Molecules that bind to CD66e polypeptide

Abstract

This document provides methods and materials involving binding of binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers and / or ADCs) to CD66e polypeptides. For example, provided are binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers and / or ADCs) that bind to CD66e polypeptides, as well as methods and materials using one or more such binding molecules to treat a mammal (e.g., a human) with cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 158,205, filed March 8, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application. [Background technology]

[0002] 1.Technical Field This document relates to methods and materials involving binding of molecules (e.g., antibodies, antibody fragments, antibody domains, chimeric antigen receptors (CARs), cell engagers, or antibody drug conjugates (ADCs)) to CD66e polypeptides. For example, this document provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to CD66e polypeptides, as well as methods and materials using such binding agents to treat cancer. This document also provides cells (e.g., host cells) engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to CD66e polypeptides, as well as methods and materials using such cells to treat cancer.

[0003] 2. Background information CD66e, also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), is a member of the carcinoembryonic antigen (CEA) gene family. CD66e is highly expressed in neuroendocrine prostate cancer and contains an N-terminal Ig variable region-like (IgV) domain and six Ig constant region type 2-like (IgC2-like) domains, designated A1-B1-A2-B2-A3-B3. The A3-B3 domain is considered to be the membrane proximal region, and these two domains are present in the splice variants of CD66e in many cancers. Summary of the Invention

[0004] This document provides methods and materials involving binding of molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) to CD66e polypeptides. For example, this document provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to CD66e polypeptides, as well as methods and materials for using one or more such binding agents to treat a mammal (e.g., a human) with cancer.

[0005] This document also provides cells (e.g., host cells) engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a CD66e polypeptide, as well as methods and materials for using such cells to treat cancer.

[0006] As described herein, a binding agent (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more CARs, one or more cell engagers, and / or one or more ADCs) can be engineered to have the ability to bind to a CD66e polypeptide. For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, or ADC) provided herein can be capable of binding to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human CD66e polypeptide set forth in SEQ ID NO: 150 or SEQ ID NO: 151 (see, e.g., FIG. 1).

[0007] In some cases, two sets of three CDRs of the antigen-binding fragments provided herein (e.g., SEQ ID NOs: 1-3 and 9-11 or SEQ ID NOs: 17-19 and 25-27) bind to CD66e + Cells (e.g., CD66e + Tumor cells and / or CD66e + CARs with the ability to target tumor vasculature + Cells (e.g., CAR + T cells, CAR + Stem cells, e.g. CAR +Induced pluripotent stem cells, or CARs + Natural killer (NK) cells) may be engineered into the CAR to generate CD66e + Cells (e.g., CD66e + Tumor cells and / or CD66e + We have developed an antibody that has the ability to target the tumor vasculature and target CD66e + It may be engineered into the antibody structure containing an Fc region to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against cells, and / or CD66e + Cells (e.g., CD66e + Tumor cells and / or CD66e + Targets tumor vasculature and targets CD66e + Fc-containing antibodies may be engineered into cell engagers, such as bispecific T cell engagers (e.g., BiTEs), bispecific killer engagers (e.g., BiKEs) and / or trispecific killer engagers (e.g., TriKEs), to create cell engagers capable of inducing one or more immune responses against the cells (e.g., a T cell immune response and / or ADCC using the cell engager in the absence of an Fc-containing antibody). Note that BiKE and TriKE mediated killing are sometimes referred to as ADCC, even though they are not initiated by an Fc domain.

[0008] In addition, as described herein, the binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, and / or one or more antibody domains) can be used to create a conjugate comprising the binding agent and a drug. For example, an ADC, such as a whole antibody drug conjugate, a Fab drug conjugate, and / or an antibody domain drug conjugate, can be designed to include an appropriate binding agent provided herein to create the conjugate. Such a conjugate can be used to bind to a target cell, such as a cancer cell (e.g., CD66e + Cancer cells) or cancer vasculature (e.g., CD66e + It can be used to deliver drug payloads to the tumor vasculature.

[0009] Also as described herein, the binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) can be used to treat a mammal (e.g., a human) with cancer. For example, cancer (e.g., CD66e + A mammal (e.g., a human) having a cancer may be administered a composition comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) described herein to reduce the number of cancer cells in the mammal, to induce ADCC against cancer cells in the mammal, and / or to extend survival of the mammal from the cancer.

[0010] Also as described herein, a cell (e.g., a host cell) can be engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a CD66e polypeptide. For example, a cell, such as a T cell (e.g., a CTL), a stem cell (e.g., an induced pluripotent stem cell), or a NK cell, can be engineered to express one or more CARs capable of binding to a CD66e polypeptide. Such cells (e.g., CD66e-specific CARs) can be engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a CD66e polypeptide. + T cells or NK cells) can be used to treat cancer.

[0011] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used to detect the presence or absence of a CD66e polypeptide. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used to detect the presence or absence of a CD66e polypeptide when a sample (e.g., a biological sample, e.g., a tumor biopsy) obtained from a mammal (e.g., a human) detects CD66e + Cells (e.g., CD66e + The CD66e polypeptide (e.g., CD66e+ Having the ability to detect the presence or absence of CD66e (cancer cells) may enable clinicians, medical professionals and patients to make better decisions regarding potential treatment options. + Detection of cancer cells is a key step for clinicians, healthcare professionals, and patients to detect CD66e + Allows selection of appropriate anti-cancer treatments to target cancer cells. CD66e + Such treatments to target cancer cells include administration of an anti-CD66e antibody, e.g., SAR408701, and / or one or more binding agents described herein capable of binding to a CD66e polypeptide, and / or one or more cells engineered to express a binding agent described herein (e.g., a CD66e-specific CAR + The method may include administration of a therapeutic agent such as a medicament for treating a disease, such as a pulmonary artery disease (PACS), a pulmonary artery disease, or a pulmonary artery disease (T cells or NK cells).

[0012] In general, one aspect of this document is a method for producing a light chain variable domain or region comprising: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The present invention features (provides) an antibody comprising (or consisting essentially of, or consisting of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.

[0013] In another aspect, this document provides a method for detecting a mutated nucleotide sequence comprising: (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The present invention features an antigen-binding fragment comprising (or consisting essentially of, or consisting of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16.The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab.

[0014] In another aspect, this document features a chimeric antigen receptor comprising (or consisting essentially of, or consisting of) an antigen binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen binding domain comprises an antibody or antigen binding fragment. The antibody comprises: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8.The antibody may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The antigen-binding domain may comprise an scFv capable of binding to a CD66e polypeptide. The hinge may comprise a hinge as set forth in FIG. 13. The transmembrane domain may comprise a transmembrane domain as set forth in FIG. 14. The chimeric antigen receptor may comprise one or more signaling domains as set forth in FIG. 15.

[0015] In another aspect, this document features a cell comprising the chimeric antigen receptor of the preceding paragraph. The cell can be a T cell, a stem cell, or a NK cell. For example, one aspect of this document features an isolated population of cells, where at least one cell of the population comprises the chimeric antigen receptor of the preceding paragraph. The cell can be a T cell, a stem cell, or a NK cell. In some embodiments, at least 50 percent, at least 75 percent, at least 95 percent, at least 99 percent, or 100 percent of the cells of the population can comprise the chimeric antigen receptor.

[0016] In another aspect, this document features a cell engager comprising (or consisting essentially of, or consisting of) a first antigen-binding domain, a linker and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody or antigen-binding fragment. The antibody comprises: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8.The antibody may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The first antigen-binding domain may comprise an scFv capable of binding to a CD66e polypeptide. The first antigen-binding domain may be an IgG capable of binding to a CD66e polypeptide. The linker may comprise a linker as set forth in FIG. 10 or FIG. 13. The second antigen-binding domain may bind to a polypeptide expressed on the surface of a T cell. The polypeptide expressed on the surface of a T cell may be a CD3 polypeptide. The second antigen-binding domain may be an antigen-binding domain as set forth in FIG. 18. The second antigen-binding domain may bind to a polypeptide expressed on the surface of a NK cell. The polypeptide expressed on the surface of the NK cell can be a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide. The second antigen-binding domain can be an antigen-binding domain described in FIG. 19. The cell engager can comprise a third antigen-binding domain. The third antigen-binding domain can bind to a polypeptide expressed on the surface of the NK cell. The polypeptide expressed on the surface of the NK cell can be a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide. The third antigen-binding domain can be an antigen-binding domain described in FIG. 19.

[0017] In another aspect, this document features a nucleic acid (e.g., isolated nucleic acid) comprising (or consisting essentially of, or consisting of) a nucleic acid sequence encoding at least a portion of an antibody or antigen-binding fragment. The antibody comprises: (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:18 (or SEQ ID NO:19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:20 (or SEQ ID NO:21 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:22 (or SEQ ID NO:22 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:23 (or SEQ ID NO:23 with one The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody may comprise a light chain variable domain or region of (i).The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The nucleic acid sequence may encode any one of the heavy chain variable domains or regions of (i) to (ii). The nucleic acid sequence may encode any one of the light chain variable domains or regions of (i) to (ii). The nucleic acid may be a viral vector. The nucleic acid may be a phagemid.

[0018] In another aspect, this document features a nucleic acid (e.g., an isolated nucleic acid) comprising (or consisting essentially of, or consisting of) a nucleic acid sequence encoding a chimeric antigen receptor as described above or a cell engager as described above. The nucleic acid can be a viral vector. The nucleic acid can be a phagemid.

[0019] In another aspect, this document features a host cell that comprises a nucleic acid of any of the two preceding paragraphs. For example, one aspect of this document features an isolated population of host cells, where at least one host cell of the population comprises a nucleic acid of any of the two preceding paragraphs. In some embodiments, at least 50 percent, at least 75 percent, at least 95 percent, at least 99 percent, or 100 percent of the cells of the population may comprise a nucleic acid of any of the two preceding paragraphs.

[0020] In another aspect, this document features a host cell that expresses the chimeric antigen receptor described above or the cell engager described above. The host cell can be a T cell, a stem cell, or a NK cell. For example, one aspect of this document features an isolated population of host cells, where at least one host cell of the population expresses the chimeric antigen receptor described above or the cell engager described above. In some embodiments, at least 50 percent, at least 75 percent, at least 95 percent, at least 99 percent, or 100 percent of the cells of the population can express the chimeric antigen receptor described above or the cell engager described above.

[0021] In another aspect, this document features an antibody drug conjugate (ADC) that comprises (or consists essentially of, or consists of) an antigen-binding domain covalently linked to a drug, where the antigen-binding domain comprises an antibody or an antigen-binding fragment. The antibody comprises: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8.The antibody may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The antigen-binding domain may comprise an scFv capable of binding to a CD66e polypeptide. The antigen-binding domain may be an IgG capable of binding to a CD66e polypeptide. The drug may be selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

[0022] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) an antibody or antigen-binding fragment, the antibody comprising: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody may comprise a light chain variable domain or region of (i).The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The composition may comprise an antibody. The composition may comprise an antigen-binding fragment. The composition may comprise a checkpoint inhibitor. The checkpoint inhibitor may be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

[0023] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) the cell engager described above. The composition can include a checkpoint inhibitor. The checkpoint inhibitor can be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

[0024] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) the cells described above. The composition can include a checkpoint inhibitor. The checkpoint inhibitor can be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

[0025] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) the ADC described above. The composition may include a checkpoint inhibitor. The checkpoint inhibitor may be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

[0026] In another aspect, this document features a method of treating a mammal having cancer. The method includes (or consists essentially of, or consists of) administering to the mammal a composition of any of the preceding four paragraphs. The mammal can be a human. The cancer is characterized by a CD66e + Could be cancer. CD66e + Cancer is CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e +The number of cancer cells in the mammal may be reduced after the administering step.

[0027] In another aspect, this document features a method of treating a mammal having cancer. The method includes (or consists essentially of, or consists of) (a) administering to the mammal a composition of any of the same four preceding paragraphs referenced in the preceding paragraphs, and (b) administering to the mammal a composition comprising a checkpoint inhibitor. The mammal can be a human. The cancer is characterized by a CD66e + Could be cancer. CD66e + Cancer is CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e + The checkpoint inhibitor may be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab. The number of cancer cells in the mammal may be reduced after administering steps (a) and (b).

[0028] In another aspect, this document features a method for conjugating a binding molecule to a CD66e polypeptide, the method comprising (or consisting essentially of, or consisting of) contacting a CD66e polypeptide with an antibody or antigen-binding fragment. The antibody comprises: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions). The antibody may comprise (or may consist essentially of, or may consist of) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). The antibody may be capable of binding to SEQ ID NO: 150 or SEQ ID NO: 151. The antibody may comprise a heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody may comprise a light chain variable domain or region of (i).The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.The antigen-binding fragment may comprise: (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions). , or may consist essentially of, or may consist of, a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 18 (or SEQ ID NO: 18 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 19 (or SEQ ID NO: 19 with 1, 2 or 3 amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with 1, 2 or 3 amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with 1, 2 or 3 amino acid additions, deletions or substitutions), and SEQ ID NO: 27 (or SEQ ID NO: 27 with 1, 2 or 3 amino acid additions, deletions or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise a light chain variable domain or region of (i). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ii).The heavy chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24. The antigen-binding fragment may comprise a light chain variable domain or region of (ii). The light chain variable domain or region may comprise an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. The contacting step may be performed in vitro. The contacting step may be performed in vivo. The contacting step may be performed in a mammal by administering the antibody or antigen-binding fragment to the mammal. The mammal may be a human.

[0029] In another aspect, this document features a method for conjugating a binding molecule to a CD66e polypeptide. The method includes (or consists essentially of, or consists of) contacting a CD66e polypeptide with a chimeric antigen receptor described above, a cell engager described above, or an ADC described above. The contacting step can be performed in vitro. The contacting step can be performed in vivo. The contacting step can be performed in a mammal by administering the chimeric antigen receptor, cell engager, or ADC to the mammal. The mammal can be a human.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods and examples are merely illustrative and are not intended to be limiting, and 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.

[0031] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows amino acid residues 1-702 of the human CD66e polypeptide (SEQ ID NO: 150). The underlined and bolded amino acid sequence of this human CD66e polypeptide (residues 501-682) represents the A3-B3 domain (SEQ ID NO: 151) and was used to identify CD66e binders. [Figure 2A] FIG. 2A shows the amino acid sequence of the heavy chain variable domain (FIG. 2A) and the light chain variable domain (FIG. 2B) of the Fab designated clone #1 (ab1). The respective CDRs, framework sequences, and constant domains are also provided and depicted. [Figure 2B] FIG. 2A shows the amino acid sequence of the heavy chain variable domain (FIG. 2A) and the light chain variable domain (FIG. 2B) of the Fab designated clone #1 (ab1). The respective CDRs, framework sequences, and constant domains are also provided and depicted. [Figure 3A] 3A and 3B show the amino acid sequences of the heavy chain variable domain (FIG. 3A) and the light chain variable domain (FIG. 3B) of the Fab designated clone #2 (ab2). The respective CDRs, framework sequences, and constant domains are also provided and depicted. [Figure 3B] 3A and 3B show the amino acid sequences of the heavy chain variable domain (FIG. 3A) and the light chain variable domain (FIG. 3B) of the Fab designated clone #2 (ab2). The respective CDRs, framework sequences, and constant domains are also provided and depicted. [Figure 4] FIG. 1 shows the nucleic acid sequences encoding the indicated strands / domains of clones #1-#2. [Diagram 5] FIG. 1 depicts an exemplary Ig structure and provides amino acid and nucleic acid sequences of exemplary hinge, CH2, and CH3 regions / domains. [Figure 6A-B]Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 6C] Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 6D] Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 6E]Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 6F] Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 6G] Figure 6A shows an exemplary scFv structure. Figures 6B and 6C show the amino acid sequences of an exemplary heavy chain variable domain (Figure 6B) and an exemplary light chain variable domain (Figure 6C) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in Figure 10. Figures 6D-6G show exemplary scFv structures and provide amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 7A]7A-7B show amino acid sequences of an exemplary heavy chain variable domain (FIG. 7A) and an exemplary light chain variable domain (FIG. 7B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. FIG. 7C shows the structure of an exemplary scFv, providing amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 7B] 7A-7B show amino acid sequences of an exemplary heavy chain variable domain (FIG. 7A) and an exemplary light chain variable domain (FIG. 7B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. FIG. 7C shows the structure of an exemplary scFv, providing amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 7C] 7A-7B show amino acid sequences of an exemplary heavy chain variable domain (FIG. 7A) and an exemplary light chain variable domain (FIG. 7B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. FIG. 7C shows the structure of an exemplary scFv, providing amino acid and nucleic acid sequences with the linker, CDR, and framework sequences depicted. [Figure 8A] FIG. 8A shows the amino acid sequences of an exemplary heavy chain variable domain (FIG. 8A) and an exemplary light chain variable domain (FIG. 8B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. [Figure 8B]FIG. 8A shows the amino acid sequences of an exemplary heavy chain variable domain (FIG. 8A) and an exemplary light chain variable domain (FIG. 8B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. [Figure 9A] 9A-9B show the amino acid sequences of an exemplary heavy chain variable domain (FIG. 9A) and an exemplary light chain variable domain (FIG. 9B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. [Figure 9B] 9A-9B show the amino acid sequences of an exemplary heavy chain variable domain (FIG. 9A) and an exemplary light chain variable domain (FIG. 9B) of an exemplary scFv. The respective CDR and framework sequences are also depicted. The heavy and light chain variable domains can be linked together to form an scFv using exemplary linker amino acid sequences, such as those set forth in FIG. 10. [Figure 10] 1 shows exemplary linker amino acid sequences that can be used to link heavy and light chain variable domains together to form scFvs. These linker sequences can also be used to create CARs and cell engagers. [Figure 11]Figure 11A shows the structure of an exemplary CAR. Figure 11B is a schematic diagram of an exemplary CAR construct designed to express a CAR. A promoter sequence (e.g., a CMV immediate early promoter sequence) may be followed by a signal peptide sequence (e.g., a GM-CSF signal peptide sequence), followed by an scFv provided herein (e.g., an scFv designed to include two sets of three CDRs of an antigen-binding fragment provided herein, e.g., CDR1, CDR2, and CDR3 of the heavy chain, and CDR1, CDR2, and CDR3 of the light chain (in either order), e.g., SEQ ID NOs: 1-3 and 9-11, or SEQ ID NOs: 17-19 and 25-27), followed by an optional linker (not shown), followed by an optional hinge (e.g., a CD8 hinge sequence, not shown), followed by a transmembrane sequence (e.g., a CD8 transmembrane sequence), followed by one or more intracellular signaling domain sequences (e.g., a 4-1BB (CD137) intracellular signaling domain sequence and a CD3 zeta intracellular signaling domain sequence). [Figure 12] FIG. 1 shows the amino acid sequences of exemplary signal peptides that can be used to design CARs. [Figure 13] FIG. 1 shows the amino acid sequences of exemplary hinges that can be used to design CARs. [Figure 14] FIG. 1 shows the amino acid sequences of exemplary transmembrane domains that can be used to design CARs. [Figure 15] FIG. 1 shows the amino acid sequences of exemplary intracellular signaling domains that can be used to design CARs. [Figure 16-1] 1 shows the amino acid sequence of a CAR (CAR #1) designed to contain an scFv created using the CDRs of the Fab of clone #1 and the nucleic acid sequence encoding that CAR. The various components of this CAR (e.g., domains and linkers) are provided and depicted. [Figure 16-2]1 shows the amino acid sequence of a CAR (CAR #1) designed to contain an scFv created using the CDRs of the Fab of clone #1 and the nucleic acid sequence encoding that CAR. The various components of this CAR (e.g., domains and linkers) are provided and depicted. [Figure 17] Figure 17A is a schematic diagram of an exemplary BiTE designed using the CDR1, CDR2, and CDR3 of the heavy chain provided herein in an Ig format (e.g., IgG1 format) and the CDR1, CDR2, and CDR3 of the light chain provided herein. A humanized anti-CD3 scFv (e.g., gOKT3-7 scFv described in U.S. Pat. No. 6,750,325) can be linked to the C-terminus of the light chain via a linker (e.g., a (G4S)3 linker). Figure 17B shows the amino acid sequence of the linker sequence (SEQ ID NO: 162, the nucleic acid sequence of this linker is SEQ ID NO: 163) followed by the gOKT3-7 scFv sequence, which can be joined to the light chain as shown in Figure 17A. Figure 17B also shows the nucleic acid sequence encoding this linker and gOKT3-7 scFv. [Figure 18] FIG. 1 shows the amino acid sequences of exemplary antigen-binding domains that can be used to design cell engagers that bind to T cells. [Figure 19-1] FIG. 1 shows the amino acid sequences of exemplary antigen-binding domains that can be used to design cell engagers that bind to NK cells, as well as exemplary BiKE amino acid and nucleic acid sequences. [Figure 19-2] FIG. 1 shows the amino acid sequences of exemplary antigen-binding domains that can be used to design cell engagers that bind to NK cells, as well as exemplary BiKE amino acid and nucleic acid sequences. [Figure 19-3] FIG. 1 shows the amino acid sequences of exemplary antigen-binding domains that can be used to design cell engagers that bind to NK cells, as well as exemplary BiKE amino acid and nucleic acid sequences. [Figure 20A]Figure 20A is a schematic diagram showing binding of an exemplary antibody provided herein to the CEACAM5 A3B3 domain. Figure 20B is a schematic diagram showing one of the rationales for using the A3B3 domain as a lead antigen for antibody discovery. [Figure 20B] Figure 20A is a schematic diagram showing binding of an exemplary antibody provided herein to the CEACAM5 A3B3 domain. Figure 20B is a schematic diagram showing one of the rationales for using the A3B3 domain as a lead antigen for antibody discovery. [Figure 21] Design of antigens and competitors for panning. [Figure 22] SDS-PAGE analysis of purified Fc-fused CEACAM5 and CEACAM6 domains. [Diagram 23] Binding of 1G9 Fab to various domains of CEACAM5 in an indirect ELISA. [Figure 24] Binding of 1G9 Fab and IgG1 to various CEACAM family members and cynomolgus CEACAM5 in indirect ELISA. [Diagram 25] Binding of 1G9 Fab to various domains of CEACAM family members in flow cytometry analysis. [Figure 26] Specificity of 1G9 IgG1 in a membrane proteome array (MPA) against over 5,800 human membrane-associated proteins. [Figure 27] This is the measurement of equilibrium dissociation constants in surface plasmon resonance (SPR) analysis using Biacore. [Figure 28] SDS-PAGE analysis of purified Fc-fused A3B3 domain with N-linked glycosylation sites and Asn to Qln mutations in the A3B3 domain of CEACAM5. [Figure 29] Binding of 1G9 Fab to mutants of the A3B3 domain. [Diagram 30]Binding of 1G9 and 1C1 hIgG1 to the A3B3 domain of CEACAM5 and the AB domain of CEACAM6. [Diagram 31] Binding of 1G9 and 1C1 hIgG1 to mutants of the A3B3 domain. [Diagram 32] Competitive ELISA of 1G9 Fab in the presence and absence of 1C1 IgG1 for binding to CEACAM5. [Diagram 33] Electron microscope (EM) images of 1G9 or 1C1 hIgG1 in complex with the CEACAM5 A3B3 domain. [Diagram 34] Binding of 1G9 and 1C1 hIgG1 to Fc gamma receptors (CD16a, CD65, and CD32). [Diagram 35] Binding of 1G9 and 1C1 hIgG1 to a CEACAM5-positive NEPC cell line (NCI-H660), a PrAd Du145 cell line stably expressing CEACAM5 (Du145-CEACAM5), and a CEACAM5-negative PrAd cell line (Du145). [Diagram 36] ADCC activity by 1G9 and 1C1 hIgG1 against a CEACAM5-positive cell line (NCI-H660 and CEACAM5-positive Du145 cells) and a CEACAM5-negative cell line (Du145) in the presence of NK cells (E:T ratio = 5:1). [Figure 37] ADCC activity by 1G9 and 1C1 hIgG1 against a CEACAM5 positive cell line (NCI-H660) in the presence of PBMC. [Figure 38] Assay of cell viability after 72 hours of treatment with 1G9 hIgG1 in the absence of immune cells in a CEACAM5 positive cell line (NCI-H660) and a CEACAM5 negative cell line (Du145). [Figure 39] Transwell migration assay following treatment with 1G9 hIgG1 in a CEACAM5 positive cell line (NCI-H660).Representative images and quantification of cells migrated through the transwell are shown. [Diagram 40]Fluorescence intensity histograms of remaining antibody levels (1G9 and 1C1 hIgG1) on the surface of NCI-H660 cells after the indicated incubation times at 37° C. and quantification of mean fluorescence intensity (MFI) for 1G9 and 1C1 hIgG1. [Diagram 41] Design of 1G9 scFv and binding of 1G9 scFv to the A3B3 domain of CEACAM5 in different VH / VL orientations and using linkers. [Diagram 42] Genetic components for exemplary CARs (e.g., second and third generation CARs). [Diagram 43] CAR expression in CD4+ T and CD8+ T populations in control T cells and anti-CEACAM5 CAR-T cells (second and third generation CAR-T cells). [Diagram 44] Cytotoxic activity of anti-CEACAM5 CAR-T against a CEACAM5-positive cell line (NCI-H660), a Du145 cell line stably expressing CEACAM5 (Du145-CEACAM5), and a CEACAM5-negative cell line (Du145). [Diagram 45] Structural model for the CEACAM5 and CEACAM6 domains with N-linked glycans added. [Figure 46] Cytokine secretion measured in cell supernatants from the indicated cell cultures. [Figure 47] Granzyme B, perforin, and IFNγ positive T cell population (%) in the supernatant after co-culture of 3rd generation anti-CEACAM5 CAR-T with NCI-H660 cells at 48 hours analyzed by intracellular staining (E:T ratio = 2:1). [Figure 48] Individual mouse tumor growth curves and body weights for the indicated hIgG1 treatment groups in Du145-CEACAM5 and Du145 tumors. [Figure 49] Individual mouse tumor growth curves and body weights for the indicated CAR-T treatment groups in Du145-CEACAM5 and Du145 tumors. [Figure 50]Overall tumor growth inhibition and survival following treatment with 1G9 hIgG1 or CAR-T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] This document provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a CD66e polypeptide (e.g., a human CD66e polypeptide). For example, this document provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a polypeptide comprising, consisting essentially of, or consisting of an amino acid set forth in SEQ ID NO: 150 or SEQ ID NO: 151 (see, e.g., FIG. 1). In some cases, a binding agent provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) can have the ability to bind to a CD66e polypeptide and can lack the ability to bind to a CD66c polypeptide. For example, a binding agent provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) can have the ability to bind to a human CD66e polypeptide and can lack the ability to bind to a human CD66c polypeptide.

[0034] The term "antibody" as used herein includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, single chain variable fragment antibodies (e.g., scFv antibodies), and tandem single chain variable fragment antibodies (e.g., taFv). Diabodies can include two chains each having a heavy chain variable domain and a light chain variable domain derived from the same or different antibodies (see, e.g., Hornig and Farber-Schwarz, Methods Mol. Biol., 907:713-27 (2012); and Brinkmann and Kontermann, MAbs., 9(2):182-212 (2017)). The two variable regions can be connected by a polypeptide linker (e.g., a polypeptide linker having a length of 5 to 10 residues or a polypeptide linker described in FIG. 10). In some cases, there may be interdomain disulfide bonds in one or both of the pairs of heavy and light chain variable domains of the diabody. scFvs are single chain polypeptide antibodies in which the heavy and light chain variable domains are connected directly or via a polypeptide linker (e.g., a polypeptide linker having a length of 8-18 residues or a polypeptide linker as described in FIG. 10). See also Chen et al., Adv. Drug Deliv. Rev., 65(10):1357-1369 (2013). scFvs can be designed with an orientation in which the heavy chain variable domain is followed by the light chain variable domain, or the light chain variable domain is followed by the heavy chain variable domain. In both cases, an optional linker can be located between the two domains. Examples of scFv structures of scFvs provided herein include, but are not limited to, the structures described in FIGS. 6A-6G, 7A-7C, 8A-8B, and 9A-9B.

[0035] The antibodies provided herein may comprise the CDRs described herein (e.g., described in Table 13) and may be configured to be human, humanized, or chimeric antibodies. In some cases, the antibodies provided herein may comprise the CDRs described herein (e.g., described in Table 13) and may be monoclonal antibodies. In some cases, the antibodies provided herein may comprise the CDRs described herein (e.g., described in Table 13) and may be configured as scFv antibodies.

[0036] As used herein, the term "antibody-binding fragment" refers to a fragment of an antibody (e.g., a fragment of a humanized antibody, a fragment of a human antibody, or a fragment of a chimeric antibody) that has the ability to bind to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', or F(ab') 2 The antigen-binding fragments provided herein may include CDRs described herein (e.g., as described in Table 13) and may be configured to be human antigen-binding fragments, humanized antigen-binding fragments, or chimeric antigen-binding fragments. In some cases, the antigen-binding fragments provided herein may include CDRs described herein (e.g., as described in Table 13) and may be monoclonal antigen-binding fragments. In some cases, the antigen-binding fragments provided herein may include CDRs described herein (e.g., as described in Table 13) and may be configured as Fab antibodies. In some cases, the Fab antibodies may include a partial hinge sequence (e.g., SEQ ID NO: 152) for disulfide bonds between the heavy and light chains of the Fab.

[0037] The term "antibody domain" as used herein refers to a domain of an antibody, e.g., a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain), in the absence of one or more other domains of the antibody. In some cases, an antibody domain may be a single antibody domain (e.g., a VH domain or a VL domain) capable of binding to an antigen. The antibody domains provided herein may comprise the CDRs described herein (e.g., as described in Table 13) and may be a human antibody domain (e.g., a human VH domain), a humanized antibody domain (e.g., a humanized VH domain), or a chimeric antibody domain (e.g., a chimeric VH domain). In some cases, the antibody domains provided herein may comprise the CDRs described herein (e.g., as described in Table 13) and may be a monoclonal antibody domain. In some cases, the antibody domains provided herein may comprise the CDRs described herein (e.g., as described in Table 13) and may be engineered as a single VH domain or a single VL domain.

[0038] The anti-CD66e antibodies, anti-CD66e antigen-binding fragments, or anti-CD66e antibody domains provided herein may be IgA-, IgD-, IgE-, IgG-, or IgM-type antibody domains, and may be IgG- or IgM-type, such as, but not limited to, IgG 1 -, IgG 2 -, IgG 3 -, IgG 4 -, IgM 1 - and IgM 2 -type. In some cases, the antibodies provided herein (e.g., anti-CD66e antibodies) can be scFv antibodies. In some cases, the antigen-binding fragments provided herein (e.g., anti-CD66e antibody fragments) can be Fab. In some cases, the antibodies provided herein (e.g., anti-CD66e antibodies) can be fully intact antibodies having the structure depicted in FIG. 5. In some cases, the antibody domains provided herein (e.g., anti-CD66e antibody domains) can be VH domains.

[0039] The term "chimeric antigen receptor" as used herein refers to a chimeric polypeptide designed to include an optional signal peptide, an antigen-binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains. As described herein, the antigen-binding domain of the CAR provided herein can be designed to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). For example, the CAR provided herein can be designed to include components (e.g., a combination of CDRs) of an antibody, antigen-binding fragment, and / or antibody domain described herein as the antigen-binding domain, provided that the antigen-binding domain has the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). In some examples, a CAR provided herein can be designed to include an antigen-binding domain (e.g., SEQ ID NOs: 1-3 and 9-11, or SEQ ID NOs: 17-19 and 25-27) that includes two sets of three CDRs of an antigen-binding fragment provided herein (e.g., CDR1, CDR2, and CDR3 of the heavy chain, and CDR1, CDR2, and CDR3 of the light chain). In some cases, the antigen-binding domain of a CAR that targets a CD66e polypeptide can be designed to include a VH domain described herein or a scFv antibody described herein.

[0040] Examples of CAR structures that can be used to generate the CARs provided herein include, but are not limited to, those depicted in Figures 11A and 11B.

[0041] In some cases, the CARs provided herein may be designed to include a signal peptide. Any suitable signal peptide may be used to design the CARs described herein. Examples of signal peptides that may be used to generate the CARs described herein include, but are not limited to, signal peptides from human IGKV1-39, IGKV1-16, IGKV1-33, IGKV3-11, IGKV4-1, or IGKV6-21. In some cases, the CARs provided herein may be designed to include a signal peptide that includes, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12. In some cases, the CARs provided herein may be designed to include a signal peptide that includes, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12, with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a CAR provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 12, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0042] In some cases, the CARs provided herein can be designed to include a hinge. Any suitable hinge can be used to design the CARs described herein. Examples of hinges that can be used to create the CARs described herein include, but are not limited to, an Ig-derived hinge (e.g., an IgG1-derived hinge, an IgG2-derived hinge, or an IgG4-derived hinge), an Ig-derived hinge containing a CD2 domain and a CD3 domain, an Ig-derived hinge containing a CD2 domain and lacking a CD3 domain, an Ig-derived hinge containing a CD3 domain and lacking a CD2 domain, an Ig-derived hinge lacking a CD2 domain and lacking a CD3 domain, a CD8α-derived hinge, a CD28-derived hinge, and a CD3ζ-derived hinge. The CARs provided herein can be designed to include any suitable length of hinge. For example, the CARs provided herein can be designed to include a hinge that is about 3 to about 75 (e.g., about 3 to about 65, about 3 to about 50, about 5 to about 75, about 10 to about 75, about 5 to about 50, about 10 to about 50, about 10 to about 40, or about 10 to about 30) amino acid residues in length. In some cases, a linker sequence may be used as a hinge to generate the CARs described herein. For example, any one of the linker sequences set forth in FIG. 10 may be used as a hinge for the CARs described herein.

[0043] In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13. In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13, with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13, with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or fewer amino acid deletions, additions, substitutions, or combinations thereof.

[0044] The CARs provided herein can be designed to include any suitable transmembrane domain. For example, the transmembrane domain of the CARs provided herein can be, but is not limited to, a CD3ζ transmembrane domain, a CD4 transmembrane domain, a CD8α transmembrane domain, a CD28 transmembrane domain, and a 4-1BB transmembrane domain. In some cases, the CARs provided herein can be designed to include a transmembrane domain that includes, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 14. In some cases, the CARs provided herein can be designed to include a transmembrane domain that includes, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 14, with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a CAR provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 14, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0045] The CARs provided herein may be designed to include one or more intracellular signaling domains. For example, the CARs provided herein may be designed to include one, two, three, or four intracellular signaling domains. Any suitable intracellular signaling domain or combination of intracellular signaling domains may be used to generate the CARs described herein. Examples of intracellular signaling domains that can be used to generate the CARs described herein include, but are not limited to, CD3ζ intracellular signaling domain, CD27 intracellular signaling domain, CD28 intracellular signaling domain, OX40 (CD134) intracellular signaling domain, 4-1BB (CD137) intracellular signaling domain, CD278 intracellular signaling domain, DAP10 intracellular signaling domain, and DAP12 intracellular signaling domain. In some cases, the CARs described herein may be designed to be first generation CARs with CD3ζ intracellular signaling domain. In some cases, the CARs described herein may be designed to be second generation CARs with CD28 intracellular signaling domain followed by CD3ζ intracellular signaling domain. In some cases, the CARs described herein can be designed to be third generation CARs having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an OX40 intracellular signaling domain, or a 4-1BB intracellular signaling domain followed by (c) a CD3ζ intracellular signaling domain. In some cases, the CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 15.In some cases, the CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 15, having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, the CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 15, having 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling.

[0046] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by SEQ ID NO:100, followed by a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0047] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:16, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a heavy chain variable domain comprising SEQ ID NO:8, followed by SEQ ID NO:100, followed by a light chain variable domain comprising SEQ ID NO:16, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0048] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by SEQ ID NO:100, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121 (see, e.g., Figure 16).

[0049] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO: 16, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a light chain variable domain comprising SEQ ID NO: 16, followed by SEQ ID NO: 100, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by SEQ ID NO: 102, followed by SEQ ID NO: 108, followed by SEQ ID NO: 119, followed by SEQ ID NO: 124, followed by SEQ ID NO: 123, followed by SEQ ID NO: 97, followed by SEQ ID NO: 121 (see, e.g., Figure 16).

[0050] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by SEQ ID NO:100, followed by a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0051] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO: 24, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO: 32, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a heavy chain variable domain comprising SEQ ID NO:24, followed by SEQ ID NO:100, followed by a light chain variable domain comprising SEQ ID NO:32, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0052] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by SEQ ID NO:100, followed by a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0053] In some cases, a CAR targeting a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO: 32, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO: 24, followed by a hinge, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g. an IgG4-derived hinge, a CD8α hinge, or a linker plus IgG4-derived hinge), followed by a transmembrane domain, such as a transmembrane domain described in FIG. 14 (e.g. a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains, such as one or more intracellular signaling domains described in FIG. 15 (e.g. a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a CD66e polypeptide can be designed to comprise an scFv having a light chain variable domain comprising SEQ ID NO:32, followed by SEQ ID NO:100, followed by a heavy chain variable domain comprising SEQ ID NO:24, followed by SEQ ID NO:102, followed by SEQ ID NO:108, followed by SEQ ID NO:119, followed by SEQ ID NO:124, followed by SEQ ID NO:123, followed by SEQ ID NO:97, followed by SEQ ID NO:121.

[0054] The term "cell engager" as used herein refers to a polypeptide that comprises two or more antigen-binding domains (e.g., two, three, or four antigen-binding domains) and has the ability to link two cells together. Examples of cell engagers include, but are not limited to, BiTE, BiKE, and TriKE. Generally, the cell engagers provided herein can be designed to comprise at least one antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) and at least one antigen-binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell). In some cases, the cell engagers described herein can be designed to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) via two or more antigen-binding domains of the cell engager. + Cells (e.g., CD66e +The cell engager may link one cell (e.g., a cancer cell) to another cell (e.g., a T cell or an NK cell). Examples of cell engager structures of the cell engagers provided herein include, but are not limited to, the structures depicted in FIG. 17A. In some cases, the anti-CD3 scFv depicted in FIG. 17A may be replaced with a different antigen binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell).

[0055] When a cell engager comprises an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) and two or more other antigen-binding domains (e.g., two, three, or four other antigen-binding domains), each of these other antigen-binding domains can bind to a different antigen expressed on the surface of a different cell type, or can bind to a different antigen expressed on the surface of the same cell type. For example, TriKE can be designed to have a first antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide), a second antigen-binding domain capable of binding to a first antigen expressed on the surface of an NK cell (e.g., a CD16 polypeptide, e.g., a CD16a polypeptide), and a third antigen-binding domain capable of binding to a second antigen expressed on the surface of an NK cell (e.g., an NKG2A polypeptide).

[0056] As described herein, at least one antigen-binding domain of a cell engager provided herein can be designed to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). For example, a cell engager provided herein can be designed to include components (e.g., combinations of CDRs) of an antibody, antigen-binding fragment, and / or antibody domain described herein as an antigen-binding domain, provided that the antigen-binding domain has the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). In some examples, a cell engager provided herein can be designed to include an antigen-binding domain that includes two sets of three CDRs (e.g., CDR1, CDR2, and CDR3 of the heavy chain, and CDR1, CDR2, and CDR3 of the light chain) of an antigen-binding fragment provided herein (e.g., SEQ ID NOs: 1-3 and 9-11, or SEQ ID NOs: 17-19 and 25-27). In some cases, the antigen-binding domain of a cell engager that targets a CD66e polypeptide can be designed to include a VH domain described herein or an scFv / Fab antibody described herein. In some cases, the antigen-binding domain of a CAR described herein that has the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) can be designed to include a CD66e polypeptide. + It can be used as an antigen-binding domain of a cell engager to target cells.

[0057] As described herein, a cell engager can be designed to include at least one antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) and at least one other antigen-binding domain. The at least one other antigen-binding domain can be capable of binding to any suitable antigen expressed on the surface of a cell. For example, a cell engager, such as a CD66e +When designing a BiTE that links cells and T cells, the cell engager may comprise an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., human CD66e polypeptide) and an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell. Examples of polypeptides expressed on the surface of a T cell that can be targeted by the antigen-binding domain of the cell engagers provided herein include, but are not limited to, CD3 polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of a T cell that can be used to generate cell engagers (e.g., BiTEs) provided herein include, but are not limited to, anti-CD3 scFv and anti-CD3 VH domains. Further examples of amino acid sequences that can be used as antigen-binding domains capable of binding to a polypeptide expressed on the surface of a T cell (e.g., CD3) are described in U.S. Pat. No. 6,750,325 (see, e.g., the sequence listing in U.S. Pat. No. 6,750,325).

[0058] In some cases, cell engagers provided herein may be designed to include an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 18. In some cases, cell engagers provided herein may be designed to include an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 18 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of a T cell. In some cases, cell engagers provided herein can be designed to comprise an antigen-binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 18, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen-binding domain has the ability to bind to a polypeptide expressed on the surface of a T cell.

[0059] Cell engagers, such as CD66e +When designing a BiKE or TriKE for linking cells to NK cells, the cell engager may comprise an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) and one or more (e.g., one, two, or three) antigen-binding domains capable of binding to a polypeptide expressed on the surface of a NK cell. Examples of polypeptides expressed on the surface of a NK cell that can be targeted by the antigen-binding domains of the cell engagers provided herein include, but are not limited to, a CD16 polypeptide (e.g., a CD16a polypeptide), an NKG2A polypeptide, an NKG2D polypeptide, an NKp30 polypeptide, an NKp44 polypeptide, and an NKp46 polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of a NK cell that can be used to generate a cell engager provided herein (e.g., BiKE or TriKE) include, but are not limited to, anti-CD16a scFv, anti-NKG2A scFv, anti-NKG2D scFv, anti-NKp30 scFv (see, e.g., BioLegend Catalog #325207), anti-NKp44 scFv, anti-NKp46 scFv, anti-CD16a VH domain, anti-NKG2A VH domain, anti-NKG2D VH domain, anti-NKp30 VH domain, anti-NKp44 VH domain, and anti-NKp46 VH domain.Further examples of amino acid sequences that can be used as antigen-binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., CD16, NKG2A, NKG2D, or NKp46) are described in McCall et al., (Mol. Immunol., 36(7):433-445 (1999); e.g., anti-CD16 scFv sequences); International Patent Application Publication No. PCT / US2017 / 048721 (see, e.g., CDRs and sequence listing for anti-CD16a binding domains); U.S. Patent Application Publication No. 2011 / 0052606 (see, e.g., CDRs and sequence listing for anti-NKG2A antibodies, e.g., Z199); U.S. Patent Application Publication No. 2011 / 0150870 (see, e.g., CDRs and sequence listing for anti-NKG2D antibodies); U.S. Patent Application Publication No. 2018 / 0369373 (see, e.g., CDRs and sequence listing for anti-NKp46 antibodies); and U.S. Patent Application Publication No. 2017 / 0368169 (see, e.g., CDRs and sequence listing for anti-NKp46 antibodies).

[0060] In some cases, cell engagers provided herein can be designed to include an antigen binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one or more of the amino acid sequences set forth in Figure 19. In some cases, cell engagers provided herein can be designed to include an antigen binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 19 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of a NK cell. In some cases, the cell engagers provided herein can be designed to comprise an antigen binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in FIG. 19, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain has the ability to bind to a polypeptide expressed on the surface of a NK cell.

[0061] In some cases, the cell engagers provided herein may be designed to include a linker located between each antigen-binding domain. Any suitable linker may be used to design the cell engagers provided herein. Examples of linkers that can be used to create the cell engagers described herein include, but are not limited to, the linker sequences described in Figure 10. The cell engagers provided herein may be designed to include a linker of any suitable length. For example, cell engagers provided herein can be designed to include a linker of about 3 to about 100 amino acid residues in length (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17). In some cases, cell engagers provided herein (e.g., BiTEs) can be designed to include a GGGGSGGGSGGGGS (SEQ ID NO: 78) linker. In some cases, the hinge of a CAR described herein can be used as a linker to generate a cell engager described herein. For example, any one of the sequences described in Figure 13 can be used as a linker of a cell engager described herein.

[0062] In some cases, cell engagers provided herein may be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13. In some cases, cell engagers provided herein may be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, cell engagers provided herein may be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Figure 10 or Figure 13, with 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less amino acid deletions, additions, substitutions, or combinations thereof.

[0063] In some cases, a cell engager (e.g., a BiTE) targeted to a CD66e polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0064] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0065] In some cases, a cell engager (e.g., a BiTE) targeted to a CD66e polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:16, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0066] In some cases, a cell engager (e.g., a BiTE) targeted to a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO: 16, followed by a linker, such as a linker described in Figure 10, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by a linker, such as a hinge / linker described in Figure 10 or Figure 13 (e.g., SEQ ID NO: 78), followed by an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0067] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0068] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0069] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:16, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0070] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to comprise a light chain variable domain comprising SEQ ID NO: 16, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO: 78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0071] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0072] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0073] In some cases, a cell engager (e.g., a BiTE) targeted to a CD66e polypeptide can be designed to comprise a heavy chain variable domain comprising SEQ ID NO:24, followed by a linker, such as a linker described in Figure 10, followed by a light chain variable domain comprising SEQ ID NO:32, followed by a linker, such as a hinge / linker described in Figure 10 or Figure 13 (e.g., SEQ ID NO:78), followed by an scFv having an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0074] In some cases, a cell engager (e.g., a BiTE) targeted to a CD66e polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO: 32, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO: 24, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO: 78), followed by an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0075] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0076] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0077] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to comprise a heavy chain variable domain comprising SEQ ID NO:24, followed by a linker, such as a linker described in FIG. 10, followed by a light chain variable domain comprising SEQ ID NO:32, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO:78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0078] In some cases, a cell engager targeting a CD66e polypeptide (e.g., BiKE or TriKE) may be designed to include a light chain variable domain comprising SEQ ID NO: 32, followed by a linker, such as a linker described in FIG. 10, followed by a heavy chain variable domain comprising SEQ ID NO: 24, followed by a linker, such as a hinge / linker described in FIG. 10 or FIG. 13 (e.g., SEQ ID NO: 78), followed by an scFv having one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., anti-human CD16a scFv for BiKE, or anti-human CD16a scFv and anti-human NKG2A scFv for TriKE).

[0079] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0080] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide can be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:8, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:16, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0081] In some cases, a cell engager (e.g., BiKE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0082] In some cases, a cell engager (e.g., BiKE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:8, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:16, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0083] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0084] In some cases, a cell engager (e.g., BiTE) targeted to a CD66e polypeptide can be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:24, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:32, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0085] In some cases, a cell engager (e.g., BiKE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0086] In some cases, a cell engager (e.g., BiKE) targeted to a CD66e polypeptide may be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:24, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:32, a constant domain (e.g., a kappa or lambda constant domain), and an antigen binding domain capable of binding to a polypeptide expressed on the surface of a NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0087] In one embodiment, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having the ability of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise: (i) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (or a variant of SEQ ID NO: 3 with one or two amino acid modifications); and / or (ii) a light chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 9 (or a variant of SEQ ID NO: 9 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 10 (or a variant of SEQ ID NO: 10 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 11 (or a variant of SEQ ID NO: 11 with one or two amino acid modifications). Examples of such antigen-binding fragments having these CDRs and the ability to bind to a CD66e polypeptide (eg, a human CD66e polypeptide) include, but are not limited to, the Fab depicted in Figures 2A and 2B.

[0088] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) having the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and having (a) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (or a variant of SEQ ID NO: 3 with one or two amino acid modifications), and / or (b) a light chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 9 (or a variant of SEQ ID NO: 9 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 10 (or a variant of SEQ ID NO: 10 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 11 (or a variant of SEQ ID NO: 11 with one or two amino acid modifications), may comprise any suitable framework region.For example, such binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers and / or ADCs) may comprise: (a) a framework region 1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 6 (or a variant of SEQ ID NO: 6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 7 (or a variant of SEQ ID NO: 7 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications); and / or (b) a heavy chain variable domain comprising framework region 4 having the amino acid sequence set forth in SEQ ID NO: 12 (or a variant of SEQ ID NO: 12 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), framework region 1 having the amino acid sequence set forth in SEQ ID NO: 13 (or a variant of SEQ ID NO: 13 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications). and a light chain variable domain comprising framework region 2 having the amino acid sequence set forth in SEQ ID NO: 14 (or a variant of SEQ ID NO: 14 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 15 (or a variant of SEQ ID NO: 15 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications).

[0089] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having any of the CDRs set forth in Figures 2A or 2B may be designed to include the framework regions set forth in Figures 2A and 2B, or may be designed to include one or more framework regions from another antibody, antibody fragment or antibody domain. For example, a Fab may be designed to include the six CDRs set forth in Figures 2A and 2B and the framework regions set forth in Figures 2A and 2B, except that framework region 1 having the amino acids set forth in SEQ ID NO:4 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:58, or framework region 1 having the amino acids set forth in SEQ ID NO:67, or framework region 1 having the amino acids set forth in SEQ ID NO:72. In some cases, a scFv may be designed to include the six CDRs set forth in Figures 2A and 2B and the framework regions set forth in Figures 2A and 2B. In some cases, an scFv can be designed to include the six CDRs set forth in Figures 2A and 2B and the framework regions set forth in Figures 2A and 2B, except that framework region 1 having the amino acids set forth in SEQ ID NO: 4 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 58, framework region 1 having the amino acids set forth in SEQ ID NO: 67, or framework region 1 having the amino acids set forth in SEQ ID NO: 72. In another example, an scFv can be designed to include the six CDRs set forth in Figures 2A and 2B and the framework regions set forth in Figures 6B and 6C, Figures 7A and 7B, Figures 8A and 8B, or Figures 9A and 9B.

[0090] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:16. For example, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:16. In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:16.

[0091] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise: (a) a heavy chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, with the proviso that it comprises the amino acid sequence set forth in SEQ ID NO:1, 2, and 3; and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:16, with the proviso that it comprises the amino acid sequence set forth in SEQ ID NO:9, 10, and 11. For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein may comprise: (a) a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:8, with the proviso that the heavy chain variable domain comprises an amino acid sequence set forth in SEQ ID NO:1, 2, and 3; and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:16, with the proviso that the light chain variable domain comprises an amino acid sequence set forth in SEQ ID NO:9, 10, and 11.

[0092] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having the ability of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise (a) a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8, or the amino acids set forth in SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), and / or (b) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16, or the amino acids set forth in SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, the antibodies or antigen-binding fragments provided herein may have the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and may comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8 with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions and / or amino acid additions), provided that they comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, and may comprise a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 16 with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions and / or amino acid additions), provided that they comprise the amino acid sequences set forth in SEQ ID NOs: 9, 10 and 11.

[0093] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise: (a) a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:2; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:3; and / or (b) a light chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:9; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:10; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:11. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:1" is a CDR1 having zero, one or two amino acid substitutions within SEQ ID NO:1, having zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:1, and / or having zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:1, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:1 include, but are not limited to, those set forth in Table 1.

[0094] [Table 1]

[0095] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:2" is a CDR2 that has zero, one or two amino acid substitutions within SEQ ID NO:2, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:2, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:2, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO:2 include, but are not limited to, those set forth in Table 2.

[0096] [Table 2]

[0097] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:3" is a CDR3 that has zero, one or two amino acid substitutions within SEQ ID NO:3, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:3, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:3, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:3 include, but are not limited to, those set forth in Table 3.

[0098] [Table 3]

[0099] As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:9" is a CDR1 having zero, one or two amino acid substitutions within SEQ ID NO:9, having zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:9, and / or having zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:9, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:9 include, but are not limited to, those set forth in Table 4.

[0100] [Table 4]

[0101] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10" is a CDR2 that has zero, one or two amino acid substitutions within SEQ ID NO: 10, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO: 10, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO: 10, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10 include, but are not limited to, those set forth in Table 5.

[0102] [Table 5]

[0103] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 11" is a CDR3 that has zero, one or two amino acid substitutions within SEQ ID NO: 11, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO: 11, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO: 11, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 11 include, but are not limited to, those set forth in Table 6.

[0104] [Table 6]

[0105] In another embodiment, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having the ability of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise: (i) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 (or a variant of SEQ ID NO: 18 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (or a variant of SEQ ID NO: 19 with one or two amino acid modifications); and / or (ii) a light chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 25 (or a variant of SEQ ID NO: 25 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 26 (or a variant of SEQ ID NO: 26 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 (or a variant of SEQ ID NO: 27 with one or two amino acid modifications). Examples of such antigen-binding fragments having these CDRs and the ability to bind to a CD66e polypeptide (eg, a human CD66e polypeptide) include, but are not limited to, the Fab depicted in Figures 3A and 3B.

[0106] In some cases, the antibody has the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and has (a) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 (or a variant of SEQ ID NO: 18 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (or a variant of SEQ ID NO: 19 with one or two amino acid modifications), and / or (b) an amino acid sequence set forth in SEQ ID NO: 25. The binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers and / or ADCs) provided herein having a light chain variable domain having a CDR1 having the amino acid sequence of SEQ ID NO: 25 (or a variant of SEQ ID NO: 25 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 26 (or a variant of SEQ ID NO: 26 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 (or a variant of SEQ ID NO: 27 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) may comprise: (a) a framework region 1 having the amino acid sequence set forth in SEQ ID NO:20 (or a variant of SEQ ID NO:20 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), an amino acid sequence set forth in SEQ ID NO:21 (or a variant of SEQ ID NO:21 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO: 22 (or a variant of SEQ ID NO: 22 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO: 22 (or a variant of SEQ ID NO: 22 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), and / or a framework region 4 having the amino acid sequence set forth in SEQ ID NO: 23 (or a variant of SEQ ID NO: 23 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications); or (b) a light chain variable domain comprising a framework region 1 having the amino acid sequence set forth in SEQ ID NO:28 (or a variant of SEQ ID NO:28 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:29 (or a variant of SEQ ID NO:29 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:30 (or a variant of SEQ ID NO:30 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:31 (or a variant of SEQ ID NO:31 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications).

[0107] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having any of the CDRs set forth in Figures 3A or 3B may be designed to include the framework regions set forth in Figures 3A and 3B, or may be designed to include one or more framework regions from another antibody or antibody fragment. For example, a Fab may be designed to include the six CDRs set forth in Figures 3A and 3B and the framework regions set forth in Figures 3A and 3B, except that framework region 1 having the amino acids set forth in SEQ ID NO:20 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:58, or framework region 1 having the amino acids set forth in SEQ ID NO:67, or framework region 1 having the amino acids set forth in SEQ ID NO:72. In some cases, a scFv may be designed to include the six CDRs set forth in Figures 3A and 3B and the framework regions set forth in Figures 3A and 3B. In some cases, an scFv can be designed to include the six CDRs set forth in Figures 3A and 3B and the framework regions set forth in Figures 3A and 3B, except that framework region 1 having the amino acids set forth in SEQ ID NO: 20 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 58, framework region 1 having the amino acids set forth in SEQ ID NO: 67, or framework region 1 having the amino acids set forth in SEQ ID NO: 72. In another example, an scFv can be designed to include the six CDRs set forth in Figures 3A and 3B and the framework regions set forth in Figures 6B and 6C, Figures 7A and 7B, Figures 8A and 8B, or Figures 9A and 9B.

[0108] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. For example, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, and / or (b) a light chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 32.

[0109] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise: (a) a heavy chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, with the proviso that it comprises the amino acid sequences set forth in SEQ ID NOs: 17, 18, and 19; and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 32, with the proviso that it comprises the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27. For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein may comprise: (a) a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, with the proviso that the heavy chain variable domain comprises an amino acid sequence set forth in SEQ ID NO: 17, 18, and 19; and / or (b) a light chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 32, with the proviso that the light chain variable domain comprises an amino acid sequence set forth in SEQ ID NO: 25, 26, and 27.

[0110] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) having the ability of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise (a) a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 24, or the amino acids set forth in SEQ ID NO: 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), and / or (b) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32, or the amino acids set forth in SEQ ID NO: 32 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, the antibodies or antigen-binding fragments provided herein may have the ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide) and may comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 24 with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions and / or amino acid additions), provided that they comprise the amino acid sequences set forth in SEQ ID NOs: 17, 18 and 19, and may comprise a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 32 with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications (e.g., amino acid substitutions, amino acid deletions and / or amino acid additions), provided that they comprise the amino acid sequences set forth in SEQ ID NOs: 25, 26 and 27.

[0111] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) may comprise (a) a heavy chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 17, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 19, and / or (b) a light chain variable domain comprising (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 25, (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 26, and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 27. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17" is a CDR1 having zero, one or two amino acid substitutions within SEQ ID NO: 17, having zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO: 17, and / or having zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO: 17, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17 include, but are not limited to, those set forth in Table 7.

[0112] [Table 7]

[0113] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 18" is a CDR2 that has zero, one or two amino acid substitutions within SEQ ID NO: 18, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO: 18, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO: 18, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 18 include, but are not limited to, those set forth in Table 8.

[0114] [Table 8]

[0115] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 19" is a CDR3 that has zero, one or two amino acid substitutions within SEQ ID NO: 19, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO: 19, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO: 19, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 19 include, but are not limited to, those set forth in Table 9.

[0116] [Table 9]

[0117] As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:25" is a CDR1 having zero, one or two amino acid substitutions within SEQ ID NO:25, having zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:25, and / or having zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:25, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:25 include, but are not limited to, those set forth in Table 10.

[0118] [Table 10]

[0119] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:26" is a CDR2 that has zero, one or two amino acid substitutions within SEQ ID NO:26, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:26, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:26, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO:26 include, but are not limited to, those set forth in Table 11.

[0120] [Table 11]

[0121] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:27" is a CDR3 that has zero, one or two amino acid substitutions within SEQ ID NO:27, has zero, one, two, three, four or five amino acid residues immediately preceding SEQ ID NO:27, and / or has zero, one, two, three, four or five amino acid residues immediately following SEQ ID NO:27, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager and / or ADC) maintains the basic ability to bind to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:27 include, but are not limited to, those set forth in Table 12.

[0122] [Table 12]

[0123] When designing a single chain antibody (e.g., scFv) having a heavy chain variable domain and a light chain variable domain, the two regions may be directly linked or may be linked using any suitable linker sequence. For example, a heavy chain variable domain having CDRs of SEQ ID NOs: 1 to 3 or 17 to 19 may be directly linked via a linker sequence to a light chain variable domain having CDRs of SEQ ID NOs: 9 to 11 or 25 to 27, respectively. Examples of linker sequences that can be used to link the heavy chain variable domain and the light chain variable domain to create an scFv include, but are not limited to, the linkers shown in FIG. 10.

[0124] As indicated herein, the amino acid sequences described herein may include amino acid modifications (e.g., a concatenated number of amino acid modifications). Such amino acid modifications may include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations. In some cases, amino acid modifications may be made to improve binding and / or contact with an antigen and / or to improve the functional activity of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein. In some cases, the amino acid substitutions in the concatenated sequence identifiers may be conservative amino acid substitutions. For example, a conservative amino acid substitution may be made by replacing one amino acid residue with another amino acid residue having a similar side chain. A family of amino acid residues having similar side chains can include 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).

[0125] In some cases, the amino acid substitutions in the concatenated sequence identifiers may be non-conservative amino acid substitutions. Non-conservative amino acid substitutions can be made by replacing one amino acid residue with another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, but are not limited to, replacing (a) a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine), (b) cysteine ​​or proline with any other residue, (c) a residue having a basic side chain (e.g., lysine, arginine, or histidine) with a residue having an acidic side chain (e.g., aspartic acid or glutamic acid), and (d) a residue having a bulky side chain (e.g., phenylalanine) with glycine or other residues having small side chains.

[0126] Methods for generating amino acid sequence variants (e.g., amino acid sequences containing one or more modifications relative to the concatenated sequence identifiers) can include site-directed or random mutagenesis (e.g., by PCR) of a nucleic acid encoding an antibody or fragment thereof. See, e.g., Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially derivatized amino acids) can be used to generate the amino acid sequence variants provided herein.

[0127] Further set forth in Table 13 are representative numbers of binding agents (eg, antibodies, antigen-binding fragments, and / or antibody domains) capable of binding to a CD66e polypeptide (eg, a human CD66e polypeptide).

[0128] [Table 13]

[0129] Table 14 contains alternative designations that can be used to refer to each of clones #1-#2.

[0130]

Table 14

[0131] The binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be produced using any suitable method. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be produced in recombinant host cells. For example, nucleic acids encoding the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be constructed, introduced into an expression vector, and expressed in a suitable host cell. Figure 4 is a sequence listing of nucleic acid sequences encoding exemplary binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains) described herein. In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) can be recombinantly produced in a prokaryotic host, such as E. coli, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei.The binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) provided herein can be expressed in eukaryotic hosts, such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), filamentous fungi of the genus Trichoderma (e.g., T. reesei), and Aspergillus (e.g., A. niger). The antibodies can also be recombinantly produced in A. niger and A. oryzae, protozoa such as Leishmania tarentolae, insect cells, or mammalian cells (e.g., mammalian cell lines such as Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, or the human embryonic kidney cell line HEK293). See, e.g., Frenzel et al., Front Immunol., 4:217 (2013).

[0132] In some cases, the antigen-binding fragments or antibody domains provided herein can be produced by proteolytic digestion of an intact antibody. For example, antigen-binding fragments can be obtained by treating an antibody with an enzyme, such as papain or pepsin. Papain digestion of a whole antibody can be used to produce F(ab) 2 Alternatively, Fab fragments can be produced, whereas pepsin digestion of whole antibody can be used to produce F(ab') fragments. 2 Alternatively, Fab' fragments can be produced.

[0133] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) may be substantially pure. The term "substantially pure" as used herein with respect to a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) refers to a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) that is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acids with which it is naturally associated. That is, a substantially pure binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) is any binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) that is removed from its natural environment and is at least 60% pure. The substantially pure binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) may be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.

[0134] This document also provides bispecific binding agents (e.g., bispecific antibodies, bispecific antigen-binding fragments, and / or bispecific antibody domains) that bind to two different epitopes, at least one of which is an epitope of a CD66e polypeptide (e.g., a human CD66e polypeptide). In some cases, the bispecific binding agents provided herein can be designed to bind to two different epitopes of the same CD66e polypeptide (e.g., a human CD66e polypeptide). In some cases, the bispecific binding agents provided herein can bind to an epitope on a CD66e polypeptide (e.g., a human CD66e polypeptide) and a different polypeptide (e.g., a CD3 polypeptide). Bispecific binding agents can be produced by chemically conjugating two different binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains) together. Bispecific binding agents can also be produced by fusing two antibody-producing cells, e.g., hybridomas, to create a hybrid cell line that produces two different heavy chains and two different light chains in the same cell, resulting in, for example, a bispecific IgG molecule. See Brinkmann and Kontermann, MAbs., 9(2):182-212 (2017).

[0135] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be fused or conjugated (e.g., covalently or non-covalently linked) to another polypeptide or other moiety to obtain a fusion protein or conjugate. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be conjugated (e.g., covalently or non-covalently linked) to a polymer (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), and / or polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, a fluorescent agent, a luminescent agent, a hapten, an enzyme, a metal chelate, a drug, a radioisotope, and / or a cytotoxic agent. Any suitable method may be used to conjugate (e.g., covalently or non-covalently link) another polypeptide or other moiety to the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers). For example, the methods described in U.S. Pat. No. 8,021,661 can be used to conjugate another polypeptide or other moiety to the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers).

[0136] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be modified with a moiety that improves stabilization and / or retention in circulation, e.g., in blood, serum, or other tissues, e.g., at least 1.5-fold, 2-fold, 5-fold, 10-fold, or 50-fold. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated (e.g., covalently or non-covalently conjugated) to a polymer, e.g., a substantially non-antigenic polymer. Examples of substantially non-antigenic polymers that can be used as described herein include, but are not limited to, polyalkylene oxides and polyethylene oxides. In some cases, the polymers used herein can have any suitable molecular weight. For example, polymers having an average molecular weight of about 200 daltons to about 35,000 daltons (e.g., about 1,000 to about 15,000 daltons or about 2,000 to about 12,500 daltons) can be used. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated (e.g., covalently or non-covalently conjugated) to a water-soluble polymer. Examples of water-soluble polymers that can be used as described herein include, but are not limited to, hydrophilic polyvinyl polymers, polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycol, polyoxyethylenated polyols, and copolymers and / or block copolymers thereof, provided that the water solubility of the copolymer or block copolymer is maintained.

[0137] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be conjugated (e.g., covalently or non-covalently bound) to one or more polyoxyalkylenes (e.g., polyoxyethylene, polyoxypropylene, or block copolymers of polyoxyethylene and polyoxypropylene), polymethacrylates, carbomers, branched or unbranched polysaccharides, or combinations thereof. For example, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be covalently bound to polyoxyethylene.

[0138] This document also provides ADCs. As used herein, the term "ADC" refers to a conjugate comprising (a) an antigen-binding domain and (b) at least one drug directly or indirectly covalently linked to the antigen-binding domain. In some cases, the ADCs described herein may comprise (a) an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) and (b) at least one drug directly or indirectly covalently linked to the antigen-binding domain. Any suitable binding agent (e.g., an antibody, an antigen-binding fragment, and / or an antibody domain) provided herein and capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) can be used as an antigen-binding domain to generate the ADCs described herein. For example, any of the binding agents listed in Table 13 can be used to generate ADCs capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide). Examples of drugs that can be used to make the ADCs described herein include, but are not limited to, auristatins (e.g., monomethylauristatin E (MMAE)), mertansine (DM-1), and pyrrolobenzodiazepine (PBD) dimers. Any suitable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) to form the ADCs provided herein. For example, a cleavable or non-cleavable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) to form the ADCs provided herein. Examples of ADC linkers that can be used to covalently attach one or more drugs to an antigen-binding domain capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) to form an ADC provided herein include, but are not limited to, an ADC disulfide linker, an ADC hydrazone linker, an ADC peptide linker, an ADC thioether linker, and an ADC PEG-containing linker.

[0139] This document also provides a nucleic acid molecule (e.g., an isolated nucleic acid molecule) having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, an antigen-binding fragment, an antibody domain, a CAR, and / or a cell engager) provided herein. For example, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence encoding a heavy chain variable domain, e.g., a heavy chain variable domain as set forth in FIG. 2A or 3A. In another example, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence encoding a light chain variable domain, e.g., a light chain variable domain as set forth in FIG. 2B or 3B. In some cases, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence encoding both (a) a heavy chain light chain variable domain and (b) a light chain variable domain, with or without encoding a linker polypeptide as set forth in FIG. 10. A nucleic acid (e.g., an isolated nucleic acid molecule) provided herein can be a single-stranded or double-stranded nucleic acid of any suitable type (e.g., DNA, RNA, or DNA / RNA hybrid).

[0140] This document also provides vectors (e.g., plasmid vectors or viral vectors) that contain one or more nucleic acids provided herein. Examples of plasmid vectors that can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, phagemids. Examples of viral vectors that can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, retroviral vectors, parvoviral vectors (e.g., adenoviral vectors and adeno-associated virus (AAV) vectors), lentiviral vectors (e.g., herpes simplex (HSV) vectors), poxviral vectors (e.g., vaccinia virus vectors and fowlpox virus vectors), and hybrid or chimeric viral vectors. For example, viral vectors having lentiviral components, such as adenoviral backbones including those described elsewhere (Zheng et al., Nat. Biotech., 18(2): 176-80 (2000); WO 98 / 22143; WO 98 / 46778; and WO 00 / 17376), or AAV components, such as adenoviral backbones including those described elsewhere (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996)), can be engineered to include one or more nucleic acids having a nucleic acid sequence encoding at least a portion of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager).

[0141] In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can include a nucleic acid sequence encoding an scFv or an antibody domain (e.g., a VH domain) provided herein. In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can include a nucleic acid sequence encoding a CAR provided herein. In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can include a nucleic acid sequence encoding a cell engager provided herein.

[0142] The vectors provided herein (e.g., plasmid vectors or viral vectors provided herein) can include any suitable promoter and other control sequences (e.g., transcription and translation start and stop codons) operably linked to a nucleic acid sequence encoding at least a portion of a binding agent provided herein (e.g., an antibody, an antigen-binding fragment, an antibody domain, a CAR, and / or a cell engager). In some cases, the promoter used to drive expression can be a constitutive promoter or a controllable promoter. Examples of controllable promoters that can be used as described herein include, but are not limited to, inducible promoters, repressible promoters, and tissue-specific promoters. Examples of viral promoters that can be used as described herein include, but are not limited to, adenovirus promoters, vaccinia virus promoters, CMV promoters (e.g., immediate early CMV promoters), and AAV promoters.

[0143] Any suitable method can be used to generate a nucleic acid molecule (or vector, e.g., a plasmid vector or a viral vector) having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, an antigen-binding fragment, an antibody domain, a CAR, and / or a cell engager) provided herein. For example, molecular cloning techniques can be used to generate a nucleic acid molecule (or vector, e.g., a plasmid vector or a viral vector) having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, an antigen-binding fragment, an antibody domain, a CAR, and / or a cell engager) provided herein, as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0144] This document also provides host cells that include a nucleic acid provided herein (e.g., a nucleic acid having a nucleic acid sequence encoding at least a portion of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager). Host cells that can be engineered to include one or more nucleic acids provided herein can be prokaryotic or eukaryotic cells. Examples of prokaryotic cells that can be engineered to include a nucleic acid provided herein include, but are not limited to, E. coli (e.g., Tb-1, TG-1, DH5α, XL-Blue MRF (Stratagene), SA2821, or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens, or Pseudomonas (e.g., P. aerugenosa) cells. Examples of eukaryotic cells that can be engineered to contain a nucleic acid provided herein include, but are not limited to, insect cells (e.g., Sf9 or Ea4 cells), yeast cells (e.g., S. cerevisiae cells), and mammalian cells (e.g., mouse, rat, hamster, monkey, or human cells). For example, VERO cells, HeLa cells, 3T3 cells, Chinese hamster ovary (CHO) cells, W138 BHK cells, COS-7 cells, and MDCK cells can be engineered to contain a nucleic acid provided herein. Any suitable method may be used to introduce one or more nucleic acids provided herein (e.g., a vector, such as a plasmid vector or a viral vector, having a nucleic acid sequence encoding at least a portion of a binding agent provided herein) into a host cell. For example, calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, electroporation, or a combination thereof can be used to introduce the nucleic acids provided herein into a host cell (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999). Harbor Laboratory, NY (1989); Davis et al., Basic Methods in Molecular Biology (1986); and Neumann et al., EMBO J., 1:841 (1982).

[0145] In some cases, cells, such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells, can be engineered to express one or more nucleic acids encoding a CAR described herein. For example, a population of T cells can be infected with a viral vector designed to express a nucleic acid encoding a CAR described herein (e.g., a CAR capable of binding to a CD66e polypeptide).

[0146] In some cases, cells, such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells, can be engineered to express one or more nucleic acids encoding a cell engager described herein. For example, a population of T cells can be infected with a viral vector designed to express a nucleic acid encoding a cell engager described herein (e.g., a cell engager capable of binding to a CD66e polypeptide).

[0147] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein can be produced using a method that includes the steps of (a) introducing a nucleic acid encoding a polypeptide into a host cell, (b) culturing the host cell in a culture medium under conditions sufficient to express the polypeptide, (c) recovering the polypeptide from the cell or the culture medium, and (d) purifying the polypeptide (e.g., to reach at least 50, 60, 70, 80, 90, 95, 97, 98, or 99 percent purity).

[0148] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) with cancer to treat the mammal. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or increase survival of a mammal afflicted with cancer. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs) capable of binding to a CD66e polypeptide (e.g., a human CD66e polypeptide) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human).In some cases, the pharmaceutical compositions provided herein may include a pharma- ceutically acceptable carrier, such as a buffer, salt, surfactant, sugar, tonicity modifier, or combinations thereof, as described elsewhere (Gervasi et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharma-ceutically acceptable carriers that can be used to make the pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, surfactants (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or sugars (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or combinations thereof. For example, a pharmaceutical composition designed to include a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) (or a nucleic acid, vector, or host cell provided herein) can be formulated to include a buffer (e.g., acetate, citrate, histidine, succinate, phosphate, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar, such as sucrose. Other components that can be included in the pharmaceutical compositions provided herein include, but are not limited to, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA), anticancer drugs such as enzalutamide, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosine, bortezomib, carfilzomib, batimastat, ganetespib, obatoclax, navitoclax, taxol, paclitaxel, or bevacizumab, or combinations thereof.For example, the pharmaceutical compositions provided herein can be used in combination with one or more checkpoint inhibitors, such as an anti-PD-1 antibody or PD-1 inhibitor (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, or AMP-514), an anti-PD-L1 antibody or PD-L1 inhibitor (e.g., avelumab, durvalumab, atezolizumab, KN The present invention can be formulated to include one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells engineered to express a CAR capable of binding to a CD66e polypeptide, one or more cell engagers, and / or one or more ADCs) in combination with an anti-CTLA-4 antibody (e.g., CD66e 2+, CD66e 035, CK-301, AUNP12, CA-170, or BMS-986189), and / or an anti-CTLA-4 antibody (e.g., ipilimumab).

[0149] In some cases, when formulating a pharmaceutical composition to include one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells engineered to express a CAR capable of binding to a CD66e polypeptide, one or more cell engagers, and / or one or more ADCs), any suitable concentration of the binding agent can be used. For example, the pharmaceutical compositions provided herein contain about 1 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR +In another example, the pharmaceutical compositions provided herein may be formulated to be a solid or semi-solid containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC). In some cases, pharmaceutical compositions containing a binding agent (e.g., an antibody, antigen-binding fragment, and / or antibody domain) provided herein may be used to treat a patient with a binding agent titer of about 1×10 5 ~Approx. 1×10 12 (For example, about 1×10 5 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 8 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 9 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 11 , or about 1 × 10 7 ~Approx. 1×10 10 ) may be formulated as a dosage form.

[0150] In some cases, when a pharmaceutical composition is formulated to include one or more nucleic acids (e.g., a vector, e.g., a viral vector) encoding at least a portion of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein, any suitable concentration of nucleic acid can be used. For example, the pharmaceutical compositions provided herein may be formulated to be liquids containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of the nucleic acid provided herein per mL. In another example, the pharmaceutical compositions provided herein may be formulated to be solid or semi-solid containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg).

[0151] In some cases, pharmaceutical compositions designed to include a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be formulated to include one or more agents that can reduce aggregation of the binding agent when formulated. Examples of such agents that can be used as described herein include, but are not limited to, methionine, arginine, lysine, aspartic acid, glycine, glutamic acid, and combinations thereof. In some cases, one or more of these amino acids can be included in the formulation at a concentration of about 0.5 mM to about 145 mM (e.g., about 1 mM to about 145 mM, about 10 mM to about 145 mM, about 100 mM to about 145 mM, about 0.5 mM to about 125 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 75 mM, or about 10 mM to about 100 mM).

[0152] The pharmaceutical compositions provided herein may be in any suitable form. For example, the pharmaceutical compositions provided herein may be designed to be liquid, semi-solid, or solid. In some cases, the pharmaceutical compositions provided herein may be a liquid solution (e.g., a solution for injection and / or infusion), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository. In some cases, the pharmaceutical compositions provided herein may be lyophilized. In some cases, the pharmaceutical compositions provided herein (e.g., the pharmaceutical compositions provided herein comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs)) may be formulated with a carrier or coating designed to protect against rapid release. For example, the pharmaceutical compositions provided herein may be formulated as controlled or regulated release formulations as described elsewhere (U.S. Patent Application Publication Nos. 2019 / 0241667, 2019 / 0233522, and 2019 / 0233498).

[0153] This document provides a method for the preparation of one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., CAR + Also provided are methods of administering a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR) provided herein) to a mammal (e.g., a human). + A composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., a CAR)) can be administered to a mammal (e.g., a human) with cancer to treat the mammal. In some cases, one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., a CAR)) can be administered to a mammal (e.g., a human) with cancer to treat the mammal. + A composition containing the cancer cells (e.g., a pharmaceutical composition provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or to increase survival of the mammal afflicted with cancer.

[0154] One or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR +Any suitable cancer can be treated using a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein. For example, a composition (e.g., a pharmaceutical composition) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein can be administered to a mammal (e.g., a human) having cancer to treat the mammal. Examples of cancers that can be treated as described herein include, but are not limited to, lung cancer, prostate cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, vaginal cancer, cervical cancer, pancreatic cancer, and breast cancer. In some cases, CD66e is a marker for a cancer that is associated with a tumor. + Cancer (e.g. CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e + A composition (e.g., pharmaceutical composition) containing one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) can be administered to a mammal (e.g., a human) having a cancer such as cervical cancer to treat the mammal (e.g., to reduce the number of cancer cells in the mammal).

[0155] Any suitable method can be used to administer a composition (e.g., a pharmaceutical composition) provided herein to a mammal (e.g., a human). For example, a composition provided herein (e.g., a pharmaceutical composition containing one or more binding agents provided herein, such as one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs provided herein) can be administered to a mammal (e.g., a human) intravenously (e.g., via intravenous injection or infusion), subcutaneously (e.g., via subcutaneous injection), intraperitoneally (e.g., via intraperitoneal injection), orally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some cases, the route and / or mode of administration of a composition (e.g., a pharmaceutical composition provided herein) can be tailored for the mammal being treated.

[0156] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR + An effective amount of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be an amount that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. In some cases, one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be an amount that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. +An effective amount of a composition (e.g., a pharmaceutical composition provided herein) containing a cellular marker (cells) can be an amount that extends the survival of a mammal having cancer compared to a control mammal having a similar level of cancer and not treated with the composition. For example, an effective amount of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be from about 0.001 mg / kg to about 100 mg / kg (e.g., from about 0.001 mg / kg to about 90 mg / kg, from about 0.001 mg / kg to about 80 mg / kg, from about 0.001 mg / kg to about 70 mg / kg, from about 0.001 mg / kg to about 60 mg / kg, from about 0.001 mg / kg to about 100 mg / kg (e.g., from about 0.001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 150 mg / kg, from about 0.001 mg / kg to about 20 mg / kg, from about 0.001 mg / kg to about 30 mg / kg, from about 0.001 mg / kg to about 40 mg / kg, from about 0.001 mg / kg to about 50 mg / kg, from about 0.001 mg / kg to about 60 mg / kg, from about 0.001 mg / kg to about 70 mg / kg, from about 0.001 mg / kg to about 80 mg / kg, from about 0.001 mg / kg to about 90 mg / kg, from about 0.001 mg / kg to about 100 mg / kg). 001mg / kg ~ approx. 50mg / kg, approx. 0.001mg / kg ~ approx. 40mg / kg, approx. 0.001mg / kg ~ approx. 30mg / kg, approx. 0.005mg / kg ~ approx. 100mg / kg, approx. 0.01m g / kg~about 100mg / kg, about 0.05mg / kg~about 100mg / kg, about 0.1mg / kg~about 100mg / kg, about 0.5mg / kg~about 100mg / kg, about 1mg / kg~about 100m g / kg, about 5 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.15 mg / kg to about 25 mg / kg, about 0.2 mg / kg~about 20mg / kg, about 0.5mg / kg~about 20mg / kg, about 1mg / kg~about 30mg / kg, about 1mg / kg~about 25mg / kg, about 1mg / kg~about 20mg / kg, about 2mg / kg The effective amount may be about 20 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 30 mg / kg, about 15 mg / kg to about 30 mg / kg, about 20 mg / kg to about 30 mg / kg, about 3 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 1 mg / kg to about 3 mg / kg. The effective amount may remain constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to the treatment. The actual effective amount used for a particular application may be affected by a variety of factors.For example, when treating a mammal with cancer, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective amount of the compositions provided herein to be administered (e.g., pharmaceutical compositions containing one or more binding agents provided herein).

[0157] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR + An effective frequency of administration of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a frequency that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. In some cases, an effective frequency of administration of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a frequency that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. +The effective frequency of administration of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (cells) may be a frequency that extends the survival of a mammal having cancer compared to a control mammal having a similar cancer and not treated with the composition. For example, the effective frequency of administration of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition containing one or more binding agents provided herein, may be from about twice daily to about once per year (e.g., from about twice daily to about once per month, from about twice daily to about once per week, from about once daily to about once per month, or from once daily to about once per week). In some cases, the frequency of administration of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition containing one or more binding agents provided herein, may be daily. The frequency of administration of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition containing one or more binding agents provided herein, may remain constant or may vary over the duration of the treatment. The actual effective frequency used for a particular application may be influenced by a variety of factors. For example, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective frequency of administration of the compositions provided herein (e.g., pharmaceutical compositions containing one or more binding agents provided herein).

[0158] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR +The effective duration of administration of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a duration that reduces the number of cancer cells in the mammal without causing significant toxicity to the mammal. In some cases, the effective duration of administration of a composition (e.g., a pharmaceutical composition provided herein) containing one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a duration that reduces the number of cancer cells in the mammal without causing significant toxicity to the mammal. + The effective duration of administration of a composition (e.g., a pharmaceutical composition provided herein) containing a cellular component (cells) may be a duration that extends the survival of a mammal having cancer compared to a control mammal having a similar cancer and not treated with the composition. For example, the effective duration of administration of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition containing one or more binding agents provided herein, may vary from a single time point administration to several weeks to several months (e.g., 4-12 weeks). The actual effective duration used for a particular application may be influenced by a number of factors. For example, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, e.g., the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an extension or shortening of the actual effective duration of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more binding agents provided herein).

[0159] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used to detect the presence or absence of a CD66e polypeptide (e.g., a human CD66e polypeptide) in vitro, in situ, or in vivo (e.g., for in vivo imaging in a mammal, e.g., a human). For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be designed to include a label (e.g., a covalently attached radioactive, enzymatic, chromogenic, or fluorescent label). The labeled binding agent can be used to detect the presence or absence of a CD66e polypeptide (e.g., a human CD66e polypeptide) in a biological sample in vitro. Examples of biological samples that can be evaluated using the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) include, but are not limited to, serum samples, plasma samples, tissue samples, biopsy samples, cell line samples, and tissue culture samples. In some cases, the biological sample that can be evaluated as described herein can include mammalian body tissues and / or cells that can express CD66e polypeptides (e.g., human CD66e polypeptides), such as white blood cells, ovarian tissues or cells, prostate tissues or cells, cardiac tissues or cells, placental tissues or cells, pancreatic tissues or cells, liver tissues or cells, splenic tissues or cells, lung tissues or cells, breast tissues or cells, head and neck tissues or cells, endometrial tissues or cells, colonic tissues or cells, colorectal tissues or cells, cervical tissues or cells, gastric tissues or cells, or umbilical cord tissues or cells. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be immobilized, for example, on a support, and retention of CD66e polypeptides (e.g., human CD66e polypeptides) from the biological sample on the support can be detected, and / or vice versa.In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used in applications such as fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and / or cell sorting (e.g., fluorescence activated cell sorting).

[0160] In some cases, binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) containing a label (e.g., a covalently attached radioactive label) can be used to detect the presence or absence of a CD66e polypeptide (e.g., a human CD66e polypeptide) in a mammal (e.g., a human). For example, a labeled (e.g., covalently labeled) binding agent provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) having a radiolabel or an MRI-detectable label can be administered to a mammal (e.g., a human), and the mammal can be evaluated using a means for detecting the detectable label. In some cases, the mammal can be scanned to evaluate the location of the labeled binding agent provided herein within the mammal. For example, the mammal can be imaged using NMR or other tomography techniques.

[0161] Examples of labels that can be attached (e.g., covalently or non-covalently attached) to the binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein include, but are not limited to, radiolabels, e.g. 131 I, 111 In, 123 I, 99m Tc, 32 P, 33 P, 125 I, 3 H, 14 C, and 188These include Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescent markers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. In some cases, short-range luminescent markers, such as isotopes detectable by short-range detector probes, can be used.

[0162] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0163] (Example) [Example 1] Obtaining a binding agent capable of binding to human CD66e polypeptide CD66e contains an N-terminal Ig variable region-like (IgV) domain attached to a GPI anchor to the plasma membrane and six Ig constant region type 2-like (IgC2-like) domains, i.e., N-A1-B1-A2-B2-A3-B3. Notably, the membrane-proximal A3B3 domain was found in the splice variants of CD66e in numerous cancers, and thus these were considered as potential therapeutic epitopes. Here, two Fab antibody fragments (clones #1 and #2, Figs. 2 and 3) were identified. To identify these Fabs, soluble CEACAM5 and CEACAM6 domains fused with Fc were designed and applied to a panning process. The isolated Fabs bound the A3B3 domain and mainly bound the B3 domain of CEACAM5 and the N-linked glycans at N612 and N650 with high affinity and specificity. Moreover, they did not cross-react with other domains of CEACAM5 or family members of CEACAM.

[0164] The sequence of clone #1 was used to generate hIgG1, which showed CEACAM5-dependent cytotoxic activity in ADCC assays using NEPC cell line NCI-H660 and PrAd cell line Du145 in the presence of primary NK cells or PBMC. Furthermore, a constructed third generation CAR containing an scFv with the CDRs of clone #1 was delivered to T cells. The T cells were shown to efficiently kill CEACAM5-positive prostate cancer cells, while no detectable cytotoxicity was found in CEACAM5-negative cells. These results demonstrate the immunotherapeutic potential of clone #1 hIgG1 for NEPC treatment with significantly lower off-target toxicity.

[0165] Preparation of soluble Fc-fused recombinant CEACAM5 and CEACAM6 domains CEACAM5 A3B3 (residues 501-682), CEACAM5 A1B1 (residues 145-322), CEACAM5 A2B2 (residues 323-500), and CEACAM6 AB (residues 145-296) fused with human immunoglobulin 1 Fc region (hIgG1 Fc) were synthesized and then cloned into pSectag2A plasmid (Invitrogen, V90020). Each plasmid DNA was complexed with PEI-Max (Polysciences, 24765-1) and fed into cultures of freestyle human embryonic kidney cell line (Gibco, R79007) for transient transfection. Seven days after transfection, the Fc-fused recombinant proteins were purified by affinity chromatography with Protein A resin (Captiva, NC0997253). Proteins bound to Protein A were eluted by adding 50 mM glycine buffer pH 3.0, and then the storage buffer was changed to phosphobuffered saline pH 7.4 (PBS) by using a PD-10 desalting column (GE, 45-000-148). The purity of the proteins was estimated by SDS-PAGE or size-exclusion chromatography packed with Superdex 200 increase 10 / 300 GL (GE healthcare, 28990944). The concentration of each protein was determined by a Nano Drop spectrophotometer 2000C (Thermo, ND2000C). N-glycosylation mutants of the A3B3 domain of CEACAM5 (N508Q, N529Q, N553Q, N560Q, N580Q, N612Q, N650Q, and N665Q) were constructed by site-directed mutagenesis using the Q5 site-directed mutagenesis kit (NEB, E0554S), and the proteins were expressed and purified in the same manner as for wild-type CEACAM5.

[0166] Fab library construction and panning A combinatorial phage-displayed human Fab library (1 × 10) was generated by grafting naturally occurring heavy chain (HC) V(D)J recombination regions and light chain (LC) VJ recombination regions into IGHV3-11 and IGKV1-39 germline frameworks, respectively. 11To amplify the complementarity determining region 3 (CDR3) and J genes for HC or LC, RNA extracted from PBMCs of 50 healthy blood donors was used for cDNA synthesis with Superscript™ IV First Strand Synthesis System (Invitrogen, 18091050), and random hexamer and oligo dT primers were applied for the annealing step. Subsequent integration of the amplified CDR3 and J genes into the selected human framework was performed by overlapping PCR with Q5 polymerase (NEB, M0491), and the prepared insert DNA and pCAT2 plasmid (in-house plasmid, modified pCom3X) were digested with NotI and ApaI restriction enzymes. The digested insert and vector were ligated at a 1:1 ratio by using T4 DNA ligase (NEB, M0202). The circularized plasmid DNA was transformed into TG1 E. coli competent cells (Lucigen, 605502) and electroporated 100 times. M13KO7 helper phage (NEB, N0315S) was amplified in TOP10F E. coli and then used for subsequent production of phage-displayed libraries. For panning, phages were preblocked with 3% (w / v) bovine serum albumin (BSA) in PBS for 1 h at room temperature. Blocked phages were incubated with 10 nM biotinylated CEACAM5 A3B3-Fc in the presence of 300 nM competitors including CEACAM5 A1B1-Fc, CEACAM5 A2B2-Fc, and CEACAM6 AB-Fc for 1 h at room temperature. Bound phages were separated by streptavidin-coated magnetic beads (Invitrogen, 11-205-D) and washed 10 times with 1 mL of PBS pH 7.4 containing 0.1% Tween-20 (w / v). Elution of bound phages was performed by adding 10 mM tris-HCl pH 8.0 containing 25 mM dithiothreitol (DTT) for 10 min. After three rounds of panning, binding of 192 individual clones was analyzed by ELISA, and selected clones were then sequenced after plasmid rescue.

[0167] Purification of scFv, Fab, and IgG pCAT2 plasmids containing scFv or Fab were transformed into HB2151 E. coli competent cells, and transformed colonies were then selected in ampicillin-containing LB plates (final concentration 100 μg / mL) overnight in a 37°C incubator. The next day, colonies were inoculated into liquid LB+ampicillin medium and cultured in a shaking incubator at 37°C. Approximately 4 × 10 8 OD of 0.4-0.6 corresponds to cells / mL 600 At 20 °C, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.1 mM. The culture was transferred to a shaking incubator set at 30 °C and 200 rpm. The next day, induced E. coli cells were harvested and resuspended in 1 / 10 volume of periplasmic extraction buffer containing polymyxin B (0.5 mg / mL in PBS, pH 7.4), then incubated on ice for 1 h. The supernatant was collected and then loaded onto pre-loaded Ni-NTA resin. Bound scFv or Fab were eluted by adding 300 mM imidazole in PBS (pH 7.4), followed by removal of the imidazole. For preparation of IgG, IgG cloned plasmid DNA was transfected into HEK239F cells and expressed 5 to 7 days after transfection. IgG was purified as previously described for preparation of Fc fusion antigens.

[0168] Binding assessment in ELISA The binding and specificity of scFv, Fab, or IgG to CEACAM-related proteins was analyzed by indirect ELISA. Briefly, Fc-fused CEACAM5 polypeptide, CEACAM6 polypeptide, extracellular domain of CEACAM5 (R&D systems, 4128-CM), CEACAM6 (R&D systems, 3934-CM), or cynomolgus CEACAM5 (Sino biological, 90891-C08H) were coated onto 96-well plates (Corning, 3690) at 200 ng / well (volume 50 μL) in PBS for 2 hours at room temperature. Blocking was performed with 3% BSA in PBS overnight at 4°C. The next day, the antigen-coated plates were treated with various concentrations of scFv, Fab, or IgG and incubated at room temperature for 1 hour. After washing three times, the plates were treated with anti-FLAG mouse antibody (M2 clone)-HRP conjugate (Sigma, A8592, 1:3000 dilution) or anti-human kappa goat antibody-HRP conjugate (Invitrogen, A18853, 1:3000 dilution) for detection of binding. The same volume of TMB (Thermo, PI34028) was added as substrate, and then the enzyme reaction was stopped by adding 2N sulfonic acid. Monomeric Fc fusion CD16a (Fc γRIIIa), CD64 (Fc γRIa), and CD32 (Fc γRIIa) were obtained from Professor Wei Li (University of Pittsburgh, PA) and used for Fc gamma receptor (FcγR) binding test.

[0169] result Isolation of clone #1 Fab that specifically targets the A3B3 domain of CEACAM5 with high specificity To develop an anti-CEACAM5 antibody (e.g., a fully human antibody), the following was performed. Briefly, CEACAM5 and CEACAM6 domains fused with human IgG1 Fc were designed and purified. The A3B3 domain was deemed to be an ideal epitope for targeting the membrane proximal region of CEACAM5 (Figures 20A and 20B). To exclude the possibility of non-specific cross-reactive binders that may cause toxicity to normal cells and to prevent binding to the membrane distal region of the extracellular portion of CEACAM5, the Fc-fused A1B2 and A2B2 domains of CEACAM5 and the AB domain of CEACAM6 were used as competitors during phage library panning, since these domains showed more than 70% primary sequence homology to the A3B3 domain of CEACAM5 (Figure 21). All recombinant proteins were purified with high quality in SDS-PAGE analysis (Figure 22). For the construction of phage-displayed Fab libraries, a strategy was established in which partial regions of VH and VL resulting from V(D)J and VJ recombination events were grafted onto the frameworks of human single germline VH3-11 and Vk1-39, respectively. After 100 electroporations, 10 11 We generated a large size Fab library showing specificity for the clone #1 Fab (also called 1G9 Fab) and successfully panned out clone #1 Fab (also called 1G9 Fab) by a competitive panning strategy using a 10-fold molar excess of competitor with biotinylated Fc-fused A3B3 domains in all panning steps. The purified 1G9 Fab showed a half maximal effective concentration (EC) of approximately 6 nM in indirect ELISA (Figure 23). 50), and CEACAM5 A3B3 domains in BIAcore experiments showed an equilibrium dissociation constant (KD) of 3 nM (Figure 27), while no significant signal was observed for CEACAM5 A1B1, A2B2 domains, or other CEACAM family members. Furthermore, 1G9 IgG1 bound to cynomolgus monkey CEACAM5, suggesting that 1G9-based immunotherapeutics could further address toxicity issues encountered in monkeys, since cross-reactivity to both human and monkey CEACAM5 with similar affinity is necessary for toxicity studies in monkeys (Figure 24). Furthermore, 1G9 Fab showed excellent specificity for CEACAM5 in flow cytometry-based analysis using HEK293F cells expressing various CEACAM family members (Figure 25), and 1G9 hIgG1 showed similar results in membrane proteome arrays (MPA) to test antibody specificity using over 5,800 human cell lines expressing various human membrane proteins (Figure 26). Taken together, these results demonstrate that the isolated 1G9 antibody binds to the membrane proximal domain of CEACAM5, i.e., the A3B3 domain, with high affinity and high specificity without any off-target binding in three separate experiments.

[0170] Epitope mapping of 1G9 Fab with N-linked glycan-deleted mutants of the A3B3 domain Because the A3B3 domain has eight N-linked glycosylation motifs (NXS / T; X is any of the 19 natural amino acids excluding proline) at N508, N529, N553, N560, and N580 in the A3 domain, and N612, N650, and N665 in the B3 domain, we hypothesized that the N-linked glycans in the A3B3 domain of CEACAM5 may be directly or indirectly involved in the binding of 1G9 Fab. These N-linked glycans may cover the exposed surface of the A3B3 domain. To explore the contribution of N-linked glycans to the generation of the epitope for 1G9 Fab, eight mutant proteins of the A3B3 domain were constructed in which asparagine (N) was replaced by glutamine (Q) in each N-glycosylation motif. The N580Q, N612Q, N650Q, and N665Q mutant proteins were successfully purified, albeit with reduced yields, whereas the other four mutants, N508Q, N529Q, N553Q, and N560Q, did not express in HEK293F (Figure 28). For these mutant proteins, 1G9 Fab showed attenuated binding to the N612Q and N650Q mutants of the B3 domain of CEACAM5 in indirect ELISA. On the other hand, the N580Q mutation slightly improved the binding of 1G9 Fab (Figure 29). These results corresponded to the images obtained by electron microscopy (EM) of the complex of 1G9 hIgG with A3B3-Fc (Figure 33). To visualize the location and orientation of the N-linked glycans of CEACAM5 and CEACAM6, the molecular structures were modeled by adding the N-linked glycans to the modeling structures. These four domains shared over 70% of their primary sequence and displayed very similar tertiary structures, as expected, since they were already known to have Ig constant region type 2-like (IgC2-like) domains. However, the addition of N-linked glycans to the structural models generated significantly different antigenic surfaces, leading to the creation of unique conformational epitopes within each domain (Figure 45). Together, these results demonstrate that 1G9 Fab binds to the A3B3 domain of CEACAM5 and that three N-linked glycans at M580, N612, and N650 are involved in the major epitopes.

[0171] [Example 2] In vitro preclinical efficacy evaluation of hIgG1 and CAR-T cell line NCI-H660, Du145, and 293T cells were purchased from ATCC. Du145 were maintained in EMEM supplemented with 10% v / v FBS (Gibco) and 1% penicillin-streptomycin (P / S, Gibco). 293T were maintained in DMEM supplemented with 10% FBS and 1% P / S. NCI-H660 were cultured in RPMI1640 (ATCC) supplemented with 5% FBS, 1x insulin-transferrin-selenium (ITS-G Giboc), 10 nM hydrocortisone, additional 2 mM L-glutamine, and 1% P / S. Du145-CEACAM5 cells stably expressing CEACAM5 were generated by stable infection with lentivirus derived from pLenti lentiviral plasmid (Origene, RC206434L3) using established methods and cultured in EMEM supplemented with 10% FBS, 1% P / S, and 1 μg / mL puromycin (Gibco).

[0172] Generation of cells expressing the CEACAM family To generate cells expressing the CEACAM family, freestyle 293F cells (Thermofisher) were used. Plasmids in which each gene for human CEACAM members was cloned were transfected into cells using PEI Max (Polysciences). Each gene for human CEACAM members with a FLAG tag at the C-terminus (RC224086 for CEACAM21, RC214882 for CEACAM20, RC215478 for CEACAM18, RC224965 for CEACAM16, RC230069 for CEACAM1, RC212214 for CEACAM7, RC204740 for CEACAM8, RC202454 for CEACAM6, and RC206434 for CEACAM5) was purchased from Origene.

[0173] Generation of third generation CAR-T cells using scFv with CDRs of clone #1 DNA for the third generation of CAR encoding an scFv with the CDRs of clone #1, human IgG4 hinge, CD28 transmembrane region, and costimulatory cytoplasmic domains of CD28, 4-1BB, and CD3 zeta was cloned into pLVX-EF1a-IRES-ZsGreen1 with EcoRI and BamHI restriction enzymes and T4 DNA ligase. For production of lentiviral supernatant, 293T cells were transduced at 4×10 per T75 flask the day before transduction. 6 The cells were seeded with 100 μg of anti-CEACAM5 CAR-encoding plasmid (8 μg), pMD.2G (2 μg), and packaging vector psPAX2 (4 μg) were co-transfected into 293T cells using a PEI-based transfection system after 24 hours to generate lentivirus. After 48 hours and 72 hours, the supernatants were collected and filtered through a 0.45 μm membrane. Pan-T cells (Precision for medicine) were then activated by Dynabeads human T activator CD3 / CD28 (Gibco), transduced with lentivirus supernatant with 8 μg / mL polybrene (Sigma), centrifuged at 800×g for 45 minutes (no acceleration or deceleration), and then incubated at 37°C. After 24 hours, the virus-containing medium was replaced and T cells were expanded in T cell medium (RPMI1640 supplemented with additional 2 mM Glutamax, 10% human serum, and 1% P / S) in the presence of hIL-2 (fed every 2 days, 50 IU / mL, Miltenyi Biotec).

[0174] Flow cytometry To determine the cell surface expression level of CEACAM5 or CEACAM family proteins, cells were stained with PE-conjugated anti-CEACAM5 IgG1 (Miltenyi Biotec, 130-114-217) or PE-conjugated anti-FLAG mouse antibody (Miltenyi Biotec, 130-101-576). To confirm cell surface binding of selected antibodies, cells were treated with hIgG1 with CDR of clone #1, hIgG1 with CDR of clone #2, or Fab of clone #1 for 1 h at 4°C, and then stained with Alexa647-conjugated goat anti-human IgG (Invitrogen, A21445) or FITC-conjugated goat anti-human kappa light chain (Invitrogen, A18854) for 0.5 h at 4°C. For internalization assays, cells (1×10 in 96-well plates) were stained with 1×10 5 Cells / well) were treated with Alexa488-labeled antibodies for 0-30 hours. After antibody incubation, cells were washed with cold PBS, and the remaining cell surface-bound antibodies were then analyzed using Alexa488 fluorescence. CAR expression in anti-CEACAM5 CAR-T cells was analyzed using CD4 antibodies conjugated with APC (ebioscience, 17-0049-42) and eFluor450-conjugated with anti-human CD8 (ebioscience, 48-0088-42). + T cells or CD8 + T cells were gated and CAR expression was examined using FITC-labeled recombinant protein L (Acrobiosystems, RPL-PF141).

[0175] In vitro cytotoxicity assay The ADCC or cytotoxicity of anti-CEACAM5 CAR-T was measured by the release of cytoplasmic LDH from target cells using the LDH-Glo Cytotoxicity Assay Kit (Promega, J2381). For ADCC, PBMCs or enriched NK cells derived from PBMCs as effector cells were cultured in the presence of hIgG1 with the CDR of clone #1 or hIgG1 with the CDR of clone #2, with NCI-H660 or Du145 (1 × 10 in 96-well plates) as target cells. 4 Cells / well) at an effector to target (E:T) ratio of 20:1 (for PBMCs) or 5:1 (for NK cells) for 4 hours. For the cell killing activity of anti-CEACAM5 CAR-T, control T cells or CAR-T cells as effector cells were incubated with NCI-H660, Du145-CEACAM5, or Du145 cells as target cells at the indicated E:T ratio in 96-well plates for 24 hours. Controls performed for calculation of percent cytotoxicity (%Cytotoxicity) were performed according to the manufacturer's instructions. %Cytotoxicity was calculated using the following formula: (experimental effector min-target min) / (target max-target min) x 100.

[0176] Cell viability was measured in the presence of hIgG1 with the CDR of clone #1 using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7571). Cells (5 × 10 3 Cells / well) were plated and cultured for 12 hours and then treated with the indicated antibodies for 72 hours. Normalized % ATP values ​​were calculated by normalizing luminescence values ​​for vehicle-treated wells.

[0177] Transwell assay Cell migration was measured using transwells (pore size 8 μm, Corning 3422). Cells (1 × 10 5Cells / well) were resuspended in serum-free medium and then added to the upper chamber in the presence of hIgG1 with the CDR of clone #1. The lower chamber contained 10% FBS. A no FBS condition was performed as a negative control. After 3 days of incubation at 37°C, cells remaining on the upper surface of the membrane were removed using a swab, while cells that had migrated to the lower membrane surface were fixed and stained with 0.5% crystal violet in 20% methanol solution. Cells migrating through the filter were quantified by dissolving the stained cells using a 10% acetic acid solution, and then the absorbance was measured at 590 nm. Normalized % migration was calculated by normalizing the absorbance values ​​for 10% FBS-treated wells in the absence of antibody.

[0178] Cytokine ELISA Control T cells and CAR-T cells were cultured in 24-well plates (2 × 10 4 The cells were plated on 100 wells (1000 x 1000 cells / well) and co-cultured with target cells at the indicated E:T ratios for 48 h at 37°C. Supernatants were subsequently analyzed for cytokine levels using ELISA kits for human IFNγ, TNFα, IL-2, IL-4, IL-13, and GM-CSF, respectively, according to the manufacturer's instructions (Thermofisher).

[0179] result 1G9 hIgG1 induces CEACAM5-dependent ADCC During the characterization of 1G9, clone #2 Fab (also referred to as 1C1 Fab) was also isolated. 1C1 hIgG1 bound specifically to the A3B3 domain of CEACAM5, similar to 1G9 hIgG1 (Figure 30), and also showed similar binding to mutants of the A3B3 domain, suggesting that N-linked glycans at N580, N612, and N650 also contribute to the epitope for 1C1 (Figure 31). In a competitive ELISA, these two antibodies competed for binding to CEACAM5, suggesting that the two antibodies bind to overlapping epitopes in the A3B3 domain (Figure 32). The binding of 1G9 and 1C1 hIgG1 to CEACAM5-positive NCI-H660 cells and CEACAM5-negative Du145 cells was then evaluated. Both 1G9 and 1C1 hIgG1 showed specific binding to NCI-H660 cells. Notably, Du145-CEACAM5 cells engineered to express CEACAM5 showed binding of 1G9 and 1C1 hIgG1 on the cell surface that was clearly different from CEACAM5-negative Du145 cells (Figure 35). Next, the ADCC activity of 1G9 and 1C1 hIgG1 against CEACAM5-positive target cells was tested using an LDH release cytotoxicity assay. Primary NK cells enriched from human peripheral blood mononuclear cells (PBMC) were used as effector cells, and NCI-H660 or Du145 cells as target cells. 1G9 hIgG1 elicited dose-dependent ADCC activity against CEACAM5-positive NCI-H660 cells and CEACAM5-positive Du145 cells, but not against CEACAM5-negative Du145 cells (Figure 36) and LNCap (Figure 37). Interestingly, no ADCC activity was observed upon treatment with 1C1 hIgG1, even though 1C1 hIgG1 displayed similar binding affinity to the A3B3 domain of CEACAM5 and cells as well as to the Fc gamma receptor (FcγR) (FIG. 34).To investigate why 1C1 hIgG1 showed different ADCC activity from 1G9, electron microscope (EM) images were examined to clarify minor differences, such as binding orientation or binding site, but 1C1 hIgG1 bound to the A3B3 domain similar to 1G9 hIgG1, and both antibodies bound mainly to the B3 domain of CEACAM5 in EM images. Secondly, the cell-killing activity of 1G9 or 1C1 hIgG1 was tested in the absence of immune cells, but no cytotoxic activity against NCI-H660 cells was found by either antibody (Figure 38). This result indicated that the cell-killing activity of 1G9 hIgG1 apparently depends on the recruitment of FcγR-expressing immune cells, including NK cells, but targeting CEACAM has no effect on cell viability in the absence of immune cells. Third, the rate of internalization for 1G9 and 1C1 hIgG1 was assessed by measuring the remaining antibody on the surface of NCI-H660 cells after antibody treatment at 37° C. in a time course by flow cytometry ( FIG. 40 ). Neither hIgG1 underwent internalization up to 30 hours, suggesting that the difference in ADCC activity of both hIgG1s is unlikely to be caused by differences in internalization kinetics.

[0180] 1G9 hIgG1 inhibits cell migration and invasion Since CEACAM5 plays a role in regulating cell adhesion and migration as a cell adhesion molecule, we used a transwell assay to explore the role of 1G9 hIgG1 in the migration of prostate cancer cells. NCI-H660 and Du145-CEACAM5 cells showed a dose-dependent decrease in migration function by 1G9 hIgG1 treatment compared to serum-treated cells, while no migration effect of 1G9 hIgG1 was observed in CEACAM5-negative Du145 cells (Figure 39). Similarly, an analog of Sanofi's antibody targeting CEACAM5 (SAR408701) inhibited cell migration. These results suggested that targeting CEACAM5 with 1G9 hIgG1 could disrupt CEACAM5-mediated cell migration.

[0181] Anti-CEACAM5 CAR-T shows strong cytotoxicity against CEACAM5-positive prostate cancer cells Based on epitope mapping and limited internalization of hIgG1 1G9, it was concluded that the 1G9 antibody could be used for CAR-T cell therapy. Lentiviral second and third generation anti-CEACAM5 CAR constructs were generated encoding the single chain variable fragment (scFv), transmembrane domain, and costimulatory intracellular domain derived from 1G9, as shown in Figure 42. T cells were transduced with the second and third generation CAR constructs, and CAR expression was confirmed by flow cytometry. The expression level of the third generation CAR in transduced T cells was 84%, of which 30% were CD4+. + T cells, 54% were CD8 + T cells, similar to the expression levels of second generation CARs, which was 78% overall (Figure 43).

[0182] To determine the cytotoxicity of anti-CEACAM5 CAR-T cells against target cells in vitro, anti-CEACAM5 CAR-T cells were co-cultured with CEACAM5-positive NCI-H660 and Du145-CEACAM5 at E:T ratios of 1:4, 1:2, 1:1, 2:1, 4:1, and 8:1 for 24 hours. The cytotoxicity (%) of second- and third-generation CAR-T cells against NCI-H660 cells was higher than that of the control T cell group at all E:T ratios. The third-generation CAR-T cells showed more potent cytotoxicity compared to the second-generation CAR-T cells (Figure 44). The cytotoxicity of the third-generation CAR-T cell group against Du145-CEACAM5 cells was observed at E:T ratios of 8:1, 4:1, and 2:1, but not against Du145 cells (Figure 44). To further explore the mechanism of cytotoxicity of anti-CEACAM5 CAR-T cells, we analyzed the production of IFN-gamma (IFNγ), TNFα, IL-2, IL-4, IL-13, and GM-CSF from the supernatants of CAR-T after 48 hours of co-culture with target cells. Secretion of IL-4 and GM-CSF was significantly enhanced by treatment of third-generation anti-CEACAM5 CAR-T cells in CEACAM5-expressing NCI-H660 and Du145-CEACAM5 cells, but not in CEACAM5-negative Du145 cells (Figure 46). Furthermore, analysis of the supernatants at 48 hours for intracellular staining of granzyme B (GrzB), perforin, and IFNγ revealed enhanced release of GrzB and perforin from third-generation anti-CEACAM5 CAR-T cells in CEACAM5-positive NCI-H660 cell cultures (Figure 47). Third-generation anti-CEACAM5 CAR-T cells produced significant cytotoxicity in CEACAM5-positive NEPC cells via secretion of IL-4, GM-CSF cytokines and release of GrzB / perforin, likely due to the membrane proximity of 1G9 binding to the A3B3 domain combined with a well-designed CAR spacer.

[0183] [Example 3] In vivo preclinical efficacy evaluation of hIgG1 and CAR-T in vivo studies For evaluation of hIgG1 1G9, Du145 and Du145-CEACAM5 cells (1 × 10 7 The 1000 cells / mouse were subcutaneously (sc) injected into the right flank of Balb / c scid mice (6-8 weeks old, male, The Jackson Laboratory). 3 Mice were randomized when they reached 100% CI, and were administered hIgG1 1G9 (20 mg / kg or 5 mg / kg) or vehicle (buffer control) intraperitoneally twice weekly. null Mice (NSG mice, 6–8 weeks old, male, The Jackson Laboratory) were treated with Du145 and Du145-CEACAM5 (1 × 10 7 The tumors were 150 mm 3 Mice were inoculated twice every 4 days via the tail vein with either control T cells or CAR-T cells (5 × 10 6 The tumor size was measured using a caliper and calculated using the formula V = 1 / 2 x length x (width). 2 Tumor volume was calculated as follows: 3 The animals were euthanized when

[0184] result Anti-CEACAM5 hIgG1 1G9 and CAR-T cells inhibited the growth of CEACAM5-positive prostate cancer xenografts. To evaluate the cytotoxic potential of hIgG1 1G9 in vivo, mouse xenograft models of Du145 and Du145-CEACAM5 were used. 3Mice bearing Du145-CEACAM5 tumors were treated with 20 or 5 mg / kg hIgG1 1G9. hIgG1 1G9 significantly delayed the growth of Du145-CEACAM5 tumors (FIG. 48) and improved mouse survival compared to that of vehicle-treated mice. For CEACAM5-negative Du145 tumors, no significant differences were observed in tumor growth and mouse survival between vehicle and hIgG1 1G9. Mouse body weight, monitored as an index of drug toxicity, was similar in the treatment and vehicle groups.

[0185] The cytotoxicity of anti-CEACAM5 CAR-T cells was also investigated using a xenograft model in mice. NSG mice were sc implanted with Du145 and Du145-CEACAM5 cells, and control T cells or anti-CEACAM5 CAR-T cells were injected intravenously twice via the tail vein of the mice. Anti-CEACAM5 CAR-T cell-treated mice had suppressed tumor growth and longer survival compared to control T cell-treated mice with Du145-CEACAM tumors, but not with Du145 tumors (Figure 49). During CAR-T cell treatment, 2 out of 5 mice with Du145-CEACAM5 tumors, but not with Du145 tumors, showed a 10% weight loss and died on days 9 and 11 after injection of CAR-T. This result suggests that anti-CEACAM5 CAR-T cells exhibited target-mediated systemic toxicity coupled with a strong anti-tumor effect in some mice. Taken together, these results demonstrate that anti-CEACAM5 hIgG1 1G9 and CAR-T cells exhibit in vivo anti-tumor efficacy that is dependent on CEACAM5 expression, consistent with the in vitro results (Figure 50).

[0186] [Example 4] Design of BiKE from binders with the ability to bind to human CD66e polypeptide The heavy and light chain variable domains of Fab from clones #1 and #2 were used together with anti-NKG2A to create vectors designed to express BiKE (BiKE #1 and BiKE #2) with the ability to bind to CD66e and NKG2A polypeptides.

[0187] Preparation of anti-CEA x NKG2A BiKE The synthetic gene for the digested gene for anti-CEA x NKG2A BiKE is digested with NotI and NheI restriction enzymes and then ligated into the pCAT2 vector with T4 DNA ligase. The recircularized plasmid DNA is transformed into HB2151 E. coli competent cells, and transformants are then selected on ampicillin-containing LB plates (final concentration 100 μg / mL) overnight in a 37 °C incubator. The next day, colonies are inoculated into liquid LB + ampicillin medium and grown in a 37 °C shaking incubator until mid-logarithmic growth phase. An OD of 0.4-0.6 is obtained. 600 Add 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture at 30 °C and continue growing in a shaking incubator at 200 rpm. The next day, induced E. coli cells are harvested by centrifugation at 6,000 g for 10 min. The cell pellet is resuspended in 1 / 10 volume of periplasmic extraction buffer containing polymyxin B (0.5 mg / mL in PBS, pH 7.4) and then incubated on ice for 1 h. The supernatant is collected by centrifugation at 12,000 g for 20 min. The filtered supernatant is loaded onto a disposable column pre-packed with Ni-NTA resin. After loading, the resin is washed with 5 bed volumes of 30 mM imidazole in PBS, pH 7.4, and then the bound BiKE molecules are eluted by the addition of 300 mM imidazole in PBS, pH 7.4. Residual imidazole is removed by passage through a PD-10 desalting column pre-filled with PBS (pH 7.4).

[0188] [Example 5] Other embodiments Embodiment 1. (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). An antibody comprising:

[0189] Embodiment 2. The antibody of embodiment 1 having the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151.

[0190] Embodiment 3. The antibody of any one of embodiments 1 to 2, comprising the heavy chain variable domain or region of (i).

[0191] Embodiment 4. The antibody of embodiment 3, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8.

[0192] Embodiment 5. The antibody of any one of embodiments 1 to 2, comprising the light chain variable domain or region of (i).

[0193] Embodiment 6 The antibody of embodiment 5, wherein the light chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:16.

[0194] Embodiment 7. The antibody of any one of embodiments 1 to 2, comprising the heavy chain variable domain or region of (ii).

[0195] Embodiment 8 The antibody of embodiment 7, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24.

[0196] Embodiment 9. The antibody of any one of embodiments 1 to 2, comprising the light chain variable domain or region of (ii).

[0197] Embodiment 10. The antibody of embodiment 9, wherein the light chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32.

[0198] Embodiment 11. (i) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:3 (or SEQ ID NO:3 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); or (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). An antigen-binding fragment comprising:

[0199] Embodiment 12. The antigen-binding fragment of embodiment 11 having the ability to bind to SEQ ID NO: 150 or SEQ ID NO: 151.

[0200] Embodiment 13. The antigen-binding fragment of any one of embodiments 11 to 12, comprising the heavy chain variable domain or region of (i).

[0201] Embodiment 14 The antigen-binding fragment of embodiment 13, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8.

[0202] Embodiment 15. The antigen-binding fragment of any one of embodiments 11 to 12, comprising the light chain variable domain or region of (i).

[0203] Embodiment 16 The antigen-binding fragment of embodiment 15, wherein the light chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:16.

[0204] Embodiment 17. The antigen-binding fragment of any one of embodiments 11 to 12, comprising the heavy chain variable domain or region of (ii).

[0205] Embodiment 18. The antigen-binding fragment of embodiment 17, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24.

[0206] Embodiment 19. The antigen-binding fragment of any one of embodiments 11 to 12, comprising the light chain variable domain or region of (ii).

[0207] Embodiment 20 The antigen-binding fragment of embodiment 19, wherein the light chain variable domain or region comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32.

[0208] Embodiment 21. The antibody of any one of embodiments 1 to 10, which is a monoclonal antibody.

[0209] Embodiment 22. The antibody of any one of embodiments 1 to 10 and 21, which is an scFv antibody.

[0210] Embodiment 23. The antigen-binding fragment of any one of embodiments 11 to 20, which is monoclonal.

[0211] Embodiment 24. The antigen-binding fragment of any one of embodiments 11 to 20 and 23, which is a Fab.

[0212] Embodiment 25. A chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0213] Embodiment 26 The chimeric antigen receptor of embodiment 25, wherein the antigen-binding domain comprises an scFv having the ability of binding to a CD66e polypeptide.

[0214] Embodiment 27. The chimeric antigen receptor of any one of embodiments 25-26, wherein the hinge comprises the hinge depicted in FIG. 13.

[0215] Embodiment 28. The chimeric antigen receptor of any one of embodiments 25 to 27, wherein the transmembrane domain comprises the transmembrane domain depicted in FIG. 14.

[0216] Embodiment 29. The chimeric antigen receptor of any one of embodiments 25 to 28, comprising one or more signaling domains as described in FIG. 15.

[0217] Embodiment 30. A cell comprising the chimeric antigen receptor of any one of embodiments 25 to 29.

[0218] Embodiment 31 The cell of embodiment 30, which is a T cell, a stem cell or a NK cell.

[0219] Embodiment 32. A cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0220] Embodiment 33 The cell engager of embodiment 32, wherein the first antigen-binding domain comprises an scFv capable of binding to a CD66e polypeptide.

[0221] Embodiment 34 The cell engager of embodiment 32, wherein said first antigen-binding domain is an IgG having the ability of binding to a CD66e polypeptide.

[0222] Embodiment 35 The cell engager of any one of embodiments 32-34, wherein the linker comprises a linker as depicted in FIG. 10 or FIG. 13.

[0223] Embodiment 36 The cell engager of any one of embodiments 32-35, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a T cell.

[0224] Embodiment 37 The cell engager of embodiment 36, wherein said polypeptide expressed on the surface of the T cell is a CD3 polypeptide.

[0225] Embodiment 38 The cell engager of embodiment 36, wherein said second antigen-binding domain is the antigen-binding domain depicted in FIG. 18.

[0226] Embodiment 39. The cell engager of any one of embodiments 32-35, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a NK cell.

[0227] Embodiment 40. The cell engager of embodiment 39, wherein said polypeptide expressed on the surface of the NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide.

[0228] Embodiment 41 The cell engager of embodiment 39, wherein the second antigen-binding domain is the antigen-binding domain depicted in FIG.

[0229] Embodiment 42 The cell engager of any one of embodiments 32 to 41, comprising a third antigen-binding domain.

[0230] Embodiment 43 The cell engager of embodiment 42, wherein the third antigen-binding domain binds to a polypeptide expressed on the surface of a NK cell.

[0231] Embodiment 44. The cell engager of embodiment 43, wherein said polypeptide expressed on the surface of the NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide.

[0232] Embodiment 45 The cell engager of embodiment 43, wherein the third antigen-binding domain is the antigen-binding domain depicted in FIG.

[0233] Embodiment 46. A nucleic acid comprising a nucleic acid sequence encoding at least a portion of the antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0234] Embodiment 47. The nucleic acid of embodiment 46, wherein the nucleic acid sequence encodes the heavy chain variable domain or region of any one of (i) to (ii) of embodiment 1.

[0235] Embodiment 48. The nucleic acid of any one of embodiments 46 to 47, wherein the nucleic acid sequence encodes the light chain variable domain or region of any one of (i) to (ii) of embodiment 1.

[0236] Embodiment 49. The nucleic acid of any one of embodiments 46 to 48, which is a viral vector.

[0237] Embodiment 50. The nucleic acid of any one of embodiments 46 to 48, which is a phagemid.

[0238] Embodiment 51. A nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of embodiments 25 to 29 or the cell engager of any one of embodiments 32 to 45.

[0239] Embodiment 52. The nucleic acid of embodiment 51, which is a viral vector.

[0240] Embodiment 53 The nucleic acid of embodiment 51, which is a phagemid.

[0241] Embodiment 54. A host cell comprising the nucleic acid of any one of embodiments 46 to 52.

[0242] Embodiment 55. A host cell expressing the chimeric antigen receptor of any one of embodiments 25 to 29 or the cell engager of any one of embodiments 32 to 45.

[0243] Embodiment 56. The host cell of any one of embodiments 54 to 55, which is a T cell, a stem cell or a NK cell.

[0244] Embodiment 57. An antibody drug conjugate (ADC) comprising an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0245] Embodiment 58 The ADC of embodiment 57, wherein the antigen-binding domain comprises an scFv capable of binding to a CD66e polypeptide.

[0246] Embodiment 59. The ADC of embodiment 57, wherein the antigen-binding domain is an IgG having the ability to bind to a CD66e polypeptide.

[0247] Embodiment 60. The ADC of any one of embodiments 57-59, wherein the drug is selected from the group consisting of an auristatin, mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

[0248] Embodiment 61. A composition comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0249] Embodiment 62. The composition of embodiment 61, comprising any one of the antibodies of embodiments 1 to 10, 21 and 22.

[0250] Embodiment 63. The composition of embodiment 61, comprising an antigen-binding fragment of any one of embodiments 11 to 20, 23 and 24.

[0251] Embodiment 64. A composition comprising a cell engager of any one of embodiments 32 to 45.

[0252] Embodiment 65. A composition comprising any one of the cells of embodiments 30, 31 and 53 to 56.

[0253] Embodiment 66. A composition comprising the ADC of any one of embodiments 57-60.

[0254] Embodiment 67. The composition of any one of embodiments 61 to 66, comprising a checkpoint inhibitor.

[0255] Embodiment 68. The composition of embodiment 67, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189 and ipilimumab.

[0256] Embodiment 69. A method of treating a mammal having cancer, comprising administering to said mammal a composition of any one of embodiments 61 to 68.

[0257] Embodiment 70 The method of embodiment 69, wherein the mammal is a human.

[0258] Embodiment 71. The cancer is CD66e + The method of any one of embodiments 69-70, wherein the cancer is cancer.

[0259] Embodiment 72. The CD66e + Cancer, CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e + 72. The method of embodiment 71, wherein the cancer is selected from the group consisting of cervical cancer.

[0260] Embodiment 73 The method of any one of embodiments 69-72, wherein the number of cancer cells in the mammal is reduced after the administering step.

[0261] Embodiment 74. A method of treating a mammal having cancer, comprising: (a) administering to the mammal a composition according to any one of embodiments 98-104; and (b) administering to the mammal a composition comprising a checkpoint inhibitor. The method includes:

[0262] Embodiment 75 The method of embodiment 74, wherein the mammal is a human.

[0263] Embodiment 76. The cancer is CD66e + The method of any one of embodiments 74-75, wherein the cancer is cancer.

[0264] Embodiment 77. The CD66e + Cancer, CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e + 77. The method of embodiment 76, wherein the cancer is selected from the group consisting of cervical cancer.

[0265] Embodiment 78. The method of any one of embodiments 74 to 77, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189 and ipilimumab.

[0266] Embodiment 79. The method of any one of embodiments 74-78, wherein the number of cancer cells in the mammal is reduced following the administering steps (a) and (b).

[0267] Embodiment 80. A method for binding a binding molecule to a CD66e polypeptide, comprising contacting said CD66e polypeptide with the antibody or antigen-binding fragment of any one of embodiments 1 to 24.

[0268] Embodiment 81 The method of embodiment 80, wherein said contacting step is carried out in vitro.

[0269] Embodiment 82 The method of embodiment 80, wherein said contacting step is performed in vivo.

[0270] Embodiment 83 The method of embodiment 82, wherein said contacting step is carried out in a mammal by administering said antibody or said antigen-binding fragment to said mammal.

[0271] Embodiment 84 The method of embodiment 84, wherein the mammal is a human.

[0272] Embodiment 85. A method for conjugating a binding molecule to a CD66e polypeptide, comprising the step of contacting said CD66e polypeptide with a chimeric antigen receptor of any one of embodiments 25-29, a cell engager of any one of embodiments 32-45, or an ADC of any one of embodiments 57-60.

[0273] Embodiment 86 The method of embodiment 85, wherein said contacting step is carried out in vitro.

[0274] Embodiment 87 The method of embodiment 85, wherein said contacting step is performed in vivo.

[0275] Embodiment 88 The method of embodiment 87, wherein said contacting step is performed in a mammal by administering said chimeric antigen receptor, said cell engager or said ADC to the mammal.

[0276] Embodiment 89. The method of embodiment 88, wherein the mammal is a human.

[0277] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion or substitution of one, two or three amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion or substitution of one, two or three amino acids) and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion or substitution of one, two or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion or substitution of one, two or three amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion or substitution of one, two or three amino acids) and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion or substitution of one, two or three amino acids); or (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions).

4. An antibody comprising:

2. 2. The antibody of claim 1, having the ability to bind to SEQ ID NO: 150 or SEQ ID NO:

151.

3. (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion or substitution of one, two or three amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion or substitution of one, two or three amino acids) and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion or substitution of one, two or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion or substitution of one, two or three amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion or substitution of one, two or three amino acids) and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion or substitution of one, two or three amino acids); or (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two or three amino acid additions, deletions or substitutions) and SEQ ID NO: 27 (or SEQ ID NO: 27 with one, two or three amino acid additions, deletions or substitutions). An antigen-binding fragment comprising:

4. 4. The antigen-binding fragment of claim 3, having the ability to bind to SEQ ID NO: 150 or SEQ ID NO:

151.

5. 5. A chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain and one or more signaling domains, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment of any one of claims 1 to 4.

6. The chimeric antigen receptor of claim 5, wherein the antigen-binding domain comprises an scFv having the ability of binding to a CD66e polypeptide.

7. The chimeric antigen receptor of any one of claims 5 to 6, wherein the hinge comprises the hinge depicted in Figure 13.

8. 8. The chimeric antigen receptor of claim 5, wherein the transmembrane domain comprises the transmembrane domain depicted in FIG.

9. 16. The chimeric antigen receptor of any one of claims 5 to 8, comprising one or more signaling domains as depicted in Figure 15.

10. A cell comprising the chimeric antigen receptor of any one of claims 5 to 9.

11. The cell of claim 10, which is a T cell, a stem cell or a NK cell.

12. 5. A cell engager comprising a first antigen-binding domain, a linker and a second antigen-binding domain, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 1 to 4.

13. The cell engager of claim 12, wherein the first antigen-binding domain comprises an scFv capable of binding to a CD66e polypeptide.

14. The cell engager of claim 12, wherein the first antigen-binding domain is an IgG having the ability to bind to a CD66e polypeptide.

15. 15. The cell engager of any one of claims 12 to 14, wherein the linker comprises a linker as depicted in Figure 10 or Figure 13.

16. The cell engager of any one of claims 12 to 15, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a T cell.

17. The cell engager of claim 16, wherein the polypeptide expressed on the surface of a T cell is a CD3 polypeptide.

18. 17. The cell engager of claim 16, wherein the second antigen-binding domain is an antigen-binding domain depicted in FIG.

18.

19. The cell engager of any one of claims 12 to 15, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a NK cell.

20. 20. The cell engager of claim 19, wherein the polypeptide expressed on the surface of a NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide.

21. 20. The cell engager of claim 19, wherein the second antigen binding domain is an antigen binding domain as depicted in FIG.

22. 22. The cell engager of any one of claims 12 to 21, comprising a third antigen-binding domain.

23. The cell engager of claim 22, wherein the third antigen-binding domain binds to a polypeptide expressed on the surface of a NK cell.

24. 24. The cell engager of claim 23, wherein the polypeptide expressed on the surface of a NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44 or NKp46 polypeptide.

25. 24. The cell engager of claim 23, wherein the third antigen-binding domain is the antigen-binding domain depicted in Figure 19.

26. A nucleic acid comprising a nucleic acid sequence encoding at least a portion of the antibody or antigen-binding fragment of any one of claims 1 to 4.

27. 26. A nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor according to any one of claims 5 to 9 or a cell engager according to any one of claims 12 to 25.

28. 28. A host cell comprising a nucleic acid according to any one of claims 26 to 27.

29. 26. A host cell expressing a chimeric antigen receptor according to any one of claims 5 to 9 or a cell engager according to any one of claims 12 to 25.

30. 30. The host cell of any one of claims 28 to 29, which is a T cell, a stem cell or a NK cell.

31. 5. An antibody drug conjugate (ADC) comprising an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment of any one of claims 1 to 4.

32. The ADC of claim 31, wherein the antigen-binding domain comprises an scFv capable of binding to a CD66e polypeptide.

33. The ADC of claim 31 , wherein the antigen-binding domain is an IgG having the ability to bind to a CD66e polypeptide.

34. 34. The ADC of any one of claims 31 to 33, wherein the drug is selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

35. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 4.

36. 26. A composition comprising a cell engager according to any one of claims 12 to 25.

37. 31. A composition comprising a cell according to any one of claims 28 to 30.

38. 35. A composition comprising the ADC of any one of claims 31 to 34.

39. 39. The composition of any one of claims 35 to 38, comprising a checkpoint inhibitor.

40. 40. The composition of claim 39, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

41. 41. A method of treating a mammal having cancer comprising administering to said mammal a composition according to any one of claims 35 to 40.

42. 42. The method of claim 41, wherein the mammal is a human.

43. The cancer is CD66e + 43. The method of any one of claims 41 to 42, wherein the cancer is cancer.

44. CD66e + Cancer, CD66e + Lung cancer, CD66e + Prostate cancer, CD66e + Esophageal cancer, CD66e + Gastric cancer, CD66e + Colorectal cancer, CD66e + Liver cancer, CD66e + Vaginal cancer or CD66e + 44. The method of claim 43, wherein the cancer is selected from the group consisting of cervical cancer.

45. 45. The method of any one of claims 41 to 44, wherein the number of cancer cells in the mammal is reduced following the administering step.

46. 1. A method of treating a mammal having cancer, comprising: (a) administering to the mammal a composition according to any one of claims 35 to 38; and (b) administering to the mammal a composition comprising a checkpoint inhibitor. The method includes:

47. 47. The method of claim 46, wherein the mammal is a human.

48. 48. The method of any one of claims 46 to 47, wherein the number of cancer cells in the mammal is reduced following the administering steps (a) and (b).

49. A method for binding a binding molecule to a CD66e polypeptide, comprising the step of contacting the CD66e polypeptide with an antibody or antigen-binding fragment of any one of claims 1 to 4.

50. 35. A method for conjugating a binding molecule to a CD66e polypeptide, the method comprising contacting the CD66e polypeptide with a chimeric antigen receptor of any one of claims 5 to 9, a cell engager of any one of claims 12 to 25, or an ADC of any one of claims 31 to 34.

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