WO2021041300A2

WO2021041300A2 - Bispecific antibodies and uses thereof

Info

Publication number: WO2021041300A2
Application number: PCT/US2020/047605
Authority: WO
Inventors: Yue Liu; Jianbo Dong
Original assignee: Ab Therapeutics, Inc.
Priority date: 2019-08-23
Filing date: 2020-08-24
Publication date: 2021-03-04
Also published as: WO2021041300A3

Abstract

This disclosure relates to bispecific antibodies or antigen-binding fragments thereof, wherein the bispecific antibodies or antigen-binding fragments thereof specifically bind to two different antigens with different binding affinities.

Description

BISPECIFIC ANTIBODIES AND USES THEREOF

TECHNICAL FIELD

This disclosure relates to bispecific antibodies or antigen-binding fragments thereof.

BACKGROUND

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, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD 19 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 to develop various therapeutics based on bispecific antibodies.

SUMMARY

This disclosure relates to imbalanced bispecific antibodies or antigen-binding fragments, wherein the bispecific antibodies or antigen-binding fragments specifically bind to two different antigens with different binding affinities.

In one aspect, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof that binds to CEACAM5 (Carcinoembryonic Antigen Related Cell Adhesion Molecule 5) and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%,

97%, 98%, or 99% identical to SEQ ID NO: 2; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 1; the second heavy chain variable region (VH) comprise SEQ ID NO: 2; the first light chain variable region (VL) comprise SEQ ID NO: 3; and the second light chain variable region (VL) comprises SEQ ID NO: 3.

In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6.

In some embodiments, the third polypeptide and the fourth polypeptide are at least 90%, 95%, 99%, or 100% identical.

In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to CEACAM5 with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the second antigen binding region specifically binds to CD3 with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the binding affinity of the first antigen binding region when it binds to CEACAM5 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

In some embodiments, the first heavy chain and the second chain associate with each other by the knobs into holes approach.

In one aspect, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof that binds to HER2 (Erb-B2 Receptor Tyrosine Kinase 2) and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 13; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 7 or 13; the second heavy chain variable region (VH) comprise SEQ ID NO: 8; the first light chain variable region (VL) comprise SEQ ID NO: 9; and the second light chain variable region (VL) comprises SEQ ID NO: 9.

In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10 or 14; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,

96%, 97%, 98%, or 99% identical to SEQ ID NO: 11; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

In some embodiments, the third polypeptide and the fourth polypeptide at least 90%,

95%, 99%, or 100% identical.

In some embodiments, the first polypeptide comprises SEQ ID NO: 10. In some embodiments, the first polypeptide comprises SEQ ID NO: 14.

In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to HER2 with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹. In some embodiments, the second antigen binding region specifically binds to CD3 with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the binding affinity of the first antigen binding region when it binds to HER2 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

In one aspect, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof that binds to Mesothelin and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 21; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16;a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 15 or 21; the second heavy chain variable region (VH) comprise SEQ ID NO: 16; the first light chain variable region (VL) comprise SEQ ID NO: 17; and the second light chain variable region (VL) comprises SEQ ID NO: 17.

In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 or 22; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,

96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20. In some embodiments, the third polypeptide and the fourth polypeptide are at least 90%, 95%, 99%, or 100% identical.

In some embodiments, the first polypeptide comprises SEQ ID NO: 18. In some embodiments, the first polypeptide comprises SEQ ID NO: 22.

In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to Mesothelin with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the binding affinity of the first antigen binding region when it binds to Mesothelin is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

In one aspect, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof that binds to PD-L1 (Programmed Cell Death 1 Ligand 1) and CD55 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23;a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25.

In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical. In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 23; the second heavy chain variable region (VH) comprise SEQ ID NO: 24; the first light chain variable region (VL) comprise SEQ ID NO: 25; and the second light chain variable region (VL) comprises SEQ ID NO: 25.

In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28.

In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to PD-L1 with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD55 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the second antigen binding region specifically binds to with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

In some embodiments, the binding affinity of the first antigen binding region when it binds to PD-L1 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD55.

In one aspect, the disclosure relates to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent.

In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent. In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the bispecific antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein, to the subject.

In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is lung cancer, stomach cancer, rectum cancer, breast cancer, mesothelioma, ovarian cancer, or pancreatic adenocarcinoma.

In one aspect, the disclosure relates 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 as described herein, or the antibody- drug conjugate as described herein, to the subject.

In one aspect, the disclosure relates 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 as described herein, or the antibody-drug conjugate as described herein, to the subject.

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

In one aspect, the disclosure relates to a pharmaceutical composition comprising the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CD3 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, in some embodiments, the selected VH 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:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 33, and 34, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 35, 36, and 37, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, and 46, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, and 49, respectively; and

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 56, 57, and 58, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CEACAM5 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 30, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 31; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 35, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 36, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 37.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to HER2 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, in some embodiments, the selected VH 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:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 38, 39, and 40, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, and 49, respectively; and

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 42, and 43, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, and 49, respectively.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to Mesothelin comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, in some embodiments, the selected VH 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:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 51, and 52, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively; and (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 53, 54, and 55, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to PD-L1 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 62, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 63, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 64; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 70.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CD55 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 65, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 66, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 67; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 70.

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.

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 only 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.

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

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing how a bispecific antibody that binds to CD3 and a cancer antigen (e.g., cancer specific antigen) can recognize and kill a tumor cell.

FIG. 2 is a schematic diagram showing how a bispecific antibody that binds to a cancer specific antigen and a cancer-associated antigen can recognize and kill a tumor cell.

FIG. 3 is a set of flow cytometry results showing anti-CEACAM5 antibody (CEACAM5 homodimer), anti-CD3 antibody (CD3 homodimer), or the V5 version of the CEACAM5/CD3 bispecific antibody (CEACAM5/CD3) binding to Jurkat or LS174T cells. Results labelled with “Cell only” and “GAH only” are negative controls. Cell percentage and mean fluorescence intensity (MFI) are provided at the upper right comer of each result.

FIG. 4 shows percentage of CD69+/CD2+ cells as determined by FACS in the CEACAM+ LS174T/T cell model and CEACAM- LS174T/T cell model. Either the CEACAM5/CD3 bispecific antibody, an isotype control antibody, or PBS was added. Schematic diagrams indicating mechanisms are inserted to each result.

FIG. 5 shows a workflow of ABS CD3-VL phage library construction. “*” indicates functional variants in the CD3 VL sequence.

FIG. 6 shows an exemplary phage library panning workflow.

FIG. 7 shows the phage ELISA assay results for the third panning output. Both Ag Mix #1 and Ag Mix #2 groups were titrated to 1E10 and 1E9 colony-forming unit (CFU).

FIG. 8 shows ELISA assay results to test binding between cancer-specific antigens (recombinant HER2 and Mesothelin proteins) with HER2-B3/CD3, HER2-D1/CD3, Meso- G7/CD3, and Meso-A4/CD3 bispecific antibodies.

FIG. 9 is a set of flow cytometry results showing anti-HER2 antibody, HER2-B3/CD3 bispecific antibody, and HER2-D1/CD3 bispecific antibody binding to SK-BR-3 expressing HER2 protein. Results labelled with “SK-BR-3 only” and “SK-BR-3 2^nd only” are negative controls. Cell percentage and mean fluorescence intensity (MFI) are provided at the upper right corner of each result.

FIG. 10 shows percentage of CD69+/CD2+ cells as determined by FACS. Either an isotype control antibody, an HER2-B3 homodimer IgG, or HER2-B3/CD3 bispecific antibody was added.

FIG. 11A shows a schematic structure of HER2-B3/CD3 (2:1).

FIG. 11B shows a schematic structure of HER2-B3V3/CD3 (1:1).

FIG. 12 shows T cells activation of HER2/CD3 bispecific antibodies in 1:1 and 2:1 formats. For CD20/CD3 positive control, B cells in PBMCs served as target cells.

FIG. 13A shows a cation exchange chromatography result. Purified HER2-B3/CD3 (2:1) bispecific antibody was recovered from A3-A7 fractions.

FIG. 13B shows an SDS-PAGE result of the HER2-B3/CD3 bispecific antibody fractions after cation exchange chromatography. Lane 1 is HER2-B3/CD3 (2:1) expressed in mammalian cell culture supernatant; lanes 2-6 are purified A3-A7 fractions (non-reduced) in FIG. 13A; and lanes 7-11 are purified A3-A7 fractions (reduced) in FIG. 13A. M is marker.

FIG. 14A shows PD-L1/CD55 bispecific antibody-mediated CDC on MDA231 cells.

11F11 is an isotype control. “PBS only” is a negative control. “Cell + Triton 100” is a positive control.

FIG. 14B shows PD-L1/CD55 bispecific antibody-mediated CDC on PANC1 cells.

FIG. 15A shows PD-L1/CD55 bispecific antibody internalization in PANC1 cells. 11F11 is an isotype control.

FIG. 15B shows PD-L1/CD55 bispecific antibody internalization in MDA231 cells.

11F11 is an isotype control.

FIG. 16 shows PD-L1/CD55 bispecific antibody internalization in MDA231 cells. 11F11 is an isotype control. PD-L1 in the figure indicates an anti-PD-Ll (Avelumab) antibody and CD55 indicates an anti-CD55 monoclonal antibody. A CF variant of 2E2 PD-L1/CD55 bispecific antibody (BsAb 2E2) contains a cysteine to phenylalanine mutation in the common VL. R-PE is a negative control. FIG. 17A shows percentage of CD69+/CD2+ cells as determined by FACS in mixed PBMC and PANC1 cells. PDL1 Ab and CD55 Ab are anti-PD-Ll (Avelumab) and anti-CD55 monoclonal antibody controls. Isotype is an isotype antibody control.

FIG. 17B shows released IL2 as determined by ELISA in mixed PBMC and PANC1 cells. PDL1 and CD55 are anti-PD-Ll (Avelumab) and anti-CD55 monoclonal antibody controls. Isotype is an isotype antibody control. RFU is relative fluorescence unit.

FIG. 17C shows released IFNy as determined by ELISA in mixed PBMC and PANC1 cells. PDL1 and CD55 are anti-PD-Ll (Avelumab) and anti-CD55 monoclonal antibody controls. Isotype is an isotype antibody control. RFU is relative fluorescence unit.

FIG. 17D shows released TNFa as determined by ELISA in mixed PBMC and PANC1 cells. PDL1 and CD55 are anti-PD-Ll (Avelumab) and anti-CD55 monoclonal antibody controls. Isotype is an isotype antibody control. RFU is relative fluorescence unit.

FIG. 17E shows percentage of CD69+/CD2+ cells as determined by FACS in pre incubated PBMC and PANC1 cells. PDL1 and CD55 are anti-PD-Ll (Avelumab) and anti-CD55 monoclonal antibody controls. Isotype is an isotype antibody control. CF, CF/B and CF/C are variants of the 2E2 PD-L1/CD55 bispecific antibody. -7(M) and -8(M) are antibody concentrations of 1 ^c 1 O ⁷ or 1 x 10⁸ M, respectively.

FIG. 18 lists sequences that are described in the present application.

FIG. 19 lists CDR sequences of several antibodies described herein.

FIG. 20 is a sequence alignment between the common VL of 2E2 PD-L1/CD55 bispecific antibody (SEQ ID NO: 71) and the common VL of the 2E2 (CF) variant (SEQ ID NO: 25).

FIG. 21 is a sequence alignment between CD55 arm of the CF, CF/B, and CF/C variants of 2E2 PD-L1/CD55 bispecific antibody. The CD55 arms of the CF, CF/B, and CF/C variants are listed as CD55VH-a (SEQ ID NO: 24), CD55VH-b (SEQ ID NO: 72), and CD55VH-C (SEQ ID NO: 73), respectively.

DETAILED DESCRIPTION

A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different types of antigens. In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms (Arms A and B). Each arm has one heavy chain variable region and one light chain variable region.

The bispecific antibody or antigen-binding fragment thereof can be IgG-like and non- IgG-like. The IgG-like bispecific antibody can have two Fab arms and one Fc region, and the two Fab arms bind to different antigens. The non-IgG-like bispecific antibody or antigen-binding fragment can be e.g., chemically linked Fabs (e.g., two Fab regions are chemically linked), and single-chain variable fragments (scFVs). For example, a scFV can have two heavy chain variable regions and two light chain variable regions.

In an imbalanced bispecific antibody or antigen-binding fragment thereof, the two arms (Arms: A and B) or the two antigen binding regions (Antigen binding regions: A and B) can bind to the respective target antigens with different affinities. The binding affinities can be expressed by the association constant (Ka):

Ka = [Antibody-Antigen] / [Antibody] [Antigen]

Antibodies with high affinity usually have Ka > 10⁷ M ¹. The Ka for one arm or one antigen binding region can be greater than 10⁵ M ¹, 10⁶ M ¹, 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ^x, or 10¹² M ¹. In some embodiments, the Ka can be less than 10⁵ M ¹, 10⁶ M ¹, 10⁷ M ¹, 10⁸ M ¹,

The binding affinity of the first arm or the first antigen binding region (A) can be greater than the binding affinity of the second arm or the second antigen binding region (B). Bispecific antibodies with imbalanced affinities can have various advantages. For example, bispecific antibodies with imbalanced affinities can be used to target a cancer specific antigen on cancer cells and CD3 on T cell. In this case, high affinity to the cancer specific antigen can lead to better capturing of cancer cells by T cells, and low affinity to CD3 can avoid triggering T-cell signaling by CD3 (FIG. 1). Only when the bispecific antibody is presented to the T cell in a multivalent fashion by a target cancer cell, can the T cell be activated and kill the target cancer cell. Furthermore, the bispecific antibodies with imbalanced affinities can also be used to target a cancer specific antigen and a cancer-associated antigen (FIG. 2). In this case, the bispecific antibody only weakly binds to non-cancer cells expressing low level of cancer-associated antigens, but strongly binds to cancer cells expressing both cancer specific antigens and high level of cancer-associated antigens.

For a bispecific antibody with imbalanced affinities, the Ka for the first arm or the first antigen binding region (A) can be greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, or 10¹² M ¹. In some embodiments, the Ka for the first arm or the first antigen binding region (A) can be 10, 100, 1000, 10000, or 100000 times greater than the Ka for the second arm or the second antigen binding region (B). Thus, in some embodiments, the Ka for the second arm or the second antigen binding region (B) can be less than 10⁵ M ¹, 10⁶ M ¹, 10⁷ M ¹, 10⁸ M ¹, or 10⁹ M ¹. In some embodiments, the Ka for the second arm or the second antigen binding region (B) still specifically binds to the target antigen with a reasonable affinity, e.g., greater than 10⁴ M ¹, 10⁵ M ¹ or 10⁶ M ¹.

The binding affinity can also be expressed by the dissociation constant (Kd).

Kd = [Antibody] [Antigen] / [Antibody-Antigen]

The Kd can be less than 10⁵ M, 10⁶ M, 10⁷ M, 10⁸ M, 10 ⁹ M, 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹² M. In some embodiments, the Kd can be greater than 10⁵ M, 10⁶ M, 10⁷ M, 10⁸ M, 10⁹ M, 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹² M.

In some embodiments, the binding affinity of the first arm or the first antigen binding region (A) is greater than the binding affinity of the second arm or the second antigen binding region (B). For example, the Kd for the second arm or the second antigen binding region (B) can be 10, 100, 1000, 10000, or 100000 times greater (thus with less affinity) than the Kd for the first arm or the first antigen binding region (A). Thus, in some embodiments, the Kd for the first arm or the first antigen binding region (A) can be less than 10⁷ M, 10⁸ M, 10⁹ M, 10 ¹⁰ M, 10 ¹¹ M, or 10 ¹² M; and the Kd for the second arm or the second antigen binding region (B) can be greater than 10⁵ M, 10⁶ M, 10⁷ M, 10⁸ M, or 10⁹ M.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two light chains and two heavy chains. Each of the two light chains has one light chain variable region (VL) and one light chain constant region (CL). Each of the two heavy chains has one heavy chain variable region (VH) and three heavy chain constant regions (CHI, CH2, and CH3). In some embodiments, the two light chains for Arm A and Arm B are the same. Thus, the CDRs in the VL of two light chains can be the same. In some embodiments, the two heavy chains in the bispecific antibody or antigen-binding fragment thereof are different. Thus, the CDRs in the VH of two heavy chains are different.

Various methods can be used to ensure that the same heavy chains do not associate with each other when making the bispecific antibodies. For example, the "knobs into holes" approach introduces a mutation for an amino acid with a large sidechain in one heavy chain, and a mutation for an amino acid with a small sidechain in the other heavy chain. Thus, the same heavy chains are less likely to associate with each other and the two different heavy chains have a higher chance to associate with each other. The “knobs into holes” approaches are described, e.g., in Ridgway, John BB, Leonard G. Presta, and Paul Carter. "‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization." Protein Engineering, Design and Selection 9.7 (1996), which is incorporated herein by reference in its entirety.

Imbalanced bispecific antibodies that bind to T cell specific antigen and cancer antigens

Bispecific antibodies (BsAb or BsMab) with a T cell specific antigen (e.g., CD3, CD4, or CD8) binding arm that can recruit and activate T cells have been widely studied for cancer therapy. However, the effector function of many of these bispecific antibodies are eliminated because of safety concerns. Because an antibody’s effector function such as ADCC and CDC have been shown to play a critical role in cancer cell killing, “safely” maintaining an antibody’s effector function would expand the mechanisms of action of an therapeutic antibody as well as improve the antibody’s cancer killing function. To “safely” maintain the effector functions and expand the applications of these bispecific antibodies, an imbalanced bispecific antibody technology platform has been developed based on computational antibody design.

In this design, the first antigen binding region targets a cancer specific antigen, and the second antigen binding region targets a T cell specific antigen (e.g., CD3, CD4, or CD8) to recruit T cell to attack cancer with the cancer specific antigen (FIG. 1).

As used herein, the term “cancer specific antigen” refers to antigens that are specifically expressed on cancer cell surfaces. These antigens can be used to identify tumor cells. Normal cells rarely express cancer specific antigens. Some exemplary cancer specific antigens include, e.g, CD20, PSA, PSCA, PD-L1, Her2, Her3, Herl, b-Catenin, CD19, CEACAM3, EGFR, c- Met, EPCAM, PSMA, CD40, METCl, and IGF1R, etc. PSA are primarily expressed on prostate cancer cells, and Her2 are primarily expressed on breast cancer cells.

An antibody with high affinity to CD3 can trigger T-cell signaling, and cause undesirable immune response. Thus, a low affinity (e.g., Ka can be less than 10⁵ M ¹, 10⁶ M ¹, or 10⁷ M ¹) to CD3 is required to reduce the risk of triggering T-cell signaling by CD3 while “safely” maintaining the antibody’s effector function. As used herein, the term “safely maintaining the antibody’s effector function” means that the antibody does not induce ADCC or CDC on normal cells (e.g., non-cancer cells). When multiple bispecific antibodies are presented on a target cancer cells (e.g., in a cluster) and bridge the interaction between cancer cell and T cell, these bispecific antibodies can trigger T-cell signaling though CD3 in a multivalent fashion, and the activated T cells will then kill the target cancer cells. Because the bispecific antibody applies different mechanism of action to treat cancer compared to therapeutic antibodies that target a cancer specific antigen alone, in some embodiments, it can be used as an alternative therapy for therapeutic monoclonal antibodies that target a cancer specific antigen, especially for those cancers which don not respond well to therapeutic monoclonal antibodies that only targets a cancer specific antigen.

(1) D1-CEACAM5/CD3 bispecific antibody

CEACAM5 (Carcinoembryonic Antigen Related Cell Adhesion Molecule 5, or CEA) is a cancer specific antigen and is expressed on e.g., lung cancer, stomach cancer and rectum cancer cells. The present disclosure provides bispecific antibodies that bind to both CEACAM5 and CD3. The bispecific antibodies can be used to treat CEACAM5 positive cancers (e.g., lung cancer, stomach cancer and/or rectum cancer) in a subject.

The bispecific antibody has two arms: a CEACAM5 arm that specifically binds to CEACAM5 and a CD3 arm that specifically binds to CD3 with a lower binding affinity. In some embodiments, the bispecific antibody has two common light chains (e.g., with identical sequence) and two different heavy chains. The sequences for the variable regions of the two heavy chains, the variable region of the common light chain, the full-length heavy chains, and the full-length light chain are shown below.

VH-CEACAM5 arm: EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKGLEWLGFIGNKANGY TTE Y S AS VKGRFTISRDKSKS SL YLQMN SLKTEDT AT YY CTRDRGLRF YFD YW GQGTTL TVSS (SEQ ID NO: 1)

VH-CD3 arm

QVQLVQSGAEVAKPGASVKMSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGY TNYNQKFKDRVTLTTDTSTSTVYMELSSLTSEDTAVYYCARYYDDHYCLDYWGQGTT LTVSS (SEQ ID NO: 2)

Common VL

QIVLSQSPAIMSASPGEKVTMTCRASSSVTYINWYQQKPGSSPKRWIYDTSNLASGVPAH F SGSGSGTS Y SLTISRVEAED AATYYCQHW S SNPFTF GGGTKLEIKR (SEQ ID NO: 3)

Heavy chain 1 (CEACAM5 arm)

EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKGLEWLGFIGNKANGY TTE Y S AS VKGRFTISRDKSKS SL YLQMN SLKTEDT AT YY CTRDRGLRF YFD YW GQGTTL TV S S AS TKGP S VFPL AP S SK S T S GGT AALGCL VKD YFPEP VT V S WN SGALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCP APE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTL PPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4)

Heavy chain 2 (CD3 arm)

QVQLVQSGAEVAKPGASVKMSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGY TNYNQKFKDRVTLTTDTSTSTVYMELSSLTSEDTAVYYCARYYDDHYCLDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEP VTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYN ST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPEVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

Common light chain

QIVLSQSPAIMSASPGEKVTMTCRASSSVTYINWYQQKPGSSPKRWIYDTSNLASGVPAH F SGSGSGT S Y SLTISRVE AED AAT YY CQHW S SNPFTF GGGTKLEIKRT VAAP S VFIFPP SD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 6)

Therefore, the disclosure provides bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and the first and the second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

In some embodiments, the first and the second light chain variable regions has a common VL sequence or a different VL sequence. In some embodiments, the first and/or the second light chain variable regions (both or individually) comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 1, the second heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 2, and the first and the second light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3.

The present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to CEACAM5. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 1, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 1. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3. When the polypeptides pair with each other, they can bind to CEACAM5.

In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to CD3. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 2, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 2. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 2. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 3. When the polypeptides pair with each other, they can bind to CD3.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to CEACAM5. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6.

The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to CEACAM5 include CDRs of the heavy chain variable domain, SEQ ID NOs: 29-31, and CDRs of the light chain variable domain, SEQ ID NOs: 35-37. The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to CD3 include CDRs of the heavy chain variable domain, SEQ ID NOs: 32-34, and CDRs of the light chain variable domain, SEQ ID NOs: 35-37.

(2) HER2/CD3 bispecific antibody HER2 (also known as Receptor tyrosine-protein kinase erbB-2, CD340, proto-oncogene Neu, HER2/neu, Erbb2, or ERBB2) is a protein that in humans is encoded by the ERBB2 gene.

It is a cancer specific antigen and is often expressed on e.g., breast cancer cells. The present disclosure provides bispecific antibodies that bind to both HER2 and CD3. The bispecific antibodies can be used to treat HER2 positive cancers (e.g., breast cancer) in a subject.

Several bispecific antibodies are designed to bind to HER2 and CD3. The bispecific antibody has two arms: a HER2 arm that specifically binds to HER2 and a CD3 arm that specifically binds to CD3 with a lower binding affinity. In some embodiments, the bispecific antibody has two common light chains (e.g., with identical sequence) and two different heavy chains. Two bispecific antibodies are selected. The sequences for the variable regions of the two heavy chains, the variable region of the common light chain, the full-length heavy chains, and the full-length light chain of the selected bispecific antibodies are shown below. a. HER2/CD3 bispecific antibody (Version HER2-B3/CD3)

VH-HER2 arm (HER2-B3 VH):

QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCASMTTPVGYWGQGTLVTV SS (SEQ ID NO: 7)

VH-CD3 arm (CD3 VH):

EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TVSS (SEQ ID NO: 8)

Common VL (Common light chain_ VL):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTS Y SLTISRVEAED AAT YY CQQWT SNPPTF GGGTKLEIK (SEQ ID NO: 9)

Heavy chain 1 (HER2 arm) (HER2-B3 heavy chain knob_v4b):

QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCASMTTPVGYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGT AALGCL VKD YFPEP VTVSWNSGALTSGVHTFP A VLQ S SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPP YREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10)

Heavy chain 2 (CD3 arm) (CD3 heavy chain full-length sequence (hole_v4b)): EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TV S S AS TKGP S VFPL AP S SK S T S GGT AALGCL VKD YFPEP VT V S WN SGALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCP APE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPEVYTL PP SREEMTKN Q VSLT CL VKGF YP SDI AVEWE SN GQPENNYKTTPP VLD SDGSFFLT SKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

Common light chain (Common light chain full-length sequence):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 12) b. HER2/CD3 bispecific antibody (version HER2-D1/CD3):

VH-HER2 arm (HER2-D1 VH):

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGN TN Y AQKLQ GRVTMTTDT S T S T A YMELRSLRSDDT A V Y Y C ARDRL Y Y YD S S GY SLQLPR YYYY GMD VW GQGTL VT V S S (SEQ ID NO: 13)

VH-CD3 arm (CD3 VH): EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TVSS (SEQ ID NO: 8)

Common VL (Common light chain_ VL):

Heavy chain 1 (HER2 arm) (HER2-D1 heavy chain full-length sequence (knob_v4b)): EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGN TN Y AQKLQ GRVTMTTDT S T S T A YMELRSLRSDDT A V Y Y C ARDRL Y Y YD S S GY SLQLPR YYYY GMD VWGQGTL VTV S SASTKGPS VFPLAPS SKSTSGGTAALGCL VKD YFPEP VTV S WN SGALT SGVHTFPAVLQ S SGLY SL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKK V EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYN STYRVV SVLTVLHQDWLNGKEYKCKV SNKALP API EKTISKAKGQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNY KTTPP VLD SDGSFFL Y SKLTVDKSRWQQGNVF SC S VMHEALHNHYT QKSLSL SPGK (SEQ ID NO: 14)

Heavy chain 2 (CD3 arm) (CD3 heavy chain full-length sequence (hole_v4b)): EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TV S S AS TKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN SGALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCP APE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKGQPREPEVYTL PP SREEMTKN Q VSLT CL VKGF YP SDI AVEWE SN GQPENN YKTTPP VLD SDGSFFLT SKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

Common light chain (Common light chain full-length sequence): QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 12)

Therefore, the disclosure provides bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 13, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and the first and the second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10 or 14; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 7 or 13, the second heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 8, and the first and the second light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9.

In some embodiments, the first and the second light chain variable regions has a common VL sequence or a different VL sequence. In some embodiments, the first and/or the second light chain variable regions (both or individually) comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

The present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to HER2. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 7 or 13, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 7 or 13. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9. When the polypeptides pair with each other, they can bind to HER2.

In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to CD3. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 8, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 8. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 8. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 9. When the polypeptides pair with each other, they can bind to CD3.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to HER2. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7 or 13, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10 or 14, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 8, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12.

In some embodiments, the CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to HER2 include CDRs of the heavy chain variable domain, SEQ ID NOs: 38-40, and CDRs of the light chain variable domain, SEQ ID NOs: 47-49. In some embodiments, the CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to HER2 include CDRs of the heavy chain variable domain, SEQ ID NOs: 41-43, and CDRs of the light chain variable domain, SEQ ID NOs: 47-49. The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to CD3 include CDRs of the heavy chain variable domain, SEQ ID NOs: 44-46, and CDRs of the light chain variable domain, SEQ ID NOs: 47-49.

(3) Mesothelin/CD3 bispecific antibody

Mesothelin is a 40 kDa protein present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. CAR T cells targeting mesothelin-expressing tumors have demonstrated safety and efficacy in a preliminary clinical evaluation in patients with malignant pleural disease.

The present disclosure provides bispecific antibodies that bind to both Mesothelin and CD3. The bispecific antibodies can be used to treat Mesothelin positive cancers (e.g., mesothelioma, ovarian cancer, and pancreatic adenocarcinoma) in a subject.

Several bispecific antibodies are designed to bind to Mesothelin and CD3. The bispecific antibody has two arms: a Mesothelin arm that specifically binds to Mesothelin and a CD3 arm that specifically binds to CD3 with a lower binding affinity. In some embodiments, the bispecific antibody has two common light chains (with identical sequence) and two different heavy chains. Two bispecific antibodies are selected. The sequences for the variable regions of the two heavy chains, the variable region of the common light chain, the full-length heavy chains, and the full- length light chain of the selected bispecific antibodies are shown below. a. Mesothelin/CD3 bispecific antibody (Version Meso-G7/CD3)

VH-Mesothelin arm (Meso-G7_VH):

QMQLVQSGAEVKKPGASVKVSCKASGYTFTSHDVNWVRQATGQGLEWMGWMNPKS GNT GY AQKFKGRV SMTRNTSINT AYMEL S SLRSEDT AVYF C ARGQGWLGAFDLW GQG TMVTVSS (SEQ ID NO: 15)

VH-CD3 arm (CD3 VH):

EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TVSS (SEQ ID NO: 16) Common VL (Common light chain_ VL):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF S GS GS GT S Y SLTI SRVEAED A AT Y Y CQ Q WT SNPPTF GGGTKLEIK (SEQ ID NO: 17)

Heavy chain 1 (Mesothelin arm) (Meso-G7 heavy chain full-length sequence (knob_v4b)): QMQLVQSGAEVKKPGASVKVSCKASGYTFTSHDVNWVRQATGQGLEWMGWMNPKS GNT GY AQKFKGRV SMTRNTSINT AYMEL S SLRSEDT AVYF C ARGQGWLGAFDLW GQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FP A VLQ S SGL Y SL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHT CPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

Heavy chain 2 (CD3 arm) (CD3 heavy chain full-length sequence (hole_v4b)): EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TV S S AS TKGP S VFPL AP S SK S T S GGT AALGCL VKD YFPEP VT V S WN SGALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCP APE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPEVYTL PP SREEMTKN Q VSLT CL VKGF YP SDI AVEWE SN GQPENN YKTTPP VLD SDGSFFLT SKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19)

Common light chain (Common light chain full-length sequence):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 20) b. Mesothelin/CD3 bispecific antibody (version Meso-A4/CD3): VH-Mesothelin arm (Meso-A4_VH):

EVQLVQSGAEVKKPGASVKV SCKASGYTFTDYYMHWVRQAPGQGLEWMGWMNPN S GGTNY AQKF QGRVTMTRDT SINT AYMEL SRLRSDDT AVYY C ARQLTTLKTGGY S Y AM DVWGQGTTVTVSS (SEQ ID NO: 21)

VH-CD3 arm (CD3 VH):

EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TVSS (SEQ ID NO: 16)

Common VL (Common light chain_ VL):

Heavy chain 1 (Mesothelin arm) (Meso-A4 heavy chain full-length sequence (knob_v4b)): EVQLVQSGAEVKKPGASVKV SCKASGYTFTDYYMHWVRQAPGQGLEWMGWMNPN S GGTNY AQKF QGRVTMTRDT SINT AYMEL SRLRSDDT AV YY C ARQLTTLKTGGY S Y AM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL T SGVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICN VNHKP SNTK VDKK VEPK S CDKT HT CPPCP APELLGGP S VFLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YVDGV EVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAK GQPREPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)

Heavy chain 2 (CD3 arm) (CD3 heavy chain full-length sequence (hole_v4b)): EVQLQESGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGEGLEWIGYINPSSGYT KYNQKFKDKATLTADKS S STAYMELS SLTSEDS AVYY C ARW QD YD VYFDYWGEGTTL TV S S AS TKGP S VFPL AP S SK S T S GGT AALGCL VKD YFPEP VT V S WN SGALT S GVHTFP A V LQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CPPCP APE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKGQPREPEVYTL PP SREEMTKN Q VSLT CL VKGF YP SDI AVEWE SN GQPENN YKTTPP VLD SDGSFFLT SKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19)

Common light chain (Common light chain full-length sequence):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 20)

Therefore, the disclosure provides bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 21, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, and the first and the second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 or 22; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 15 or 21, the second heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 16, and the first and the second light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17.

In some embodiments, the first and the second light chain variable regions has a common VL sequence or a different VL sequence. In some embodiments, the first and/or the second light chain variable regions (both or individually) comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

The present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to Mesothelin. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 15 or 21, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 15 or 21. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17. When the polypeptides pair with each other, they can bind to Methothelin.

In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to CD3. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 16, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 16. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 16. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 17. When the polypeptides pair with each other, they can bind to CD3.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to Mesothelin. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 15 or 21, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 17. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 18 or 22, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 17. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20.

In some embodiments, the CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to Mesothelin include CDRs of the heavy chain variable domain, SEQ ID NOs: 50-52, and CDRs of the light chain variable domain, SEQ ID NOs: 59-61. In some embodiments, the CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to Mesothelin include CDRs of the heavy chain variable domain, SEQ ID NOs: 53-55, and CDRs of the light chain variable domain, SEQ ID NOs: 59-61.

The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to CD3 include CDRs of the heavy chain variable domain, SEQ ID NOs: 56-58, and CDRs of the light chain variable domain, SEQ ID NOs: 59-61.

Imbalanced bispecific antibodies that bind to cancer specific antigens and cancer- associated antigen.

The present disclosure also provides imbalanced bispecific antibodies that have the first antigen binding region targets a cancer specific antigen, and the second antigen binding region targets a cancer-associated antigen.

As used herein, the term “cancer-associated antigen” refers to antigens that are expressed at a relatively high level on cancer cells but may be also expressed at a relatively low level on normal cells. CD55, CD59, CD46 and many adhesion molecules such as N-cadherin, VE- cadherin, NCAM, Mel-CAM, ICAM, NrCAM, VCAM1, ALCAM, MCAM, etc., are cancer- associated antigens. While both cancer specific antigen and cancer-associated antigen are expressed on cancer cell surface, the difference between a cancer specific antigen and a cancer- associated antigen is that the cancer-associated antigen is also expressed on normal cells, but at a relative low level as compared to the level on cancer cells. In contrast, a cancer specific antigen is rarely expressed on normal cells, and even if it is expressed on normal cells, the amount is extremely low. An antibody that targets cancer specific antigen usually will not induce Antibody-dependent Cellular Cytotoxicity (ADCC) or Complement-dependent cytotoxicity (CDC) on normal cells. In contrast, an antibody that targets a cancer-associated antigen with a high affinity may cause cytotoxic effects among normal cells. Thus, it is important that the bispecific antibody binds to a cancer-associated antigen with a relatively low affinity (FIG. 2).

A bispecific antibody that binds to PD-L1 and CD55 is provided. This antibody can be used for treating a subject with PD-L1 and CD55 positive cancers though ADCC or CDC as well as blocking the PD-L1/PD1 interaction to activate T cell dependent immune response and to decrease CD55’s repression on CDC. Furthermore, as cancer cells may become resistant to PD- L1 antibodies, the binding between the second arm and CD55 on the cancer cells can provide additional therapeutic effects.

The PD-L1/CD55 bispecific antibody has two arms: a PD-L1 arm that specifically binds to PD-L1 and a CD55 arm that specifically binds to CD55. In some embodiments, the bispecific antibody has two common light chains (with identical sequence) and two different heavy chains. The sequences for the variable regions of the two heavy chains, the variable region of the common light chain, the full-length heavy chains, and the full-length light chain of the bispecific antibody are shown below.

VH-PD-L1 arm (PD-L1-2E2 VH):

QVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGST YY AD S VKGRFTISRDN SKNTL YLQMS SLRAEDT AVY Y C VKGRRRLRGF YYY GMD VW G QGTTVTVSS (SEQ ID NO: 23)

VH-CD55 arm (VH-CD55a):

QVKLQESGGGLVKPGGSLKLSCAASGFTFSGYGMSWIRQTPGKRLEWVATINSGGSYT YY SD SVKGRFTISRDNVKNTL YLQMS SLKSEDT AMY Y C ARRNGTL YYYLMD YW GRGT LVTVSS (SEQ ID NO: 24)

Common VL (Common light chain_ VL):

DIVLTQTPGTL SL SPGERATL SCRASQ S VN SNYL AW YQQKPGQ APRLLMF GASTRATGIP DRFRGSGSGTDFTLTISRLEPEDF AVYY CQ YY GRSRTF GQGTKVEIKRR (SEQ ID NO: 25)

Heavy chain 1 (PD-L1 arm) (PD-L1 2E2 heavy chain full-length sequence): QVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGST YY AD S VKGRFTISRDN SKNTL YLQMS SLRAEDT AVY Y C VKGRRRLRGF YYY GMD VW G QGTT VT V S S ASTKGP S VFPL AP S SK ST SGGT AALGCLVKD YFPEP VT V S WNSGALT SGV HTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VEPK S CDKTHT CP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNK ALP APIEKTISKAKGQPR EPQVYTLPPYREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFL Y SKLTVDK SRWQQGNVF SC S VMHE ALHNHYTQKSL SL SPGK (SEQ ID NO: 26)

Heavy chain 2 (CD55 arm) (CD55a heavy chain full-length sequence): QVKLQESGGGLVKPGGSLKLSCAASGFTFSGYGMSWIRQTPGKRLEWVATINSGGSYT YY SD S VKGRFTISRDNVKNTL YLQMS SLKSEDT AMY Y C ARRNGTL YYYLMD YW GRGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQ S SGL Y SLS S VVTVP S S SLGTQT YICNVNHKP SNTK VDKKVEPKSCDKTHTCPPCP A PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNST YRVV S VLTVLHQDWLNGKEYKCK V SNKALP APIEKTISKAKGQPREPEVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)

Common light chain (Common light chain full-length sequence):

DIVLTQTPGTL SL SPGERATL SCRASQ S VN SNYL AW YQQKPGQ APRLLMF GASTRATGIP DRFRGSGSGTDFTLTISRLEPEDF AVYY CQ YY GRSRTF GQGTKVEIKRRTVAAPS VFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 28)

Therefore, the disclosure provides bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24, 72, or 73, and the first and the second light chain variable regions comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25 or 71.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26; a second heavy chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; a first light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; and a second light chain amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are identical.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 23, the second heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 24, 72, or 73, and the first and the second light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71.

In some embodiments, the first and the second light chain variable regions has a common VL sequence or a different VL sequence. In some embodiments, the first and/or the second light chain variable regions (both or individually) comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25 or 71.

The present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to PD-L1. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 23, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 23. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71. When the polypeptides pair with each other, they can bind to PD-L1.

In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to CD55. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 24, 72, or 73, and the light chain variable regions comprise 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 24, 72, or 73. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71. Thus, in some aspects, the disclosure provides a polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises 3 heavy chain CDRs that are identical to the CDRs in SEQ ID NO: 24, 72, or 73. In some aspects, the disclosure provides a polypeptide comprising a light chain variable region, wherein the light chain variable region comprises 3 light chain CDRs that are identical to the CDRs in SEQ ID NO: 25 or 71. When the polypeptides pair with each other, they can bind to CD55.

The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to PD-L1. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 23, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25 or 71. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 26, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28. The disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. The antibodies or antigen-binding fragments thereof contains a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 24, 72, or 73, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25 or 71. In some embodiments, the antibodies or antigen-binding fragments thereof contains a heavy chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27, and a light chain comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28.

The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to PD-L1 include CDRs of the heavy chain variable domain, SEQ ID NOs: 62-64, and CDRs of the light chain variable domain, SEQ ID NOs: 68-70.

The CDR sequences for the antibodies or antigen-binding fragments (e.g., humanized antibodies) thereof that specifically bind to CD3 include CDRs of the heavy chain variable domain, SEQ ID NOs: 65-67, and CDRs of the light chain variable domain, SEQ ID NOs: 68-70.

Antibodies and Antigen Binding Fragments

The present disclosure provides antibodies and antigen-binding fragments thereof that comprise complementary determining regions (CDRs), heavy chain variable regions, light chain variable regions, heavy chains, or light chains described herein. In some embodiments, the antibodies and antigen-binding fragments thereof are imbalanced bispecific antibodies and antigen-binding fragments thereof.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting 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. An antibody can comprise two identical copies of a light chain and/or two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

These 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 antigen-binding region.

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 Rabat 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 Rabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Rabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(l-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); Rontermann, R., & Diibel, S. (Eds.). (2010). Antibody engineering: Volume 2. Springer; each of which is incorporated herein by reference in its entirety. In some embodiments, the CDRs are based on Rabat definition. In some embodiments, the CDRs are based on the Chothia definition. In some embodiments, the CDRs are the longest CDR sequences as determined by Rabat, Chothia, AbM, IMGT, or contact definitions.

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.

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, differ 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., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; 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.

The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “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 an antigen binding fragment thereof can be, 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 multi-specific 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 antigen binding 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. In some embodiments, the scFV 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.

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4 IBB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

In some embodiments, the antibodies or antigen-binding fragments thereof can bind to two different antigens or two different epitopes.

Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGl molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody mul timers.

Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen binding fragment thereof in a subject or 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 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).

In some embodiments, the antibodies or antigen-binding fragments (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment 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., 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, dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein increases T cell activation (e.g., as indicated by percentage of CD69+CD2+ cells, or specific killing of target cells) by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 4 folds, 5 folds, 6 folds, 7 folds, 8 folds, 9 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 100 folds, or more, as compared to an isotype control antibody, or a monoclonal antibody comprising the same VH or VL of the bispecific antibody. In some embodiments, the T cells are selected from Jurkat cells or PBMCs. In some embodiments, the target cells are selected from LS174T cells, SK-BR-3 cells, MDA231 cells, or PANC1 cells. In some embodiments, the target cells expresses cancer-specific antigens (e.g., CEACAM5, HER2, Mesothelin, or PD-L1) and/or cancer-associate antigens (e.g., CD55) endogenously or by transient transfection. In some embodiments, the T cells activation is mediated by the bispecific antibody or antigen-binding fragment at a concentration that is at least or about 0.1 pg/ml, 0.2 pg/ml, 0.3 pg/ml, 0.4 pg/ml, 0.5 pg/ml, 1 pg/ml, 2 pg/ml, 3 pg/ml, 4 pg/ml, 5 pg/ml, 6 pg/ml, 7 pg/ml, 8 pg/ml, 9 pg/ml, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, or higher. In some embodiments, the T cells, target cells, and the bispecific antibody are incubated for at least or about 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours. In some embodiments, the ratio of the T cells and target cells is about 1:1, about 2:1, about 3:1, about 5:1, about 10:1, about 20:1, or about 25:1.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein (e.g., a CEACAM5/CD3 bispecific antibody) binds to an antigen (e.g., CEACAM5) with a binding affinity that is about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, or about 200% to that of a monoclonal antibody (e.g., an anti-CEACAM5 monoclonal antibody) comprising the same VH or VL of the bispecific antibody. In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein (e.g., a PD-L1/CD55 bispecific antibody) mediates complement-dependent cytotoxicity (CDC) to at least or about 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 6 folds, 7 folds, 8 folds, 9 folds, 10 folds, 11 folds, 12 folds, 13 folds, 14 folds, 15 folds, 16 folds, 17 folds, 18 folds, 19 folds, 20 folds, 30 folds, 40 folds, or 50 folds as compared to that mediated by an isotype control antibody.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein (e.g., a PD-L1/CD55 bispecific antibody) is internalized at a percentage that is about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, or about 200% to that of a monoclonal antibody (e.g., an anti-PD-Ll antibody, an anti-CD55 antibody, or an isotype antibody control).

In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 19.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 52 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 62 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 64 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 65 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 66 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 61 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 68 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 69 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 70 with zero, one or two amino acid insertions, deletions, or substitutions.

Antibody Characteristics

The antibodies or antigen-binding fragments thereof (e.g., bispecific antibodies) as described herein can increase immune response. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of T cells (e.g., CD3+ cells, CD8+ and/or CD4+ cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds .

In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can decrease the activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%,

60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies or antigen-binding fragments thereof as described herein does not induce immune response in normal cells (e.g., non-tumor cells) or in the absence of tumor cells.

In some embodiments, the antibodies or antigen-binding fragments thereof (e.g., bispecific antibodies) can bind to PD-L1 or PD-L2. Thus, the antibodies or antigen-binding fragments thereof described herein can block the binding between PD-1 and PD-L1 and/or the binding between PD-1 and PD-L2. In some embodiments, by binding to PD-L1 or PD-L2, the antibody can inhibit PD-1 signaling pathway and upregulates the immune response. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof as described herein are PD-1 antagonist. In some embodiments, the antibodies or antigen-binding fragments thereof are PD-1 agonist.

In some embodiments, the antibodies or antigen-binding fragments thereof (e.g., bispecific antibodies) can bind to CD3. Thus, the antibodies or antigen-binding fragments thereof described herein can recruit T cells to a target cell.

In some embodiments, the antibody (or antigen-binding fragments thereof) specifically binds to an antigen (e.g., a human protein, a monkey protein, and/or a mouse protein) 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. 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.

Affinities can be deduced from the quotient of the kinetic rate constants (Kd=koff7kon). In some embodiments, Kd is less than 1 x 10⁴M, less than 1 x 10⁵M, less than 1 x 10⁶M, less than 1 x 10⁷M, less than 1 x 10⁸ M, less than 1 x 10⁹ M, or less than 1 x 10 ¹⁰ M. In some embodiments, the Kd is less than 50nM, 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 10⁴M, greater than 1 x 10⁵M, greater than 1 x 10⁶M, greater than 1 x 10⁷M, greater than 1 x 10⁸M, greater than 1 x 10⁹ M, greater than 1 x 10 ¹⁰ M, greater than 1 x 10 ¹¹ M, or greater than 1 x 10 ¹² M. Furthermore, Ka can be deduced from Kd by the formula Ka=l/Kd.

General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR).

In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, or three transitions, with a first denaturation temperature, Tm Dl, and a second denaturation temperature Tm D2, and optionally a third denaturation temperature Tm D3.

In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm Dl greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D2 greater than 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D3 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95

C.

In some embodiments, Tm, Tm Dl, Tm D2, Tm D3 are less than 60, 61, 62, 63, 64, 65,

66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91

92, 93, 94, or 95 °C.

In some embodiments, the antibodies or antigen binding fragments as described herein do not start to form aggregation when the temperate is less than 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, Tagg266 or Tagg473 is less than 60, 61, 62, 63, 64, 65, 66, 67,

In some embodiments, the antibodies or antigen binding fragments as described herein have a pi greater than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,

8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9. In some embodiments, the antibodies or antigen binding fragments as described herein have a pi less than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,

9.8, or 9.9.

In some embodiments, the antibody has a tumor growth inhibition 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 antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. 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, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

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

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. In some embodiments, the antibodies or antigen binding fragments can increase complement dependent cytotoxicity (CDC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies or antigen binding fragments can increase antibody- dependent cell-mediated cytotoxicity (ADCC) by at least 10%, 20%, 30%, 40%, 50%, 60%,

70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies or antigen binding fragments can increase internalization rate by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies or antigen binding fragments can increase phagocytosis rate by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds,

3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies or antigen binding fragments can enhance T cell function, for example, by increasing effector T cell proliferation and/or increasing gamma interferon production by the effector T cell (e.g., as compared to proliferation and/or cytokine production prior to treatment with the antibodies or antigen binding fragments).

In some embodiments, the antibodies or antigen binding fragments enhance CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (e.g., as compared to proliferation and/or cytokine production prior to treatment with the antibodies or antigen binding fragments). In some embodiments, the cytokine is gamma interferon. In some embodiments, the antibodies or antigen binding fragments increase number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells prior to treatment with antibodies or antigen binding fragments. In some embodiments, the antibodies or antigen binding fragments increase number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells that express gamma interferon prior to treatment. In some embodiments, the antibodies or antigen binding fragments increase number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD8+ T effector cells prior to treatment. In some embodiments, the antibodies or antigen binding fragments increase number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells), e.g., compared to number of intratumoral (infiltrating) CD8+ T cells that express gamma interferon prior to treatment with the antibody.

In some embodiments, the antibodies or antigen binding fragments enhance memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine (e.g., gamma interferon) production by the memory cell.

In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent 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.

In some embodiments, the antibodies or antigen binding fragments can induce apoptosis.

In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F(ab’)2, and Fv fragments.

In some embodiments, the antibodies or antigen binding fragments are humanized antibodies. The humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. In some embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage is known in the art, and is described, e.g., in Jones, Tim D., et al. "The INNs and outs of antibody nonproprietary names." MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects. In some embodiments, the antibodies or antigen binding fragments are human antibodies.

In some embodiments, the bispecific antibody described herein (e.g., a HER2/CD3, CEACAM/CD3, Meso/CD3, or PD-L1/CD55 bispecific antibody) has an asymmetric structure comprising: 2, 3, 4, 5, or 6 antigen binding sites. In some embodiments, the bispecific antibody described herein comprises 2, 3, 4, 5, or 6 antigen binding sites (e.g., antigen binding Fab domains, scFV, or naonbody (VHH)) that target a cancer-specific antigen (e.g., HER2) or a cancer associated antigen. In some embodiments, the bispecific antibody described herein comprises 2, 3, 4, 5, or 6 antigen binding sites (e.g., antigen binding Fab domains, scFV, or naonbody (VHH)) that target a T cell specific antigen (e.g., CD3). In some embodiments, the bispecific antibody described herein (e.g., a HER2/CD3 bispecific antibody) comprises at least 2, 3, 4, 5, 6, or 7 common light chains. In some embodiments, the at least 2, 3, 4, 5, 6, or 7 common light chains have the same VL sequence. In some embodiments, the at least 2, 3, 4, 5, 6, or 7 common light chains have different VL sequences. In some embodiments, the cancer-specific antigen (e.g., HER2) binding Fab domain comprises the same VH sequence. In some embodiments, the cancer-specific antigen (e.g., HER2) binding Fab domain comprises different VH sequences. In some embodiments, the C-terminus of a cancer-specific antigen binding Fab domain is connected (e.g., covalently connected or chemically connected) to the N-terminus of a neighboring cancer-specific antigen binding Fab domain within the same bispecific antibody.

The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein. The cross- competing assay is known in the art, and is described e.g., in Moore et ak, "Antibody cross competition analysis of the human immunodeficiency virus type 1 gpl20 exterior envelope glycoprotein." Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein. The epitope binning assay is known in the art, and is described e.g., in Estep et al. "High throughput solution-based measurement of antibody- antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein reference in its entirety. Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed 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 recombinant antibody polypeptides or fragments thereof by recombinant techniques.

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.

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.

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; Rolls 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 ak, 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.

For expression, the DNA insert comprising an antibody-encoding or 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 SV40 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.

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.

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. Non-limiting bacterial promoters suitable for use include the E. coli lad 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.

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 etal, Methods Enzymol, 153: 516-544 (1997).

Introduction of the construct into the host cell can be effected 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.

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.

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.

The polypeptide (e.g., antibody) 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.

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.

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 nucleotide 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.

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.

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. 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.

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 length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. 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 purposes of the present invention, 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.

The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine percentage of sequence homology is known in the art. In some embodiments, amino acid residues conserved with similar physicochemical properties (percent homology), e.g. leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The homology percentage, in many cases, is higher than the identity percentage.

Methods of Making Antibodies

An isolated fragment of human protein (e.g., CEACAM5, HER2, Mesothelin, PD-L1, CD55, CD3, cancer specific antigen or cancer-associated antigen) can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of the protein) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody -producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (. Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

Phage display (panning) can be used to optimize antibody sequences with desired binding affinities. In this technique, a gene encoding single chain Fv (comprising VH or VL) can be inserted into a phage coat protein gene, causing the phage to "display" the scFv on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against target antigens, in order to detect interaction between the displayed antigen binding sites and the target antigen. Thus, large libraries of proteins can be screened and amplified in a process called in vitro selection, and antibodies sequences with desired binding affinities can be obtained.

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.

A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et ah, Nature, 321:522-525 (1986); Riechmann et ah, Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

In some embodiments, a covalent modification can be made to the antibody or antigen binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

In some embodiments, 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 gly costructures 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 Rabat 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 gly can heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).

In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.

In some embodiments, the methods described here are designed to make a bispecific antibody. 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.

In some embodiments, one or more amino acid residues in the CH3 portion of the IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W. The other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V. Furthermore, a substitution (-ppcpScp— >- ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG. In some embodiments, one heavy chain has a T366Y (knob) substitution, and the other heavy chain has a Y407T (hole) substation.

Furthermore, an anion-exchange chromatography can be used to purify bispecific antibodies. Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl- aminoethyl groups (DEAE). In solution, the resin is coated with positively charged counter-ions (cations). Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pi). The isoelectric point is defined as the pH at which a protein has no net charge. When the pH > pi, a protein has a net negative charge and when the pH < pi, a protein has a net positive charge. Thus, in some embodiments, different amino acid substitution can be introduced into two heavy chains, so that the pi for the homodimer comprising two Arm A and the pi for the homodimer comprising two Arm B is different. The pi for the bispecific antibody having Arm A and Arm B will be somewhere between the two pis of the homodimers. Thus, the two homodimers and the bispecific antibody can be released at different pH conditions. The present disclosure shows that a few amino acid residue substitutions can be introduced to the heavy chains to adjust pi.

Thus, in some embodiments, the amino acid residue at Kabat numbering position 83 is lysine, arginine, or histidine. In some embodiments, the amino acid residues at one or more of the positions 1, 6, 43, 81, and 105 (Kabat numbering) is aspartic acid or glutamic acid.

In some embodiments, the amino acid residues at one or more of the positions 13 and 105 (Kabat numbering) is aspartic acid or glutamic acid. In some embodiments, the amino acid residues at one or more of the positions 13 and 42 (Kabat numbering) is lysine, arginine, histidine, or glycine.

Bispecific antibodies can also include e.g., cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’

TNB derivative to form the bispecific antibody. Methods of Treatment

The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of engineered bispecific antibodies (e.g., imbalanced bispecific antibodies) of antigen-binding fragments thereof as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

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 (e.g., imbalanced bispecific antibodies) for the treatment of a condition associated with cancer will result in decreased number of cancer cells and/or alleviated symptoms.

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.

In some embodiments, the cancer is Rituximab (Rituxan®) resistant cancer.

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 inhibit 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.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or an antibody drug conjugate disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.

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 of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. 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.

In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

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.

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 subject 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.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody, an antigen binding fragment, or an antibody-drug conjugate 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 delay proliferation of a cell (e.g., abiopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody, antigen binding fragment, or antibody- drug conjugate may vary, depending on, inter alia , patient history as well as other factors such as the type (and/or dosage) of antibody used.

Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, 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 antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, antibody-drug conjugates, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et ah, Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 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 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.

In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, antibody-drug conjugates, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody, antigen binding fragment, antibody-drug conjugates, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

In some embodiments, the subject can be administered the at least one antibody, antigen binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks,

1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer). As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments, antibody-drug conjugates (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, 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 PBK/mTOR 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 -di oxygenase- 1) (IDOl) (e.g., epacadostat).

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, 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.

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, Vmflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

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.

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

In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti- PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti -LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

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 pharmaceutically 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. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Compositions containing one or more of any of the antibodies, antigen-binding fragments, 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). 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.

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 one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (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 antigen-binding fragments 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).

Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments, or 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 1 pg/kg to about 50 pg/kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen binding fragments thereof, 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 antibody or antibody fragment in vivo.

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 thereof, or antibody-drug conjugates for various uses as described herein.

EXAMPLES

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

Example 1. CEACAM5/CD3 bispecific antibody

Antibody Design

Five versions (VI, V2, V3, V4, and V5) of the CEACAM5/CD3 bispecific antibody were designed. Each bispecific antibody contains a CEACAM5 arm, a CD3 arm, and two identical common light chains. As shown in Table 1, the heavy chain variable region (VH) of each arm, and the common light chain (LC) are listed. Specifically, only the V5 version has knobs-into- holes mutations, i.e., the heavy chain of the CEACAM5 arm has knob mutations and the heavy chain of the CD3 arm has hole mutations. The sequence for 020618H3 VH is shown in SEQ ID NO: 1. The sequence for CCN196326 is shown in SEQ ID NO: 2. And the sequence for CCN209901 common light chain VL is shown in SEQ ID NO: 3. The V5 was selected for further experiments.

Table 1

Antigen Binding Test

Antigen binding test was performed by incubating a testing antibody at 12 pg/ml with Jurkat or LS174T cells, staining cells with a secondary antibody (Alexa Fluor 488, goat-anti human (GAH) at 1:500), and followed by flow cytometry analysis. An anti-CEACAM5 antibody (homodimer with the same VH sequence as 020618H3 and the same VL sequence as CCN209901 in Table 1), an anti-CD3 antibody (homodimer with the same VH sequence as CCN196326 and the same VL sequence as CCN209901 in Table 1), and the various versions of the CEACAM5/CD3 bispecific antibody in Table 1 were tested. The Jurkat cells express endogenous CD3 protein. The LS174T cells are colorectal cancer cells expressing CEACAM5 protein. As shown in FIG. 3 and Table 1, the V5 version of the CEACAM5/CD3 bispecific antibody exhibited comparable binding to Jurkat cells as the anti-CD3 antibody and comparable binding to LS174T cells as the anti-CEACAM5 antibody.

T cell Activation Test

T cell activation of Jurkat cells was measured in the presence and absence of cancer cells expressing CEACAM5. Specifically, 5 x 10⁴ LS174T cells (either expressing CEACAM5 (CEACAM5+) or not expressing CEACAM5 (CEACAM5-)) were incubated with 5 x 10⁴ Jurkat cells in the presence of different testing antibodies overnight. The testing antibodies included the CEACAM5/CD3 bispecific antibody and an isotype control antibody at 6 pg/ml. Equal volume of phosphate buffered saline (PBS) was used as a negative control. T cell activation was measured by determining percentage of CD69+/CD2+ cells by fluorescence-activated cell soring (FACS). As shown in FIG. 4, the CEACAM5/CD3 bispecific antibody alone induced T cell activation as compared to the isotype control antibody and PBS control. In addition, the presence of CEACAM5+ cancer cells further increased T cell activation induced by the CEACAM5/CD3 bispecific antibody.

Example 2. HER2/CD3 bispecific antibody and Mesothelin/CD3 bispecific antibody

Construction of Panning Phage Library and Panning Strategy

ABS CD3-VL phage library with human naive VH repertoire was constructed as shown in FIG. 5. Specifically, the VL repertoire of ABS human naive phage library was replaced with ABS anti-CD3 VLs and a few of its functional variants. The constructed library comprised about 10⁸ sequences.

A phage library panning workflow is shown in FIG. 6 and specific phage binders were enriched after the third panning with antigen mixtures. Specifically, 5 cancer antigens were selected and pooled into two groups (Ag Mix #1 and Ag Mix #2 group, respectively). The ABS- CD3-VL library was then panned with each antigen mixture. After 3 rounds of panning, the output phage was tested for specific binding by ELISA. The phage ELISA assay results for the third panning output is shown in FIG. 7.

Specific binders were effectively recovered from the ABS-CD3-VL library by phage clone screening. Specifically, after 3 rounds of panning, single colony was picked to one 96-well plate for each antigen mixture, and screened by ELISA with individual antigen. As a result, 5 unique sequences were recovered to bind HER2 and 6 unique sequences were recovered to bind Mesothelin. It was also observed that some binders can cross react with other antigens in the same group.

The positive clones were assembled into HER2/CD3 or Mesothelin/CD3 bispecific antibodies. The cancer-specific antigen (CSA) arms of 4 lead bispecific antibodies were tested by ELISA binding assays with HER2 and Mesothelin recombinant proteins. As shown in FIG. 8, the four bispecific antibodies showed high CSA specific binding by ELISA. In particular, the HER2-B3/CD3 bispecific antibody showed a higher HER2 binding compared to the HER2- D1/CD3 bispecific antibody. The Meso-G7/CD3 bispecific antibody showed a higher Mesothelin binding compared to the Meso-A4/CD3 bispecific antibody. No cross reaction was observed in the four lead bispecific antibodies.

For the two HER2/CD3 bispecific antibodies, the sequence for the HER2-B3 arm VH is shown in SEQ ID NO: 7. The sequence for the HER2-D1 arm VH is shown in SEQ ID NO: 13. The sequence for the CD3 arm VH is shown in SEQ ID NO: 8. The sequence for the common VL is shown in SEQ ID NO: 9. Similarly, with respect to the two Meso/CD3 bispecific antibodies, the sequence for the Meso-G7 arm VH is shown in SEQ ID NO: 15. The sequence for the Meso-A4 arm VH is shown in SEQ ID NO: 21. The sequence for the CD3 arm VH is shown in SEQ ID NO: 16. The sequence for the common VL is shown in SEQ ID NO: 17.

Antigen Binding Test

Antigen binding test was performed by incubating SK-BR-3 cells with a testing antibody at 10 pg/ml with, staining the cells with a secondary antibody (Alexa Fluor 488, goat-anti-human (GAH) at 1 : 1000), detaching the cells with 2 nM EDTA, and followed by flow cytometry analysis. An anti-HER2 antibody was used as a positive control. HER2-B3/CD3 and HER2- D1/CD3 bispecific antibodies were tested. The SK-BR-3 cells express HER2 protein endogenously. As shown in FIG. 9, the HER2-B3/CD3 bispecific antibody exhibited comparable binding to SK-BR-3 as the anti-HER2 antibody. However, HER2-D1/CD3 did not bind to SK- BR-3. Thus, HER2-B3/CD3 was selected as a lead HER2/CD3 bispecific antibody.

T cell Activation Test

T cell activation and SK-BR-3 cell killing mediated by HER2/CD3 bispecific antibodies in the presence of peripheral blood mononuclear cells (PBMCs) were measured as follows. A frozen aliquot of PBMC was thawed overnight and mixed with SK-BR-3 cells at 25:1 ratio. The cells were seeded in a U-bottom 96-well plate. Antibodies were diluted to 10 pg/ml and co incubated with the mixed cells at 37 °C for 24 hours. Afterwards, the plate was centrifuged at 1200 rpm for 5 minutes. The cells were washed with 120 pi PBS. Supernatant was then discarded and 200 pi EDTA (2 mM) was added to digest the cells for 10 minutes. After the digestion, cells were centrifuged at 1200 rpm for 5 minutes, and then washed twice with FACS buffer (PBS supplemented with 2% BSA). Next, PE conjugated anti-human CD69 antibody and APC conjugated anti -human CD2 antibody were added at 10 pl/well and 2 pl/well, respectively, followed by an incubation at room temperature for 30 minutes. After the incubation, the cells were washed twice with FACS buffer. After washing by FACS buffer twice, the cells were subjected to flow cytometry analysis. As shown in FIG. 10, HER2-B3/CD3 bispecific antibody exhibited higher T cell activation than an isotype control and a HER2-B3 homodimer IgG at both 10 pg/ml and 1 pg/ml The specific CD69+/CD2+ cell percentages (T cell activation) are listed in the table below.

Table 2

In a different experiment, T cell activation mediated by HER2-B3/CD3 (1:1) and HER2- B3/CD3 (2:1) in the presence of PBMC was measured. Schematic structures of HER2-B3/CD3 (2:1) and HER2-B3V3 /CD3 (1:1) are shown in FIG. 11A and FIG. 11B, respectively. As shown in the figure, in the 2:1 format, the HER2 arm has two antigen binding sites.

Briefly, PMBCs were co-incubated with SK-BR-3 cells and HER2-B3V3/CD3 (1:1) or HER2-B3/CD3 (2:1) bispecific antibodies. After 24 hours, the cells were stained with PE conjugated anti-human CD69 and APC conjugated anti-human CD2 antibodies, followed by flow cytometry analysis.

The experiment was performed as follows. A frozen aliquot of PBMC was thawed overnight. On the next day, both SK-BR-3 cells and PMBCs were diluted to a cell density of 5 ^c 10⁵ cells/ml, and then mixed in a 1:10 ratio, i.e., 5 x 10⁴ SK-BR-3 cells mixed with 5 x 10⁵ PBMCs in each well. The mixed cells were seeded in a flat-bottom 96-well plate at 100 mΐ/well. Next, HER2-B3V3/CD3 (1:1), HER2-B3/CD3 (2:1), CD20/CD3 bispecific antibodies, and an isotype control antibody (11F11) were diluted to 20 pg/ml in cell medium. Then, 100 mΐ of each antibody was added to corresponding wells in the 96-well plate to have final antibody concentration of 10 pg/ml. Next, the plate was incubated in a 37 °C, 5% CO2 incubator for 24 hours. After the incubation, the cells and supernatant were transferred to a new V-bottom plate, which was then centrifuged at 1200 rpm for 5 minutes to form a pellet. The supernatant was gently flicked, and the cell pellet was washed twice with FACS buffer. Next, the stock PE conjugated anti-human CD69 antibody was diluted at 1 : 50 dilution ratio to a working concentration, and 100 mΐ of diluted CD69-PE was added to corresponding wells, followed by an incubation for 30 minutes. After the incubation, cells were washed twice, and resuspended in 120 mΐ FACS buffer for flow cytometry analysis. Unstained SK-BR-3 cells mixed with PBMCs were used for gating purposes.

As shown in FIG. 12, the percent of CD69/CD2 positive cells ranged from 0-28% depending on the treatment. As expected, 10 pg/ml 11F11 treatment achieved the lowest CD69/CD2 positive cell percentage. In addition, the positive control 10 pg/ml of CD20/CD3 bispecific antibody treatment achieved the highest CD69/CD2 positive cell percentage of about 28%. T cell activation induced by HER2-B3/CD3 (2: 1) bispecific antibody was similar to that of the CD20/CD3 bispecific antibody positive control. HER2-B3V3/CD3 (1:1) bispecific antibody achieved a slightly lower T cell activation as compared to that of HER2-B3/CD3 (2:1) bispecific antibody, which was about 14% of CD69/CD2 positive cells. Based on these results, HER2/CD3 (2: 1) format activates T cells at a higher level as compared to the 1:1 format using PBMCs.

Purification of HER2-B3/CD3 (2:1) Bispecific Antibody

HER2-B3/CD3 (2:1) bispecific antibody was purified using cation exchange (CEX) chromatography, as shown in FIG. 13A. Briefly, 3.2 mg of protein A column-purified sample was loaded to a cation exchange column, and 1.43 mg purified HER2-B3/CD3 (2:1) bispecific antibody was recovered from the A3-A7 fractions. Thus, the recovery rate was 1.43/3.2 = 44.7%. The A3-A7 fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) after the cation exchange chromatography. As shown in FIG. 13B, the non-reduced and reduced bands confirmed that the recovered HER2-B3/CD3 (2: 1) bispecific antibody was of high purity and had expected molecular weight.

Example 3. PD-L1/CD55 bispecific antibody

Antibody Design

A human naive scFv phage library with human naive VH repertoire and VL repertoire was constructed. Panning was performed with human PD-L1. The antibodies were purified and then screened to test their capability to block PD-L1 and PD1 interaction. A total of 14 anti-PD- L1 monoclonal antibodies were selected for downstream analysis.

Light chains of the selected anti-PD-Ll antibodies were used to replace the light chain of an anti-CD55 monoclonal antibody. The resulting hybrid antibodies were screened to test their binding with CD55. Results are shown in the table below. Table 3

Specifically, a rituximab-resistant cell line (RRCL) was co-transfected with a plasmid encoding the heavy chain of the anti-CD55 monoclonal antibody and a plasmid encoding a light chain of the 14 anti-PD-Ll monoclonal antibodies, respectively. The transfected cells expressing a hybrid antibody were screened to test their binding with CD55. Eight hybrid antibodies exhibited high CD55 binding, three hybrid antibodies exhibited medium CD55 binding, and three hybrid antibodies exhibited low CD55 binding. Those anti-PD-Ll antibodies whose light chain could pair with the anti-CD55 antibody heavy chain without significant loss of CD55 binding were redesigned to produce IgG-like PD-L1/CD55 bispecific antibodies with a common light chain. One PD-L1/CD55 bispecific antibody was selected for further experiments.

The sequence for the PD-L1 arm VH is shown in SEQ ID NO: 23. The sequence for the CD55 arm VH is shown in SEQ ID NO: 24. The sequence for the common VL is shown in SEQ ID NO: 71.

Complement-dependent cytotoxicity Test

Complement-dependent cytotoxicity (CDC) mediated by the 2E2, 1E6, 2B2, 2D1, 1G11, and IG4 PD-L1/CD55 bispecific antibodies was determined as follows. A reference PD- L1/CD55 bispecific antibody was used for comparison reasons, and details can be found, e.g., in PCT/US2018/044778, which is incorporated herein by reference in its entirety. Specifically, target cells (MDA231 or PANC1 cells) were seeded in a 96-well plate (5 ^c 10⁴ cells/well) and testing antibodies were added to a final concentration of 10 pg/ml. The plate was then incubated at 37 °C for 40 hours. Afterwards, the cells were trypsinized and then washed with plain medium (without FBS). Next, The cells were resuspended in a solution with 10 pg/ml antibody and 5% complement enriched serum, followed by an incubation at 37 °C for 4 hours. After the incubation, the cells were trypsinized again and washed with FACS buffer twice. Next, the cells were stained with 7-aminoactinomycin D (7-AAD) at a 1:50 dilution ratio and incubated for 15 minutes in dark at room temperature. Afterwards, the cells were washed twice with FACS buffer and subjected to FACS analysis. As shown in FIGS. 14A-14B, the 2E2, 1E6, 2B2, 2D1, 1G11, and IG4 PD-L1/CD55 bispecific antibodies exhibited comparable CDC activity as compared to that of the reference PD-L1/CD55 bispecific antibody. In particular, some of the testing antibodies (e.g., 2E2) exhibited a higher CDC activity than that of the reference bispecific antibody.

Internalization Test

Pep-ZAP was used to measure antibody internalization. Pep-ZAP is a Fc binding peptide conjugated to a translation inhibitor protein and cell death is the readout of antibody internalization. Details of Pep-ZAP can be found, e.g., in PCT/US2019/042314, which is incorporated herein by reference in its entirety. Specifically, target cells (MDA231 or PANC1 cells) were seeded in a 96-well plate (5 ^c 10⁴ cells/well) and antibodies were added to a final concentration of 10 pg/ml. The plate was then incubated at 37 °C for 30 minutes. Afterwards, Pep-ZAP was added to a final concentration of 10 pg/ml and the plate was incubated at 37 °C for 18 hours. After the incubation, cells were trypsinized and washed twice with FACS buffer. Next, the cells were stained with 7-aminoactinomycin D (7-AAD) at a 1:50 dilution ratio and incubated for 15 minutes in dark at room temperature. Afterwards, the cells were washed twice with FACS buffer and subjected to FACS analysis. The results are shown in FIGS. 15A-15B, in which the 2E2, 1E6, 2B2, 2D1, 1G11, and IG4 PD-L1/CD55 bispecific antibodies exhibited comparable internalization ability as compared to that of the reference PD-L1/CD55 bispecific antibody. In particular, some of the testing antibodies (e.g., 2E2) exhibited a higher internalization ability than that of the reference bispecific antibody. In a separate experiment, flow cytometry was used to measure antibody internalization. Specifically, goat anti human IgG was labelled with pHrodo Red. Target cells (MDA231 cells) were seeded in a 96-well plate (5 ^c 10⁴ cells/well) and testing antibodies were added to a final concentration of 10 pg/ml. The plate was then incubated at 37 °C for 30 minutes. Next, pHrodo Red labelled goat anti human IgG was added to a final concentration of 10 pg/ml, and the plate was incubated at 37 °C for 24 hours. After the incubation, the cells were trypsinized, washed twice with FACS buffer, and subjected to FACS analysis. As shown in FIG. 16, both the 2E2 PD-L1/CD55 bispecific antibody (with common light chain VL comprising SEQ ID NO: 71) and its CF variant (with common light chain VL comprising SEQ ID NO: 25) exhibited comparable internalization as compared to that of the reference PD-L1/CD55 bispecific antibody. In addition, the CF variant has better antibody developability.

T cell Activation Test

In order to test whether the PD-L1/CD55 bispecific antibodies could activate T cell at a higher level than anti-PD-Ll monoclonal antibodies, the following experiment was performed. PANC-1 cells were incubated with PBMCs in the presence of different testing antibodies at 1.5 pg/ml, 0.15 pg/ml, or 0 pg/ml, as indicated in FIG. 17A. Specifically, PANC1 cells were incubated with PBMCs at a 1:3 ratio in the presence of different testing antibodies at 37 °C for 48 hours. T cell activation was determined by measuring percentage of CD69+/CD2+ cells by FACS. The results showed that 2E2 (CF) PD-L1/CD55 bispecific antibody exhibited higher T cell activation than anti-PD-Ll and anti-CD55 monoclonal antibodies. In addition, the 2E2 (CF) PD-L1/CD55 bispecific antibody exhibited comparable T cell activation level as compared to that of the reference PD-L1/CD55 bispecific antibody. Released cytokines (e.g., IL2, IFNy, and TNFa) were also measured by ELISA and results are shown in FIGS. 17B-17D, in which the 2E2 (CF) PD-L1/CD55 bispecific antibody induced comparable level of released cytokines as compared to that of the reference PD-L1/CD55 bispecific antibody. In particular, the 2E2 (CF) PD-L1/CD55 bispecific antibody induced higher level of released TNFa as compared to anti-PD- Ll or anti-CD55 monoclonal antibodies and an isotype control antibody.

In a separate experiment, PANC-1 cells and PBMCs were pre-incubated at a 1:3 ratio for 24 hours before the testing antibodies were added. After the pre-incubation, different testing antibodies were added to a final concentration of 1 ^c 1 O ⁷ or 1 ^c 10⁸ M, followed by an incubation at 37 °C for 48 hours. T cell activation was determined by measuring percentage of CD69+/CD2+ cells by FACS. As shown in FIG. 17E, 2E2 (CF) PD-L1/CD55 bispecific antibody exhibited higher T cell activation than anti-PD-Ll and anti-CD55 monoclonal antibodies. In addition, the 2E2 (CF) PD-L1/CD55 bispecific antibody exhibited comparable T cell activation level as compared to that of the reference PD-L1/CD55 bispecific antibody. However, the CF/B and CF/C variant exhibited a lower T cell activation level as compared to the 2E2 (CF) PD-L1/CD55 bispecific antibody.

A sequence alignment between the common VL of 2E2 PD-L1/CD55 bispecific antibody and the common VL of the 2E2 (CF) variant is shown in FIG. 20. The CF variant contains a cysteine to phenylalanine mutation at position 83 (Kabat numbering). In addition, a sequence alignment between the CD55 arm of the CF, CF/B, and CF/C variants of 2E2 PD-L1/CD55 bispecific antibody is shown in FIG. 21.

OTHER EMBODIMENTS

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

WHAT IS CLAIMED IS:

1. A bispecific antibody or antigen-binding fragment thereof that binds to CEACAM5 (Carcinoembryonic Antigen Related Cell Adhesion Molecule 5) and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

2. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

3. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 1; the second heavy chain variable region (VH) comprise SEQ ID NO: 2; the first light chain variable region (VL) comprise SEQ ID NO: 3; and the second light chain variable region (VL) comprises SEQ ID NO: 3.

4. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6.

5. The bispecific antibody or antigen-binding fragment thereof of claim 4, wherein the third polypeptide and the fourth polypeptide are at least 90%, 95%, 99%, or 100% identical.

6. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to CEACAM5 with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

7. The bispecific antibody or antigen-binding fragment thereof of claim 6, wherein the second antigen binding region specifically binds to CD3 with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹ 10⁵ M ¹ or 10⁴ M ¹

8. The bispecific antibody or antigen-binding fragment thereof of claim 6, wherein the binding affinity of the first antigen binding region when it binds to CEACAM5 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

9. The bispecific antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the first heavy chain and the second chain associate with each other by the knobs into holes approach.

10. A bispecific antibody or antigen-binding fragment thereof that binds to HER2 (Erb-B2 Receptor Tyrosine Kinase 2) and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 13; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

11. The bispecific antibody or antigen-binding fragment thereof of claim 10, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

12. The bispecific antibody or antigen-binding fragment thereof of claim 10, wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 7 or 13; the second heavy chain variable region (VH) comprise SEQ ID NO: 8; the first light chain variable region (VL) comprise SEQ ID NO: 9; and the second light chain variable region (VL) comprises SEQ ID NO: 9.

13. The bispecific antibody or antigen-binding fragment thereof of claim 10, wherein the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10 or 14; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

14. The bispecific antibody or antigen-binding fragment thereof of claim 13, wherein the third polypeptide and the fourth polypeptide at least 90%, 95%, 99%, or 100% identical.

15. The bispecific antibody or antigen-binding fragment thereof of claim 13, , wherein the first polypeptide comprises SEQ ID NO: 10.

16. The bispecific antibody or antigen-binding fragment thereof of claim 13, , wherein the first polypeptide comprises SEQ ID NO: 14.

17. The bispecific antibody or antigen-binding fragment thereof of claim 10, wherein the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to HER2 with a binding affinity greater than 10⁷ M^·1, 10⁸ M^-1, 10⁹ M^-1, IO^ M ¹, lO¹^ ¹, 10¹² M^-1, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

18. The bispecific antibody or antigen-binding fragment thereof of claim 17, wherein the second antigen binding region specifically binds to CD3 with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹ 10⁵ M ¹ or 10⁴ M ¹

19. The bispecific antibody or antigen-binding fragment thereof of claim 17, wherein the binding affinity of the first antigen binding region when it binds to HER2 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

20. The bispecific antibody or antigen-binding fragment thereof of any one of claims 10-19, wherein the first heavy chain and the second chain associate with each other by the knobs into holes approach.

21. A bispecific antibody or antigen-binding fragment thereof that binds to Mesothelin and CD3 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 21; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

22. The bispecific antibody or antigen-binding fragment thereof of claim 21, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

23. The bispecific antibody or antigen-binding fragment thereof of claim 21, wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 15 or 21; the second heavy chain variable region (VH) comprise SEQ ID NO: 16; the first light chain variable region (VL) comprise SEQ ID NO: 17; and the second light chain variable region (VL) comprises SEQ ID NO: 17.

24. The bispecific antibody or antigen-binding fragment thereof of claim 21, wherein the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 or 22; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20.

25. The bispecific antibody or antigen-binding fragment thereof of claim 24, wherein the third polypeptide and the fourth polypeptide are at least 90%, 95%, 99%, or 100% identical.

26. The bispecific antibody or antigen-binding fragment thereof of claim 24, , wherein the first polypeptide comprises SEQ ID NO: 18.

27. The bispecific antibody or antigen-binding fragment thereof of claim 24, , wherein the first polypeptide comprises SEQ ID NO: 22.

28. The bispecific antibody or antigen-binding fragment thereof of claim 21, wherein the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to Mesothelin with a binding affinity greater than 10⁷ M ¹, 10⁸ M ¹, 10⁹ M ¹, 10¹⁰ M ¹, 10¹¹ M ¹, 10¹² M ¹, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD3 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

29. The bispecific antibody or antigen-binding fragment thereof of claim 28, wherein the second antigen binding region specifically binds to CD3 with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹ 10⁵ M ¹ or 10⁴ M ¹

30. The bispecific antibody or antigen-binding fragment thereof of claim 28, wherein the binding affinity of the first antigen binding region when it binds to Mesothelin is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD3.

31. The bispecific antibody or antigen-binding fragment thereof of any one of claims 21-30, wherein the first heavy chain and the second chain associate with each other by the knobs into holes approach.

32. A bispecific antibody or antigen-binding fragment thereof that binds to PD-L1 (Programmed Cell Death 1 Ligand 1) and CD55 comprising a first polypeptide comprising a first heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23; a second polypeptide comprising a second heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; a third polypeptide comprising a first light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25; and a fourth polypeptide comprising a second light chain variable region (VL) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25.

33. The bispecific antibody or antigen-binding fragment thereof of claim 32, wherein the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

34. The bispecific antibody or antigen-binding fragment thereof of claim 32, wherein the first heavy chain variable region (VH) comprises SEQ ID NO: 23; the second heavy chain variable region (VH) comprise SEQ ID NO: 24; the first light chain variable region (VL) comprise SEQ ID NO: 25; and the second light chain variable region (VL) comprises SEQ ID NO: 25.

35. The bispecific antibody or antigen-binding fragment thereof of claim 32, wherein the first polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26; the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; the third polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28; and the fourth polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28.

36. The bispecific antibody or antigen-binding fragment thereof of claim 35, wherein the third polypeptide and the fourth polypeptide are at least 90%, 95%, 99%, or 100% identical.

37. The bispecific antibody or antigen-binding fragment thereof of claim 32, wherein the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to PD-L1 with a binding affinity greater than 10⁷ M^·1, 10⁸ M^-1, 10⁹ M^-1, IO^ M ¹, lO¹^ ¹, 10¹² M^-1, and the second heavy chain variable region and the second light chain variable region associate with each other, forming a second antigen binding region that specifically binds to CD55 with a binding affinity less than 10⁹ M ¹, 10⁸ M ¹, 10⁷ M ¹, 10⁶ M ¹, 10⁵ M ¹, or 10⁴ M ¹.

38. The bispecific antibody or antigen-binding fragment thereof of claim 37, wherein the second antigen binding region specifically binds to with a binding affinity greater than 10⁷ M ¹, 10⁶ M ¹ 10⁵ M ¹ or 10⁴ M ¹

39. The bispecific antibody or antigen-binding fragment thereof of claim 37, wherein the binding affinity of the first antigen binding region when it binds to PD-L1 is at least 100, 1000, or 10000 times greater than the binding affinity of the second antigen binding region when it binds to CD55.

40. The bispecific antibody or antigen-binding fragment thereof of any one of claims 32-39, wherein the first heavy chain and the second chain associate with each other by the knobs into holes approach.

41. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-40 covalently bound to a therapeutic agent.

42. The antibody drug conjugate of claim 41, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

43. A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the bispecific antibody or antigen-binding fragment thereof of any one of claims 1-40, or the antibody-drug conjugate of claims 41 or 42, to the subject.

44. The method of claim 43, wherein the subject has a solid tumor.

45. The method of claim 43, wherein the cancer is lung cancer, stomach cancer, rectum cancer, breast cancer, mesothelioma, ovarian cancer, or pancreatic adenocarcinoma.

46. 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 1-40, or the antibody-drug conjugate of claims 41 or 42, to the subject.

47. 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 of any one of claims 1-40, or the antibody-drug conjugate of claims 41 or 42, to the subject.

48. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-40, and a pharmaceutically acceptable carrier.

49. A pharmaceutical composition comprising the antibody drug conjugate of claim 41 or 42, and a pharmaceutically acceptable carrier.

50. An antibody or antigen-binding fragment thereof that binds to CD3 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, 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, 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:

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, and 46, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, and 49, respectively; and (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 56, 57, and 58, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively.

51. An antibody or antigen-binding fragment thereof that binds to CEACAM5 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 30, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 31; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 35, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 36, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 37.

52. An antibody or antigen-binding fragment thereof that binds to HER2 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, 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, 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: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 38, 39, and 40, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, and 49, respectively; and

53. An antibody or antigen-binding fragment thereof that binds to Mesothelin comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, 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, 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:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 51, and 52, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively; and

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 53, 54, and 55, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 59, 60, and 61, respectively.

54. An antibody or antigen-binding fragment thereof that binds to PD-L1 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 62, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 63, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 64; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 70.

55. An antibody or antigen-binding fragment thereof that binds to CD55 comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 65, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 66, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 67; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 70.

56. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 50-55.