Molecules that bind to b7 homolog 3 polypeptides

Abstract

This provides methods and materials involved in binding a molecule (e.g., an antibody, a fragment of an antibody, an antibody domain, a chimeric antigen receptor (CAR), a cell engager, or an antibody-drug conjugate (ADC)) to a B7 homolog 3 (B7-H3) polypeptide (e.g., a human B7-H3 polypeptide). For example, binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to a B7-H3 polypeptide as well as methods and materials for using such binders to treat cancer are provided. Also provided are cells (e.g., host cells) designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to a B7-H3 polypeptide as well as methods and materials for using such cells to treat cancer.

Description

[0001] MOLECULES THAT BIND TO B7 HOMOLOG 3 POLYPEPTIDES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U. S. Patent Application Serial No. 63 / 713,270, filed on October 29, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2339W01.xml.” The XML file, created on October 8, 2025, is 135,515 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials involved in binding a molecule (e.g., an antibody, a fragment of an antibody, an antibody domain, a chimeric antigen receptor (CAR), a cell engager, or an antibody-drug conjugate (ADC)) to a B7 homolog 3 (B7-H3) polypeptide (e.g., a human B7-H3 polypeptide). For example, this document provides binders (e g., antibodies, antigen binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to a B7-H3 polypeptide and methods and materials for using such binders to treat cancer. This document also provides cells (e.g., host cells) designed to express one or more binders (e.g., antibodies, antigen binding fragments, antibody domains, CARs, or cell engagers) having the ability to bind to a B7-H3 polypeptide and methods and materials for using such cells to treat cancer.

[0008] BACKGROUND

[0009] Recent advances in cancer immunotherapy demonstrate that the immune system can be harnessed to eradicate cancer (Topalian et al., Cancer Cell, 27:450-461 (2015)). One of the most successful immunotherapies targets the PD-1 / PD-L1 pathway by using blocking antibodies, which enhances the immune response to tumors (Dong et al., J. Mol. Med., 81:281-287 (2003).). However, not all cancers respond to immunotherapy or checkpoint blockade targeting PD-1 or CTLA-4 (Dong et al., Nat. Med., 8:793-800 (2002); and Kather et al., Semin. Cancer. Biol., 52(Pt 2): 189-197 (2018)).

[0010] B7-H3 is highly expressed on many different tumors and generally is associated with poorer outcomes (Kontos etal., Clin. Cancer Res., 27:1227-1235 (2021)). In addition, B7-H3 on tumor cells has been shown to modulate the immune response. While some studies have shown a positive role for B7-H3 in activating immune cells in the tumor microenvironment (Hashiguchi et al., Proc. Natl. Acad. Sci. USA, 105:10495-10500 (2008); andKobori etal., Immunology, 130:363-373 (2010)), the majority of recent studies have indicated that B7-H3 on tumors inhibits the local immune response (Cai etal., Cell. Mol. Immunol., 17:227-236 (2020); Wang etal.. Cell Stem Cell, 28:1597-1613 (2021); and Yonesaka etal., Clin. Cancer Res., 24:2653-2664 (2018)). Lack of clarity for the receptors that bind B7-H3 have hampered rational design to block B7-H3 function, such as with monoclonal antibodies.

[0011] SUMMARY

[0012] This document provides methods and materials involved in binding a molecule (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) to a B7-H3 polypeptide. For example, this document provides binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, cell engagers, and ADCs) that bind to a B7-H3 polypeptide and methods and materials for using one or more such binders to treat a mammal (e.g., a human) having cancer (e.g., a leukemia such as acute myeloid leukemia (AML)).

[0013] As described herein, binders (e.g., antibody domains, antigen binding fragments, and antibodies) can be designed to have the ability to bind to a B7-H3 polypeptide. For example, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein can include an antigen binding domain having the ability to bind to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human B7-H3 polypeptide as set forth in any one of SEQ ID NOs:61-64 (see, e.g., Example 3). In some cases, a binder provided herein and having the ability to bind to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human B7-H3 polypeptide can include the ability to inhibit the ability of a human B7-H3 polypeptide to bind to human Siglec-9 and activate Siglec-9 immunoinhibitory activity. In some cases, a binder provided herein and having the ability to bind to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human B7-H3 polypeptide can include at least one set of three CDRs of an antigen binding domain provided herein (e.g., SEQ ID NOs: 1-3, SEQ ID NOs:9-11, SEQ ID NOs: 17-19, SEQ ID NO:25-27, SEQ ID NOs:33-35, or SEQ ID NOs:41-43). In some cases, a binder provided herein and having the ability to bind to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of a human B7-H3 polypeptide can include two sets of three CDRs (e.g., CDR1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain) of an antigen binding domain provided herein (e.g., SEQ ID NOs: 1-3 and 9-11, SEQ ID NOs:17-19 and 25-27, or SEQ ID NOs:33-35 and 41-43).

[0014] In some cases, binders (e.g., antibody domains, antigen binding fragments, and antibodies) provided herein can be engineered into a CAR to create CAR+cells (e.g., CAR+T cells, CAR+stem cells such as CAR+induced pluripotent stem cells, or CAR+natural killer (NK) cells) having the ability to target B7-H3+cells (e.g., B7-H3+cancer cells), can be engineered into an antibody structure that includes an Fc region to create antibodies having the ability to target B7-H3+cells (e.g., B7-H3+cancer cells) and induce antibody-dependent cell-mediated cytotoxicity (ADCC) against the targeted B7-H3+cells, and / or can be engineered into a cell engager such as a bi-specific T cell engager (e.g., a BiTE), a bi-specific killer engager (e.g., a BiKE), and / or a tri-specific killer engager (e.g., a TriKE) to create cell engagers having the ability to target B7-H3+cells (e.g., B7-H3+cancer cells) and induce one or more immune responses (e.g., T cell immune responses and / or ADCC using a cell engager in the absence of an Fc-containing antibody) against the targeted B7-H3+cells. It is noted that BiKE- and TriKE-mediated killing can be referred to ADCC even though it is not initiated by an Fc domain.

[0015] In addition, as described herein, binders (e.g., antibody domains, antigen binding fragments, and antibodies) provided herein can be used to create conjugates that include the binder and a drug (e.g., ADCs). For example, ADCs such as full antibody-drug conjugates, Fab-drug conjugates, and / or antibody domain-drug conjugates can be designed to include an appropriate binder provided herein to create the conjugate. Such conjugates can be used to deliver the drug payload to target B7-H3+cells (e g., B7-H3+cancer cells). As also described herein, cells (e.g., host cells) can be designed to express one or more binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, and / or cell engagers) having the ability to bind to a B7-H3 polypeptide. For example, cells such as T cells (e.g., CTLs), stem cells (e.g., induced pluripotent stem cells), or NK cells can be engineered to express one or more CARs having the ability to bind to a B7-H3 polypeptide. Such cells (e.g., B7-H3-specific CAR+T cells or NK cells) can be used to treat cancer (e.g., a leukemia such as AML).

[0016] This document also provides methods for using binders (e.g., antibody domains, antigen binding fragments, antibodies, cell engagers, and / or ADCs) having the ability to bind to a B7-H3 polypeptide or cells expressing one or more binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, and / or cell engagers) having the ability to bind to a B7-H3 polypeptide to treat cancer (e.g., a leukemia such as AML). As described herein, binders (e.g., antibody domains, antigen binding fragments, antibodies, cell engagers, and / or ADCs) provided herein can be used to treat a mammal (e.g., a human) having cancer (e.g., a leukemia such as AML). For example, a mammal (e.g., a human) having cancer (e.g., a B7-H3+cancer) can be administered a composition comprising one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, a vector, or a host cell (e g., CAR+cells) provided herein) to reduce the number of cancer cells within the mammal (e.g., B7-H3+cancer cells), to induce ADCC against cancer cells within the mammal, and / or to increase the survival duration of the mammal from cancer. In some cases, a mammal (e.g., a human) having cancer (e.g., a B7-H3+cancer) can be administered a composition comprising one or more binders (e g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more cell engagers, and / or one or more ADCs) provided herein to bind said binder to a human B7-H3 polypeptide and to inhibit the ability of that human B7-H3 polypeptide to (a) bind to human Siglec-9 and (b) activate Siglec-9 immunoinhibitory activity.

[0017] In some cases, a binder provided herein having the ability to bind to a human B7-H3 polypeptide and lacking a functional Fc domain (e.g., an Fab fragment or F(ab’)2 fragment) can be used to bind to a human B7-H3 polypeptide and to inhibit the ability of that human B7-H3 polypeptide to (a) bind to human Siglec-9 and (b) activate Siglec-9 immunoinhibitory activity, thereby inhibiting Siglec-9 immunoinhibitory activity within the mammal (e.g., within the tumor microenvironment) and reducing the number of tumor cells within the mammal without promoting ADCC. For example, a mammal (e.g., a human) having cancer (e g., a B7-H3+cancer) can be administered a composition comprising one or more binders lacking a functional Fc domain (e.g., an Fab fragment or F(ab’)2 fragment) to inhibit the ability of a human B7-H3 polypeptide to (a) bind to human Siglec-9 and (b) activate Siglec-9 immunoinhibitory activity without promoting ADCC, thereby reducing the number of tumor cells within the mammal without promoting ADCC.

[0018] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a cell engager, or an ADC) provided herein can be used to detect the presence or absence of a B7-H3 polypeptide. For example, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a cell engager, or an ADC) provided herein can be used to determine whether or not a sample (e.g., a biological sample such tumor biopsy) obtained from a mammal (e.g., a human) contains B7-H3+cells (e.g., B7-H3+cancer cells). Having the ability to detect the presence or absence of a B7-H3 polypeptide (e.g., B7-H3+cancer cells) can allow clinicians, health professionals, and patients to make better decisions about possible treatment options. For example, detection of B7-H3+cancer cells within a mammal can allow clinicians, health professionals, and patients to select an appropriate anti-cancer treatment that targets the B7-H3+cancer cells. Such treatments that target the B7-H3+cancer cells can include administration of one or more of the binders described herein having the ability to bind to a B7-H3 polypeptide and / or administration of one or more cells (e.g., B7-H3-specific CAR+T cells or NK cells) designed to express a binder described herein.

[0019] In general, one aspect of this document features binding molecules 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); (ii) a heavy 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), 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 sequences 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); or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The binding molecule can have the ability to bind to a polypeptide sequence set forth in any one of SEQ ID NOs:61-64. The binding molecule can include (i). For example, the heavy chain variable domain can have an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO: 16. The binding molecule can include (ii). For example, the heavy chain variable domain can have an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO:32. The binding molecule can include (iii). For example, the heavy chain variable domain can have an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:40, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO:48. The binding molecule can be a monoclonal antibody. The binding molecule can be an scFv antibody. This document also features nucleic acid including a nucleic acid sequence encoding such a binding molecule. The nucleic acid sequence can encode said heavy chain variable domain or region of (i). The nucleic acid sequence can encode said light chain variable domain or region of (i). The nucleic acid sequence can encode said heavy chain variable domain or region of (ii). The nucleic acid sequence can encode said light chain variable domain or region of (ii). The nucleic acid sequence can encode said heavy chain variable domain or region of (iii). The nucleic acid sequence can encode said light chain variable domain or region of (iii). The nucleic acid can be a viral vector. This document also features compositions including such binding molecules.

[0020] In another aspect, this document features CARs including an antigen binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein said antigen binding domain includes a binding molecule 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 NOTO (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); (ii) a heavy 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), 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 sequences 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); or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The antigen binding domain can include a scFv having the ability to bind to a B7-H3 polypeptide. The hinge can be a hinge set forth in any one of SEQ ID NOs: 88-95. The hinge can be a CD8a hinge. The transmembrane domain can be a transmembrane domain set forth in any one of SEQ ID NOs:97-104. The transmembrane domain can be a CD8a transmembrane domain. The CAR can include one or more signaling domains set forth in any one of SEQ ID NOs:106-115. The CAR can include a 4-1BB intracellular signaling domain and a CD3(^ intracellular signaling domain. This document also features nucleic acid including a nucleic acid sequence encoding such CARs. The nucleic acid can be a viral vector. This document also features host cells including a nucleic acid including a nucleic acid sequence encoding such CARs. This document also features host cells that express such CARs. The host cell can be a T cell, stem cell, or NK cell. This document also features compositions including such host cells.

[0021] In another aspect, this document features cell engagers including a first antigen binding domain, a linker, and a second antigen binding domain, wherein said first antigen binding domain comprises a binding molecule 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); (ii) a heavy 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), 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 sequences 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); or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The first antigen binding domain can include a scFv having the ability to bind to a B7-H3 polypeptide. The linker can be a linker set forth in any one of SEQ ID NOs:71-74 or SEQ ID NOs:88-95. The second antigen binding domain can bind to a polypeptide expressed on the surface of T cells. The polypeptide expressed on the surface of T cells can be a CD3 polypeptide. The second antigen binding domain can be an antigen binding domain set forth in Example 15. The second antigen binding domain can bind to a polypeptide expressed on the surface of NK cells. The polypeptide expressed on the surface of NK cells can be a CD 16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide. The second antigen binding domain can be an antigen binding domain set forth in Example 16. This document also features compositions including such cell engagers. This document also features nucleic acid including a nucleic acid sequence encoding such cell engagers. The nucleic acid can be a viral vector. This document also features host cells including a nucleic acid including a nucleic acid sequence encoding such cell engagers. This document also features host cells that expresses such cell engagers. The host cell can be a T cell, stem cell, or NK cell. This document also features compositions including such host cells.

[0022] In another aspect, this document features ADCs including an antigen binding domain covalently linked to a drug, wherein said antigen binding domain comprises a binding molecule 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); (ii) a heavy 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), 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 sequences 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);or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The binding molecule can have the ability to bind to a B7-H3 polypeptide. The drug can be an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer. This document also features compositions including such ADCs.

[0023] In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, administering, to a mammal having cancer, a composition including a a binding molecule 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); (ii) a heavy 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), 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 sequences 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);or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions), or a composition including a CAR, a cell engager, or an ADC including such a binding molecule. The mammal can be a human. The cancer can be a B7-H3+cancer. The cancer can be a leukemia, a breast cancer, a non-small cell lung cancer, a bladder cancer, a colorectal cancer, a kidney cancer, a lymphoma, a melanoma, a pancreatic cancer, an ovarian cancer, a brain cancer, a gastric cancer, an esophageal cancer, or a squamous cell carcinoma. The number of cancer cells within said mammal can be reduced following said administering step.

[0024] In another aspect, this document features methods for binding a binding molecule to a B7-H3 polypeptide. The methods can include, or consist essentially of, contacting a B7-H3 polypeptide with a binding molecule 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); (ii) a heavy 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), 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 sequences 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); or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The contacting can be performed in vitro. The contacting can be performed in vivo. The contacting can be performed within a mammal by administering said binding molecule to said mammal. The mammal can be a human.

[0025] In another aspect, this document features methods for binding a binding molecule to a B7-H3 polypeptide. The methods can include, or consist essentially of, contacting a B7-H3 polypeptide with a CAR, a cell engager, or an ADC including a binding molecule 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); (ii) a heavy 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), 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 sequences 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);or (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions). The contacting can be performed in vitro. The contacting can be performed in vivo. The contacting can be performed within a mammal by administering said CAR, said cell engager, or said ADC to said mammal. The mammal can be a human.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0027] 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.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figures 1A-1C. B7-H3 bound to recombinant Siglec-9 (rec-Siglec-9) but not recombinant Siglec-7 (rec-Siglec-7) in trans. Figure 1 A) Schematic of experiment to examine Siglec binding to B7-H3. Figure IB) 293T cells were transfected either with empty vector, 4Ig-B7-H3 or 2Ig-B7-H3. After two days, cells were examined for binding of either rec-Siglec-9 or rec-Siglec-7. Lines in histogram are to aid in observing the shift between samples. Figure 1C) Quantification of percent positive for binding to rec-Siglec-9 or rec-Siglec-7 across three independent experiments. ***p<0.001, ****p<0.0001, ns, not significant.

[0030] Figures 2A-2B. Binding of Siglec-9 to B7-H3 required sialylation. Figure 2A) Transfections were performed as in Figure 1, except that prior to rec-Siglec-9 binding the transfected cells were either left untreated in PBS or treated with sialidase. The line in the histogram is to aid in observing the shift between samples. Figure 2B) Quantitation of percent positive for binding to rec-Siglec-9 across three independent experiments.

[0031] ***p<0.001, ****p<0.0001, ns, not significant.

[0032] Figures 3A-3B. F7 and G7 monoclonal antibodies bound to both 41g- and 2Ig-B7H3.

[0033] 293T cells were transfected with empty vector control, or Flag-tagged 2Ig-B7-H3 or 4Ig-B7-H3. After two days, transfected cells were examined using mouse monoclonal antibodies F7 (Figure 3 A) or G7 (Figure 3B), prior to staining with an anti-mouse secondary AF488.

[0034] Intracellular staining for Flag was performed and gated for analysis on B7-H3 expressing cells. MFI was quantified across three independent experiments at right. * p<0.05;

[0035] ****p<0.0001, ns, not significant.

[0036] Figure 4. F7 anti-B7-H3 mAh blocked binding of rec-Siglec-9 to B7-H3. 293T cells were transiently transfected as in Figure 1, except that cells were incubated with F7 anti-B7- H3 mAb or control prior to binding to Rec-Siglec-9. Representative data shown at left and quantified across three independent experiments at right. ****p<0.0001, ns, not significant.

[0037] Figure 5. Other commercial antibodies did not block the binding of F7 to B7-H3. Nalm6 cells stably expressing B7-H3 were pre-incubated with commercial antibodies against B7-H3 (MIH42, DCN70 or 376.96) or PBS for 30 minutes prior to binding of our IgG2b F7 mAb. F7 binding was detected with an anti-IgG2b secondary that does not cross react to a mixture of the commercial antibodies (last lane). Data were quantified across three independent experiments at right. **p<0.01, ns, not significant.

[0038] Figure 6. The 376.96 anti-B7-H3 antibody did not block Siglec-9 binding to B7-H3. Nalm6 stably overexpressing B7-H3 (Nalm6-B7H3OE) were pre-incubated with either F7, 376.96, or PBS control prior to binding with Rec-Siglec-9 as in Figure 1. Nalm6 without stable expression of B7-H3 is shown as a control (Nalm6 WT). *** p<0.001; ****p<0.0001, ns, not significant.

[0039] Figure 7. Binding of F7 or G7 did not depend on B7-H3 sialylation. Nalm6 B cells stably overexpressing B7-H3 (Nalm6-B7H3OE) were left untreated or pre-treated with sialidase as in Figure 2, prior to binding F7 or G7 mAb against B7-H3. An isotype negative control was used. A secondary mouse IgG was used to detect antibody binding. Data are representative of three independent experiments. ****p<0.0001, * p<0.05, ns, not significant.

[0040] Figure 8. B7-H3 expression inhibited NK cell-mediated killing. Stable 293 T B7-H3 KO cells or cells overexpressing (“O / E”) 4Ig-B7-H3 were labelled with Cr51for use in killing assays with purified freshly isolated human NK cells at various EffectorTarget ratios as denoted in the figure. Two-way ANOVA was used to determine statistical differences between the two curves. Data are representative of three independent experiments.

[0041] ****p<0.0001.

[0042] Figures 9A-9B. F7 blocking B7-H3 antibody increased NK cell killing of B7-H3 bearing target cells. Human NK cells were co-cultured at a 1:1 effector to target cell ratio for 12 hours with stably transduced mRaspberry-NLS+B7-H3+HEK293T cells with either an IgG2b isotype control or blocking F7 antibody. 1x106HEK293T cells were seeded 24 hours before co-culture with 1x106NK cells and 10 pg of control IgG of F7 B7-H3 antibody.

[0043] Figure 9A) Representative killing assay analyzed every three hours over a 12-hour timecourse using the CellCyte Live Cell Analyzer. Data are expressed as the percent of HEK293T cells remaining, compared to the control, at the indicated timepoints. Figure 9B) Quantification of Figure 9A at 3 hours with N=7 NK cell donors. Statistics were generated using the paired T test in Graphpad Prism. ***p < 0.001.

[0044] Figure 10. Preferred ligands for Siglec-9 from published glycan arrays from the Consortium for Functional Glycomics.

[0045] Figure 11. Di-sialic addition by ST8Sia6 reduced binding of Siglec-9 to B7-H3. 293T cells were transfected with empty vector (EV), myc-tagged ST8Sia6 and / or Flag-tagged 41g-B7-H3. Two days later, binding of Rec-Siglec-9 was assessed in transfected cells.

[0046] Representative histograms shown at left with quantitation across four independent experiments shown at left (normalized between experiments to the binding to B7-H3).

[0047] *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

[0048] Figures 12A-12C. Single and combined effects of B7-H3 (CD276) and Siglec-9 expression in AML from TCGA. The Kaplan-Meier survival curves were generated using AML samples from TCGA (The Cancer Genome Atlas database), n=l 17. Figure 12A) AML patient survival stratified by CD276 expression level high and low. Figure 12B) AML patient stratified by high and low Siglec-9 expression. Figure 12C) combining assigned CD276 and Siglec-9 expression level where CD276 high and lower patients were further stratified into Siglec-9 high and low expression. The stratification criteria remain the same as in (Figure 12 A) and (Figure 12B).

[0049] Figure 13. Titration curves comparing the binding of humanized and original murine F7 and G7 antibodies to Nalm6 cells with stable expression of B7-H3. Data are normalized to maximum binding =100, as different secondary antibodies were used.

[0050] Figure 14. Human NK cells were co-cultured at a 1: 1 effector to target cell ratio with stably transduced mRaspberry-NLS+B7-H3+HEK293T cells along with humanized IgGl isotype control or a humanized F7 B7-H3 blocking antibody. Quantification at 3 hours with N=7 NK cell donors. Statistics were generated by paired T test using Graphpad Prism* ***p < 0.0001.

[0051] Figure 15. B7H3 overexpressing (OE) HEK293T cells were pretreated with either PBS, 3 pg of mouse F3 or 3 pg of humanized F7 F(ab’)2. Then treated with recombinant Siglec-9 to assess the binding. Quantitation of percent positive for binding to rec-Siglec9 across four independent experiments. ****p<0.0001, ***p<0.001. Figure 16. Combination of anti-B7H3 G7 (IgG) and E8 (IgM) induced death in B7H3 overexpressing cells. B7H3 negative Nalm6 WT cells and B7H3 overexpressing cells were incubated with either mouse IgG and IgM isotype controls, anti-B7H3 IgG clone G7, anti-B7H3 IgM clone E8 or both G7 and E8 in triplicate in 24 well plates for 24 hours in culture. Post 24-hour incubation, cells were stained with a viability dye and examined via flow cytometry.

[0052] Figure 17. Combination of anti-B7H3 G7 (IgG) and E8 (IgM) induce death in B7H3 overexpressing cells. Raji and Nalm6 cells were cultured with anti-B7H3 antibodies in culture for 24 hours. HEK293T cells were incubated on ice for 2 hours. At the end of the incubation cells were stained with a viability dye and examined via flow cytometry.

[0053] Figures 18A-18C. Anti-B7H3 antibodies synergize with CAR19 in vivo. Figure 18A) A schematic for the experiment. NSG mice were injected with IxlO6luc+Nalm6-B7-H3 cells. Luminescence was measured one week later and randomized into groups for equivalent tumor burden. The following day received a single injection of 1 x 106CART 19 or untransduced (UTD) human T cells, along with either 10 mg / kg F7 mAb or IgG2b isotype control which was continued 3x / week. Figure 18B) Luminescence was measured once per week. Figure 18C) Quantified luminescence from Figure 18B shown as a logio plot.

[0054] Figures 19A-19B. Affinity of Siglec-9, Siglec-7, and anti-B7-H3 F7 and G7 antibodies. Figure 19A) Biacore Plasmon Resonance measurement of affinity of recombinant B7-H3 for Siglec-9 and Siglec-7 proteins. Figure 19B) Biacore Plasmon Resonance measurement of affinity of anti-B7-H3 F7 and G7 antibodies for human B7-H3 (Figure 19B).

[0055] DETAILED DESCRIPTION

[0056] This document provides binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). A binder provided herein can be any appropriate binder including an antigen binding domain having the ability to bind to a B7-H3 polypeptide. For example, this document provides antibody domains, antigen binding fragments, antibodies, CARs, cell engagers, and ADCs that bind (e.g., specifically bind) to a polypeptide comprising, consisting essentially of, or consisting of the amino acid set forth in any one of SEQ ID NOs:61-64 (see, e.g., Example 3). In some cases, a binder provided herein can have the ability to bind to a B7-H3 polypeptide and can lack the ability to bind to other members of the B7 family (e.g., B7-1 polypeptides, B7-2 polypeptides, B7-DC polypeptides, B7-H1 polypeptides, B7-H2 polypeptides, B7-H4 polypeptides, B7-H5 polypeptides, B7-H6 polypeptides, and B7-H7 polypeptides). For example, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can have the ability to bind to a human B7-H3 polypeptide and can lack the ability to bind to one or more other human B7 polypeptides (e.g., a human B7-1 polypeptide, a human B7-2 polypeptide, a human B7-DC polypeptide, a human B7-H1 polypeptide, a human B7-H2 polypeptide, a human B7-H4 polypeptide, a human B7-H5 polypeptide, a human B7-H6 polypeptide, and a human B7-H7 polypeptide).

[0057] The term “antibody domain” as used herein refers to a domain of an antibody such as 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 an antibody. In some cases, an antibody domain can be a single antibody domain (e.g., a VH domain or a VL domain) having the ability to bind to an antigen. An antibody domain provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can 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, an antibody domain provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be a monoclonal antibody domain. In some cases, an antibody domain provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be engineered as a single VH domain or a single VL domain.

[0058] The term “antigen binding fragment” as used herein 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) having the ability to bind to an antigen. Examples of antigen binding fragments include, without limitation, Fab, Fab’, or F(ab’)2 antigen binding fragments. An antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be configured to be a human antigen binding fragment, a humanized antigen binding fragment, or a chimeric antigen binding fragment. In some cases, an antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be a monoclonal antigen binding fragment. In some cases, an antigen binding fragment provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be configured as an Fab antibody. In some cases, a Fab antibody can include a hinge sequence to allow for disulfide bonding between heavy and light chains of the Fab.

[0059] The term “antibody” as used herein includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, single-domain antibodies (sdAbs; also referred to as nanobodies), multi-specific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, singlechain variable fragment antibodies (e.g., scFv antibodies), and tandem single-chain variable fragments antibody (e.g., taFv). A diabody can include two chains, each having a heavy chain variable domain and a light chain variable domain, either from the same or from 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 domains can be directly connected or can be connected using any appropriate linker sequence (e.g., a polypeptide linker such as a polypeptide linker having five to ten residues in length). For example, a heavy chain variable domain can be directly connected to a light chain variable domain, or a heavy chain variable domain can be connected to a light chain variable domain via a linker sequence. In some cases, a linker can be a flexible linker (e g., such that the linker can function as a hinge). Examples of linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain (e.g., to create a scFv) include, without limitation, those linkers set forth in Example 9. In some cases, an interdomain disulfide bond can be present in one or both of the heavy chain variable domain and light chain variable domain pairs of the diabody. A scFv is a single-chain polypeptide antibody in which the heavy chain variable domain and the light chain variable domain are directly connected or connected via a polypeptide linker (e.g., a polypeptide linker having eight to 18 residues in length or a polypeptide linker as set forth in Example 9). See, also, Chen et al., Adv. Drug Deliv. Rev, 65(10): 1357-1369 (2013). A scFv can be designed to have an orientation with the heavy chain variable domain being followed by the light chain variable domain or can be designed to have an orientation with the light chain variable domain being followed by the heavy chain variable domain. In both cases, the optional linker can be located between the two domains. Examples of scFv structures of scFv’s provided herein include, without limitation, those structures set forth in Example 4.

[0060] An antibody provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be configured to be a human antibody, a humanized antibody, or a chimeric antibody. In some cases, an antibody provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be a monoclonal antibody. In some cases, an antibody provided herein can include the CDRs as described herein (e.g., as described in Table 4) and can be configured as a scFv antibody.

[0061] An anti-B7-H3 antibody domain, anti-B7-H3 antigen binding fragment, or anti-B7-H3 antibody provided herein can be of the IgA-, IgD-, IgE-, IgG-, or IgM-type, including IgG- or IgM-types such as, without limitation, IgGi-, IgG2-, IgGs-, IgG4-, IgMi-, and IgNE-types. In some cases, an antibody domain provided herein (e.g., an anti-B7-H3 antibody domain) can be a VH domain. In some cases, an antibody domain provided herein (e.g., an anti-B7-H3 antibody domain) can be a sdAb (a nanobody). In some cases, an antigen binding fragment provided herein (e.g., an anti-B7-H3 antibody fragment) can be a Fab. In some cases, an antibody provided herein (e.g., an anti-B7-H3 antibody) can be a scFv antibody. In some cases, an antibody provided herein (e.g., an anti-B7-H3 antibody) can be a fully intact antibody.

[0062] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein can include the CDRs as described herein (e.g., as described in Table 4). In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:1, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:3, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:9, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 10, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0063] In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:1, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:3, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:9, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 10, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 11. In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include CDRs that comprise, consist essentially of, or consist of the CDR amino acid sequences set forth in Table 1.

[0064] Table 1. Exemplary CDR sequences for a binder that can bind a B7-H3 polypeptide.

[0065]

[0066] A CDR that can be included in a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) that consists essentially of a CDR amino acid sequence set forth in any one of SEQ ID NOs: 1-3 or 9-10 is a CDR that has zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 1-3 or 9-10), has zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 1-3 or 9-10), and / or has zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:1-3 or 9-10), provided that the binder retains the ability to bind to aB7-H3 polypeptide (e.g., a human B7-H3 polypeptide).

[0067] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:3 includes, without limitation, the VH domain set forth in SEQ ID NO:8 (see, e.g., Example 5).

[0068] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 3 can include any appropriate framework regions. For example, such a binder can include a heavy chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:5 (or a variant of SEQ ID NO:5 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:6 (or a variant of SEQ ID NO:6 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:7 (or a variant of SEQ ID NO: 7 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications).

[0069] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8. For example, a binder provided herein can include a heavy chain variable domain that includes 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. In some cases, a binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:8.

[0070] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3. For example, a binder provided herein can include a heavy chain variable domain that includes 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, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:1, 2, and 3.

[0071] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:9, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 10, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 11 includes, without limitation, the VH domain set forth in SEQ ID NO: 16 (see, e.g., Example 5).

[0072] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:9, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 10, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 11 can include any appropriate framework regions. For example, such a binder can include a light chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO: 12 (or a variant of SEQ ID NO: 12 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO: 13 (or a variant of SEQ ID NO: 13 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO: 14 (or a variant of SEQ ID NO: 14 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO: 15 (or a variant of SEQ ID NO: 15 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications).

[0073] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e g., a human B7-H3 polypeptide) can include a light chain variable domain that includes 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 binder provided herein can include a heavy chain variable domain that includes 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 binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:16.

[0074] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO: 16, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:9, 10, and 11. For example, a binder provided herein can include a heavy chain variable domain that includes 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, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:9, 10, and 11.

[0075] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 17, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 18, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 19, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:25, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:27.

[0076] In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 17, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 18, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 19, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:25, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:27. In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include CDRs that comprise, consist essentially of, or consist of the CDR amino acid sequences set forth in Table 2.

[0077] Table 2. Exemplary CDR sequences for a binder that can bind a B7-H3 polypeptide.

[0078]

[0079] VL domain CDR3

[0080]

[0081] A CDR that can be included in a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) that consists essentially of a CDR amino acid sequence set forth in any one of SEQ ID NOs: 17-19 or 25-26 is a CDR that has zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 17-19 or 25-27), has zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 17-19 or 25-27), and / or has zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 17-19 or 25-27), provided that the binder retains the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide).

[0082] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 17, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 18, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 19 includes, without limitation, the VH domain set forth in SEQ ID NO:24 (see, e g., Example 5).

[0083] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 17, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 18, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 19 can include any appropriate framework regions. For example, such a binder can include a heavy chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:20 (or a variant of SEQ ID NO:20 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:21 (or a variant of SEQ ID NO:21 with one, two, three, four, five, six, seven, eight, nine, ten, 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 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:23 (or a variant of SEQ ID NO:23 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications).

[0084] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24. For example, a binder provided herein can include a heavy chain variable domain that includes 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. In some cases, a binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:24.

[0085] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:17, 18, and 19. For example, a binder provided herein can include a heavy chain variable domain that includes 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, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:17, 18, and 19.

[0086] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:25, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:27 includes, without limitation, the VH domain set forth in SEQ ID NO:32 (see, e.g., Example 5).

[0087] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:25, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:27 can include any appropriate framework regions. For example, such a binder can include a light chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO: 28 (or a variant of SEQ ID NO:28 with one, two, three, four, five, six, seven, eight, nine, ten, 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 with one, two, three, four, five, six, seven, eight, nine, ten, 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 with one, two, three, four, five, six, seven, eight, nine, ten, 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 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications).

[0088] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a light chain variable domain that includes 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 binder provided herein can include a heavy chain variable domain that includes 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 binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO 32. In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:32, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:25, 26, and 27. For example, a binder provided herein can include a heavy chain variable domain that includes 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, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:25, 26, and 27.

[0089] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:33, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:34, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:35, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:41, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:42, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:43.

[0090] In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:33, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:34, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:35, and / or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:41, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:42, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:43. In some cases, a binder (e.g., a CAR) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include CDRs that comprise, consist essentially of, or consist of the CDR amino acid sequences set forth in Table 3.

[0091] Table 3. Exemplary CDR sequences for a binder that can bind a B7-H3 polypeptide.

[0092]

[0093] A CDR that can be included in a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) that consists essentially of a CDR amino acid sequence set forth in any one of SEQ ID NOs:33-35 or 41-43 is a CDR that has zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:33-35 or 41-43), has zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:33-35 or 41-43), and / or has zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:33-35 or 41-43), provided that the binder retains the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide).

[0094] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:33, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:34, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:35 includes, without limitation, the VH domain set forth in SEQ ID NO:40 (see, e.g., Example 5). In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:33, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:34, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:35 can include any appropriate framework regions. For example, such a binder can include a heavy chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:36 (or a variant of SEQ ID NO:36 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:37 (or a variant of SEQ ID NO:37 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:38 (or a variant of SEQ ID NO:38 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:39 (or a variant of SEQ ID NO:39 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications).

[0095] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:40. For example, a binder provided herein can include a heavy chain variable domain that includes 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:40. In some cases, a binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:40.

[0096] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:40, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:33, 34, and 35. For example, a binder provided herein can include a heavy chain variable domain that includes 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:40, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:33, 34, and 35.

[0097] An example of a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:41, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:42, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:43 includes, without limitation, the VH domain set forth in SEQ ID NO:48 (see, e.g., Example 5).

[0098] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:41, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:42, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:43 can include any appropriate framework regions. For example, such a binder can include a light chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:44 (or a variant of SEQ ID NO:44 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:45 (or a variant of SEQ ID NO:45 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:46 (or a variant of SEQ ID NO:46 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:47 (or a variant of SEQ ID NO:47 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a light chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:48. For example, a binder provided herein can include a heavy chain variable domain that includes 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:48. In some cases, a binder provided herein can include a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:48.

[0099] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can include a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:48, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:41, 42, and 43. For example, a binder provided herein can include a heavy chain variable domain that includes 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:48, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:41, 42, and 43.

[0100] A representative number of binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, cell engagers, and / or ADCs) having the ability to bind to a B7- H3 polypeptide (e.g., a human B7-H3 polypeptide) are further described in Table 4.

[0101] Table 4. Representative binders having the ability to bind to a B7-H3 polypeptide.

[0102]

[0103] Table 5 includes an alternative designation that can be used to refer to each of Clones #1 - #3.

[0104] Table 5. Alternative nomenclature for Clones #1 - #3.

[0105]

[0106] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein 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 set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:11. For example, a binder provided herein can be designed to include a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO: 16.

[0107] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein 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 set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3. For example, a binder provided herein can be designed to include a light chain variable domain comprising SEQ ID NO: 16, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO: 8.

[0108] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein 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 set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. For example, a binder provided herein can be designed to include a heavy chain variable domain comprising SEQ ID NO:24, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:32.

[0109] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein 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 set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. For example, a binder provided herein can be designed to include a light chain variable domain comprising SEQ ID NO:32, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:24.

[0110] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein can be designed to include a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. For example, a binder provided herein can be designed to include a heavy chain variable domain comprising SEQ ID NO:40, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:48.

[0111] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, or an antibody) provided herein can be designed to include a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. For example, a binder provided herein can be designed to include a light chain variable domain comprising SEQ ID NO:48, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:40.

[0112] In some cases, binders (e.g., antibody domains, antigen binding fragments, and antibodies) provided herein can be used to create chimeric antigen receptors (CARs). The term “chimeric antigen receptor” as used herein refers to a chimeric polypeptide that is 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 a CAR provided herein can be designed to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). For example, a CAR provided herein can be designed to include the components of a binder (e.g., an antibody domain, antigen binding fragment, or antibody) provided herein (e.g., a combination of CDRs) as an antigen binding domain provided that that antigen binding domain has the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). In some cases, a CAR provided herein can be designed to include an antigen binding domain that includes at least one set of three CDRs (e.g., CDR1, CDR2, and CDR3) of an antigen binding domain provided herein (e g., SEQ ID NOs: 1-3, SEQ ID NOs:9-11, SEQ ID NOs: 17-19, SEQ ID NO:25-27, SEQ ID NOs:33-35, or SEQ ID NOs:41-43). For example, an antigen binding domain of a CAR targeting a B7-H3 polypeptide can be designed to include a VH domain and / or a VL domain described herein. In some cases, a CAR 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 a heavy chain and CDR1, CDR2, and CDR3 of a light chain) of an antigen binding domain provided herein (e.g., SEQ ID NOs: 1-3 and 9-11, SEQ ID NOs: 17-19 and 25-27, or SEQ ID NOs:33-35 and 41-43). For example, an antigen binding domain of a CAR targeting a B7-H3 polypeptide can be designed to include a scFv antibody described herein.

[0113] In some cases, a CAR provided herein can be designed to have a structure set forth in Example 10.

[0114] In some cases, a CAR provided herein can be designed to include a signal peptide. Any appropriate signal peptide can be used to design a CAR described herein. Examples of signal peptide that can be used to make a CAR described herein include without limitation, human CD-8-derived signal peptides (e.g., human CD-8alpha-derived signal peptides), chronodroitin sulphate proteoglycan 4 (CSPG4)-derived signal peptides, and dickkopf-related protein 3 (DKK3)-derived signal peptides. 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 Example 11 (e.g., a signal peptide that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:79). 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 Example 11 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 Example 11 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 less amino acid deletions, additions, substitutions, or combinations thereof.

[0115] In some cases, a CAR provided herein can be designed to include a hinge. Any appropriate hinge can be used to design a CAR described herein. Examples of hinges that can be used to make a CAR described herein include, without limitation, CD8a-derived hinges, CD4-derived hinges, CD28-derived hinges, and IgG-derived hinges. A CAR provided herein can be designed to include a hinge of any appropriate length. For example, a CAR provided herein can be designed to include a hinge that is from about 3 to about 75 (e.g., from about 3 to about 65, from about 3 to about 50, from about 5 to about 75, from about 10 to about 75, from about 5 to about 50, from about 10 to about 50, from about 10 to about 40, or from about 10 to about 30) amino acid residues in length. In some cases, a linker sequence can be used as a hinge to make a CAR described herein. For example, any one of the linker sequences set forth in Example 9 can be used as a hinge of a CAR described herein. In some cases, a CAR 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 Example 9 or Example 12. In some cases, a CAR 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 Example 9 or Example 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 hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 9 or Example 12 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 less amino acid deletions, additions, substitutions, or combinations thereof.

[0116] A CAR provided herein can be designed to include any appropriate transmembrane domain. Examples of transmembrane domains that can be used to make a CAR described herein include, without limitation, CD3C transmembrane domains, CD4 transmembrane domains, CD8a transmembrane domains, CD28 transmembrane domains, 4-1BB transmembrane domains, CD28 transmembrane domains, and 0x40 transmembrane domains. 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 Example 13. 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 Example 13 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 Example 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 less amino acid deletions, additions, substitutions, or combinations thereof.

[0117] A CAR provided herein can be designed to include one or more intracellular signaling domains. For example, a CAR provided herein can be designed to include one, two, three, or four intracellular signaling domains. Any appropriate intracellular signaling domain or combination of intracellular signaling domains can be used to make a CAR described herein. Examples of intracellular signaling domains that can be used to make a CAR described herein include, without limitation, CD3(^ intracellular signaling domains, CD27 intracellular signaling domains, CD28 intracellular signaling domains, 0X40 (CD 134) intracellular signaling domains, 4-1BB (CD137) intracellular signaling domains, CD278 intracellular signaling domains, DAP10 intracellular signaling domains, DAP12 intracellular signaling domains, and ICOS intracellular signaling domains. In some cases, a CAR described herein can be designed to be a first-generation CAR having a CD3(^ intracellular signaling domain. In some cases, a CAR described herein can be designed to be a second-generation CAR having a CD28 intracellular signaling domain followed by a CD3 intracellular signaling domain. In some cases, a CAR described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an 0X40 intracellular signaling domains, or a 4-1BB intracellular signaling domain followed by (c) a CD3(^ intracellular signaling domain. In some cases, a CAR 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 Example 14. In some cases, a CAR 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 Example 14 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, a CAR 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 Example 14 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 less amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling.

[0118] In some cases, a CAR targeting a B7-H3 polypeptide can be designed to include a heavy chain variable domain comprising SEQ IDNO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as a linker set forth in Example 9, 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 set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e.g., a human 4- IBB intracellular signaling domain followed by a human CD3^ intracellular signaling domain).

[0119] In some cases, a CAR targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e.g., a human 4- IBB intracellular signaling domain followed by a human CD3^ intracellular signaling domain).

[0120] In some cases, a CAR targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO: 97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3^ intracellular signaling domain).

[0121] In some cases, a CAR targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO: 97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e g., a human 4-1BB intracellular signaling domain followed by a human CD3(^ intracellular signaling domain).

[0122] In some cases, a CAR targeting a B7-H3 polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a hinge such as a hinge / linker set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO: 97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e g., a human 4-1BB intracellular signaling domain followed by a human CD3(^ intracellular signaling domain).

[0123] In some cases, a CAR targeting a B7-H3 polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a hinge such as a hinge / linker set forth in Example 9 or Example 12 (e.g., a CD8a hinge such as a CD8a hinge that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO:88), followed by a transmembrane domain such as a transmembrane domain set forth in Example 13 (e.g., a CD8a transmembrane domain such as a CD8a transmembrane domain that comprises, consists of, or consisting of the amino acid set forth in SEQ ID NO: 97), followed by one or more intracellular signaling domains such as one or more intracellular signaling domain set forth in Example 14 (e g., a human 4-1BB intracellular signaling domain followed by a human CD3t^ intracellular signaling domain).

[0124] In some cases, binders (e.g., antibody domains, antigen binding fragments, and antibodies) provided herein can be used to create cell engagers. The term “cell engager” as used herein refers to a polypeptide that includes 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, without limitation, BiTEs, BiKEs, TriKEs, and TetraKEs. In general, a cell engager provided herein can be designed to include at least one binder (e.g., an antibody domain, antigen binding fragment, or antibody) provided herein (e.g., a combination of CDRs) as an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and at least one antigen binding domain having the ability to bind to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell). In some cases, a cell engager described herein can link a B7-H3+cell (e.g., a B7- H3+cancer cell) to another cell (e.g., a T cell or an NK cell) via the two or more antigen binding domains of the cell engager.

[0125] When a cell engager includes an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and two or more other antigen binding domains (e.g., two, three, or four other antigen binding domains), each of those other antigen binding domains can bind to different antigens expressed on the surface of different cell types or can bind to different antigens expressed on the surface of the same cell type. For example, a TriKE can be designed to have a first antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide), a second antigen binding domain having the ability to bind to a first antigen expressed on the surface of an NK cell (e.g., a CD16 polypeptide such as a CD16a polypeptide), and a third antigen binding domain having the ability to bind to a second antigen expressed on the surface of an NK cell (e.g., an NKG2A polypeptide).

[0126] As described herein, at least one antigen binding domain of a cell engager provided herein can be designed to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). For example, a cell engager provided herein can be designed to include the components of an antibody, antigen binding fragment, and / or antibody domain described herein (e.g., a combination of CDRs) as an antigen binding domain provided that that antigen binding domain has the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). In some examples, a cell engager provided herein can be designed to include an antigen binding domain that includes at least one set of three CDRs (e.g., CDR1, CDR2, and CDR3) of an antigen binding fragment provided herein (e.g., SEQ ID NOs:1-3, SEQ ID NOs:9-11, SEQ ID NOs: 17-19, SEQ ID NO:25-27, SEQ ID NOs:33-35, or SEQ ID NOs:41-43). In some cases, an antigen binding domain of a cell engager targeting a B7-H3 polypeptide can be designed to include a VH domain described herein or a scFv or Fab antibody described herein. In some cases, an antigen binding domain of a CAR described herein that has the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can be used as an antigen binding domain of a cell engager that targets B7-H3+cells.

[0127] As described herein, a cell engager can be designed to include at least one antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and at least one other antigen binding domain. That at least one other antigen binding domain can have the ability to bind to any appropriate antigen expressed on the surface of a cell. For example, when designing a cell engager such as a BiTE to link a B7-H3+cell and a T cell, the cell engager can include an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell. Examples example of polypeptides expressed on the surface of a T cell that can be targeted by an antigen binding domain of a cell engager provided herein include, without limitation, CD3 polypeptides. Examples of antigen binding domains having the ability to bind to a polypeptide expressed on the surface of a T cell that can be used to make a cell engager provided herein (e.g., a BiTE) include, without limitation, anti-CD3 scFvs, and anti-CD3 VH domains. Additional examples of amino acid sequences that can be used as antigen binding domains having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., CD3) are described in U. S. Patent No. 6,750,325 (see, e.g., the sequence listing of U. S. Patent No. 6,750,325).

[0128] In some cases, a cell engager provided herein can 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 Example 15. In some cases, a cell engager provided herein can 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 Example 15 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, a cell engager provided herein can 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 Example 15 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 less 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.

[0129] When designing a cell engager such as a BiKE or a TriKE to link a B7-H3+cell and an NK cell, the cell engager can include an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and one or more (e.g., one, two, or three) antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell. Examples of polypeptides expressed on the surface of an NK cell that can be targeted by an antigen binding domain of a cell engager provided herein include, without limitation, CD16 polypeptides (e.g., CD16a polypeptides), NKG2A polypeptides, NKG2D polypeptides, NKp30 polypeptides, NKp44 polypeptides, NKp46 polypeptides, and IL-15 polypeptides. Examples of antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell that can be used to make a cell engager provided herein (e.g., a BiKE or TriKE) include, without limitation, anti-CD16a scFvs, anti-NKG2A scFvs, anti-NKG2D scFvs, anti-NKp30 scFvs (see, e.g., BioLegend Catalog #325207), anti-NKp44 scFvs, anti-NKp46 scFvs, anti-CD 16a VH domains, anti-NKG2AVH domains, anti-NKG2D VH domains, anti-NKp30 VH domains, anti-NKp44 VH domains, anti-NKp46 VH domains, anti-IL-15 VH domains, and anti-IL-15 VH domains. Additional examples of amino acid sequences that can be used as antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., CD 16, NKG2A, NKG2D, or NKp46) are described in McCall et al. (Mol. Immunol., 36(7):433-445 (1999); see, e.g., anti-CD16 scFv sequences); International Patent Application Publication No.

[0130] PCT / US2017 / 048721 (see, e.g., the CDRs and sequence listing for anti-CD16a binding domains); U. S. Patent Application Publication No. 2011 / 0052606 (see, e.g., the CDRs and the sequence listing for anti-NKG2A antibodies such as Z199); U. S. Patent Application Publication No. 2011 / 0150870 (see, e.g., the CDRs and sequence listing for anti-NKG2D antibodies); U. S. Patent Application Publication No. 2018 / 0369373 (see, e.g., the CDRs and sequence listing for anti-NKp46 antibodies); and U. S. Patent Application Publication No. 2017 / 0368169 (see, e.g., the CDRs and sequence listing for anti-NKp46 antibodies).

[0131] In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a VH or a scFv) that comprises, consists essentially of, or consists of one or more of the amino acid sequences set forth in Example 16. In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a VH or a scFv) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 16 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 an NK cell. In some cases, a cell engager provided herein can be designed to include an antigen binding domain (e.g., a VH or a scFv) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 16 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 less 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 an NK cell.

[0132] In some cases, a cell engager provided herein can be designed to include a linker located between each antigen binding domain. Any appropriate linker can be used to design a cell engager provided herein. Examples of linkers that can be used to make a cell engager described herein include, without limitation, the linker sequences set forth in Example 9. A cell engager provided herein can be designed to include a linker of any appropriate length. For example, a cell engager provided herein can be designed to include a linker that is from about 3 to about 100 amino acid residues in length. In some cases, a cell engager provided herein (e.g., a BiTE) can be designed to include a GGGGSGGGGSGGGGS (SEQ ID NO:71) linker. In some cases, a hinge of a CAR described herein can be used as a linker to make a cell engager described herein. For example, any one of the sequences set forth in Example 9 can be used as a linker of a cell engager described herein.

[0133] In some cases, a cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 9 or Example 12. In some cases, a cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 9 or Example 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 cell engager provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 9 or Example 12 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 less amino acid deletions, additions, substitutions, or combinations thereof.

[0134] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0135] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0136] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti -human CD3 scFv).

[0137] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti -human CD3 scFv).

[0138] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a linker such as a linker set forth in Example 9, followed by a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a hinge such as a hinge / linker set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti -human CD3 scFv).

[0139] In some cases, a cell engager (e.g., a BiTE) targeting a B7-H3 polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a hinge such as a hinge / linker set forth in Example 9 or Example 12, followed by an antigen binding domain having the ability to bind to a polypeptide expressed on the surface of a T cell (e.g., an anti -human CD3 scFv).

[0140] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 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 set forth in Example 9, followed by a light chain domain comprising SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11, followed by a linker such as a hinge / linker set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD 16a scFv for a BiKE or an anti-human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0141] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti -human CD 16a scFv for a BiKE or an anti -human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0142] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 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 set forth in Example 9, followed by a light chain domain comprising SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO: 27, followed by a linker such as a hinge / linker set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD 16a scFv for a BiKE or an anti-human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0143] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 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 set forth in Example 9, 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 set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti -human CD 16a scFv for a BiKE or an anti -human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0144] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a linker such as a linker set forth in Example 9, followed by a light chain domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a linker such as a hinge / linker set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD 16a scFv for a BiKE or an anti-human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0145] In some cases, a cell engager (e.g., a BiKE or a TriKE) targeting a B7-H3 polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, followed by a linker such as a linker set forth in Example 9, followed by a heavy chain variable domain comprising SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, followed by a linker such as a hinge / linker set forth in Example 9 or Example 12, followed by one or more antigen binding domains having the ability to bind to a polypeptide expressed on the surface of an NK cell (e.g., an anti -human CD 16a scFv for a BiKE or an anti -human CD 16a scFv and an anti -human NKG2A scFv for a TriKE).

[0146] In some cases, binders (e.g., antibody domains, antigen binding fragments, and antibodies) provided herein can be used to create antibody-drug conjugates (ADCs). The term “ADC” as used herein refers to a conjugate that includes (a) an antigen binding domain and (b) at least one drug covalently linked directly or indirectly to that antigen binding domain. In some cases, an ADC described herein can include (a) an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) and (b) at least one drug covalently linked directly or indirectly to that antigen binding domain. Any appropriate binder (e.g., an antibody domain, antigen binding fragment, antibody, CAR, or cell engager) provided herein (e.g., a combination of CDRs) can be used as an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can be used as an antigen binding domain to make an ADC described herein. For example, any of the binders set forth in Table 4 can be used to make an ADC having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). Examples of drugs that can be used to make an ADC described herein include, without limitation, maytansines, auristatins (e.g., monomethyl auristatin E (MMAE)), mertansine (DM-1), pyrrolobenzodiazepine (PBD) dimers), claicheamicins, anthracyclines, duocarmcins, and pyrrolobenzodiazepines. Any appropriate ADC linker can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) to form an ADC provided herein. For example, cleavable or non-cleavable ADC linkers can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) to form an ADC provided herein. Examples of ADC linkers can be used to covalently attach one or more drugs to an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) to form an ADC provided herein include, without limitation, ADC disulfide linkers, ADC hydrazone linkers, ADC peptide linkers, ADC thioether linkers, and ADC PEG-containing linkers. In some cases, an ADC can be a radioconjugate (e.g., can include one or more radioisotopes covalently attach one or more drugs to an antigen binding domain having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide)).

[0147] As indicated herein, the amino acid sequences described herein can include amino acid modifications (e.g., the articulated number of amino acid modifications). Such amino acid modifications can include, without limitation, amino acid substitutions, amino acid deletions, amino acid additions, and combinations. In some cases, an amino acid modification can be made to improve the binding and / or contact with an antigen and / or to improve a functional activity of a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein. In some cases, an amino acid substitution within an articulated sequence identifier can be a conservative amino acid substitution. For example, conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a similar side chain. Families 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), non-polar 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).

[0148] In some cases, an amino acid substitution within an articulated sequence identifier can be a non-conservative amino acid substitution. Non-conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, without limitation, substituting (a) a hydrophilic residue (e.g., serine or threonine) for a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) a cysteine or proline for any other residue; (c) a residue having a basic side chain (e.g., lysine, arginine, or histidine) for 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) for glycine or other residue having a small side chain.

[0149] Methods for generating an amino acid sequence variant (e.g., an amino acid sequence that includes one or more modifications with respect to an articulated sequence identifier) can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of a nucleic acid encoding the antibody or fragment thereof. See, for example, Zoller, Curr Opin. Biotechnol.

[0150] 3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially-derivatized amino acids) can be used to generate an amino acid sequence variant provided herein.

[0151] The binders (e.g., antibody domains, antigen binding fragments, antibodies, CARs, cell engagers, and / or ADCs) provided herein can be produced using any appropriate method. For example, the binders provided herein can be produced in recombinant host cells. For example, a nucleic acid encoding a binder provided herein can be constructed, introduced into an expression vector, and expressed in suitable host cells. Example 6 is a sequence listing of nucleic acid sequences encoding exemplary binders described herein. In some cases, a binder provided herein can be recombinantly produced in prokaryotic hosts such as E. coli, Bacillus brevis Bacillus suhlihs. Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei. A binder provided herein also can be recombinantly produced in eukaryotic hosts such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Sclnvanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytic ), filamentous fungi of the genera Trichoderma (e.g., T. reesei) and Aspergillus (e.g., 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 NSO cells, baby hamster kidney (BHK) cells, or human embryonic kidney cell line HEK293). See, for example, the Frenzel et al. reference (Front Immunol., 4:217 (2013)).

[0152] In some cases, an antigen binding fragment or antibody domain provided herein can be produced by proteolytic digestion of an intact antibody. For example, an antigen binding fragment can be obtained by treating an antibody with an enzyme such as papain or pepsin. Papain digestion of whole antibodies can be used to produce F(ab’)2 or Fab fragments, while pepsin digestion of whole antibodies can be used to produce F(ab’)2 or Fab’ fragments.

[0153] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be substantially pure. The term “substantially pure” as used herein with reference to a binder refers to the binder as being substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure binder provided herein is any binder that is removed from its natural environment and is at least 60 percent pure. A substantially pure binder provided herein can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.

[0154] This document also provides bispecific binders (e.g., bispecific antibody domains, bispecific antigen binding fragments, bispecific antibodies, bispecific CARs, bispecific cell engagers, and / or bispecific ADCs) that bind to two different epitopes with at least one being an epitope of a CLDN18.2 polypeptide (e.g., a human CLDN18.2 polypeptide). In some cases, a bispecific binder provided herein can be designed to bind to two different epitopes of the same CLDN18.2 polypeptide (e.g., a human CLDN18.2 polypeptide). In some cases, a bispecific binder provided herein can bind to a CLDN18.2 polypeptide (e.g., a human CLDN18.2 polypeptide) and to an epitope on a different polypeptide (e.g., a CD3 polypeptide). Bispecific binders can be produced by chemically conjugating two different binders together. Bispecific binders also can be produced by fusing two antibody-producing cells, e.g., hybridomas, to make a hybrid cell line that produces two different heavy and two different light chains within the same cell, which can result in, for example, bispecific IgG molecules. See, Brinkmann and Kontermann, MAbs., 9(2): 182-212 (2017).

[0155] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be fused or conjugated (e.g., covalently or non-covalently attached) to another polypeptide or other moiety to provide a fusion protein or conjugate. For example, a binder provided herein can be conjugated (e.g., covalently or non-covalently attached) to a polymer (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), and / or polyglutamic acid (PGA) (N-(2 -Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, a fluorescent substance, a luminescent substance, a hapten, an enzyme, a metal chelate, a drug, a radioisotope, and / or a cytotoxic agent. Any appropriate method can be used to conjugate (e.g., covalently or non-covalently attach) another polypeptide or other moiety to a binder provided herein. For example, another polypeptide or other moiety can be conjugated to a binder provided herein using the methods described in U. S. Patent No. 8,021,661.

[0156] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be modified with a moiety that improves its stabilization and / or retention in circulation, for example, in blood, serum, or other tissues by, for example, at least 1.5-, 2-, 5-, 10-, or 50-fold. For example, a binder provided herein can be attached (e.g., covalently or non-covalently attached) to a polymer such as a substantially non-antigenic polymer. Examples of substantially non-antigenic polymers that can be used as described herein include, without limitation, polyalkylene oxides, and polyethylene oxides. In some cases, a polymer used herein can have any appropriate molecule weight. For example, a polymer having an average molecular weight from about 200 Daltons to about 35,000 Daltons (e.g., from about 1,000 to about 15,000 Daltons or from about 2,000 to about 12,500 Daltons) can be used. In some cases, a binder provided herein can be attached (e.g., covalently or non-covalently) to a water-soluble polymer. Examples of water-soluble polymers that can be used as described herein include, without limitation, hydrophilic polyvinyl polymers, polyvinylalcohol, polyvinylpyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycols, and polyoxyethylenated polyols and copolymers thereof and / or block copolymers thereof provided that the water solubility of the copolymer or block copolymers is maintained.

[0157] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be attached (e.g., covalently or non-covalently attached) 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 binder provided herein can be covalently attached to polyoxyethylene.

[0158] This document also provides nucleic acid molecules (e.g., isolated nucleic acid molecules) having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein. For example, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence encoding a heavy chain variable domain such as a heavy chain variable domain as set forth in Example 6. In another example, an isolated nucleic acid molecule provided herein can include a nucleic acid sequence set forth in Example 8. A nucleic acid provided herein (e.g., an isolated nucleic acid molecule) can be single stranded or double stranded nucleic acid of any appropriate type (e.g., DNA, RNA, or DNA / RNA hybrids).

[0159] This document also provides vectors (e.g., plasmid vectors or viral vectors) containing one or more nucleic acids provided herein. An example of a plasmid vector that can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein includes, without limitation, phagemids. Examples of viral vectors that can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder provided herein include, without limitation, retroviral vectors, parvovirus-based vectors (e.g., adenoviral- based vectors and adeno-associated virus (AAV)-based vectors), lentiviral vectors (e.g., herpes simplex (HSV)-based vectors), poxviral vectors (e.g., vaccinia virus-based vectors and fowlpox virus-based vectors), and hybrid or chimeric viral vectors. For example, a viral vector having an adenoviral backbone with lentiviral components such as those described elsewhere (Zheng et al., Nat. Biotech., 18(2): 176-80 (2000); WO 98 / 22143; WO 98 / 46778; and WO 00 / 17376) or viral vectors having an adenoviral backbone with AAV components such as those described elsewhere (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996)) can be designed to include one or more nucleic acids having a nucleic acid sequence encoding at least part of a binder provided herein.

[0160] In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding an antibody domain (e.g., a VH domain) provided herein. In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding CAR provided herein. In some cases, a vector (e.g., a plasmid vector or a viral vector) provided herein can include a nucleic acid sequence encoding cell engager provided herein.

[0161] A vector provided herein (e.g., a plasmid vector or viral vector provided herein) can include any appropriate promoter and other regulatory sequence (e.g., transcription and translation initiation and termination codons) operably linked the nucleic acid sequence encoding at least part of a binder (e g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein. In some cases, a promoter used to drive expression can be a constitutive promotor or a regulatable promotor. Examples of regulatable promoters that can be used as described herein include, without limitation, inducible promotors, repressible promotors, and tissue-specific promoters. Examples of viral promotors that can be used as described herein include, without limitation, adenoviral promoters, vaccinia virus promotors, CMV promotors (e.g., immediate early CMV promoters), and AAV promoters.

[0162] Any appropriate method can be used to make a nucleic acid molecule (or vector such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least part of a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein. For example, molecule cloning techniques can be used to make a nucleic acid molecule (or vector such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least part of a binder provided herein as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, N. Y. (1994)).

[0163] 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 part of a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein). Host cells that can be designed to include one or more nucleic acids provided herein can be prokaryotic cells or eukaryotic cells. Examples of prokaryotic cells that can be designed to include a nucleic acid provided herein include, without limitation, E. coli (e.g., Tb-1, TG-1, DH5a, XL-Blue MRF (Stratagene), SA2821, or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens, or Pseudomonas (e.g., P. aeruginosa)' cells. Examples of eukaryotic cells that can be designed to include a nucleic acid provided herein include, without limitation, 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 designed to include a nucleic acid provided herein. Any appropriate method can be used to introduce one or more nucleic acids provided herein (e.g., a vector such as a plasmid vector or viral vector having a nucleic acid sequence encoding at least part of a binder 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 combinations thereof can be used to introduce a nucleic acid provided herein into a host cell (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Davis et al., Basic Methods in Molecular Biology (1986); and Neumann et al., EMBO J., 1:841 (1982)).

[0164] 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 designed to express one or more nucleic acids encoding a CAR described herein. For example, a population of T cells can be infected with viral vectors designed to express nucleic acid encoding a CAR described herein (e.g., a CAR having the ability to bind to a B7-H3 polypeptide).

[0165] 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 designed to express one or more nucleic acids encoding a cell engager described herein. For example, a population of T cells can be infected with viral vectors designed to express nucleic acid encoding a cell engager described herein (e.g., a cell engager having the ability to bind to a B7-H3 polypeptide).

[0166] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be produced using a method that includes (a) introducing nucleic acid encoding the polypeptide into a host cell; (b) culturing the host cell in culture medium under conditions sufficient to express the polypeptide; (c) harvesting the polypeptide from the cell or 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).

[0167] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein, a nucleic acid provided herein (e.g., nucleic acid encoding an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC provided herein), a vector provided herein (e.g., a viral vector designed to express an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC provided herein), and / or a host cell provided herein (e.g., a host cell designed to express an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC provided herein) can be formulated as a pharmaceutical composition for administration to a mammal (e.g., a human) having cancer (e.g., a leukemia such as AML) to treat that mammal. In some cases, a binder provided herein, a nucleic acid provided herein, a vector provided herein, and / or a host cell provided herein can be formulated as a pharmaceutical composition for administration to a mammal (e.g., a human) to reduce the number of cancer cells within the mammal and / or to increase the survival of the mammal suffering from cancer. For example, a binder provided herein having the ability to bind to a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) can be formulated as a pharmaceutical composition for administration to a mammal (e.g., a human). In some cases, a pharmaceutical composition provided herein can include a pharmaceutically acceptable carrier such as a buffer, a salt, a surfactant, a sugar, a tonicity modifier, or combinations thereof as, for example, described elsewhere (Gervasi, et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharmaceutically acceptable carriers that can be used to make a pharmaceutical composition provided herein include, without limitation, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or a sugar (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or combinations thereof. For example, a pharmaceutical composition designed to include a binder provided herein (or a nucleic acid, a vector, or a host cell provided herein) can be formulated to include a buffer (e.g., an 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 ingredients that can be included within a pharmaceutical composition provided herein include, without limitation, amino acids such as glycine or arginine, and antioxidants such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA).

[0168] In some cases, when a pharmaceutical composition is formulated to include one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein), any appropriate concentration of the binder can be used.

[0169] In some cases, a pharmaceutical composition designed to include one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) can be formulated to include one or more agents capable of reducing aggregation of the binder when formulated. Examples of such agents that can be used as described herein include, without limitation, methionine, arginine, lysine, aspartic acid, glycine, glutamic acid, and combinations thereof. In some cases, one or more of these amino acids can be included within the formulation at a concentration from about 0.5 mM to about 145 mM (e.g., from about 1 mM to about 145 mM, from about 10 mM to about 145 mM, from about 100 mM to about 145 mM, from about 0.5 mM to about 125 mM, from about 0.5 mM to about 100 mM, from about 0.5 mM to about 75 mM, or from about 10 mM to about 100 mM).

[0170] A pharmaceutical composition provided herein can be in any appropriate form. For example, a pharmaceutical composition provided herein can designed to be a liquid, a semisolid, or a solid. In some cases, a pharmaceutical composition provided herein can be a liquid solution (e.g., an injectable and / or infusible solution), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository. In some cases, a pharmaceutical composition provided herein can be lyophilized. In some cases, a pharmaceutical composition provided herein (e.g., a pharmaceutical composition that includes one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein)) can be formulated with a carrier or coating designed to protect against rapid release. For example, a pharmaceutical composition provided herein can be formulated as a controlled release formulation or as a regulated release formulation as described elsewhere (U. S. Patent Application Publication Nos. 2019 / 0241667;

[0171] 2019 / 0233522; and 2019 / 0233498).

[0172] This document also provides methods for administering a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) to a mammal (e.g., a human). For example, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR cells) provided herein) can be administered to a mammal (e.g., a human) having cancer (e g., a leukemia such as AML) to treat that mammal. In some cases, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR+cells) provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells within the mammal and / or to increase the survival of the mammal. In some cases, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, 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 cells) provided herein) can be administered to a mammal (e.g., a human) having cancer (e.g., a B7-H3+cancer) to reduce or eliminate one or more symptoms of the cancer. Examples of symptoms of a cancer (e.g., a leukemia such as AML) that can be reduced using a composition comprising one or more binders described herein include, without limitation, fever, bone pain, lethargy, fatigue, shortness of breath, pale skin, frequent infections, easy bruising, unusual bleeding (e.g., frequent nosebleeds and bleeding from the gums), thrombocytopenia, and anemia.

[0173] In some cases, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, 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+cells) provided herein) can be administered to a mammal (e.g., a human) having cancer (e.g., a B7-H3+cancer) to reduce the size of cancer within the mammal. For example, a mammal having cancer and in need of treatment thereof can be administered one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e g., CAR? cells) provided herein) to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In another example, a mammal having cancer and in need of treatment thereof can be administered one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR+cells) provided herein) to reduce the volume of one or more solid tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0174] In some cases, a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, 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? cells) provided herein) can be administered to a mammal (e.g., a human) having cancer (e.g., a B7-H3+cancer) to improve survival of the mammal. For example, a mammal having cancer and in need of treatment thereof can be administered one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR? cells) provided herein) to improve the survival of a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In another example, a mammal having cancer and in need of treatment thereof can be administered one or more binders provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR? cells) provided herein) to improve the survival of a mammal having cancer by, for example, at least 6 months (e g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

[0175] Any appropriate mammal having cancer can be treated as described herein (e.g., by administering a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, 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+cells) provided herein)). Examples of mammals that can have cancer and can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having cancer can be treated by administering one or more provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR? cells) provided herein) to the human.

[0176] Any appropriate cancer can be treated using a composition (e.g., a pharmaceutical composition provided herein) containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein). For example, a mammal (e.g., a human) having cancer can be treated by administering a composition (e.g., a pharmaceutical composition) containing one or more binders provided herein to that mammal. In some cases, a cancer that can be treated as described herein can include one or more B7-H3+cancer cells. In some cases, a cancer that can be treated as described herein can include one or more solid tumors. In some cases, a cancer that can be treated as described herein can be a blood cancer. Examples of cancers that can be treated as described herein include, without limitation, leukemias (e.g., AML), breast cancers, non-small cell lung cancers, bladder cancers, colorectal cancers, kidney cancers, lymphomas, melanomas, pancreatic cancers, ovarian cancers, brain cancers (e.g. meningiomas, glioblastomas, diffuse intrinsic pontine gliomas, astrocytomas, and medullablastomas), gastric cancers, esophageal cancers, and squamous cell carcinomas. In some cases, a mammal (e.g., a human) having aB7-H3+cancer can be administered a composition (e.g., a pharmaceutical composition) containing one or more binders provided herein to treat that mammal.

[0177] Any appropriate 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 binders provided herein such as one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more cells designed to express a CAR having the ability to bind a B7-H3 polypeptide, 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 an intravenous injection or infusion), subcutaneously (e.g., via a subcutaneous injection), intraperitoneally (e.g., via an 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 adjusted for the mammal being treated.

[0178] In some cases, an effective amount (e.g., an effective dose) of a composition containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be an amount that reduces the number of cancer cells within a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective amount of a composition containing one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be an amount that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition. For example, an effective amount of a binder provided herein can be about 10 mg / kg. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the severity of cancer when treating a mammal having cancer (e.g., a leukemia such as AML), the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as 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 a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein) that is administered.

[0179] In some cases, an effective frequency of administration of a composition containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a frequency that reduces the number of cancer cells within a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective frequency of administration of a composition containing one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) can be a frequency that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition. For example, an effective frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be from about twice daily to about once a year (e g., from about twice daily to about once a month, from about twice daily to about once a week, from about once daily to about once a month, or from one once daily to about once a week). In some cases, the frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can be daily. The frequency of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as 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 a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein).

[0180] In some cases, an effective duration of administration of a composition containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be a duration that reduces the number of cancer cells within a mammal without producing significant toxicity to the mammal. In some cases, an effective duration of administration of a composition containing one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) can be a duration that increases the survival time of a mammal having cancer as compared to a control mammal having comparable cancer and not treated with the composition. For example, an effective duration of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more binders provided herein can vary from a single time point of administration to several weeks to several months (e.g., 4 to 12 weeks). Multiple factors can influence the actual effective duration used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as 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 duration of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more binders provided herein).

[0181] In some cases, one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR cells) provided herein) (e.g., a pharmaceutical composition provided herein) can be administered to a mammal (e.g., a human) having cancer as the sole active agent(s) to treat the cancer.

[0182] In some cases, methods for treating a mammal (e.g., a human) as described herein (e g., by administering a composition containing one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR? cells) provided herein) (e.g., a pharmaceutical composition provided herein)) also can include administering to the mammal one or more (e.g., one, two, three, or more) additional agents used to treat cancer and / or performing one or more (e.g., one, two, three, or more) therapies used to treat cancer. For example, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) and administering to the mammal one or more (e.g., one, two, three, or more) additional agents used to treat cancer. In some cases, an additional anti-cancer agent that can be administered to a mammal can be a chemotherapeutic agent. In some cases, an additional anti-cancer agent that can be administered to a mammal can be a cytotoxic agent. In some cases, an additional anti-cancer agent that can be administered to a mammal can be an immune-checkpoint inhibitor (e.g., anti-PD-1 antibodies, PD-1 inhibitors, anti-PD-Ll antibodies, PD-L1 inhibitors and anti-CTLA-4 antibodies). Examples of additional anti-cancer agents that can be administered to a mammal (e.g., a human) having cancer (e.g., a leukemia such as AML) to treat the mammal include, without limitation, enzalutamide, imanitib, gefitinib, erlotini, 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, bevacizumab, cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP -224, AMP-514, avelumab, durvalumab, atezolizumab, KNO35, CK-301, AUNP12, CA-170, BMS-986189, ipilimumab, and any combinations thereof.

[0183] In cases where one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR cells) provided herein) (e.g., a pharmaceutical composition provided herein) are used in combination with additional anti-cancer agents, the one or more additional anticancer agents can be administered at the same time (e.g., in a single composition containing one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR? cells) provided herein) and containing the one or more additional agents) or independently. For example, a composition including one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR cells) provided herein) can be administered first, and the one or more additional agents administered second, or vice versa.

[0184] In some cases, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal one or more binders (e.g., one or more antibody domains, one or more antigen binding fragments, one or more antibodies, one or more CARs, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR? cells) provided herein) (e.g., a pharmaceutical composition provided herein), and can include performing one or more (e.g., one, two, three, or more) therapies used to treat cancer. Examples of additional therapies that can be used to treat a mammal (e.g., a human) having cancer include, without limitation, radiation therapies, surgeries, and / or stem cell transplants (e g., bone marrow transplants). In cases where one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR? cells) provided herein) and containing the one or more additional agents) are used in combination with one or more therapies used to treat a mammal (e.g., a human) having cancer, the one or more additional therapies can be performed at the same time or independently of the administration of the one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR+cells) provided herein) and containing the one or more additional agents). For example, one or more binders provided herein (or a nucleic acid, vector, or host cell (e.g., CAR? cells) provided herein) and containing the one or more additional agents) can be administered before, during, or after the one or more additional therapies are performed.

[0185] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein can be used to detect the presence or absence of a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) in vitro in situ, or in vivo (e.g., in vivo imaging within a mammal such as a human). For example, a binder provided herein can be designed to include a label (e.g., a covalently attached radioactive, enzymatic, colorimetric, or fluorescent label). The labelled binder can be used to detect the presence or absence of a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) within a biological sample in vitro. Examples of biological samples that can be assessed using a binder provided herein include, without limitation, serum samples, plasma samples, tissue samples, biopsy samples, cell line samples, and tissue culture samples. In some cases, a biological sample that can be assessed as described herein can include mammalian body tissues and / or cells such as leukocytes, ovary tissue or cells, prostate tissue or cells, heart tissue or cells, placenta tissue or cells, pancreas tissue or cells, liver tissue or cells, spleen tissue or cells, lung tissue or cells, breast tissue or cells, head and neck tissue or cells, endometrium tissue or cells, colon tissue or cells, colorectal tissue or cells, cervix tissue or cells, stomach tissue or cells, or umbilical tissue or cells that may express a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide). In some cases, a binder provided herein can be immobilized, e.g., on a support, and retention of aB7-H3 polypeptide (e.g., a human B7-H3 polypeptide) from a biological sample on the support can be detected, and / or vice versa. In some cases, a binder provided herein can be used in applications such as fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and / or cell sorting (e.g., fluorescence activated cell sorting).

[0186] In some cases, a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein containing a label (e.g., a covalently attached radioactive label) can be used to detect the presence or absence of a B7-H3 polypeptide (e.g., a human B7-H3 polypeptide) within a mammal (e.g., a human). For example, a binder provided herein that is labelled (e.g., covalently labelled) with a radiolabel or an MRI detectable label can be administered to a mammal (e.g., a human), and that mammal can be assessed using a means for detecting the detectable label. In some cases, a mammal can be scanned to evaluate the location(s) of a labelled binder provided herein within the mammal. For example, the mammal can be imaged using NMR or other tomographic techniques.

[0187] Examples of labels that can be attached (e.g., covalently or non-covalently attached) to a binder (e.g., an antibody domain, an antigen binding fragment, an antibody, a CAR, a cell engager, or an ADC) provided herein include, without limitation, radiolabels such as131I,11’in,123I, "mTc,32P,33P,125I,3H,14C, and188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, and enzymatic markers such as a peroxidase or a phosphatase. In some cases, short-range radiation emitters such as isotopes detectable by short-range detector probes can be used.

[0188] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0189] EXAMPLES

[0190] Example 1: Targeting B7-H 3 to Treat Cancer

[0191] Although B7-H3 was cloned in 2001 (Chapoval et al., Nat. Immunol., 2:269-274 (2001)), the inability to identify the inhibitory receptors through which it exerts its immunomodulatory effects during tumor responses has greatly impeded progress toward developing effective therapeutics (Zhao etal., J. HematoL Oncol., 15(1): 153 (2022); Liu et al., Cancer Immunol. Immunother., 71:1549-1567 (2022); and Kontos et al., Clin. Cancer Res., 27:1227-1235 (2021)).

[0192] This Example describes the design and generation of molecules that can bind to, and inhibit, a human B7-H3 polypeptide (also referred to as anti-B7-H3 antibodies).

[0193] B7-H3 generates ligands for the human inhibitory receptor Siglec-9 in trans

[0194] As B7-H3 has been shown to have both costimulatory and inhibitory roles in immune cell activation, whether modification of the extracellular domain by sialylation could engage inhibitory receptors belonging to the Siglec (Sialic acid binding immunoglobulin like lectin) family on immune cells was evaluated.

[0195] With the lack of knowledge as to the inhibitory receptors engaged by B7-H3, it was evaluated whether sialylated B7-H3 could serve as a ligand for inhibitory Siglecs. To test this, human 293T cells were transiently transfected with human Flag-tagged 4Ig-B7-H3 or 2Ig-B7-H3 expression plasmids (Figure 1). The Flag tag was attached to the cytoplasmic tail, to preclude any interference with binding to extracellular ligands. Transfected cells were examined by flow cytometry using recombinant (rec)-Siglec-7 or rec-Siglec-9 chimeric proteins that were fused to human IgGi-Fc (R& D Systems). Binding of the recombinant Siglecs was detected using PE-labelled anti-human IgGi (Southern Biotechnology). Cells were fixed and examined with fluorescently labelled anti-Flag to discriminate between B7-H3 expressing and non-expressing transfected cells. As shown in Figure 1, rec-Siglec-9, but not rec-Siglec-7, bound strongly in trans to 4Ig-B7-H3 but less well to 2Ig-B7-H3 which may simply reflect more potential binding sites in the larger form. When transfected cells were pre-treated with sialidase (neuraminidase) to remove sialic acid from the cell surface, the binding of rec-Siglec-9 to 4Ig-B7-H3 was abrogated (Figure 2). Thus, the inhibitory receptor through which B7-H3 may exert its immunomodulatory effect is Siglec-9, which depends on B7-H3 sialylation.

[0196] Generation of monoclonal antibodies that bind B7-H3

[0197] Antibodies that target B7-H3 and block the interaction with Siglecs were developed using B7-H3 immunogens. Five fusions were performed and a total of approximately 6000 hybridoma supernatants were screened by ELISA for reactivity against B7-H3 but not the myc-his tag. 654 supernatants positive by ELISA were tested for binding to B7-H3 by flow cytometry, and 13 antibodies recognized B7-H3 on the cell surface of 293T cells by flow cytometry. Of these, two were chosen for further study (see below) which recognize both the 41g- and 21g- forms of B7-H3, called “F7” and “G7” (Figure 3). Neither F7 nor G7 bound to murine B7-H3.

[0198] Anti-B7-H3 antibodies block the interaction between B7-H3 and Siglec-9

[0199] The F7 and G7 anti-B7-H3 antibodies were tested for their ability to block the interaction of B7-H3 with rec-Siglec-9. 293T cells were transiently transfected with either Flag-tagged 41g-B7-H3 or empty vector control. The transfected cells were incubated with either F7, G7 or Mouse IgG control, prior to staining with rec-Siglec-9 and anti-human IgG-Fc-PE. Cells were fixed and intracellularly stained for transfected cells using anti-Flag. F7 (Figure 4) and G7 anti-B7-H3 antibodies blocked the binding of rec-Siglec-9 to 4-Ig B7-H3. F7 and G7 also block binding of Siglec-9 to 2Ig-B7-H3. It was next analyzed whether F7 bound to a unique part of B7-H3 as compared to other commercially available antibodies. F7 is an IgG2b, while the commercially available antibodies are of other isotypes. B7-H3 expressing 293T cells were pre-incubated with the commercial antibodies prior toF7. As shown in Figure 5, none of the these commercially available antibodies blocked the binding of F7, indicating that F7 uniquely bound to a different part of B7-H3. One of these, 376.96, has been used to generate CAR-T cells that are in clinical trials at UNC and has been used to generate TRiKEs (Tri-specific killer engagers) (Du et al., Cancer Cell, 35:221-237 (2019); Lichtman etal., Clin. Cancer Res., 27:3141-3153 (2021); and Vallera etal., Cancers, 12(9):2659 (2020)). It was examined whether 376.96 could block rec-Siglec-9 binding to B7-H3. As shown in Figure 6, the F7 antibody inhibited this association but 376.96 did not. This experiment was performed in B7-H3 overexpressing Nalm6 B cell line, demonstrating that the association of rec-Siglec-9 with B7-H3 was not specific to 293T cells. Additionally, using sialidase as in Figure 2 above, it was demonstrated that the binding of F7 or G7 to B7-H3 does not depend upon sialylation (Figure 7), indicating that the binding site of F7 and G7 must be adjacent to the sialylation site but does not include the sialylation site.

[0200] B7-H3 expression on tumor cells inhibits NK cell killing

[0201] Primary human NK cells express both Siglec-7 and Siglec-9 (Jandus et al., J. Clin. Invest., 124:1810-1820 (2014)). Consequently, the expression of B7-H3 on target cells could diminish cell-mediated killing through the engagement of the Siglec-9 inhibitory receptor in trans. 293T cells express low levels of cell surface B7-H3. 293T cells with a CRISPR-mediated deletion of B7-H3 (“293T B7-H3 KO”) or stably overexpressing B7-H3 (“293T B7-H3 O / E”) were loaded with Cr51and used in Cr51-release assays using freshly isolated human NK cells from anonymized normal donor apheresis cones as described elsewhere (Phatarpekar et al., J. Cell. Biol., 219(1 l):e202002145 (2020)). Prior to usage, NK cells were examined to confirm expression of Siglec-9. NK cells were incubated with target cells at different Effector to Target ratios from 20: 1 to 1.25: 1, and Cr51measured after 3 hours. As shown in Figure 8, NK cell killing was significantly inhibited when B7-H3 was overexpressed in 293T cells as compared to B7-H3-deficient 293T cells. This was performed using 3 different donors, and there was a significant decrease across all 3 donors. Thus, B7-H3 expression inhibited NK cell-mediated lysis. Anti-B7-H3 antibodies block B7-H3 binding to Siglec-9

[0202] It was examined whether blockade of the B7-H3 / Siglec-9 interaction enhanced NK cell killing. To do this, purified human NK cells were co-cultured at a 1:1 effector to target cell ratio for 12 hours with stably transduced mRaspberry-NLS1B7-H31HEK293T cells with either an IgG2b isotype control or blocking F7 antibody. IxlO6HEK293T cells were seeded 24 hours before co-culture with 1x106NK cells and 10 mg of control IgG or F7 B7-H3 blocking antibody. A representative experiment from one donor measured every three hours over a 12-hour time course using CellCyte Live Cell Analyzer is shown in Figure 9A. The three-hour time point for 7 independent NK cell donors comparing IgG2b isotype control to F7 is shown in Figure 9B. Thus, the F7 blocking antibody enhanced NK-cell mediated lysis of target cells expressing B7-H3.

[0203] Sialyltransferases modify B7-H3 to generate ligands for Siglecs

[0204] B7-H3 sialylation was required for binding to Siglec-9, as stripping off sialic acid from the cell surface with sialidase disrupted Siglec-9 binding to B7-H3 (Figure 2). Thus, B7-H3 expression alone was not sufficient for Siglec-9 binding, and sialic acid transferases that generate the terminal sialic acid on B7-H3 recognized by Siglec-9 must also be present.

[0205] Siglec-9 can bind to both a2,3- and a2,6 linked sialic acids, although it preferred ligands which carry core fucose in addition to sialic acid (Figure 10). Thus, sialyltransferases that can add terminal a2,3-linked or a2,6-linked sialic acid may be generating ligands for Siglec-9 on B7-H3. There are 20 mammalian sialic acid transferases that can be broadly divided into four families based upon the glycan it modifies and how it adds the sialic acid to that glycan. Members of the ST3Gal family (ST3Gall through 6) add sialic acid via an a2,3 linkage onto an existing galactose, while members of the ST6Gal family (ST6Gall and ST6Gal2) add sialic acid via an a2,6 linkage onto an existing galactose. Members of the ST6GalNac family (ST6GalNacl through 6) adds sialic acid via an a2,6 linkage onto an existing N-acetylgalactosamine (GalNac). Members of the ST8Sia family of enzymes (ST8Sial through 6) add sialic acid(s) onto existing sialic acids via an a.2,8 linkage, creating di-sialic through polysialic acids. Q-PCR was performed on 293T cells and all members of the ST3Gal family were expressed, as well as ST6Gall. Members of the ST8Sia family of sialyltransferases add one or more sialic acids onto an existing sialic acid, myc-tagged ST8Sia6 and Flag-tagged B7-H3 were co-transfected into cells and binding of rec-Siglec-9 was examined. The expression of ST8Sia6 blocked the ability of Siglec-9 to bind to B7-H3 (Figure 11).

[0206] Anti-B7-H3 antibodies and anti-tumor immunity

[0207] To understand whether B7-H3 is working through Siglec-9 in AML, the AML dataset from The Cancer Genome Atlas (TCGA) was analyzed. AML patients with higher levels of B7-H3 had lower survival (Figure 12A). AML patients with higher levels of Siglec-9 also exhibited lower survival (Figure 12B). Tn patients with high B7-H3, survival was worse if patients also had high Siglec-9 (Figure 12C). However, in patients with low B7-H3, there was no difference in survival whether patients had high or low levels of Siglec-9 (Figure 12D). This indicated that B7-H3 may work in part through Siglec-9 in AML.

[0208] The F7 and G7 anti-B7-H3 antibodies were humanized by grafting their CDR1, CDR2, and CDR3 loops onto human IgGl. The binding curves for the highest affinity humanized F7 and G7 antibodies are shown in Figure 13. Human IgGl-Fc portion activates NK cells through CD 16. Thus, these humanized anti-B7-H3 IgGl antibodies not only blocked B7-H3 inhibitory signals through Siglec-9 engagement, but also activated NK cells through CD16 which will activate ADCC.

[0209] The efficacy of the humanized F7 mAb in NK-cell mediated killing of B7-H3 expressing 293T cells was tested as performed in testing the murine F7 mAb (Figure 9B). Briefly, human NK cells were co-cultured at a 1:1 effector to target cell ratio with stably transduced mRaspberry-NLS1B7-H31HEK293T cells along with humanized IgGl isotype control or a humanized F7 B7-H3 blocking antibody (Figure 14). After 3 hours, the viability of the mRaspberry-NLS+B7-H3+HEK293T cells was significantly reduced. Thus, humanized F7 was effective at mediating NK cell-killing of B7-H3 expressing target in vitro.

[0210] The efficacy of the humanized F(ab’)2 fragment of F7 to block the binding Siglec-9 to B7-H3 was confirmed. As the humanized F7 antibody was generated with human IgGl and the recombinant Siglec-9 was fused to human IgGl Fc as well, both could not be used in the same assay. This necessitated the generation of the F(ab’)2 from humanized F7 to quantify blocking. The humanized F7 F(ab’)2 blocked Siglec-9 binding similarly to the original murine F7 anti-B7-H3 antibody (Figure 15).

[0211] Example 2: Anti-B7-H3 Antibodies to Treat Cancer

[0212] Cell death induced by anti-B7-H3 antibody E8

[0213] The mAb E8 in combination with the mAb G7 can induce cell death dependent on the expression of B7-H3. Nalm6 (without B7-H3 expression) or B7-H3 expressing Nalm6 (Nalm6 B7-H3 O / E) cell lines were incubated either with 5 pg isotype control, 5 pg mAb G7, 5 pg mAb E8 or a combination of 5 pg mAb G7 with 5 pg mAb E8 for twenty four hours in culture. The cells were then incubated with a fixable viability dye to examine the percentage of cell death in each culture under each condition.

[0214] There was no effect on cell viability of Nalm6 cells incubated with either isotype control, mAb G7, mAb E8, or the combination of mAb G7 and mAb E8 (Figure 16, top row). However, Nalm6 B7-H3 O / E cells incubated with the combination of mAb G7 and mAb E8 exhibited decreased viability as compared to isotype control, or mAb G7 alone or mAb E8 alone (Figure 16, bottom row). Thus, the combination of the mAb G7 and mAb E8 led to decreased viability of B7-H3 overexpressing Nalm6 cells but not Nalm6 cells that lack B7-H3 expression.

[0215] The same experiment was repeated in Raji cells without B7-H3 expression and in B7-H3 expressing Raji cells (Raji B7-H3 O / E), where they were also incubated with 5 pg isotype control, 5 pg mAb G7, 5 pg mAb E8, or a combination of 5 pg mAb G7 with 5 pg mAb E8 for twenty -four hours in culture. Similarly, the combination of mAb G7 and mAb E8 led to decreased viability of Raji B7-H3 O / E cells, but not of Raji cells without B7-H3 expression or isotype control incubated Raji B7-H3 O / E cells or Raji cells (Figure 17). The combination of mAb G7 and mAb E8 was also examined for the effect on viability of 293T cells or 293T cells that overexpress B7-H3 after two hours on ice. Similarly, the combination of 0.5 pg mAb G7 and 0.5 pg mAb E8 led to decreased viability of 293T B7-H3 O / E cells, but not of 293T cells or 293T cells with or without B7-H3 overexpression incubated with isotype controls (Figure 17). Thus, the combination of mAb G7 and mAb E8 led to decreased viability of Nalm6, Raji and 293T cells that express B7-H3. Anti-B7-H3 mAb F7 synergizes with CART19 to lower tumor burden of luciferase* Nalm6-B7-H3 cells

[0216] The ability of mAb F7 to alter growth of B7-H3 expressing tumor cells in vivo was examined. A schematic of the experiment is shown in Figure 18A. CART 19 T cells express a CAR T receptor against CD19, which is present on the surface of Nalm6 B7-H3 O / E cells. NSG mice were injected with IxlO6luc+Nalm6-B7-H3 cells that express luciferase to quantify tumor burden by IVIS imaging. Seven days later (which is day = -1) mice were imaged by IVIS, and mice were randomized into groups with similar tumor burden. The following day (day = 0) mice received a single injection of 1 x 106CART 19 or untransduced (UTD) human T cells, along with either 10 mg / kg F7 mAb or IgG2b isotype control which was continued 3x / week as indicated in the schematic.

[0217] Luminescence was measured once per week (Figure 18B), with individual mouse data shown and quantified at bottom (Figure 18C).

[0218] The curves were censored after the loss of the first mouse per group. Statistics were performed by two-way ANOVA of the curves at 21 days between UTD+F7, CAR19+isotype and CAR19+F7. **p<0.01, **** p<0.0001.

[0219] Affinity of Siglec-9, Siglec-7, and anti-B7-H3 F7 and G7 mAb

[0220] The affinity of recombinant B7-H3 for Siglec-9 and Siglec-7 proteins was measured using Biacore surface plasmon resonance. Recombinant B7-H3 was bound to the chip, and the association and dissociation of recombinant Siglec-9 and Siglec-7 at several concentrations was examined. From these results, the equilibrium dissociation constant (KD) was calculated for three independent experiments involving three independent isolates of B7-H3 produced in human 293T cells. The sample tracing from one experiment is shown. The KD for Siglec-9 binding to B7-H3 was 130 nM while the KD for Siglec-7 was 80-300 times higher (approximately 10 pM) (Figure 19A). Thus, Siglec-9 but not the closely related family member Siglec-7 has a high affinity for B7-H3.

[0221] Biacore surface plasmon resonance was performed as above, and both of these antibodies have high affinity for human B7-H3 (1.7 nM for F7 and 7.2 nM for G7) (Figure 19B). Example 3: Exemplary B7-H3 Polypeptides

[0222] This Example provides amino acid sequences of a human B7-H3 polypeptides. The boxed| amino acid sequence of each human B7-H3 polypeptide depicts the B7-H3 extracellular domain.

[0223] 41g form of human B7-H3 MLRRRGSPGMGVHVGAALGALWFCLTGA[LEVQVPEDPVVALVGTDATLCCSFSPE PGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQR VRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQ GYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRN PVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLN LIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSF TCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVF WQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDA HGSVTITGQPMTFPPEA^WVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQ DGEGEGSKTALQPLKHSDSKEDDGQEIA

[0224] (SEQ ID NO:61)

[0225] SEQ ID NO 62

[0226]

[0227] extracellular

[0228]

[0229] of the above 41g form of human B7-H3 polypeptide (SEQ ID NO:61)

[0230] 21g form of human B7-H3 MLRRRGSPGMGVHVGAALGALWFCLTGA|LEVQVPEDPVVALVGTDATLCCSFSPE PGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQR VRVADEGSFTCFVS1RDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVT1TCSSYR GYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVR NPVLQQDAHGSVTITGQPMTFPPEA|LWVTVGLSVCLIALLVALAFVCWRKIKQSCEE ENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA

[0231] (SEQ ID NO:63) SEQ ID NO:64 is the [extracellular domain] of the above 21g form of human B7-H3 polypeptide (SEQ ID NO:63)

[0232] Example 4: Exemplary scFv Structures

[0233] This Example provides structures of exemplary scFv’s. An exemplary linker amino acid sequence such as a linker amino acid sequence set forth in Example 9 can be used to link the heavy chain variable domain and the light chain variable domain together to form a scFv. Structures of exemplary scFv’s are shown.

[0234] Exemplary scFv structure:

[0235] Heavy Chain Variable Domain / Region + Linker + Light Chain Variable Domain / Region

[0236] Exemplary scFv structure:

[0237] Light Chain Variable Domain / Region + Linker + Heavy Chain Variable Domain / Region

[0238] Example 5: Exemplary B7-H3-Binding Molecules

[0239] This Example provides the amino acid sequences of exemplary binding molecules (e.g., scFvs) having the ability to bind a B7-H3 polypeptide. The CDRs and framework sequences of each also are provided and delineated

[0240] Anti-B7-H3 clone #1 (also referred to as 3.1BE8 or E8)

[0241] VH domain:

[0242] QVQLQQSGPELVKPGASVKMSCKASGYTFT|DYVIS|WVKQRTGQGLEWIG|EIYPGSGST YYNEKFKG|KATLTADKSSNTAYMQLSSLTSEDSAVYFCAR|GGIGNNFPFAY|WGQGTLV TVS A (SEQ ID NO: 8)

[0243] Framework Region 1 of VH domain:

[0244] QVQLQQSGPELVKPGASVKMSCKASGYTFT (SEQ ID NO:4) CDR1 of VH domain:

[0245] DYVIS (SEQ ID NO: 1)

[0246] Framework Region 2 of VH domain:

[0247] WVKQRTGQGLEWIG (SEQ ID NO: 5)

[0248] CDR2 of VH domain:

[0249] EIYPGSGSTYYNEKFKG (SEQ ID NO:2)

[0250] Framework Region 3 of VH domain:

[0251] KATLTADKSSNTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO:6)

[0252] CDR3 of VH domain:

[0253] GGIGNNFPFAY (SEQ ID NO:3)

[0254] Framework Region 4 of VH domain:

[0255] WGQGTLVTVSA (SEQ ID NO: 7)

[0256] VL domain:

[0257] DIQMTQSPASLSASVGETVTITC|RASENIYSYLA|WYQQKQGK SPQLLVY|NAKALAE GVPSRFSGSRSGTQFSLKINSLQPEDFGSYYC^HHYVTPRT[FGGGTKLEIK (SEQ ID NO: 16)

[0258] Framework Region 1 of VL domain:

[0259] DIQMTQSPASLSASVGETVTITC (SEQ ID NO: 12)

[0260] CDR1 of VL domain:

[0261] RASENIYSYLA (SEQ ID NO: 9)

[0262] Framework Region 2 of VL domain:

[0263] WYQQKQGKSPQLLVY (SEQ ID NO: 13)

[0264] CDR2 of VL domain:

[0265] NAKALAE (SEQ ID NO: 10) Framework Region 3 of VL domain:

[0266] GVPSRFSGSRSGTQFSLKINSLQPEDFGSYYC (SEQ ID NO: 14)

[0267] CDR3 of VL domain:

[0268] QHHYVTPRT (SEQ ID NO: 11)

[0269] Framework Region 4 of VL domain:

[0270] FGGGTKLEIK (SEQ ID NO: 15)

[0271] Anti-B7-H3 clone #2 (also referred to as 6.2F7 or F7)

[0272] VH domain:

[0273]

[0274] Framework Region 1 of VH domain:

[0275] EVQLEQSGPELVKPGASVKISCKASGYTFT (SEQ ID NO 20)

[0276] CDR1 of VH domain:

[0277] ESVIH (SEQ ID NO: 17)

[0278] Framework Region 2 of VH domain:

[0279] WVKQNHERSLEWIG (SEQ ID NO:21)

[0280] CDR2 of VH domain:

[0281] GNNPASGGTTLNQKFKD (SEQ ID NO: 18)

[0282] Framework Region 3 of VH domain:

[0283] RATLTVDKSTSTAYMELRSLTSDDSAIYFCAR (SEQ ID NO:22)

[0284] CDR3 of VH domain:

[0285] LGGNFAHYGLDY (SEQ ID NO: 19) Framework Region 4 of VH domain:

[0286] WGQGTSLTVSS (SEQ ID NO:23)

[0287] VL domain:

[0288] QIVLTQSPAIMSASPGEKVTMTC|RASSVSYMN|WYQQKSGTSPKRWIY|DTSKLAS|GV PPRFSGSGSGTSYSLTISSFEAEDAATYYC|QQWNSNPWT]FGGGTKLEIK (SEQ ID NO:32)

[0289] Framework Region 1 of VL domain:

[0290] QIVLTQSPAIMSASPGEKVTMTC (SEQ ID NO:28)

[0291] CDR1 of VL domain:

[0292] RASSVSYMN (SEQ ID NO:25)

[0293] Framework Region 2 of VL domain:

[0294] WYQQKSGTSPKRWIY (SEQ ID NO:29)

[0295] CDR2 of VL domain:

[0296] DTSKLAS (SEQ ID NO:26)

[0297] Framework Region 3 of VL domain:

[0298] GVPPRFSGSGSGTSYSLTISSFEAEDAATYYC (SEQ ID NO:30)

[0299] CDR3 of VL domain:

[0300] QQWNSNPWT (SEQ ID NO:27)

[0301] Framework Region 4 of VL domain:

[0302] FGGGTKLEIK (SEQ ID NO:31)

[0303] Anti-B7-H3 clone #3 (also referred to as 3.1.2G7 or G7)

[0304] VH domain:

[0305]

[0306] Framework Region 1 of VH domain:

[0307] EIQLQQTGPVLVKPGASVKISCKASGYSFT (SEQ ID NO:36)

[0308] CDR1 of VH domain:

[0309] DYILV (SEQ IDNO:33)

[0310] Framework Region 2 of VH domain:

[0311] WVKQSHGRSLEWIG (SEQ ID NO:37)

[0312] CDR2 of VH domain:

[0313] NINPYYGGTTYNLKFKG (SEQ ID NO:34)

[0314] Framework Region 3 of VH domain:

[0315] KATLTVDKSSSTAYMQLSNLTSEDSSVYYCAK (SEQ ID NO:38)

[0316] CDR3 of VH domain:

[0317] GASYGSTSFYYGMDY (SEQ ID NO:35)

[0318] Framework Region 4 of VH domain:

[0319] WGQGTSVTVSS (SEQ ID NO: 39)

[0320] VL domain:

[0321] QIVLTQSPAIMSASPGEKVTMTC^ASSSVNYMI WYQQKSGTPPRIWIY^)TSKLTS|GV S ARF SGRGSGT S YSLTI STM EA ED A AT Y YC|QQW S SN P LT|FGAGTKLELR (SEQ ID NO:48)

[0322] Framework Region 1 of VL domain:

[0323] QIVLTQSPAIMSASPGEKVTMTC (SEQ ID NO:44)

[0324] CDR1 of VL domain:

[0325] SASSSVNYMH (SEQ ID NO:41)

[0326] Framework Region 2 of VL domain:

[0327] WYQQKSGTPPRIWIY (SEQ ID NO:45) CDR2 of VL domain:

[0328] DTSKLTS (SEQ ID NO:42)

[0329] Framework Region 3 of VL domain:

[0330] GVSARFSGRGSGTSYSLTISTMEAEDAATYYC (SEQ ID NO:46)

[0331] CDR3 of VL domain:

[0332] QQWSSNPLT (SEQ ID NO:43)

[0333] Framework Region 4 of VL domain:

[0334] FGAGTKLELR (SEQ ID NO:47)

[0335] Example 6: Nucleic Acids Encoding Exemplary B7-H3 Binding Molecules

[0336] This Example provides the nucleic acid sequences encoding the indicated chains / domains of Clones #1 - #3.

[0337] Clone #1

[0338] Nucleic acid encoding SEQ ID NO:8 (Clone #1; VH domain):

[0339] CAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTATAAGCTGGG TGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAAATTTATCCTGGAA GTGGTAGTACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAG ACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACT CTGCGGTCTATTTCTGTGCAAGAGGGGGGATCGGGAATAACTTCCCTTTTGCTTA CTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO 49)

[0340] Nucleic acid encoding SEQ ID NO: 16 (Clone #1; VL domain):

[0341] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTG TCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCA GCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAGCCTTAGC AGAAGGTGTGCCATCAAGGTTCAGTGGCAGTAGATCAGGCACACAGTTTTCTCT

[0342]

[0343] TATGTTACTCCTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA (SEQ ID NO:50)

[0344] Clone #2

[0345] Nucleic acid encoding SEQ ID NO:24 (Clone #2; VH domain):

[0346] GAGGTCCAACTGGAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCAGTG AAGATATCCTGCAAGGCTTCTGGATACACTTTCACTGAATCTGTCATACACTGGG TGAAACAGAACCATGAAAGGAGCCTTGAGTGGATTGGTGGTAATAATCCTGCCA GTGGTGGGACTACCCTCAATCAGAAGTTCAAGGACAGGGCCACATTGACTGTTG ACAAGTCCACCAGCACAGCCTATATGGAGCTCCGCAGCCTGACTTCTGACGACT CTGCAATCTATTTCTGTGCAAGATTGGGAGGTAACTTCGCCCACTATGGACTGGA CTATTGGGGTCAGGGAACCTCACTCACCGTCTCCTCA (SEQ ID NO 51)

[0347] Nucleic acid encoding SEQ ID NO:32 (Clone #2; VL domain):

[0348] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCGTCTCCAGGGGAGAAGG TCACCATGACCTGCAGAGCCAGCTCCGTAAGTTACATGAACTGGTACCAACAGA AGTCAGGCACCTCCCCCAAAAGGTGGATTTATGACACATCCAAACTGGCTTCTG GAGTCCCTCCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACGAT CAGCAGCTTTGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAATAGT AATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 52)

[0349] Clone #3

[0350] Nucleic acid encoding SEQ ID NO:40 (Clone #3; VH domain):

[0351] GAGATCCAGCTGCAGCAGACTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTG AAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGACTACATCTTGGTTTGGGT GAAACAGAGCCATGGAAGGAGCCTTGAGTGGATTGGAAATATTAATCCTTACTA TGGTGGTACTACCTACAATCTGAAGTTCAAGGGCAAGGCCACATTGACTGTTGAC AAATCTTCCAGCACAGCCTACATGCAGCTCAGCAATCTGACATCTGAGGACTCTT CAGTCTATTACTGTGCAAAGGGGGCTTCCTACGGTAGTACCTCCTTTTACTATGG TATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ IDNO:53) Nucleic acid encoding SEQ ID NO:48 (Clone #3; VL domain):

[0352] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGG TCACCATGACCTGCAGTGCCAGCTCAAGTGTGAATTACATGCACTGGTACCAGC AGAAGTCAGGCACCCCCCCCAGAATCTGGATTTATGACACATCCAAACTGACTT CTGGAGTCTCTGCTCGCTTCAGTGGCAGAGGGTCTGGGACCTCTTACTCTCTCAC AATCAGCACCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAG TAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGA (SEQ ID NO:54)

[0353] Example 7: Exemplary Immunoglobulins Having the Ability to Bind a B7-H3 Polypeptide This Example provides the amino acid sequences of exemplary immunoglobulins (e.g., IgGs and IgMs) having the ability to bind a B7-H3 polypeptide. The |boxed| amino acid sequence depicts an optional signal peptide. The bold amino acid sequence depicts the constant region.

[0354] Clone #1 Heavy Chain MEWRIFLFILSGTAGVHS|QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVISWVKQRT GQGLEWIGEIYPGSGSTYYNEKFKGKATLTADKS SNTAYMQLS SLTSEDS AVYFC ARGG IGNNFPFAYWGQGTLVTVSAESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTIS FTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGK NRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKD GKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLK NVSSTCAASPSTDILTFTIPPSFAD1FLSKSANLTCLVSNLATYETLN1SWASQSGEPLE TKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEV HKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTS APMPEPGAPGFYFTHSILTVTEEEWNSGETYTCWGHEALPHLVTERTVDKSTGKP TLYNVSLIMSDTGGTCY (SEQ ID NO 55)

[0355] Clone #1 Light Chain

[0356]

[0357] GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQIDNO:56)

[0358] Clone #2 Heavy Chain

[0359]

[0360] HERSLEWIGGNNPASGGTTLNQKFKDRATLTVDKSTSTAYMELRSLTSDDSAIYFCARL GGNFAHYGLDYWGQGTSLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPE SVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTV DKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCW VDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRWSTLPIQHQDWMSGKEF KCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLWGFNPGDI SVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLK NYYLKKTISRSPGK (SEQ ID NO: 57)

[0361] Clone #2 Light Chain MDFQVQIFSLLLISASVIMSRG|QIVLTQSPAIMSASPGEKVTMTCRASSVSYMNWYQQKS GTSPKRWIYDTSKLASGVPPRFSGSGSGTSYSLTISSFEAEDAATYYCQQWNSNPWTFGG GTKLEIKRADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNG VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0362] (SEQ ID NO:58)

[0363] Clone #3 Heavy Chain

[0364]

[0365] GRSLEWIGNINPYYGGTTYNLKFKGKATLTVDKS SSTAYMQLSNLTSEDS S VYYCAKGA SYGSTSFYYGMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGY FPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASS TKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDP EVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSA AFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQW NGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTE KSLSHSPGK (SEQ ID NO: 59) Clone #3 Light Chain MDFQVQIFSFLLISASVMISRGlQIVLTQSPAIMSASPGEKVTMTCSASSSVNYMHWYQQK SGTPPRIWIYDTSKLTS GVS ARFSGRGSGTS YSLTISTMEAED AATYYCQQWS SNPLTFG AGTKLELRRADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQN GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPTVKSFNRNEC

[0366] (SEQ ID NO: 60)

[0367] Example 8: Nucleic Acids Encoding Exemplary B7-H3 Immunoglobulins

[0368] This Example provides the nucleic acid sequences encoding immunoglobulin Clones #1 - #3.

[0369] Clone #1

[0370] Nucleic acid encoding SEQ ID NO:55 (Clone #1; Heavy Chain):

[0371] ATGGAATGGAGGATCTTTCTCTTCATCCTGTCAGGAACTGCAGGTGTCCACTCCC AGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGA AGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTATAAGCTGGGT GAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAAATTTATCCTGGAAG TGGTAGTACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGA CAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC TGCGGTCTATTTCTGTGCAAGAGGGGGGATCGGGAATAACTTCCCTTTTGCTTAC TGGGGCCAGGGGACTCTGGTCACTGTCTCTGCAGAGAGTCAGTCCTTCCCAAATG TCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCAT GGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTAC CAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACA GGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTG AAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAG ATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTT CGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATC TGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGAT GGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAA GGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATC GACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCT TCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAAC CTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGA CCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTC TCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGCCATCCCAA TGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAAC AGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAG AAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGC TGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCC TGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGC AACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGG CCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGA ACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGG TGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCT CCCTGATCATGTCTGACACAGGCGGCACCTGCTATTGA (SEQ ID NO:65)

[0372] Nucleic acid encoding SEQ ID NO:56 (Clone #1; Light Chain):

[0373] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCA GATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGA AACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGG TATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAGCC TTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTAGATCAGGCACACAGTTT TCTCTGAAGATCAATAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAAC ATCATTATGTTACTCCTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAAC GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAAC ATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATC AATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACG TTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCAC AAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 66) Clone #2

[0374] Nucleic acid encoding SEQ ID NO:57 (Clone #2; Heavy Chain):

[0375] ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGACCTGCAGGTGTCCTCTC TGAGGTCCAACTGGAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCAGT GAAGATATCCTGCAAGGCTTCTGGATACACTTTCACTGAATCTGTCATACACTGG GTGAAACAGAACCATGAAAGGAGCCTTGAGTGGATTGGTGGTAATAATCCTGCC AGTGGTGGGACTACCCTCAATCAGAAGTTCAAGGACAGGGCCACATTGACTGTT GACAAGTCCACCAGCACAGCCTATATGGAGCTCCGCAGCCTGACTTCTGACGAC TCTGCAATCTATTTCTGTGCAAGATTGGGAGGTAACTTCGCCCACTATGGACTGG ACTATTGGGGTCAGGGAACCTCACTCACCGTCTCCTCAGCCAAAACAACACCCC CATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCCGTGAC TCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAAC TCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGAC TCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGAC CGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACT TGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGT CACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTC CAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGT GGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAA CGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTAC TATCCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAA GGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAAC CATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCC ACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGG CTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGA GAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATAT AGCAAGCTCAATATGAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGC AACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGG TCTCCGGGTAAATGA (SEQ ID NO:67)

[0376] Nucleic acid encoding SEQ ID NO:58 (Clone #2; Light Chain): ATGGATTTTCAAGTGCAGATTTTCAGCCTCCTGCTAATCAGTGCCTCAGTCATCA TGTCTAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCGTCTCC AGGGGAGAAGGTCACCATGACCTGCAGAGCCAGCTCCGTAAGTTACATGAACTG GTACCAACAGAAGTCAGGCACCTCCCCCAAAAGGTGGATTTATGACACATCCAA ACTGGCTTCTGGAGTCCCTCCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTAC TCTCTCACGATCAGCAGCTTTGAGGCTGAAGATGCTGCCACTTATTACTGCCAAC AGTGGAATAGTAATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAAC ATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATC AATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACG TTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCAC AAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO:68)

[0377] Clone #3

[0378] Nucleic acid encoding SEQ ID NO:59 (Clone #3; Heavy Chain):

[0379] ATGGGAGGGATCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGCCCACT CTGAGATCCAGCTGCAGCAGACTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAG TGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGACTACATCTTGGTTTGG GTGAAACAGAGCCATGGAAGGAGCCTTGAGTGGATTGGAAATATTAATCCTTAC TATGGTGGTACTACCTACAATCTGAAGTTCAAGGGCAAGGCCACATTGACTGTTG ACAAATCTTCCAGCACAGCCTACATGCAGCTCAGCAATCTGACATCTGAGGACT CTTCAGTCTATTACTGTGCAAAGGGGGCTTCCTACGGTAGTACCTCCTTTTACTAT GGTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACG ACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCA TGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGAC CTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAG TCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCA GCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACA AGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGA AGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACT CTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAG GTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAA CCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCA TGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAG CTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGG CTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAG TCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTG GCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGG ACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACT GGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACC ACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO:69)

[0380] Nucleic acid encoding SEQ ID NO:60 (Clone #3; Light Chain):

[0381] ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATGA TATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCC AGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTGAATTACATGCA CTGGTACCAGCAGAAGTCAGGCACCCCCCCCAGAATCTGGATTTATGACACATC CAAACTGACTTCTGGAGTCTCTGCTCGCTTCAGTGGCAGAGGGTCTGGGACCTCT TACTCTCTCACAATCAGCACCATGGAGGCTGAAGATGCTGCCACTTATTACTGCC AGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGA GACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT AACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGAC ATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTC ACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACT CACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0382] (SEQ ID NO:70) Example 9: Exemplary Linkers

[0383] This Example provides exemplary linker amino acid sequences, and nucleic acid sequences encoding such linkers, that can be used to link a heavy chain variable domain and a light chain variable domain together to form a scFv. These linker sequences also can be used to create CARs and cell engagers.

[0384] Linker sequence

[0385] GGGGSGGGGSGGGGS (SEQ ID NO: 71)

[0386] Nucleic acid sequence encoding a linker sequence set forth in SEQ ID NO: 71 GGCGGTGGGGGGTCAGGTGGCGGCGGGAGCGGGGGAGGAGGCTCC (SEQ ID NO:75)

[0387] Linker sequence

[0388] SSGGGGSGGGGSSGGGS (SEQ ID NO 72)

[0389] Nucleic acid sequence encoding a linker sequence set forth in SEQ ID NO: 72 TCTAGCGGAGGCGGCGGATCTGGCGGAGGGGGATCTAGTGGCGGAGGCTCT

[0390] (SEQ ID NO:76)

[0391] Linker sequence

[0392] GSTSGSGKPGSGEGSTKG (SEQ ID NO:73)

[0393] Nucleic acid sequence encoding a linker sequence set forth in SEQ ID NO: 73 GGCTCCACTAGCGGTTCCGGCAAACCTGGCAGCGGAGAAGGCAGCACCAAAGG

[0394] G(SEQ ID NO: 77)

[0395] Linker sequence

[0396] AAA (SEQ ID NO: 74)

[0397] Nucleic acid sequence encoding a linker sequence set forth in SEQ ID NO: 74 GCGGCCGCA (SEQ ID NO:78) Example 10: Exemplary CAR Structures

[0398] This Example provides structures of exemplary CARs. An exemplary linker amino acid sequence such as a linker amino acid sequence set forth in Example 9 or Example 12 can be used to link the sdAb or scFv to the transmembrane domain. Structures of exemplary CARs are shown.

[0399] Exemplary CAR structures:

[0400] Signal Peptide + sdAb + (Linker / Hinge)m+ Transmembrane Domain + (Intracellular Signaling Domain)n

[0401] m = 0, 1, 2, or 3

[0402] n = 1, 2, 3, 4, or 5 with or without a linker located between consecutive intracellular signaling domains

[0403] Signal Peptide + sdAb + Linker + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0404] Signal Peptide + sdAb + Linker + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0405] Signal Peptide + sdAb + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0406] Signal Peptide + sdAb + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Intracellular Signaling Domain

[0407] Signal Peptide + sdAb + Linker + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Intracellular Signaling Domain

[0408] Signal Peptide + scFv + (Linker / Hinge)m+ Transmembrane Domain + (Intracellular Signaling Domain)n

[0409] m = 0, 1, 2, or 3 n = 1, 2, 3, 4, or 5 with or without a linker located between consecutive intracellular signaling domains

[0410] Signal Peptide + scFv + Linker + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0411] Signal Peptide + scFv + Linker + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0412] Signal Peptide + scFv + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Linker + Intracellular Signaling Domain

[0413] Signal Peptide + scFv + Hinge + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Intracellular Signaling Domain

[0414] Signal Peptide + scFv + Linker + Transmembrane Domain + Intracellular Signaling Domain + Intracellular Signaling Domain + Intracellular Signaling Domain

[0415] Example 11: Exemplary Signal Peptides

[0416] This Example provides the amino acid sequences of exemplary signal peptides, and nucleic acid sequences encoding such signal peptides, that can be used to design a CAR.

[0417] Exemplary CDS alpha (CD8a) signal peptide

[0418] MALPVTALLLPLALLLHAARP (SEQ ID NO: 79)

[0419] Nucleic acid sequence encoding a CD8a signal peptide set forth in SEQ ID NO:79 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCG (SEQ ID NO: 80)

[0420] Exemplary human IGKVl-39-derived signal peptide:

[0421] MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 81) Nucleic acid sequence encoding a human IGKVl-39-derived signal peptide set forth in SEQ ID NO:81 ATGGATATGAGGGTCCCCGCACAATTGCTCGGTCTCCTTCTGCTTTGGCTCCGGG GCGCGCGGTGC (SEQ ID NO 82)

[0422] Exemplary human IGKVl-16-derived signal peptide:

[0423] MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO:83)

[0424] Exemplary human IGKV3-ll-derived signal peptide:

[0425] MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 84)

[0426] Exemplary human IGKV4-1 -derived signal peptide:

[0427] MVLQTQVFISLLLWISGAYG (SEQ ID NO:85)

[0428] Exemplary human IGKV6-21 -derived signal peptide:

[0429] MLPSQLIGFLLLWVPASRG (SEQ ID NO:86)

[0430] Exemplary human IGKVl-33-derived signal peptide:

[0431] MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO:87)

[0432] Example 12: Exemplary Hinges

[0433] This Example provides the amino acid sequences of exemplary hinges, and nucleic acid sequences encoding such hinges, that can be used to design a CAR.

[0434] Exemplary CD8a hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO 88)

[0435] Nucleic acid sequence encoding a CD8a hinge set forth in SEQ ID NO:88 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO:96) Exemplary human IgGl-derived hinge:

[0436] EPKSCDKTHTCPPCP (SEQ ID NO: 89)

[0437] Exemplary human IgGl-derived hinge:

[0438] DKTHTCPPCPAPPVA (SEQ ID NO:90)

[0439] Exemplary human IgG4-derived hinges:

[0440] ESKYGPPCPPCP (SEQ ID NO:91)

[0441] ESKYGPPCPSCP (SEQ ID NO:92)

[0442] Exemplary human CD28-derived hinges:

[0443] IEVMYPPPYLDNERSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:93) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 94) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:95)

[0444] Example 13: Exemplary Transmembrane Domains

[0445] This Example provides the amino acid sequences of exemplary transmembrane domains, and nucleic acid sequences encoding such transmembrane domains, that can be used to design a CAR.

[0446] Exemplary CD8a transmembrane domain

[0447] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:97) 1Y1WAPLAGTCGVLLLSLV1T (SEQ ID NO: 98)

[0448] IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 99)

[0449] IWAPLAGTCGVLLLSLVIT (SEQ ID NO: 100)

[0450] Nucleic acid encoding a CD8a transmembrane domain set forth in SEQ ID NO:97 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTACTGC (SEQ ID NO: 105)

[0451] Exemplary human CD3 transmembrane domain:

[0452] LCYLLDGILFIYGVILTALFL (SEQ ID NO: 101) Exemplary human CD4 transmembrane domain:

[0453] MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 102)

[0454] Exemplary human CD28 transmembrane domain:

[0455] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 103)

[0456] Exemplary human CD278 transmembrane domain:

[0457] FWLPIGCAAFVVVCILGCILI (SEQ ID NO: 104)

[0458] Example 14: Exemplary Intracellular Signaling Domains

[0459] This Example provides the amino acid sequences of exemplary intracellular signaling domains, and nucleic acid sequences encoding such intracellular signaling domains, that can be used to design a CAR.

[0460] 4-1BB signaling domains KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 106) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE (SEQ ID NO: 107)

[0461] Nucleic acid encoding a 4-1BB signaling domain set forth in SEQ ID NO: 106 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG (SEQ ID NO: 116)

[0462] Exemplary CD3 zeta (Q signaling domains RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO 108)

[0463] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR (SEQ ID NO: 109) Nucleic acid encoding a CD3^ signaling domain set forth in SEQ ID NO: 108 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 117)

[0464] Exemplary human CD28 intracellular signaling domain:

[0465] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 110)

[0466] Exemplary human 0X40 (CD 134) intracellular signaling domain:

[0467] ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 111)

[0468] Exemplary human CD278 intracellular signaling domain:

[0469] CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 112)

[0470] Exemplary human DAP10 intracellular signaling domain:

[0471] LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 113)

[0472] Exemplary human DAP12 intracellular signaling domain:

[0473] YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 114)

[0474] Exemplary human CD27 intracellular signaling domain:

[0475] QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:115)

[0476] Example 15: Exemplary Antigen Binding Domains Having the Ability to Bind a T Cell This Example provides the amino acid sequences of exemplary antigen binding domains that can be used to design cell engagers that bind to T cells. Exemplary anti-human CD3 scFv (clone OKT3):

[0477] VH domain:

[0478] QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSR GYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWG QGTPVTVSS (SEQ ID NO: 118)

[0479] VL domain:

[0480] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGV PSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 119)

[0481] Exemplary anti-human CD3 scFv (clone UCHT1):

[0482] VH domain:

[0483] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYK GVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFD VWGQGTTLTVFS (SEQ ID NO: 120)

[0484] VL domain:

[0485] MDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK (SEQ ID NO:121)

[0486] Example 16: Exemplary Antigen Binding Domains Having the Ability to Bind a NK Cell This Example provides the amino acid sequences of exemplary antigen binding domains that can be used to design cell engagers that bind to NK cells.

[0487] Exemplary anti-human CD16a scFv:

[0488] VH domain:

[0489] EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNG GSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQG TLVTVSR (SEQ ID NO: 122) VL domain:

[0490] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVG (SEQ ID NO: 123)

[0491] Exemplary anti-human NKG2A scFv:

[0492] VH domain:

[0493] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPY DSETHYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLY WFFDVWGQGTTVTVSS (SEQ ID NO: 124)

[0494] VL domain:

[0495] DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIK (SEQ ID NO: 125)

[0496] Exemplary anti-human NKG2A scFv:

[0497] VH domain:

[0498] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEISS GGSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGDYPRFF DVWGQGTTVTVSS (SEQ ID NO: 126)

[0499] VL domain:

[0500] EIVLTQSPATLSLSPGERATLSCSASSSVSSYIYWYQQKPGQAPRLLIYLTSNLASGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSGNPYTFGQGTKLEIK (SEQ ID NO: 127) Exemplary anti-human NKG2D scFv:

[0501] VH domain:

[0502] QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDY WGQGTTVTVSS (SEQ ID NO: 128)

[0503] VL domain:

[0504] QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPS GVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL (SEQ ID NO: 129)

[0505] Exemplary anti-human NKp30 scFv:

[0506] VH domain:

[0507] QVQLVQSGAEVKKPGASVKVSCKASGHTFTSYFMHWVRQAPGQGLEWMGIINPSD DYANYAQKFQGRVTMTRDTST STVYMELS SLRSEDT AVYYCATAIFDYWGQGTLV TVSS (SEQ ID NO: 130)

[0508] alternative VH domain:

[0509] QVQLVQSGAEVKKPGASVKVSCKASGHTFTSYFMHWVRQAPGQGLEWMGIINPSD DYANYAQKFQGRVTMTRDTST STVYMELS SLRSEDT AVYYC AT AIFDYWGQGTPV TVSS (SEQ ID NO: 131)

[0510] VL domain:

[0511] DIQMTQ SP S SL S AS VGDRVTITCRASQ SIS S YLNWYQQKPGK APKLLIYAAS SLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK (SEQ ID NO: 132) Exemplary anti-human NKp46 scFv

[0512] VH domain:

[0513] EIQLQQSGAELVKPGASVKLSCTASGFNIKDTYFHWVKQRPEQGLEWIGRIDPANGN TKYDPKFHDKATIIADISSNTAYLQFSSLTSEDTAVYYCAANRYGYWGQGTTLTVSS

[0514] (SEQ ID NO: 133)

[0515] VL domain:

[0516] DIVMTQAAPSIPVTPGESVSISCRSSKSLLYINGNTHLFWFLQRPGQSPQLLIYRMSNL ASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK (SEQ ID NO: 134)

[0517] Exemplary anti-human NKp46 scFv

[0518] VH domain:

[0519] QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSG TNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWG QGTSVTVSS (SEQ ID NO: 135)

[0520] VL domain:

[0521] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSG VPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK (SEQ ID NO: 136)

[0522] Exemplary anti-human CD16a VH domain:

[0523] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWIGSIYYSGS TNYNPSLKSLVTISRDNSKNTLYLQMNSLRAEDTATYYCARESIDYWGQGTLVTVSS

[0524] (SEQ ID NO: 137)

[0525] Example 17: Treating a B7-H3+Cancer

[0526] One or more binders (e.g., antibodies, antigen binding fragments, antibody domains, cell engagers, or ADCs) having the ability to bind to a B7-H3 polypeptide are administered to a human identified as having cancer. The one or more binders having the ability to bind to a B7-H3 polypeptide are administered using intravenous injection. After the administration of one or more binders having the ability to bind to a B7-H3 polypeptide, the number of cancer cells (e.g., B7-H3+cancer cells) within the human is reduced. After the administration of the one or more binders having the ability to bind to a B7-H3 polypeptide, the size of one or more tumors (e.g., tumors having one or more B7-H3+cancer cells) within the human is reduced.

[0527] Example 18: Generation of B 7 -H 3 -specific CAR+T cells

[0528] T cells are obtained from a human. Nucleic acid designed to express one or more CARs having the ability to bind to a B7-H3 polypeptide provided herein is introduced into the T cells by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection such that the T cells express the CAR(s) having the ability to bind to a B7-H3 polypeptide.

[0529] Example 19: Treating a B7-H3+Cancer

[0530] Cells (e.g., T cells) designed to express one or more CARs having the ability to bind to a B7-H3 polypeptide are administered to a human identified as having a B7-H3+cancer. The cells (e.g., T cells) engineered to express one or more binders having the ability to bind to a B7-H3 polypeptide are administered using intravenous injection. After the administration of cells (e.g., T cells) engineered to express one or more binders having the ability to bind to a B7-H3 polypeptide, the number of cancer cells (e g., B7-H3+cancer cells) within the human is reduced. After the administration of the cells (e.g., T cells) engineered to express one or more binders having the ability to bind to a B7-H3 polypeptide, the size of one or more tumors (e.g., tumors having one or more B7-H3+cancer cells) within the human is reduced.

[0531] Example 20: Exemplary Embodiments

[0532] Embodiment 1. A binding molecule comprising:

[0533] (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);

[0534] (ii) a heavy 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), 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 sequences 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); or

[0535] (iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions).

[0536] Embodiment 2. The binding molecule of Embodiment 1, wherein said binding molecule comprises the ability to bind to a polypeptide sequence set forth in any one of SEQ ID NOs:61-64. Embodiment 3. The binding molecule of any one of Embodiments 1-2, wherein said binding molecule comprises said (i).

[0537] Embodiment 4. The binding molecule of Embodiment 3, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO: 16.

[0538] Embodiment 5. The binding molecule of any one of Embodiments 1-2, wherein said binding molecule comprises said (ii).

[0539] Embodiment 6. The binding molecule of Embodiment 5, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO: 32.

[0540] Embodiment 7. The binding molecule of any one of Embodiments 1-2, wherein said binding molecule comprises said (iii).

[0541] Embodiment 8. The binding molecule of Embodiment 7, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:40, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO:48.

[0542] Embodiment 9. The binding molecule of any one of Embodiments 1-8, wherein said binding molecule is an antigen binding fragment. Embodiment 10. The binding molecule of any one of Embodiments 1-8, wherein said binding molecule is a Fab, a Fab’, F(ab’)2, or an scFv antibody.

[0543] Embodiment 11. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein said antigen binding domain comprises a binding molecule of any one of Embodiments 1-10.

[0544] Embodiment 12. The CAR of Embodiment 11, wherein said antigen binding domain comprises a scFv having the ability to bind to a B7 homolog 3 (B7-H3) polypeptide.

[0545] Embodiment 13. The CAR of any one of Embodiments 11-12, wherein said hinge comprises a hinge set forth in any one of SEQ ID NOs:88-95.

[0546] Embodiment 14. The CAR of any one of Embodiments 11-12, wherein said hinge is a CD8a hinge.

[0547] Embodiment 15. The CAR of any one of Embodiments 11-14, wherein said transmembrane domain comprises a transmembrane domain set forth in any one of SEQ ID NOs:97-104.

[0548] Embodiment 16. The CAR of any one of Embodiments 11-14, wherein said transmembrane domain is a CD8a transmembrane domain.

[0549] Embodiment 17. The CAR of any one of Embodiments 11-16, wherein said CAR comprises one or more signaling domains set forth in any one of SEQ ID NOs:106-115.

[0550] Embodiment 18. The CAR of any one of Embodiments 11-16, wherein said CAR comprises a 4-1BB intracellular signaling domain and a CD3(^ intracellular signaling domain.

[0551] Embodiment 19. A cell comprising a CAR of any one of Embodiments 11-18. Embodiment 20. The cell of Embodiment 19, wherein said cell is a T cell, a stem cell, or an NK cell.

[0552] Embodiment 21. A cell engager comprising a first antigen binding domain, a linker, and a second antigen binding domain, wherein said first antigen binding domain comprises a binding molecule of any one of Embodiments 1-10.

[0553] Embodiment 22. The cell engager of Embodiment 21, wherein said first antigen binding domain comprises a scFv having the ability to bind to a B7-H3 polypeptide.

[0554] Embodiment 23. The cell engager of any one of Embodiments 21-22, wherein said linker comprises a linker set forth in any one of SEQ ID NOs:71-74 or SEQ ID NOs:88-95.

[0555] Embodiment 24. The cell engager of any one of Embodiments 21-23, wherein said second antigen binding domain binds to a polypeptide expressed on the surface of T cells.

[0556] Embodiment 25. The cell engager of Embodiment 24, wherein said polypeptide expressed on the surface of T cells is a CD3 polypeptide.

[0557] Embodiment 26. The cell engager of Embodiment 24, wherein said second antigen binding domain is an antigen binding domain set forth in Example 15.

[0558] Embodiment 27. The cell engager of any one of Embodiments 21-23, wherein said second antigen binding domain binds to a polypeptide expressed on the surface of NK cells.

[0559] Embodiment 28. The cell engager of Embodiment 27, wherein said polypeptide expressed on the surface of NK cells is a CD 16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide.

[0560] Embodiment 29. The cell engager of Embodiment 27, wherein said second antigen binding domain is an antigen binding domain set forth in Example 16. Embodiment 30. A nucleic acid comprising a nucleic acid sequence encoding at least part of a binding molecule of any one of Embodiments 1-10.

[0561] Embodiment 31. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (i) of Embodiment 1.

[0562] Embodiment 32. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (i) of Embodiment 1.

[0563] Embodiment 33. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (ii) of Embodiment 1.

[0564] Embodiment 34. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (ii) of Embodiment 1.

[0565] Embodiment 35. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (iii) of Embodiment 1.

[0566] Embodiment 36. The nucleic acid of Embodiment 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (iii) of Embodiment 1.

[0567] Embodiment 37. The nucleic acid of any one of Embodiments 30-36, wherein said nucleic acid is a viral vector.

[0568] Embodiment 38. A nucleic acid comprising a nucleic acid sequence encoding a CAR of any one of Embodiments 11-18 or a cell engager of any one of Embodiments 21-29.

[0569] Embodiment 39. The nucleic acid of Embodiment 38, wherein said nucleic acid is a viral vector. Embodiment 40. A host cell comprising a nucleic acid of any one of Embodiments 30-39.

[0570] Embodiment 41. A host cell that expresses a CAR of any one of Embodiments 11-18 or a cell engager of any one of Embodiments 21-29.

[0571] Embodiment 42. The host cell of any one of Embodiments 40-41, wherein said host cell is a T cell, stem cell, or NK cell.

[0572] Embodiment 43. An antibody-drug conjugate (ADC) comprising an antigen binding domain covalently linked to a drug, wherein said antigen binding domain comprises a binding molecule of any one of Embodiments 1-10.

[0573] Embodiment 44. The ADC of Embodiment 43, wherein said binding molecule has the ability to bind to a B7-H3 polypeptide.

[0574] Embodiment 45. The ADC of any one of Embodiments 43-44, wherein said drug is selected from the group consisting of auristatins, mertansines, and pyrrolobenzodiazepine (PBD) dimers.

[0575] Embodiment 46. A composition comprising a binding molecule of any one of Embodiments 1-10.

[0576] Embodiment 47. A composition comprising a cell engager of any one of Embodiments 21-29.

[0577] Embodiment 48. A composition comprising a cell of any one of Embodiments 19-20 or 40-42.

[0578] Embodiment 49. A composition comprising an ADC of any one of Embodiments 43-45. Embodiment 50. A method of treating a mammal having cancer, wherein said method comprises administering, to said mammal, a composition of any one of Embodiments 46-49.

[0579] Embodiment 51. The method of Embodiment 50, wherein said mammal is a human.

[0580] Embodiment 52. The method of any one of Embodiments 50-51, wherein said cancer is a B7-H3+cancer.

[0581] Embodiment 53. The method of any one of Embodiments 50-52, wherein said cancer is selected from the group consisting of a leukemia, a breast cancer, a non-small cell lung cancer, a bladder cancer, a colorectal cancer, a kidney cancer, a lymphoma, a melanoma, a pancreatic cancer, an ovarian cancer, a brain cancer, a gastric cancer, an esophageal cancer, and a squamous cell carcinoma.

[0582] Embodiment 54. The method of any one of Embodiments 50-53, wherein the number of cancer cells within said mammal is reduced following said administering step.

[0583] Embodiment 55. A method for binding a binding molecule to a B7-H3 polypeptide, wherein said method comprises contacting said B7-H3 polypeptide with a binding molecule of any one of Embodiments 1-10.

[0584] Embodiment 56. The method of Embodiment 55, wherein said contacting is performed in vitro.

[0585] Embodiment 57. The method of Embodiment 55, wherein said contacting is performed in vivo.

[0586] Embodiment 58. The method of Embodiment 57, wherein said contacting is performed within a mammal by administering said binding molecule to said mammal.

[0587] Embodiment 59. The method of Embodiment 58, wherein said mammal is a human. Embodiment 60. A method for binding a binding molecule to a B7-H3 polypeptide, wherein said method comprises contacting said B7-H3 polypeptide with a CAR of any one of Embodiments 11-18, a cell engager of any one of Embodiments 21-29, or an ADC of any one of Embodiments 43-45.

[0588] Embodiment 61. The method of Embodiment 60, wherein said contacting is performed in vitro.

[0589] Embodiment 62. The method of Embodiment 60, wherein said contacting is performed in vivo.

[0590] Embodiment 63. The method of Embodiment 62, wherein said contacting is performed within a mammal by administering said CAR, said cell engager, or said ADC to said mammal.

[0591] Embodiment 64. The method of Embodiment 63, wherein said mammal is a human.

[0592] Embodiment 65. A composition comprising an antibody, a fragment of an antibody, an antibody domain, a cell engager, or an antibody-drug conjugate comprising an ability to bind to a human B7-H3 polypeptide and inhibit binding of said human B7-H3 polypeptide to a human Siglec-9.

[0593] Embodiment 66. The composition of Embodiment 65, wherein said composition comprising said antibody.

[0594] Embodiment 67. The composition of any one of Embodiments 65-66, wherein said composition comprising said fragment.

[0595] Embodiment 68. The composition of any one of Embodiments 65-67, wherein said composition comprising said antibody domain. Embodiment 69. The composition of any one of Embodiments 65-68, wherein said composition comprising said cell engager.

[0596] OTHER EMBODIMENTS

[0597] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT Is CLAIMED IS:

1. A binding molecule 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);(ii) a heavy 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), 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 sequences 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); or(iii) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:33 (or SEQ ID NO:33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:34 (or SEQ ID NO:34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:35 (or SEQ ID NO:35 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:41 (or SEQ ID NO:41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ IDNO:42 (or SEQ ID NO:42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:43 (or SEQ ID NO:43 with one, two, or three amino acid additions, deletions, or substitutions).

2. The binding molecule of claim 1, wherein said binding molecule comprises the ability to bind to a polypeptide sequence set forth in any one of SEQ ID NOs:61-64.

3. The binding molecule of any one of claims 1-2, wherein said binding molecule comprises said (i).

4. The binding molecule of claim 3, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:8, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO: 16.

5. The binding molecule of any one of claims 1-2, wherein said binding molecule comprises said (ii).

6. The binding molecule of claim 5, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:24, and wherein said light chain variable region comprises an amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO:32.

7. The binding molecule of any one of claims 1-2, wherein said binding molecule comprises said (iii).

8. The binding molecule of claim 7, wherein said heavy chain variable domain comprises an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:40, and wherein said light chain variable region comprisesan amino acid sequence having at least 90 percent identify to the amino acid sequence set forth in SEQ ID NO:48.

9. The binding molecule of any one of claims 1-2, wherein said binding molecule is an antigen binding fragment.

10. The binding molecule of any one of claims 1-2, wherein said binding molecule is a Fab, a Fab’, F(ab’)2, or an scFv antibody.

11. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein said antigen binding domain comprises a binding molecule of any one of claims 1-10.

12. The CAR of claim 11, wherein said antigen binding domain comprises a scFv having the ability to bind to a B7 homolog 3 (B7-H3) polypeptide.

13. The CAR of any one of claims 11-12, wherein said hinge comprises a hinge set forth in any one of SEQ ID NOs:88-95.

14. The CAR of any one of claims 11-12, wherein said hinge is a CD8a hinge.

15. The CAR of any one of claims 11-12, wherein said transmembrane domain comprises a transmembrane domain set forth in any one of SEQ ID NOs:97-104.

16. The CAR of any one of claims 11-12, wherein said transmembrane domain is a CD8a transmembrane domain.

17. The CAR of any one of claims 11-12, wherein said CAR comprises one or more signaling domains set forth in any one of SEQ ID NOs: 106-115.

18. The CAR of any one of claims 11-12, wherein said CAR comprises a 4-1BB intracellular signaling domain and a CD3^ intracellular signaling domain.

19. A cell comprising a CAR of any one of claims 11-18.

20. The cell of claim 19, wherein said cell is a T cell, a stem cell, or an NK cell.

21. A cell engager comprising a first antigen binding domain, a linker, and a second antigen binding domain, wherein said first antigen binding domain comprises a binding molecule of any one of claims 1-10.

22. The cell engager of claim 21, wherein said first antigen binding domain comprises a scFv having the ability to bind to a B7-H3 polypeptide.

23. The cell engager of any one of claims 21-22, wherein said linker comprises a linker set forth in any one of SEQ ID NOs:71-74 or SEQ ID NOs:88-95.

24. The cell engager of any one of claims 21-22, wherein said second antigen binding domain binds to a polypeptide expressed on the surface of T cells.

25. The cell engager of claim 24, wherein said polypeptide expressed on the surface of T cells is a CD3 polypeptide.

26. The cell engager of claim 24, wherein said second antigen binding domain is an antigen binding domain set forth in Example 15.

27. The cell engager of any one of claims 21-22, wherein said second antigen binding domain binds to a polypeptide expressed on the surface of NK cells.

28. The cell engager of claim 27, wherein said polypeptide expressed on the surface of NK cells i29. The cell engager of claim 27, wherein said second antigen binding domain is an antigen binding domain set forth in Example 16.

30. A nucleic acid comprising a nucleic acid sequence encoding at least part of a binding molecule of any one of claims 1-10.

31. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (i) of claim 1.

32. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (i) of claim 1.

33. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (ii) of claim 1.

34. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (ii) of claim 1.

35. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said heavy chain variable domain or region of said (iii) of claim 1.

36. The nucleic acid of claim 30, wherein said nucleic acid sequence encodes said light chain variable domain or region of said (iii) of claim 1.

37. The nucleic acid of any one of claims 30-36, wherein said nucleic acid is a viral vector.

38. A nucleic acid comprising a nucleic acid sequence encoding a CAR of any one of claims 11-18 or a cell engager of any one of claims 21-29.

39. The nucleic acid of claim 38, wherein said nucleic acid is a viral vector.

40. A host cell comprising a nucleic acid of any one of claims 30-39.

41. A host cell that expresses a CAR of any one of claims 11-18 or a cell engager of any one of claims 21-29.

42. The host cell of any one of claims 40-41, wherein said host cell is a T cell, stem cell, or NK cell.

43. An antibody-drug conjugate (ADC) comprising an antigen binding domain covalently linked to a drug, wherein said antigen binding domain comprises a binding molecule of any one of claims 1-10.

44. The ADC of claim 43, wherein said binding molecule has the ability to bind to a B7-H3 polypeptide.

45. The ADC of any one of claims 43-44, wherein said drug is selected from the group consisting of auristatins, mertansines, and pyrrolobenzodiazepine (PBD) dimers.

46. A composition comprising a binding molecule of any one of claims 1-10.

47. A composition comprising a cell engager of any one of claims 21-29.

48. A composition comprising a cell of any one of claims 19-20 or 40-42.

49. A composition comprising an ADC of any one of claims 43-45.

50. A method of treating a mammal having cancer, wherein said method comprises administering, to said mammal, a composition of any one of claims 46-49.

51. The method of claim 50, wherein said mammal is a human.

52. The method of any one of claims 50-51, wherein said cancer is a B7-H3+cancer.

53. The method of any one of claims 50-51, wherein said cancer is selected from the group consisting of a leukemia, a breast cancer, a non-small cell lung cancer, a bladder cancer, a colorectal cancer, a kidney cancer, a lymphoma, a melanoma, a pancreatic cancer, an ovarian cancer, a brain cancer, a gastric cancer, an esophageal cancer, and a squamous cell carcinoma.

54. The method of any one of claims 50-51, wherein the number of cancer cells within said mammal is reduced following said administering step.

55. A method for binding a binding molecule to a B7-H3 polypeptide, wherein said method comprises contacting said B7-H3 polypeptide with a binding molecule of any one of claims 1-10.

56. The method of claim 55, wherein said contacting is performed in vitro.

57. The method of claim 55, wherein said contacting is performed in vivo.

58. The method of claim 57, wherein said contacting is performed within a mammal by administering said binding molecule to said mammal.

59. The method of claim 58, wherein said mammal is a human.

60. A method for binding a binding molecule to a B7-H3 polypeptide, wherein said method comprises contacting said B7-H3 polypeptide with a CAR of any one of claims 11-18, a cell engager of any one of claims 21-29, or an ADC of any one of claims 43-45.

61. The method of claim 60, wherein said contacting is performed in vitro.

62. The method of claim 60, wherein said contacting is performed in vivo.

63. The method of claim 62, wherein said contacting is performed within a mammal by administering said CAR, said cell engager, or said ADC to said mammal.

64. The method of claim 63, wherein said mammal is a human.

65. A composition comprising an antibody, a fragment of an antibody, an antibody domain, a cell engager, or an antibody-drug conjugate comprising an ability to bind to a human B7-H3 polypeptide and inhibit binding of said human B7-H3 polypeptide to a human Siglec-9.

66. The composition of claim 65, wherein said composition comprising said antibody.

67. The composition of any one of claims 65-66, wherein said composition comprising said fragment.

68. The composition of any one of claims 65-66, wherein said composition comprising said antibody domain.

69. The composition of any one of claims 65-66, wherein said composition comprising said cell engager.

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