacy of different myeloid cell engager EpCAM / CLEC5A bispecific antibodies in M2 macrophages at different E:T ratios was assessed. Human monocytes were isolated from PBMCs (donors #DC233432) by EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058). Isolated monocytes were induced by 50 ng / mL M-CSF (StemCell Technologies, Cat#: 78057). On Day 6, 25ng / ml IL- 10 (StemCell Technologies, Cat# 78024) was used to polarize the cell. Then, the next day M2 macrophages were obtained. Different E:T ratios of M2 macrophages and SK-BR-3 cells were incubated with the myeloid cell engagers to test their functions in M2 macrophages. The myeloid cell engagers (EpCAM / CLEC5A bispecific antibodies) were serially diluted. SK-BR-3 cells wereAttorney Docket No.: 55679-0015WO3

[0592] stained with CFSE first, then spiked into M2 macrophages (E:T=3 : 1, or E:T=1 : 1) in a 96-well-round-bottom plate (Fixed SK-BR-3 counts: 2 / IO4cells / well), which were further incubated with serially diluted EpCAM / CLEC5A bispecific antibodies (1 to 3 dilutions from 10 nM). After 24 hours of incubation, the plate was spun down. Cells were harvested by enzyme free cell dissociation buffer (Sigma, Cat#: S-014-B). Then, cells were stained with live / dead staining dye, and then analyzed on a CytoFLEX LX flow cytometer. Percentage of cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in nontreatment group X 100. As shown in the table below and FIGs. 21A-21B , All the tested myeloid engagers showed potent SK-BR-3 cell killing effects in M2 macrophages in vitro at different E:T ratios. The maximum specific tumor cell killing was around 95%-99% at E:T=3:1, 90%-95% at E:T=1:1 for SK-BR-3 cells at 24 hours, indicating that myeloid cell engagers can promote target cell killing efficiently at low E: T ratios of M2 macrophages to target cells.

[0593] Table 18: EpCAM / CLEC5Abispecific antibodies mediated SKBR3 cell killing by M2 macrophages

[0594]

[0595] Atorney Docket No.: 55679-0015WO3

[0596] Example 25. EpCAM / CLEC5A Bispecific Antibodies Mediated MCF7 Cell Killing by MO Macrophages

[0597] To test the abilities of EpCAM / CLEC5Abispecific antibodies to kill breast cancer cells, MCF7 cells killing by effector macrophages (CLEC5A+) were evaluated. CD14+ monocytes (purified from human PBMC donors #654, #859 with EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058)) were differentiated to MO macrophages with 50 ng / mL of macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. MCF7 cells were labeled with CFSE (ThermoFisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+ MCF7 (E:T ratio of 3:1) in the presence of serially diluted antibodies in complete RPMI media (with 10% heat-inactivated FBS and 5% penicillin / streptomycin) for 24 hours at 37°C. After incubation, the cells were washed with PBS-Calcium / magnesium and treated with 0.25% trypsin for 15 minutes on ice. The cells were collected and washed with PBS for one time, then cells were stained with NIR live and dead dye for 15 minutes on ice and were resuspended in 100 pl of FACS buffer and analyzed with a Cytek® cytometer. MCF7 cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume for all treatment conditions. Percentage of target cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100.. As shown in the table below and FIGs.22A-22B, Bispecific antibodies with EpCAM arm of EpCAM3 and EpCAMl showed higher killing efficacy. Table 19: EpCAM / CLEC5Abispecific antibodies mediated MCF7 cell killing by M0 macrophages

[0598]

[0599] Atorney Docket No.: 55679-0015WO3

[0600] Example 26. EpCAM / CLEC5A Bispecific Antibodies Mediated HCT116 Cell Killing by MO Macrophages

[0601] EpCAM / CLEC5A bispecific antibody-mediated cancer cell killing of HCT116 (ATCC, Cat# CCL-247) cancer cells by MO monocyte derived macrophage (MDM) was assessed. Monocytes were isolated from human PBMC donor 120 (Stanford Blood Center) using StemCell’s EasySep monocyte isolation kit without CD16 depletion (cat # 19058) following the manufacturer’s protocol. Monocytes were seeded at l*106per mL to tissue culture (TC) treated plates in RPMI with 10% HI-FBS, Ixglutamax and 50 ng / mL of human macrophage colony stimulating factor (M-CSF, StemCell cat # 78057) and cultured at 37°C in a 5% CO2 humidified incubator for 7 days for differentiation. On day 7, MDMs were harvested with enzyme free dissociation buffer (Millipore cat # S-014-M), resuspended in RPMI with 10% HI-FBS and 100,000 MDMs were added to 96-well u-bottom TC treated plates and incubated at 37°C in a 5% CO2 humidified incubator for one hour. Target cancer cell line HCT116 were harvested and stained with 0.25 pM CFSE (Invitrogen cat # C34554) following the manufacturer’s protocol. Target cells were resuspended in RPMI with 10% HI-FBS and about 20,000 target cells were added to 96-well u-bottom TC treated plates containing MDMs for an E:T ratio of 5:1. A3-fold 11 -point serial dilution of EpCAM / CLEC5 A antibodies starting at lO nM in RPMI with 10% HI-FBS were added to 96-well u-bottom TC treated plates containing MDMs and target cancer cells and incubated at 37°C in a 5% CO2 humidified incubator overnight. Following overnight incubation, cells were harvested using 0.05% trypsin (Gibco, cat# 25300-054) for 10 min at 37°C, stained with L / D NIR (Invitrogen cat # L34975A, diluted 1:2000 in PBS) on ice in the dark for 15 minutes, resuspended in 100 pL of FACS buffer (PBS with 0.5% BSA) and analysed on a Cytek® flow cytometer. Total killing was calculated from live CFSE+ target cell counts in EpCAM / CLEC5Atreated wells compared to untreated wells in FlowJo using the following formula: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in nontreatment group X 100. As shown in the tables below and FIGs. 23A-23B, EC50s ranged from 5 - 60 pM and max killing from 86 - 100%.

[0602] Table 20A: EpCAM / CLEC5 A bispecific antibodies mediated HCT116 cell killing by M0 macrophages

[0603]

[0604] Atorney Docket No.: 55679-0015WO3

[0605]

[0606] Table 20B: EpCAM / CLEC5Abispecific antibodies mediated HCT116 cell killing by MO macrophages

[0607]

[0608] Example 27. EpCAM / CLEC5A Bispecific Antibodies Mediated T47D Cell Killing by MO Macrophages

[0609] The effects of EpCAM / CLEC5 A bispecific antibodies with different EpCAM antibody clones were evaluated in MO monocyte derived macrophage (MDM) killing of target cancer cell line T47D. Monocytes were isolated from human PBMC donor 112 using StemCell’s EasySep monocyte isolation kit without CD16 depletion (cat # 19058) following the manufacturer’s protocol. Monocytes were seeded at 1 x 106per mL to tissue culture (TC) treated plates in RPMI with 10% HI-FBS, 1 xglutamax and 50 ng / mL of human macrophage colony stimulating factor (M-CSF, StemCell cat # 78057) and cultured at 37 °C in a 5% CO2 humidified incubator for 7 days for differentiation. On day 7, MDMs were harvested with enzyme free dissociation buffer (Millipore cat # S-014-M), resuspended in RPMI with 10% HI-FBS and 1 xglutamax, 100,000 MDMs were added to 96-well u-bottom TC treated plates and incubated at 37 °C in a 5% CO2 humidified incubator for one hour. Target cancer cell line T47D were harvested and stained with 0.25 pM CFSE (Invitrogen cat # C34554) following the manufacturer’s protocol. Target cells were resuspended in RPMI with 10% HI-FBS and 1 xglutamax and about 20,000 target cells were added to 96-well u-bottom TC treated plates containing MDMs for an E:T ratio of 5:1. A3-fold 11-point serial dilution of EpCAM / CLEC5A antibodies starting at 10 nM in RPMI with 10% HI-FBS and 1 xglutamax were added to 96-well u-bottom TC treated plates containing MDMs and target cancer cells and incubated at 37 °C in a 5% CO2 humidified incubator overnight. Following overnight incubation, cells were harvested, stained with L / D NIR (Invitrogen catAttorney Docket No.: 55679-0015WO3

[0610] # L34975A, diluted 1 :2000 in PBS) on ice in the dark for 15 minutes, resuspended in 100 pL of FACS buffer (PBS with 2% HI-FBS and 0.5% BSA) and analysed on Attune Cytpix flow cytometer. Total killing was calculated from live CFSE+ target T47D cell counts in EpCAM / CLEC5A treated wells compared to untreated wells in FlowJo using the following formula: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIG. 24A, EC50s ranged from 20-70 pM with max killing ranging from 62-94%. EpCAM2 / 5C72+2AFc-E and EpCAM3 / 5C72+2AFc-E had the highest max killing at 93.9% and 90.5%, respectively.

[0611] Table 21 : EpCAM / CLEC5A bispecific antibodies mediated T47D cell killing by M0 macrophages

[0612]

[0613] To test the abilities of EpCAM / CLEC5 Abispecific antibodies to kill breast cancer cells, MCF7 cells killing by effector macrophages (CLEC5A+) were evaluated. CD14+ monocytes (purified from human PBMC donors #654, #859 with EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058)) were differentiated to M0 macrophages with 50 ng / mL of macrophage colony-stimulating factor (M-CSF, StemCell Technologies, Cat#: 78057) for 7 days. MCF7 cells were labeled with CFSE (ThermoFisher, Cat#: C34554). About 100,000 macrophages were incubated with 20,000 CFSE+ MCF7 (E:T ratio of 3:1) in the presence of serially diluted antibodies in complete RPMI media (with 10% heat-inactivated FBS and 5%Atorney Docket No.: 55679-0015WO3

[0614] penicillin / streptomycin) for 24 hours at 37°C. After incubation, the cells were washed with PBS-Calcium / magnesium and treated with 0.25% trypsin for 15 minutes on ice. The cells were collected and washed with PBS for one time, then cells were stained with NIR live and dead dye for 15 minutes on ice and were resuspended in 100 pl of FACS buffer and analyzed with a Cytek® cytometer. MCF7 cells were gated as CFSE+ by FACS, and the absolute cell count of CFSE+ cells was obtained by collecting a fixed volume for all treatment conditions. Percentage of target cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table above and FIGs.24B-24C, bispecific antibodies with EpCAM arm of EpCAM3 and EpCAMl showed higher killing efficacy.

[0615] Example 28. EpCAM / CLEC5A Bispecific Antibodies Mediated LNCaP Cell Killing by M0 Macrophages

[0616] The effects of different myeloid cell engager EpCAM / CLEC5A bispecific antibodies on cell killing of prostate cancer cell (LNCaP) by M0 macrophages were assessed. Human monocytes were isolated from PBMCs (donors #DS21806) by Easy Sep™ Human Monocyte Enrichment Kit without CD16 Depletion (StemCell Technologies, Cat#: 19058). Isolated monocytes were induced by 50 ng / mLM-CSF (StemCell Technologies, Cat#: 78057). On Day 7, M0 macrophages were obtained. The E:T ratio of M0 macrophages and LNCaP cells is 5:1. M0 macrophages and LNCaP cells were incubated with the myeloid cell engagers to test their functions in M0 macrophages. The myeloid cell engagers (EpCAM / CLEC5A bispecific antibodies) were serially diluted. LNCaP cells were stained with CFSE first, then spiked into M0 macrophages in a 96-well-round-bottom plate (Fixed LNCaP counts: 2 x io4cells / well), which were further incubated with serially diluted EpCAM / CLEC5A bispecific antibodies (1 to 3 dilutions from 10 nM). After 24 hours of incubation, the plate was spun down. Cells were harvested by enzyme free cell dissociation buffer (Sigma, Cat#: S-014-B), then, cells were stained with live / dead staining dye, and then processed on a CytoFLEX LX flow cytometer. Percentage of cell killing was calculated as: (live target cell number in non-treatment group-live target cell number in treatment group) / live target cell number in non-treatment group X 100. As shown in the table below and FIG. 25, All the tested myeloid engagers showed potentAttorney Docket No.: 55679-0015WO3

[0617] LNCaP cell killing effects in MO macrophages in vitro at 5:1 ratio. The maximum specific tumor cell killing was around 68%-72% for LNCaP cells at 24 hours.

[0618] Table 22: EpCAM / CLEC5Abispecific antibodies mediated LNCaP cell killing by MO macrophages

[0619]

[0620] Example 29: Efficacy evaluation of EpCAM / CLEC5A bispecific antibody in the treatment of B-hEpCAM MC38 colon carcinoma mouse model (Study 1)

[0621] The anti -turn or effects of an EpCAM / CLEC5Abispecific antibodies were examined in a mouse model using B-hEpCAM MC38 colon carcinoma cells and C57BL / 6 mice. Female C57BL / 6 mice, 6-8 weeks old, were obtained and used in the experiments. The MC38 murine colon carcinoma cells were genetically modified to express human EpCAM and named B-hEpCAM MC38. Cells were maintained in vitro as a monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. A total of 70 C57BL / 6 mice were subcutaneously injected with B-hEpCAM MC38 tumor cells (5*105cells / mouse) in 0.1ml PBS in the right front flank to initiate tumor development. When the average tumor volume reaches approximately 80-100 mm3, 37 mice were selected based on tumor volume and body weight, and randomly assigned into 6 study groups, with 8 mice in the first 4 groups and 5 mice in the last group. On the grouping day (Day 0), treatment was initiated, and the dosing regimen was shown in the table below:

[0622] Table 23 : dosing regimen of Study 1

[0623]

[0624] Atorney Docket No.: 55679-0015WO3

[0625]

[0626] The tumors were measured twice per week in two dimensions using a caliper, and the volume were calculated using the formula: V = (L * W * W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Graphing and statistical analysis was performed using GraphPad Prism program, two-way ANOVA with Dunnett multiple comparisons test for tumor growth. As shown in the FIG.27, the tumor volume in the PBS control group and the groups treated with CLEC5AFc-S surrogate, EpCAM Fc-S surrogate, or EpCAM Fc surrogate increased substantially over the 20-day observation period, showing a marked trend of rapid tumor growth, while the group treated with EpCAM / CLEC5AFc-S surrogate exhibited potent and significant tumor growth inhibition. A complete remission was observed in some mice treated with EpCAM / CLEC5A Fc-S surrogate. At the end of the study (Day 48), the treatment with EpCAM / CLEC5AFc-S surrogate led to 4 tumor-free mice, while no tumor-free mouse was found in other groups (FIG 28A-28E). The results indicated that EpCAM / CLEC5AFc-S surrogate exerts a far superior anti-tumor effect.

[0627] Example 30: B-hEpCAM MC38 colon carcinoma mouse model rechallenge testing after treatment of EpCAM / CLEC5A bispecific antibody

[0628] Following Example 29, after treatment with the EpCAM / CLEC5A bispecific antibody, 2 tumor-free mice were selected. Subsequently, tumor-free C57BL / 6 mice were subcutaneously injected with B-hEpCAM MC38 tumor cells (5 x 105cells per mouse) in 0.1 mL PBS into the left front flank to initiate tumor development (rechallenge procedure). As shown in FIG 29, 28 days after rechallenging, no tumor growth was observed in the rechallenged group. In contrast, the control group (without prior EpCAM / CLEC5A bispecific antibody treatment) showed a marked increase in tumor volume over time. These results demonstrate that the anti-tumor activity induced by the EpCAM / CLEC5Abispecific antibody is long-lasting and potent.Atorney Docket No.: 55679-0015WO3

[0629] Example 31: Efficacy evaluation of EpCAM / CLEC5A bispecific antibody in the treatment of B-hEpCAM MC38 colon carcinoma mouse model with large tumors (Study 2)

[0630] The anti -tumor effects of an EpC AM / CLEC5 A bispecific antibody were examined in a mouse model using B-hEpCAM MC38 colon carcinoma cells and C57BL / 6 mice with large tumors. Female C57BL / 6 mice, 6-8 weeks old, were obtained and used in the experiments. The MC38 murine colon carcinoma cells were genetically modified to express human EpCAM and named B-hEpCAM MC38. Cells were maintained in vitro as a monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. C57BL / 6 mice were subcutaneously injected with B-hEpCAM MC38 tumor cells (5><105 / mouse) in 0.1ml PBS in the right front flank to initiate tumor development. When the average tumor volume reaches approximately 80-100 mm3, 32 mice were selected based on tumor volume and body weight, and randomly assigned into 4 study groups, with 8 mice in each group. Treatment dosing regimen was shown in the table below:

[0631] Table 24: dosing regimen of Study 2

[0632]

[0633] The tumors were measured twice per week in two dimensions using a caliper, and the volume were calculated as described in Example 29. As shown in FIG. 30, tumor volume in the PBS control group (Gl) increased rapidly over time, reaching -1400 mm3by day 25 after the first treatment. In contrast, mice treated with EpCAM / CLEC5A Fc-S surrogate (G2, G3, G4) showed significantly suppressed tumor growth. Tumor-free mice were observed in each of the G2, G3, and G4 groups (FIGs.Atorney Docket No.: 55679-0015WO3

[0634] 31A-31D). The results indicate that EpCAM / CLEC5 A bispecific antibody exhibits excellent efficacy against large tumors.

[0635] Example 32: Efficacy evaluation of EpCAM / CLEC5A bispecific antibody in the treatment of B-hEpCAM MC38 colon carcinoma mouse model at different dose levels (Study 3)

[0636] The anti-tumor effects of an EpCAM / CLEC5 A bispecific antibodies were examined in a mouse model using B-hEpCAM MC38 colon carcinoma cells and C57BL / 6 mice at different dose levels. Female C57BL / 6 mice, 6-8 weeks old, were obtained and used in the experiments. The MC38 murine colon carcinoma cells were genetically modified to express human EpCAM and named B-hEpCAM MC38. Cells were maintained in vitro as a monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. A total of 70 C57BL / 6 mice were subcutaneously injected with B-hEpCAM MC38 tumor cells (5*105cells / mouse) in 0.1ml PBS in the right front flank to initiate tumor development. When the average tumor volume reaches approximately 80-100 mm3, mice with tumors were randomly assigned to four groups: PBS group, 0.133 MPK group, 1.33 MPK group, and 13.3 MPK group. Tumor volume and body weight were measured at multiple time points post treatment.

[0637] The tumors were measured twice per week in two dimensions using a caliper, and the volume were calculated as described in Example 29. As shown in FIG. 31, FIGs. 32A-32D, treatment with EpCAM / CLEC5A bispecific antibody resulted in a dose-dependent inhibition of tumor growth. The highest dose (13.3 MPK) demonstrated the most potent anti-tumor activity, effectively stalling or reducing tumor volume throughout the observation period. As shown in FIG.33, no significant bodyweight loss was observed at any dose, implying that the tested doses of EpCAM / CLEC5 A bispecific antibody did not induce notable toxicity.

[0638] Example 33: EpCAM / CLEC5A bispecific antibody mediated tumor cells killing in malignant ascites

[0639] To estimate ascites endogenous tumor cells killing efficacy in malignant ascites by EpCAM / CLEC5Abispecific antibody. The endogenous tumor cells in ascites co-express both EpCAM and EGFR. Frozen ascites cells were recovered and resuspended in complete media (RPMI 1640+10% FBS). EpCAM / CLEC5Abispecific antibody were serially diluted. Recovered ascites cells were spiked into 96-well-round-bottom plate (Fixed ascites cells counts: 2xl05 / well), further incubated with serial diluted EpCAM / CLEC5A bispecific antibody, after 24hrs incubation, the plate were spin down,Attorney Docket No.: 55679-0015WO3

[0640] supernatant were aspirated, ascites cells were stained with live / dead staining, then blocked by Human TruStain FcX™ (Biolegend, CAT#422302), stained with APC anti-EGFR and FITC anti-CD324(E-Cadherin), then poccessed on CytoFLEX LX flow cytometry. Percent of ascites tumor cells killing was calculated as (live tumor cell (EGFR+ or CD324+) number in nontreatment group-live tumor cell (EGFR+ or CD324+) number in treatment group) / live tumor cell number in nontreatment group)* 100. The EpCAM / CLEC5A bispecific antibody showed potent endogenous ascites tumor cells killing effects.

[0641] Table 25. Some of the amino acid sequences described in the present disclosure.

[0642]

[0643] Atorney Docket No.: 55679-0015WO3

[0644]

[0645] Atorney Docket No.: 55679-0015WO3

[0646]

[0647] Atorney Docket No.: 55679-0015WO3

[0648]

[0649] Atorney Docket No.: 55679-0015WO3

[0650]

[0651] Atorney Docket No.: 55679-0015WO3

[0652]

[0653] Atorney Docket No.: 55679-0015WO3

[0654]

[0655] Atorney Docket No.: 55679-0015WO3

[0656]

[0657] Atorney Docket No.: 55679-0015WO3

[0658]

[0659] Atorney Docket No.: 55679-0015WO3

[0660]

[0661] Attorney Docket No.: 55679-0015WO3

[0662]

[0663] OTHER EMBODIMENTS

[0664] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 55679-0015WO3WHAT IS CLAIMED IS:

1. An antibody or antigen-binding fragment thereof, comprising:i) a first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is epithelial cell adhesion molecule (EpCAM); andii) a second antigen-binding domain that specifically binds to C-type lectin domain family 5 member A (CLEC5A).

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the first antigenbinding 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).

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the second antigen-binding domain is a single-chain fragment variable (scFv) domain, wherein the VH2 and VL2 are linked by a first linker.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the second antigen-binding domain is connected to the C-terminus of a light chain through a second linker.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the second antigen-binding domain is connected to the C-terminus of a heavy chain through a second linker.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, further comprising an Fc region.Attorney Docket No.: 55679-0015WO38. The antibody or antigen-binding fragment thereof of claim 7, wherein the C-terminus of the VH1 of the first antigen-binding domain is connected to the Fc region, optionally through a CHI domain.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the C-terminus of the VH1 of the first antigen-binding domain is connected to the N-terminus of a Fc region, and the N-terminus of the second antigen-binding domain is connected to the C-terminus of the Fc region.

10. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody comprises a first heavy chain comprising the VH1 and a first light chain comprising the VL1, and a second heavy chain comprising the second antigen-binding domain.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the C-terminus of the second antigen-binding domain is connected to the Fc region, optionally through a CHI domain.

12. The antibody or antigen-binding fragment thereof of claim 10, wherein the N-terminus of the second antigen-binding domain is connected to the Fc region, optionally through a second linker.

13. The antibody or antigen-binding fragment thereof of any one of claims 10-12, wherein: the first heavy chain comprises one or more knob mutations; andthe second heavy chain comprises one or more hole mutations.

14. The antibody or antigen-binding fragment thereof of any one of claims 10-12, wherein: the first heavy chain comprises one or more hole mutations; andthe second heavy chain comprises one or more knob mutations.

15. The antibody or antigen-binding fragment thereof of any one of claims 7-14, wherein the Fc region is an Fc region of human IgGl, IgG2, IgG3, or IgG4.Attorney Docket No.: 55679-0015WO316. The antibody or antigen-binding fragment thereof of claim 15, wherein the Fc region is an Fc region of human IgGl.

17. The antibody or antigen-binding fragment thereof of any one of claims 7-16, wherein the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering):an Alanine (A) at position 236;a Leucine (L) at position 330; anda Glutamic acid (E) at position 332.

18. The antibody or antigen-binding fragment thereof of any one of claims 7-16, wherein the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering):an Alanine (A) at position 234;an Alanine (A) at position 235;and a Glycine (G) at position 329.

19. The antibody or antigen-binding fragment thereof of any one of claims 7-16, wherein the Fc region comprises one or more the following amino acid residues (all numbering is according to EU numbering):an Aspartic acid (D) at position 239; andan glutamic acid (E) at position 332.

20. The antibody or antigen-binding fragment thereof of any one of claims 7-16, wherein the Fc region is afucosylated.

21. The antibody or antigen-binding fragment thereof of any one of claims 1-20, wherein the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises anAttorney Docket No.: 55679-0015WO3amino acid sequence that is at least 80% identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH1 CDR3 amino acid sequence; andthe first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL1 CDR3 amino acid sequence, wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively;(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively;(5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; and(6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively.Attorney Docket No.: 55679-0015WO322. The antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH2 CDR3 amino acid sequence; andthe second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL2 CDR3 amino acid sequence, wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:(1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;(2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;(3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; and(4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively.

23. The antibody or antigen-binding fragment thereof of any one of claims 20-22, wherein (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ IDAttorney Docket No.: 55679-0015WO3NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;(5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 33, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38, respectively;(6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 34, and 35, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 36, 37 and 38, respectively;(7) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 3, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ IDAttorney Docket No.: 55679-0015WO3NOs: 6, 7, 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;(8) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 5, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;(9) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 11, 13, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively;(10) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 12, 14, and 15, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively;(11) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 21, 23, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 43, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, and 48, respectively; or(12) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 24, and 25, respectively, the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, and 28, respectively; the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 42, 44, and 45, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47 and 48, respectively.Attorney Docket No.: 55679-0015WO324. The antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the Fc region has increased complement-dependent cytotoxicity (CDC), increased antibodydependent cellular cytotoxicity (ADCC) or increased half-life.

25. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-24.

26. A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-25 to the subject.

27. The method of claim 26, wherein the cancer is a solid tumor or a blood tumor.

28. The method of claim 27, 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, cancer of the small intestine, pancreatic cancer and / or liver cancer.

29. The method of any one of claims 26-28, wherein the subject is further treated with an effective amount of an anti-4-lBB antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, or an anti-PD-Ll antibody.

30. A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-25.

31. A method of killing a tumor cell, the method comprisingcontacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-25.Attorney Docket No.: 55679-0015WO332. A method of increasing immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-25.

33. A pharmaceutical composition comprising the antibody or antigen-binding fragment hereof of any one of claims 1-25, and a pharmaceutically acceptable carrier.

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