Molecules that bind to B-cell activating factor receptor polypeptides

Abstract

The present application provides methods and materials for conjugating binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) to BAFF-R polypeptides. For example, provided are binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) that bind to BAFF-R polypeptides, as well as methods and materials for treating a mammal (e.g., a human) with cancer (e.g., one or more B-cell cancers) using one or more such binding molecules.

Description

[Technical Field]

[0001] Cross-references to related fields This application claims the benefit of U.S. Patent Application No. 63 / 448,713, filed February 28, 2023, and U.S. Patent Application No. 63 / 553,951, filed February 15, 2024. The disclosures of the prior applications are considered part of the disclosure of this application and are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled "07039-2188WO1_SL.xml." This XML file, created on February 27, 2024, is 57,000 bytes. The contents of this XML file are incorporated by reference in their entirety into this application.

[0003] Technical Field This application relates to methods and materials involving binding of molecules (e.g., antibodies, antibody fragments, antibody domains, chimeric antigen receptors (CARs), cell engagers, or antibody-drug conjugates (ADCs)) to B-cell activating factor receptor (BAFF-R) polypeptides. For example, this application provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to BAFF-R polypeptides, and methods and materials for using such binding agents to treat cancer. This application also provides cells (e.g., host cells) engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to BAFF-R polypeptides, and methods and materials for using such cells to treat cancer. [Background technology]

[0004] Immunotherapy can be nonspecific (e.g., interleukins or cytokines) or specific (e.g., monoclonal antibodies or CAR-expressing T cells (CAR-T cells)). Immunotherapy is currently being applied to cancer treatment. However, refractory and recurrent disease has been observed after CAR-T cell therapy, with rates reaching as high as 50% in some malignancies. Both antigen-positive and antigen-negative recurrent cancers (e.g., loss or reduction of antigens expressed on malignant cells) have been observed. Summary of the Invention

[0005] The present application provides methods and materials related to binding of molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) to BAFF-R polypeptides. For example, the present application provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to BAFF-R polypeptides, and methods and materials for treating a mammal (e.g., a human) with cancer using one or more of the binding agents.

[0006] The present application also provides cells (e.g., host cells) engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R polypeptide, and methods and materials for using such cells to treat cancer.

[0007] As provided herein, binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more CARs, one or more cell engagers, and / or one or more ADCs) can be designed to have the ability to bind to a BAFF-R polypeptide. For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, or ADC) provided herein can have the ability to bind to a polypeptide that can comprise, consist essentially of, or consist of the amino acid sequence of the human BAFF-R polypeptide set forth in SEQ ID NO: 42 (see, e.g., Example 2).

[0008] In some cases, two sets of three complementarity determining regions (CDRs) of the antigen-binding fragments provided herein (e.g., SEQ ID NOS: 1-3 and 9-11) are incorporated into a CAR to form a BAFF-R. + Cells (e.g., BAFF-R + CAR with the ability to target tumor cells + Cells (e.g., CAR + T cells, CAR + CARs such as induced pluripotent stem cells + Stem cells, or CARs + BAFF-R can be incorporated into antibody structures containing an Fc region, which can be used to generate natural killer (NK) cells. + Cells (e.g., BAFF-R + Targeting BAFF-R + Antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against BAFF-R cells can be generated and / or incorporated into cell engagers, such as bispecific T cell engagers (e.g., BiTEs), bispecific killer engagers (e.g., BiKEs), and / or trispecific killer engagers (e.g., TriKEs), to inhibit BAFF-R. + Cells (e.g., BAFF-R + tumor) and target BAFF-R +Cell engagers can be generated that have the ability to induce one or more immune responses against cells (e.g., a T cell immune response and / or ADCC using cell engagers in the presence or absence of Fc-containing antibodies). Note that BiKE- and TriKE-mediated killing is sometimes referred to as ADCC even though it is not initiated by an Fc domain.

[0009] Additionally, as provided herein, the binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, and / or one or more antibody domains) can be used to generate conjugates comprising the binding agent and a drug. For example, ADCs, such as full-length antibody-drug conjugates, Fab-drug conjugates, and / or antibody domain-drug conjugates, can be engineered to include an appropriate binding agent provided herein to generate the conjugate. Such conjugates can deliver a drug payload to cancer cells (e.g., BAFF-R). + It can be used to deliver the drug to target cells, such as cancer cells.

[0010] Also, as provided herein, the binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) can be used to treat a mammal (e.g., a human) with cancer (e.g., one or more B-cell cancers). For example, cancers (e.g., BAFF-R) can be treated with the binding agents provided herein. + A composition comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) described herein may be administered to a mammal (e.g., a human) having a cancer (e.g., a tumor) to reduce the number of cancer cells in the mammal, induce ADCC against cancer cells in the mammal, and / or extend the mammal's survival from cancer.

[0011] Also, as provided herein, cells (e.g., host cells) can be engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R 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 capable of binding to BAFF-R. Such cells (e.g., BAFF-R-specific CAR-positive T cells or NK cells) can be used to treat cancer (e.g., one or more B-cell cancers).

[0012] In general, one aspect herein features an antibody comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of one, two, or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of one, two, or three amino acids). The antibody may have the ability to bind to SEQ ID NO:42. The heavy chain variable domain or region may comprise an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:8. The light chain variable domain or region may comprise an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody.

[0013] In another aspect, the present specification features an antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions). The antigen-binding fragment may have the ability to bind to SEQ ID NO:42. The heavy chain variable domain or region may comprise an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:8. The light chain variable domain or region may comprise an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab.

[0014] In another aspect, the document features a chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of one, two, or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of one, two, or three amino acids). The antigen-binding domain may comprise an scFv capable of binding to a BAFF-R polypeptide. The hinge may comprise the amino acid sequence set forth in SEQ ID NO: 30. The transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO: 32. The chimeric antigen receptor comprises one or more signaling domains set forth in any one of SEQ ID NOs: 34 and 36.

[0015] In another aspect, the description features a cell comprising a chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions). The cell can be a T cell, a stem cell, or a NK cell.

[0016] In another aspect, the description features a cell engager including a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody or antigen-binding fragment including a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions). The first antigen-binding domain may comprise an scFv capable of binding to a BAFF-R polypeptide. The first antigen-binding domain may be an IgG capable of binding to a BAFF-R polypeptide. The linker may comprise any one of SEQ ID NOs: 25, 26, and 30. The second antigen-binding domain may bind to a polypeptide expressed on the surface of a T cell (e.g., a CD3 polypeptide). The second antigen-binding domain may bind to a polypeptide expressed on the surface of an NK cell (e.g., a CD16a, NKG2A, NKG2D, NKp30, NKp44, NKp46, or CRTAM polypeptide). The cell engager may comprise a third antigen-binding domain. The third antigen-binding domain may bind to a polypeptide expressed on the surface of an NK cell (e.g., a CD16a, NKG2A, NKG2D, NKp30, NKp44, NKp46, or CRTAM polypeptide).

[0017] In another aspect, the description features a nucleic acid comprising a nucleic acid sequence encoding at least a portion of an antibody or antigen-binding fragment, the nucleic acid comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions). The nucleic acid sequence may encode a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The nucleic acid sequence may encode a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The nucleic acid may be a viral vector. The nucleic acid may be a phagemid.

[0018] In another aspect, the present specification provides a chimeric antigen receptor that is an antibody or antigen-binding fragment comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions). The present invention features a nucleic acid comprising a nucleic acid sequence encoding a cell engager comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The nucleic acid may be a viral vector. The nucleic acid may be a phagemid.

[0019] In another aspect, the present specification provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof, wherein the chimeric antigen receptor comprises an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions); The present invention features a host cell comprising a nucleic acid sequence encoding a cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions).

[0020] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof, the chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of one, two, or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of one, two, or three amino acids). The present invention also features a host cell capable of expressing an antibody or cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions). The host cell may be a T cell, stem cell, or NK cell.

[0021] In another aspect, the description features an ADC comprising an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions). The antigen-binding domain may comprise an scFv capable of binding to a BAFF-R polypeptide. The antigen-binding domain can be an IgG capable of binding to a BAFF-R polypeptide.The drug can be selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

[0022] In another aspect, this document features a composition comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions). The composition can comprise an antibody. The composition can comprise an antigen-binding fragment. The composition can comprise a checkpoint inhibitor. The checkpoint inhibitor can be cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, or ipilimumab.

[0023] In another aspect, the description features a composition including a cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody or antigen-binding fragment including a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions). The composition may include a checkpoint inhibitor. The checkpoint inhibitor can be cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, or ipilimumab.

[0024] In another aspect, this description features a composition comprising a cell expressing a chimeric antigen receptor described herein. The composition can include a checkpoint inhibitor, such as cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, or ipilimumab.

[0025] In another aspect, this description features a composition comprising an ADC described herein. The composition can include a checkpoint inhibitor, such as cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, or ipilimumab.

[0026] In another aspect, this document features a method of treating a mammal with cancer, comprising administering to the mammal with cancer an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions), or a first antigen-binding domain, a linker, and a second antigen-binding fragment. The method may comprise, or consist essentially of, administering a composition comprising a cell engager comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The mammal may be a human. The cancer is characterized by the administration of a BAFF-R + It could be cancer. BAFF-R +The cancer may be chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, follicular lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), or intravascular large B-cell lymphoma. After the administering step, the number of cancer cells in the mammal may be reduced.

[0027] In another aspect, this document features a method for treating a mammal with cancer, including: (a) administering to the mammal with cancer an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions), or a cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain The method may comprise, or consist essentially of, (a) administering to a mammal a composition comprising a cell engager comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and (b) administering to the mammal a composition comprising a checkpoint inhibitor. The mammal may be a human. The cancer is characterized by BAFF-R. + It's cancer. BAFF-R +The cancer may be CLL, ALL, hairy cell leukemia, follicular lymphoma, NHL, Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, DLBCL, or intravascular large B-cell lymphoma. The checkpoint inhibitor may be cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, or ipilimumab. Following administration steps (a) and (b), the number of cancer cells in the mammal may be reduced.

[0028] In another aspect, the document features a method for attaching a binding molecule to a BAFF-R polypeptide. The method can comprise, or consist essentially of, contacting the BAFF-R polypeptide with an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of one, two, or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of one, two, or three amino acids). The contacting can be performed in vitro. The contacting can be performed in vivo. The contacting can be performed in a mammal by administering the antibody or antigen-binding fragment to the mammal. The mammal can be a human.

[0029] In another aspect, this document features a method of conjugating a binding molecule to a BAFF-R polypeptide. The method includes producing a chimeric antigen receptor comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), or a chimeric antigen receptor comprising a first antigen-binding domain, a linker, and a second antigen-binding domain. a cell engager comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids); and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids); or an ADC covalently linked to a drug, wherein the antigen-binding domain is SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids),The method may comprise, or consist essentially of, contacting a BAFF-R polypeptide with an ADC comprising an antigen-binding domain comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The contacting may be performed in vitro. The contacting may be performed in vivo. The contacting may be performed inside a mammal by administering the chimeric antigen receptor, cell engager, or ADC to the mammal. The mammal may be a human.

[0030] In another aspect, this document features a method of treating a mammal having a transplanted organ, the method including administering to the mammal having the transplanted organ an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions), or a cellular engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain. The method may comprise administering, or consist essentially of, a composition comprising a cell engager comprising an antibody or antigen-binding fragment, wherein the first antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids). The mammal may be a human. The transplant organ may be a kidney, lung, liver, heart, pancreas, or bone marrow.

[0031] In another aspect, this document features a method of delaying or preventing organ transplant rejection in a mammal that has received or is preparing to receive an organ transplant. The method includes administering to a mammal that has undergone an organ transplant or is being prepared to undergo an organ transplant an antibody or antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of one, two, or three amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of one, two, or three amino acids), or a cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, The method may comprise administering a composition comprising a cell engager comprising an antibody or antigen-binding fragment, wherein the first antigen-binding domain comprises a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), thereby delaying or preventing organ transplant rejection in a mammal. The mammal may be a human.The organ transplant can be a kidney transplant, a lung transplant, a liver transplant, a heart transplant, a pancreas transplant, or a bone marrow transplant.

[0032] 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 in the practice of the present 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 the case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0033] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0034] [Figure 1-1] Figures 1A-1E show the generation of anti-BAFF-R antibodies. Figures 1A-1B) NIH / 3T3 or 293FT cells infected with BAFF-R-pCDH lentivirus were selected with puromycin and then sorted. Flow cytometry confirmed BAFF-R expression in BAFF-R-NIH / 3T3 single-cell clones (Figure 1A) or BAFF-R-293FT cell clones (Figure 1B). Figure 1C) Crude hybridoma supernatants were screened at serial dilutions for antigen-specific binding affinity to BAFF-R-positive and BAFF-R-negative 293FT cells, generating representative data. Figure 1D) Antigen-specific binding to crude C21 supernatant. Figure 1E) Antigen-specific binding affinity of purified mAb C21 to BAFF-R-positive and BAFF-R-negative 293FT cells (0.002 mg mAb / 106 cells). Data are representative of three independent experiments. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 2-1] Figures 2A–2E show the initial characterization of the MC10029 CAR construct and MC10029 CAR-T cell production. Figure 2A) Schematic diagram showing the order of functional elements in the CAR design. In addition to the scFv of the novel BAFF-R antibody, the following elements were incorporated into this construct: promoter (EF1P), signal peptide (SP), BAFF-R scFv, hinge, CD28 transmembrane (TM) domain, CD28 costimulatory domain (CD28QQ), CD3ζ, T2A (a ribosomal skipping site that stops translation), and truncated epidermal growth factor receptor (tEGFR or EGFR). Figure 2B) The fold expansion of CAR-T cells from days 1 to 14 was calculated by measuring the number of viable CAR-T cells using trypan blue staining with a Bio-Rad Cell Counter. Untransduced T cells from the same donor were used as a control. Figure 2C-2D) MC10029 CAR-T cells were stained with antibodies to measure surface expression of CD3 (Figure 2C) or EGFR (Figure 2D) for potency, and CAR-T cell characteristics were assessed using the data shown in these representative flow cytometry dot plots. Figure 2E) Quality control assays were performed on three production batches of MC10029 CAR-T cells used in various in vitro assays. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 3]Figures 3A-3B show the characterization of antigen expression in the malignant B cell lines used to characterize MC10029 CAR-T cells. BAFF-R surface expression was tested for the three cell lines used to evaluate the antigen-specific cytotoxicity of MC10029 CAR-T cells. (Figure 3A) Flow cytometry histograms using anti-BAFF-R-AF647 antibody show BAFF-R expression in Nalm-6, Z-138, or MEC-1 cells. (Figure 3B) These cell lines were also evaluated for CD19 surface expression, and CD19 knockout Nalm-6 (CD19 KO Nalm-6) cells were generated and evaluated for antigen surface expression. Flow cytometry histograms show BAFF-R or CD19 expression in wild-type Nalm-6, Z-138, or MEC-1 cell lines, and the CD19 knockout (KO) Nalm-6 cell line. Cells were stained with BAFF-R-AF647 or CD19 allophycocyanin (APC). [Figure 4-1]Figures 4A-4D show the antigen-specific cytotoxicity of MC10029 CAR-T cells targeting acute lymphoblastic leukemia (ALL) cell lines. Figures 4A and 4B show flow cytometry contour plots of CAR-T cell functional potency measured by surface expression of CD107a in a degranulation assay. MC10029 CAR-T cells were incubated with wild-type Nalm-6 cells (Nalm-6 WT) or Nalm-6 BAFF-R knockout (CD19 KO BAFF-R) cells at an E:T ratio of 2:1. Analysis was gated on CD4+ BAFF-R CAR-T cells (Figure 4A) or CD8+ BAFF-R CAR-T cells (Figure 4B). Gating based on epidermal growth factor receptor (EGFR) was used as a surrogate for BAFF-R. Non-transduced (non-CAR) T cells from the same donor were used as a negative control. Figure 4C) Direct target cell cytolysis was measured by incubating CAR-T cells with Nalm-6 cells expressing green fluorescent protein (GFP). MC10029 CAR-T cells were co-incubated with Nalm-6 WT cells or Nalm-6 BAFF-R KO cells at an E:T ratio of 20:1 for 24 hours. GFP-expressing target cells were then quantified using flow cytometry. Results shown here are representative of three independent experiments. Figure 4D) Granzyme B enzyme-linked immunosorbent assay (ELISA) further confirmed the antigen-specific potency of MC10029 CAR-T cells targeting Nalm-6 cells. MC10029 CAR-T cells or untransduced T cells generated from the same donor were co-incubated with Nalm-6 WT cells or Nalm-6-BAFF-R KO cells at an E:T ratio of 4:1. After 72 hours, supernatants were collected and granzyme B was measured by ELISA. The graphed data are the average of quadruplicate samples. Results shown are representative of three independent experiments. [Figure 4-2] Continued from Figure 4-1. [Figure 5]Figures 5A-5B show that MC10029 CAR-T cells induce antitumor effects against Nalm-6 ALL tumors in vivo. Figure 5A) The therapeutic efficacy of BAFF-R CAR-T cells was tested in a NOD scid gamma (NSG) mouse model by challenging mice with luciferase-labeled human Nalm-6 tumor cells (Nalm-6-Luc, 0.25 × 10 cells). Bioluminescence imaging was used to observe changes in luciferase-expressing Nalm-6 ALL tumor cells. Six days after tumor cell injection, tumor-bearing mice were randomly divided into three groups (N = 5 per group) and received a single intravenous (IV) injection of either vehicle (PBS), non-CAR-T cells (10 × 10 cells) generated from the same donor, or BAFF-R CAR-T cells (2 × 10 cells). Images were taken weekly, but representative images over time are shared to highlight changes due to treatment. These data are representative of two independent experiments using different donor T cells. (Figure 5B) Kaplan-Meier plots show overall survival versus days after tumor challenge, and log-rank analysis identified statistical differences between treatment groups. [Figure 6-1]Figures 6A-6D show the in vitro and in vivo cytotoxicity properties of MC10029 CAR-T cells targeting the CD19-deficient Nalm-6 cell line. Figure 6A) Flow cytometry contour plots showing the functional potency of CAR-T cells against target cells with surface-expressed CD107a in a degranulation assay. Untransduced T cells, MC10029 CAR-T cells, or CD19 CAR-T cells were generated from the same donor and incubated with CD19 KO-Nalm-6 cells at an E:T ratio of 2:1 to identify CD19 antigen-specific cytotoxicity. Analysis was gated on the CD8+ CAR-T cell population. Figure 6B) Granzyme B ELISA shows the functional potency of MC10029 CAR-T cells targeting CD19 KO-Nalm-6 cells. Untransduced T cells, MC10029 CAR-T cells, or CD19 CAR-T cells were incubated with WT or Nalm-6-CD19 KO cells at an E:T ratio of 4:1 for 72 hours, and supernatants were collected for subsequent ELISA. Graphed data are the average of quadruplicate samples. Data are representative of three independent experiments. Figure 6C) Bioluminescence imaging catalog showing changes in a luciferase-expressing, CD19-deficient Nalm-6 model (CD19 KO-Nalm6-Luc, 0.25 x 106 cells) injected into NSG mice and treated with one of four treatment regimens. Six days after tumor cell injection, tumor-bearing mice were randomly divided into four groups (N = 5 per group). On day 7, mice received a single injection of either PBS (vehicle), untransduced T cells (10 x 10 cells), MC10029 CAR-T cells (2 x 10 cells), or CD19 CAR-T cells (2 x 10 cells) from the same donor. Tumor burden was quantified by bioluminescence intensity. Results shown are representative of two independent experiments using T cells from different donors. (Figure 6D) Kaplan-Meier plots show overall survival versus days after tumor challenge. Log-rank analysis revealed statistically significant differences between treatment groups. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 7-1]Figures 7A-7D show in vitro and in vivo cytotoxicity characterization of MC10029 CAR-T cells targeting the lymphoma cell line Z138. Figure 7A) Flow cytometry contour plots showing the functional potency of CAR-T cells against target cells by surface expression of CD107a in a degranulation assay. Untransduced T cells and MC10029 CAR-T cells were generated from the same donor and incubated with Z138 cells at an E:T ratio of 2:1 to characterize antigen-specific cytotoxicity. Analysis was gated on the CD8+ CAR-T cell population. Figure 7B) Granzyme B ELISA showing the functional potency of MC10029 CAR-T cells targeting Z138 cells. Untransduced T cells or MC10029 CAR-T cells were cocultured with Z138 cells at an E:T ratio of 4:1 for 72 hours, and then supernatants were collected for ELISA. Graphed data are the average of quadruplicate samples. Data are representative of three independent experiments. Figure 7C) Bioluminescence imaging catalog showing changes following injection of a luciferase-expressing Z138 model (Z138-Luc, 0.5x106 cells) into NSG mice followed by treatment with MC10029 CAR-T cells. Six days after tumor cell injection, tumor-bearing mice were randomly divided into three groups (N=5 per group). On day 7, mice received a single injection of either PBS (vehicle), non-CAR-T cells (untransduced T cells) from the same donor (10x106 cells), or MC10029 CAR-T cells (2x106 cells). Tumor burden was quantified by bioluminescence intensity. Results shown are representative of two independent experiments using T cells from different donors. Figure 7D) Kaplan-Meier plot showing overall survival versus days after Z138 tumor challenge, and log-rank analysis identified statistical differences between treatment groups. [Figure 7-2] Continued from Figure 7-1. [Figure 8-1]Figures 8A-8D show the in vitro and in vivo cytotoxicity characteristics of MC10029 CAR-T cells targeting the chronic lymphocytic leukemia (CLL) cell line MEC-1. Figure 8A) Flow cytometry contour plots showing the functional potency of CAR-T cells against target cells with surface-expressed CD107a in a degranulation assay. Untransduced T cells and MC10029 CAR-T cells were generated from the same donor and incubated with MEC-1 cells at an E:T ratio of 2:1 to assess antigen-specific cytotoxicity. Analysis was gated on the CD8+ CAR-T cell population. Figure 8B) Granzyme B ELISA assay showing the functional potency of MC10029 CAR-T cells targeting MEC-1. Untransduced T cells or MC10029 CAR-T cells were cocultured with MEC-1 cells at an E:T ratio of 4:1 for 72 hours, and then supernatants were collected for ELISA. Graphed data are the average of quadruplicate samples. Data are representative of three independent experiments. Figure 8C) Bioluminescence imaging catalog showing changes in a luciferase-expressing MEC-1 model (MEC-1-Luc, 1.0 x 106 cells) injected into NSG mice and subsequently treated with one of three treatment regimens. Six days after tumor cell injection, tumor-bearing mice were randomly divided into three groups (N = 5 per group). On day 7, mice received a single injection of either PBS (vehicle), untransduced T cells (10 x 106 cells) from the same donor, or MC10029 CAR-T cells (2 x 106 cells). Tumor burden was quantified by bioluminescence intensity. Results shown are representative of two independent experiments using T cells from different donors. Figure 8D) Kaplan-Meier plot showing overall survival versus days after MEC-1 tumor challenge, and log-rank analysis identified statistical differences between treatment groups. [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 9-1]Figures 9A-9C show that MC10029 CAR-T cells elicited cytotoxicity against primary CLL tumors in vitro. Figure 9A) Flow cytometry assay showing cell surface expression of BAFF-R peripheral blood mononuclear cells (PBMCs) collected from six CLL patients. Fluorescence minus 1 (FMO) staining was used as the gating method. Figure 9B) Enriched primary B-cell tumor samples from six subjects were incubated with MC10029 CAR-T cells from two different healthy donors at an E:T ratio of 2:1. The CD107a degranulation assay was used to visualize the cytotoxicity of MC10029 CAR-T cells by flow cytometry. Analysis was gated on the CD8+ CAR-T cell population. Untransduced T cells from the same donor were used as a negative control. Figure 9C) Granzyme B release confirmed the functional efficacy of MC10029 CAR-T cells targeting primary CLL tumor cells. MC10029 CAR-T cells or non-transduced T cells were co-cultured with enriched primary tumor cells isolated from CLL patients for 72 hours (E:T ratio 4:1). Granzyme B was measured from the harvested supernatant. Graphed data are the average of quadruplicate samples. [Figure 9-2] Continued from Figure 9-1. [Figure 10] Control experiments were performed to confirm the quality of both the B cells used and the generated CAR-T cells. To confirm that the activity of MC10029 CAR-T cells targeted primary CLL tumor cells, PBMCs were enriched for tumor cells and CLL B cells. To confirm the depletion of endogenous T cells, flow cytometry was used to characterize CD3+ cell populations collected from six CLL patients in the original PBMC samples (Figure 10, top panel) and after B cell enrichment (Figure 10, bottom panel). Cells were stained with CD3-BV605. [Figure 11-1]Figures 11A-11C show the characterization of MC10029 CAR-T cells, which targeted primary CLL tumor cells and elicited the responses shown in Figures 9B-9C. MC10029 CAR-T cells were generated from two healthy donors. Two production batches of MC10029 CAR-T cells were characterized using standard QC assays. The identity (CD3-positive cells) and potency (EGFR abundance) of MC10029 CAR-T cells from donor A (Figure 11A) were similar to those from donor B (Figure 11B). Although the fold expansion (final total number of MC10029 CAR-T cells) differed between the two batches (Figure 11C), both fold expansion rates allowed for comparison of the two batches of MC10029 CAR-T cells in in vitro assays. [Figure 11-2] Continued from Figure 11-1. [Figure 12-1] Figures 12A-12B show the quality control assay results for three engineered clinical-grade MC10029 CAR-T cells. Figure 12A) Quality control product release criteria for CAR-T cell batches are shown. Three batches of MC10029 CAR-T cells and corresponding non-CAR-T cell controls were evaluated for cell quality by fold expansion (>25x) and viability (>70%), and for CAR-T cell-specific characteristics by identity (>80%) and potency (>10%). To demonstrate that the lentiviral vector is noninfectious and safe against adventitious viral agents, lentiviral copy number (WPRE <5 copies per cell) was determined by real-time qPCR, and vesicular stomatitis virus G glycoprotein (VSVG) was assayed by qPCR (<5 copies / 50 ng DNA). Figure 12B) Flow cytometry contour plot of CAR-T cell functional potency measured by CD107a degranulation assay. MC10029 CAR-T cells (characterized in Figure 12A) from three different healthy donors were incubated with Nalm-6 WT or Nalm-6 BAFF-R KO at an E:T ratio of 2:1. Analysis was gated on the CD8+ T cell population. Non-transduced T cells from the same donors were used as a negative control. [Figure 12-2]Continued from Figure 12-1. [Figure 13] FIG. 13 is a schematic diagram of an exemplary vector that can encode the C21-BAFF-R 10029 CAR. [Figure 14] 14 is a schematic diagram of an exemplary BiTE designed in an Ig format (e.g., an IgG1 format) using the heavy chain CDR1, CDR2, and CDR3 shown herein and the light chain CDR1, CDR2, and CDR3 shown herein. A humanized anti-CD3 scFv (e.g., the gOKT3-7 scFv shown in U.S. Pat. No. 6,750,325) can be linked to the C-terminus of the light chain via a linker (e.g., a linker shown in Example 10, such as the (G4S)3 (SEQ ID NO: 25) linker). [Figure 15-1] Figures 15A-15C show that subject-derived MC10029 CAR-T cells induced ex vivo cytotoxicity against autologous CLL tumors. Figure 15A) Growth curves of MC10029 CAR-T cells derived from T cells isolated from the peripheral blood of three CLL subjects and their corresponding non-CAR-T cells were plotted side-by-side to compare growth patterns. Figure 15B) The identity (>80% CD3-positive cells) and potency (>10% EGFR-positive cells) of subject-derived MC10029 CAR-T cells were assessed. Figure 15C) The cytotoxicity of MC10029 CAR-T cells derived from three CLL subjects was assessed against autologous tumor cells using a CD107a degranulation assay. MEC-1 cells were used as a positive control, and corresponding non-CAR-T cells were used as a negative control. Cytotoxicity analysis was gated on the CD8+ CAR-T cell population. [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 16-1]Figures 16A-16B show the characteristics of three qualification products of clinical-grade MC10029 CAR-T cells. Figure 16A) Product release criteria for CAR-T cells. Three batches of MC10029 CAR-T cells were assessed for cell quality by fold expansion (>25x) and viability (>70% as measured by trypan blue staining), and for CAR-T cell-specific characteristics by identity (>80% as measured by flow cytometry for CD3-positive cells) and potency (>10% as measured by flow cytometry for EGFR (transgene)-positive T cells). Matched non-CAR-T cells served as controls. To demonstrate that the lentiviral vector is noninfectious and safe against adventitious viral agents, lentiviral copy number (WPRE <5 copies per cell) and VSVG (<5 copies per 50 ng of DNA) were measured by qPCR assay. Figure 16B) Flow cytometry contour plot of CAR-T cell functional potency as measured by CD107a degranulation assay. MC10029 CAR-T cells (characterized in panel A) from three different healthy donors were incubated with WT Nalm-6 or BAFF-R KO Nalm-6 at an E:T ratio of 2:1. Analysis was gated on the CD8+ CAR-T cell population, and no differences were observed between batches (Figure 20F). Non-transduced T cells (non-CAR-T) from the same donors were used as a negative control. [Figure 16-2] Continued from Figure 16-1. [Figure 17-1]Figures 17A-17C show a comparison of BAFF-R CAR-T cell constructs with different costimulatory domains. Using the option of complementing CD3ζ with either the CD28 costimulatory domain (MC10029) or the 4-1BB costimulatory domain (MC10023), both CARs were generated and transduced into T cells to determine which BAFF-R CAR-T cells exhibited superior antigen-specific cytotoxicity. Figure 17A) MC10023 and MC10029 CAR-T cell proliferation showed comparable increases by day 14. Figure 17B) The T cell content (CD3) and CAR-T cell percentage (using EGFR as a proxy) in the final MC10029 and MC10023 CAR-T cell populations were also found to be comparable. (Figure 17C) Antigen-specific cytotoxicity was assayed by testing MC10023 or MC10029 CAR-T cells against Nalm-6 and Nalm-6 BAFF-R KO cell lines. This degranulation assay demonstrated the enhanced activity of MC10029 CAR-T cells compared to MC10023 CAR-T cells, providing the rationale for introducing MC10029 CAR-T cells into further experiments. [Figure 17-2] Continuation of Figure 17-1. [Figure 18-1]Figures 18A-18F show the in vitro cytotoxicity of MC10029 CAR-T cells against CD19-deficient malignant B-cell tumor lines. Antigen expression was tested for the three cell lines used to evaluate the cytotoxicity of MC10029 CAR-T cells. Figure 18A) Flow cytometry histograms using anti-BAFF-R-AF647 antibody show BAFF-R expression on Nalm-6, Z-138, or MEC-1. Figure 18B) These cell lines were also evaluated for CD19 surface expression using anti-CD19-APC antibody. CD19 knockout (KO) Nalm-6, Z-138, and MEC-1 variants were generated and evaluated for antigen surface expression. Flow cytometry histograms show BAFF-R expression on wild-type Nalm-6, Z-138, or MEC-1 cell lines and their CD19-deficient counterparts. Figures 18C and 18E) Flow cytometry plots show the functional efficacy of CAR-T cells against CD19-deficient tumor cells by surface-expressed CD107a in a degranulation assay. Non-CAR-T cells, MC10029 CAR-T cells, or CD19 CAR-T cells were generated from the same donor and incubated with CD19-deficient Z-138 cells (Figure 18C) or CD19-deficient MEC-1 cells (Figure 18E) at an E:T ratio of 2:1, and the cytotoxicity of MC10029 CAR-T cells against these CD19-deficient tumor cells was analyzed. Figures 18D and 18F) Granzyme B ELISA shows the functional efficacy of MC10029 CAR-T cells against CD19-deficient tumor cells. Non-CAR-T cells, MC10029 CAR-T cells, or CD19 CAR-T cells were co-incubated with CD19-deficient Z-138 cells (Figure 18D) or CD19-deficient MEC-1 cells (Figure 18F) at an E:T ratio of 4:1 for 72 hours, and supernatants were collected and subjected to ELISA. Graphed data are the average of quadruplicate samples. Data are representative of three independent experiments. [Figure 18-2] Continuation of Figure 18-1. [Figure 18-3] Continuation of Figure 18-2. [Figure 18-4] Continued from Figure 18-3. [Figure 19-1]Figures 19A-19D show the activity of MC10029 CAR-T cells against primary CLL tumor cells. Figure 19A) Basic demographic data for nine B-CLL subjects evaluated with MC10029 CAR-T cells. Figure 19B) Subject PBMCs were enriched for CLL tumor cells, and characterization of CD3-positive T cells in the original PBMC samples (top row) and the enriched B cell population (bottom row) confirmed depletion of endogenous T cells in these six samples. Figure 19C) Statistical analysis of the degranulation data in Figure 9B shows statistical significance in cytotoxicity against primary CLL tumor cells between MC10029 CAR-T cells and non-CAR-T cells (*p<0.05). However, no difference was observed between donor A and donor B. Figure 19D) Release of multiple granule proteins / cytokines from MC10029 CAR-T cells incubated with primary CLL tumor cells isolated from selected subjects. A significant increase in granule protein release was observed in the CAR-T cell group compared to the non-CAR-T cell group (**p<0.01). [Figure 19-2] Continuation of Figure 19-1. [Figure 19-3] Continuation of Figure 19-2. [Figure 20-1] Figures 20A-20F show analyses of CD107a degranulation assays. Figure 20A) Statistical analysis of degranulation data for MC10029 CD4 CAR-T cells in Figure 4A. Figure 20B) Statistical analysis of degranulation data for MC10029 CD8 CAR-T cells in Figure 4B. Figure 20C) Statistical analysis of degranulation data for MC10029 CAR-T cells on CD19 KO Nalm-6 cells in Figure 6A. Figure 20D) Statistical analysis of degranulation data for MC10029 CAR-T cells on Z-138 cells in Figure 7A. Figure 20E) Statistical analysis of degranulation data for MC10029 CAR-T cells on MEC-1 cells in Figure 8A. Figure 20F) Statistical analysis of degranulation data for the three production batches of MC10029 CAR-T cells shown in Figure 16B. (**p<0.01; ns: not significant). [Figure 20-2] Continuation of Figure 20-1. [Figure 20-3] Continued from Figure 20-2. [Figure 21-1]Figures 21A-21C show the antigen-specific cytotoxicity of patient-derived MC10029 CAR T cells against autologous B cells. Figure 21A) The B cell enrichment process from the peripheral blood of a representative patient efficiently depleted T cells (CD3-positive cells) and significantly enriched CD20-positive B cells (95.9%). These enriched B cells showed a strong positive signal for BAFF-R expression (shown in the upper histogram). Enriched B cells from a healthy donor were used as a positive control for T and B cell immunostaining. Figure 21B) Antigen-specific cytotoxicity was assessed by measuring the surface expression of CD107a, a degranulation marker. Autologous B cells isolated from a representative patient activated patient-derived MC10029 CAR T cells (MC10029 CAR-T) and induced degranulation. To verify the antigen-specific functionality of CAR T cells, we paired BAFF-R-positive and BAFF-R-deficient Nalm-6 cells (Nalm-6 BAFF-R KO). Figure 21C) Incubation of MC10029 CAR T cells with BAFF-R-expressing autologous B cells resulted in a significant release of the cytolytic protein granzyme B compared with non-CAR T cells (P = 0.0123). Data are plotted as the mean ± SEM of triplicate samples and analyzed using Student's T-test (*P < 0.05, **P < 0.01, ns = not significant). [Figure 21-2] Continuation of Figure 21-1. [Figure 22]Figure 22 shows a schematic diagram of an exemplary method for generating CAR T cells from a patient blood sample. (1) Blood is collected from a sensitized kidney transplant patient and PBMCs are isolated. (2) B cells are enriched using the EasySep™ Direct Human B Cell Isolation Kit and characterized by surface markers to confirm purity and BAFF-R expression. (3) T cells are isolated using a pan T cell isolation kit and transduced with the MC10029 CAR lentivirus. (5) The resulting MC10029 CAR T cells are expanded, characterized, and subjected to a product release assay. (7) A second blood collection is performed at least two weeks after the first collection to isolate B cells. (9) B cells are enriched using the EasySep™ Human B Cell Isolation Kit. (10) Patient-derived MC10029 CAR T cells are incubated with autologous B cells. (11) Antigen-specific cytotoxicity is measured using flow cytometry (CD107a degranulation assay) and ELISA (cytokine release assay). [Figure 23] Figure 23 shows confirmation of antigen-specific functionality of patient-derived MC10029 CAR T cells from patients 1 to 6. Patient-derived MC10029 CAR T cells were stimulated with BAFF-R-positive Nalm-6 cells to induce degranulation. Degranulation was measured by assessing surface expression of CD107a, an established marker of degranulation. To confirm antigen-specific functionality of CAR T cells, BAFF-R-deficient Nalm-6 cells (Nalm-6 BAFF-R KO) were used as an antigen-negative control. [Figure 24]Figures 24A-24B show that MC10029 CAR T cells from a sensitized patient who had a failed lung transplant exhibited antigen-specific cytotoxicity against autologous B cells. Figure 24A) Enriched autologous B cells activated patient-derived MC10029 CAR T cells (MC10029 CAR-T) and induced degranulation. BAFF-R-positive Nalm-6 cells and BAFF-R-deficient Nalm-6 cells (Nalm-6 BAFF-R KO) were used as controls. Figure 24B) Significant granzyme B release was detected when MC10029 CAR T cells from patient 10 were incubated with autologous B cells or BAFF-R-positive Nalm-6 cells, but not when incubated with non-CAR-T cells (***P<0.001; ns = not significant). [Figure 25] Figure 25 shows the antigen-specific cytotoxicity of MC10029 CAR T cells from patient 7. Stimulation of patient-derived MC10029 CAR T cells with BAFF-R-positive Nalm-6 cells resulted in degranulation. Degranulation was measured by assessing surface expression of CD107a, an established marker of degranulation. To confirm the antigen-specific functionality of the CAR T cells, BAFF-R-deficient Nalm-6 cells (Nalm-6 BAFF-R KO) were used as an antigen-negative control. [Figure 26] Figures 26A-26B show direct killing of B cells by MC10029 CAR T cells derived from a primed kidney transplant patient. Figure 26A) A direct killing assay was developed using MC10029 CAR T cells derived from a healthy donor with B cells as target cells. Non-CAR T cells or MC10029 CAR T cells were co-cultured with freshly isolated autologous B cells at an E:T ratio of 10:1 for 1 hour. Cells were then assessed by flow cytometry to quantitate viable CD20+ B cells. Non-CAR T cells were used as a negative control. Figure 26B) MC10029 CAR T cells obtained from a primed kidney transplant patient (patient 5) demonstrated the ability to induce cell killing in B cells freshly isolated from the PBMCs of a healthy donor. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present application provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). For example, the present specification provides binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a polypeptide comprising, consisting essentially of, or consisting of a BAFF-R amino acid sequence set forth in SEQ ID NO: 42 (see, e.g., Example 2).

[0036] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, single-chain variable fragment antibodies (e.g., scFv antibodies), and tandem single-chain variable fragment antibodies (e.g., taFv). Diabodies may contain two chains, each with a heavy chain variable domain and a light chain variable domain, derived from the same or different antibodies (see, e.g., Hornig and Farber-Schwarz, Methods Mol. Biol., 907:713-27 (2012); Brinkmann and Kontermann, MAbs., 9(2):182-212 (2017)). The two variable regions may be linked by a polypeptide linker (e.g., a polypeptide linker of 5 to 10 amino acids in length, or the polypeptide linker described in Example 10). In some cases, interdomain disulfide bonds may exist in one or both of the heavy and light chain variable domain pairs of the diabody. scFvs are single-chain polypeptide antibodies in which the heavy and light chain variable domains are linked directly or via a polypeptide linker (e.g., an 8- to 18-amino acid long polypeptide linker, or a polypeptide linker as described in Example 10). See also Chen et al., Adv. Drug Deliv. Rev., 65(10):1357-1369 (2013). scFvs can be designed with a heavy chain variable domain followed by a light chain variable domain, or a light chain variable domain followed by a heavy chain variable domain. In either case, an optional linker can be placed between the two domains. Examples of scFv structures described herein include, but are not limited to, the structures shown in Examples 7, 8, and 9.

[0037] The antibodies described herein may comprise the CDRs described herein (e.g., an antibody comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be configured as human antibodies, humanized antibodies, or chimeric antibodies. In some cases, the antibodies described herein may comprise the CDRs described herein (e.g., an antibody comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be monoclonal antibodies. In some cases, the antibodies provided herein may comprise the CDRs described herein (e.g., an antibody comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be configured as scFv antibodies.

[0038] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody (e.g., a fragment of a humanized antibody, a fragment of a human antibody, or a fragment of a chimeric antibody) that has the ability to bind to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', or F(ab')2 antigen-binding fragments. The antigen-binding fragments provided herein may contain the CDRs described herein (e.g., an antigen-binding fragment comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be configured to be human, humanized, or chimeric antigen-binding fragments. In some cases, the antigen-binding fragments provided herein may contain the CDRs described herein (e.g., an antigen-binding fragment comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be monoclonal antigen-binding fragments. In some cases, the antigen-binding fragments described herein may contain the CDRs described herein (e.g., an antigen-binding fragment comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3 and a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be configured as a Fab antibody. In some cases, the Fab antibody may contain a hinge sequence for disulfide bonding between the heavy and light chains of the Fab.

[0039] As used herein, the term "antibody domain" 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 the antibody. In some cases, the antibody domain may be a single antibody domain (e.g., a VH domain or a VL domain) capable of binding to an antigen. The antibody domains provided herein may comprise the CDRs described herein (e.g., an antibody domain comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3, or a light chain variable domain having the CDRs of SEQ ID NOs: 9-11), and may be a human antibody domain (e.g., a human VH domain), a humanized antibody domain (e.g., a humanized VH domain), or a chimeric antibody domain (e.g., a chimeric VH domain). In some cases, the antibody domains described herein may comprise the CDRs described herein (e.g., an antibody domain comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3, or a light chain variable domain having the CDRs of SEQ ID NOs: 9-11), and may be a monoclonal antibody domain. In some cases, the antibody domains described herein may comprise the CDRs described herein (e.g., an antibody domain comprising a heavy chain variable domain having the CDRs of SEQ ID NOs: 1-3, or a light chain variable domain having the CDRs of SEQ ID NOs: 9-11) and may be produced as a single VH domain or a single VL domain.

[0040] The anti-BAFF-R antibodies, anti-BAFF-R antigen-binding fragments, or anti-BAFF-R antibody domains described herein can be, but are not limited to, IgA, IgD, IgE, IgG, or IgM types, including IgG or IgM types such as IgG1, IgG2, IgG3, IgG4, IgM1, and IgM2. In some cases, the antibodies (e.g., anti-BAFF-R antibodies) described herein can be scFv antibodies. In some cases, the antigen-binding fragments (e.g., anti-BAFF-R antibody fragments) described herein can be Fab. In some cases, the antibodies (e.g., anti-BAFF-R antibodies) provided herein can be complete, intact antibodies having the structure described in Example 6. In some cases, the antibody domains (e.g., anti-BAFF-R antibody domains) provided herein can be VH domains.

[0041] The term "chimeric antigen receptor" as used herein refers to a chimeric polypeptide designed to include an optional signal peptide, an antigen-binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains. As described herein, the antigen-binding domain of the CARs provided herein can be designed to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). For example, the CARs provided herein can be designed to include components of an antibody, antigen-binding fragment, and / or antibody domain (e.g., a combination of CDRs) described herein as an antigen-binding domain, which has the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). In some examples, the CARs provided herein can be designed to include an antigen-binding domain comprising two sets of three CDRs (e.g., CDR1, CDR2, and CDR3 of the heavy chain and CDR1, CDR2, and CDR3 of the light chain) of the antigen-binding fragments provided herein (e.g., SEQ ID NOS: 1-3 and 9-11). In some cases, the antigen binding domain of a CAR-T targeting a BAFF-R polypeptide can be designed to comprise a VH domain described herein or an scFv antibody described herein. Examples of CAR structures that can be used to generate the CARs described herein include, but are not limited to, those shown in Figure 2A.

[0042] In some cases, the CARs provided herein can be designed to include a signal peptide. Any suitable signal peptide can be used to design the CARs provided herein. In some cases, the signal peptide that can be used to generate the CARs provided herein can be derived from a human polypeptide. Examples of signal peptides that can be used to generate the CARs provided herein include, but are not limited to, a GMCSF-derived signal peptide (e.g., a human GMCSF-derived signal peptide), a CD3-derived signal peptide, a CD4-derived signal peptide, and a CD8-derived signal peptide. In some cases, the CARs provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences provided in Example 11. In some cases, the CARs provided herein can be designed to include a signal peptide that includes, consists essentially of, or consists of one of the amino acid sequences provided in Example 11, including one with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, the CARs provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences shown in Example 11 with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0043] In some cases, the CARs described herein can be designed to include a hinge. Any suitable hinge can be used to design the CARs described herein. Examples of hinges that can be used to create the CARs described herein include, but are not limited to, Ig-derived hinges (e.g., IgG4-derived hinges, such as human IgG4-derived hinges), CD8-derived hinges (e.g., partial CD8 extracellular domains), and CD28-derived hinges (e.g., partial CD28 extracellular domains). The CARs described herein can be designed to include hinges of any suitable length. For example, the CARs provided herein can be designed to include a hinge having a length of about 3 to about 250 amino acid residues (e.g., about 3 to about 200, about 3 to about 150, about 3 to about 100, about 3 to about 50, about 5 to about 250, about 25 to about 250, about 50 to about 250, about 75 to about 250, about 100 to about 250, about 150 to about 250, about 5 to about 200, about 10 to about 150, about 20 to about 100, about 25 to about 75, about 10 to about 40, about 20 to about 50, about 30 to about 60, about 50 to about 100, or about 80 to about 120). In some cases, a linker sequence can be used as a hinge to produce the CARs described herein. For example, any one of the linker sequences described in Example 10 can be used as a hinge in the CARs described herein. In some cases, the CARs described herein can be designed to include a hinge comprising, consisting essentially of, or consisting of one of the amino acid sequences shown in Example 10 or Example 12. In some cases, the CARs described herein can be designed to include a hinge comprising, consisting essentially of, or consisting of one of the amino acid sequences shown in Example 10 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, the CARs provided herein can be designed to include a hinge comprising, consisting essentially of, or consisting of one of the amino acid sequences shown in Example 10 or Example 12, with two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, or ten or fewer amino acid deletions, additions, substitutions, or combinations thereof.

[0044] The CARs provided herein can be designed to include any suitable transmembrane domain. Examples of transmembrane domains that can be used to generate the CARs described herein include, but are not limited to, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD3 zeta transmembrane domain, and an ICOS transmembrane domain. In some cases, the CARs provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences shown in Example 13. In some cases, the CARs provided herein can be designed to include a transmembrane domain that includes, consists essentially of, or consists of one of the amino acid sequences shown in Example 13, including those with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, the CARs provided herein can be designed to include a transmembrane domain that consists essentially of, or consists of, including one of the amino acid sequences shown in Example 13, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0045] The CARs described herein can be designed to include one or more intracellular signaling domains. For example, the CARs described herein can be designed to include one, two, three, or four intracellular signaling domains. Any suitable intracellular signaling domain or combination of intracellular signaling domains can be used to generate the CARs described herein. Examples of intracellular signaling domains that can be used to generate the CARs described herein include, but are not limited to, a CD3ζ intracellular signaling domain, a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, and an ICOS intracellular signaling domain. In some cases, the CARs described herein can be designed to be first-generation CARs having a CD3ζ intracellular signaling domain. In some cases, the CARs described herein can be designed to be second-generation CARs having a CD28 intracellular signaling domain followed by a CD3ζ intracellular signaling domain. In some cases, the CARs described herein can be designed to be third-generation CARs having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an OX40 intracellular signaling domain, or a 4-1BB intracellular signaling domain followed by (c) a CD3ζ intracellular signaling domain. In some cases, the CARs described 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, the CARs provided herein can be designed to include at least one intracellular signaling domain that includes, consists essentially of, or consists of one of the amino acid sequences set forth in Example 14 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling.In some cases, the CARs provided herein can be designed to include at least one intracellular signaling domain that consists essentially of, or consists of, including one of the amino acid sequences set forth in Example 14 with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling.

[0046] In some cases, a CAR targeting a BAFF-R polypeptide can be designed to include an scFv having a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as the linker shown in Example 10, and 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 the hinge / linker shown in Example 10 or Example 12 (e.g., an IgG4-derived hinge), followed by a transmembrane domain such as the transmembrane domain shown in Example 13 (e.g., a human CD28 transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domains shown in Example 14 (e.g., a human CD28 intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR targeting a BAFF-R 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 light chain variable domain comprising SEQ ID NO:25, followed by SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by an scFv having SEQ ID NO:30, followed by SEQ ID NO:32, followed by SEQ ID NO:34, followed by SEQ ID NO:36, followed by SEQ ID NO:38.

[0047] In some cases, a CAR targeting a BAFF-R polypeptide can be designed to comprise an scFv having a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker such as that shown in Example 10, a light chain variable domain comprising SEQ ID NO:16, followed by a hinge (e.g., an IgG4-derived hinge) such as the hinge / linker shown in Examples 10 or 12, followed by a transmembrane domain (e.g., a human CD28 transmembrane domain) such as that shown in Example 13, followed by one or more intracellular signaling domains (e.g., a human CD28 intracellular signaling domain, followed by a human CD3ζ intracellular signaling domain), such as one or more intracellular signaling domains shown in Example 14. For example, a CAR targeting a BAFF-R polypeptide can be designed to comprise a heavy chain variable domain comprising SEQ ID NO:8, followed by SEQ ID NO:25, followed by a light chain variable domain comprising SEQ ID NO:16, followed by SEQ ID NO:30, followed by SEQ ID NO:32, followed by SEQ ID NO:34, followed by SEQ ID NO:36, followed by an scFv having SEQ ID NO:38.

[0048] In some cases, a CAR targeting a BAFF-R polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker such as that shown in Example 10, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a hinge (e.g., an IgG4-derived hinge), such as a hinge / linker such as that shown in Example 10 or Example 12, followed by a transmembrane domain (e.g., a human CD28 transmembrane domain) such as that shown in Example 13, followed by one or more intracellular signaling domains (e.g., a human CD28 intracellular signaling domain followed by a human CD3ζ intracellular signaling domain), such as that shown in Example 14. For example, a CAR targeting a BAFF-R polypeptide can be designed to contain a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by SEQ ID NO:25, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by SEQ ID NO:30, followed by SEQ ID NO:32, followed by SEQ ID NO:34, followed by SEQ ID NO:36, followed by an scFv having SEQ ID NO:38 (see, e.g., Example 16).

[0049] In some cases, a CAR targeting a BAFF-R polypeptide can be designed to include an scFv having a light chain variable domain comprising SEQ ID NO: 16, followed by a linker such as that shown in Example 10, a heavy chain variable domain comprising SEQ ID NO: 8, followed by a hinge (e.g., an IgG4-derived hinge) such as the hinge / linker shown in Examples 10 or 12, followed by a transmembrane domain (e.g., a human CD28 transmembrane domain) such as that shown in Example 13, followed by one or more intracellular signaling domains (e.g., a human CD28 intracellular signaling domain, followed by a human CD3ζ intracellular signaling domain) such as that shown in Example 14. For example, a CAR targeting a BAFF-R polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO: 16, followed by SEQ ID NO: 25, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by SEQ ID NO: 30, followed by SEQ ID NO: 32, followed by SEQ ID NO: 34, followed by SEQ ID NO: 36, followed by an scFv having SEQ ID NO: 38 (see, e.g., Example 16).

[0050] In some cases, the CARs provided herein may comprise (e.g., may be designed to comprise) one or more additional components. Examples of additional components that may be included in the CARs provided herein include, but are not limited to, a detectable marker, a suicide switch, and a surface marker that aids in enrichment. When the CARs provided herein comprise a detectable marker, the detectable marker can be any suitable detectable marker. Examples of detectable markers that may be included in the CARs provided herein include, but are not limited to, a non-functional polypeptide (e.g., a truncated epidermal growth factor receptor (tEGFR) polypeptide), a bioluminescent polypeptide (e.g., a luciferase polypeptide), a fluorescent polypeptide (e.g., a green fluorescent polypeptide (GFP)), and a ribosomal skipping polypeptide (e.g., a T2A polypeptide). For example, the CARs provided herein may comprise a tEGFR polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 38 (see, e.g., Example 15).

[0051] As used herein, the term "cell engager" refers to a polypeptide that comprises two or more antigen-binding domains (e.g., two, three, or four antigen-binding domains) and has the ability to link two cells together. Examples of cell engagers include, but are not limited to, BiTE, BiKE, and TriKE. Generally, the cell engagers provided herein can be designed to comprise at least one antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and at least one antigen-binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell). In some cases, the cell engagers described herein can bind to BAFF-R via the two or more antigen-binding domains of the cell engager. + Cells (e.g., BAFF-R + The cell engager may link a specific cell (e.g., a cancer cell) to another cell (e.g., a T cell or an NK cell). Examples of cell engager structures for the cell engagers provided herein include, but are not limited to, the structure shown in Figure 14. In some cases, the anti-CD3 scFv shown in Figure 14 can be replaced with a different antigen-binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell).

[0052] When a cell engager comprises an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R 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 may bind to a different antigen expressed on the surface of a different cell type, or may bind to a different antigen expressed on the surface of the same cell type. For example, TriKE can be designed to have a first antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide), a second antigen-binding domain capable of binding to a first antigen expressed on the surface of an NK cell (e.g., a CD16 polypeptide, such as a CD16a polypeptide), and a third antigen-binding domain capable of binding to a second antigen expressed on the surface of an NK cell (e.g., an NKG2A polypeptide).

[0053] As described herein, at least one antigen-binding domain of a cell engager described herein can be designed to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). For example, a cell engager described herein can be designed to include components of an antibody, antigen-binding fragment, and / or antibody domain (e.g., a combination of CDRs) described herein as an antigen-binding domain, which antigen-binding domain has the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). In some examples, a cell engager described herein can be designed to include an antigen-binding domain comprising two sets of three CDRs (e.g., CDR1, CDR2, and CDR3 of the heavy chain and CDR1, CDR2, and CDR3 of the light chain) of an antigen-binding fragment provided herein (e.g., SEQ ID NOS: 1-3 and 9-11). In some cases, the antigen-binding domain of a cell engager targeting a BAFF-R polypeptide can be designed to include a VH domain described herein, or an scFv or Fab antibody described herein. In some cases, the antigen-binding domain of a CAR described herein that has the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) can be expressed as a BAFF-R polypeptide. + It can be used as an antigen-binding domain of a cell engager to target cells.

[0054] As described herein, a cell engager can be designed to include at least one antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and at least one other antigen-binding domain. The at least one other antigen-binding domain can be capable of binding to any suitable antigen expressed on the cell surface. For example, a BAFF-R polypeptide can be designed to bind to a BAFF-R polypeptide. +When designing a cell engager, such as a BiTE, for linking cells and T cells, the cell engager may comprise an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell. Examples of polypeptides expressed on the surface of T cells that can be targeted by the antigen-binding domain of the cell engagers described herein include, but are not limited to, CD3 polypeptide and 4-1BB polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of T cells that can be used to generate the cell engagers (e.g., BiTEs) provided herein include, but are not limited to, anti-CD3 scFv, anti-CD3 VH domain, anti-4-1BB scFv, and anti-4-1BB VH domain. Further examples of amino acid sequences that can be used as antigen-binding domains capable of binding to a polypeptide expressed on the surface of T cells (e.g., CD3) are described in U.S. Patent No. 6,750,325 (see, e.g., the sequence listing in U.S. Patent No. 6,750,325).

[0055] BAFF-R +When designing a cell engager such as BiKE or TriKE to link cells with NK cells, the cell engager may comprise an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and one or more (e.g., one, two, or three) antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell. Examples of polypeptides expressed on the surface of NK cells that can be targeted by the antigen-binding domains of the cell engagers described herein include, but are not limited to, CD16a polypeptide, NKG2A polypeptide, NKG2D polypeptide, NKp30 polypeptide, NKp44 polypeptide, NKp46 polypeptide, and CRTAM polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell that can be used to generate a cell engager (e.g., BiKE or TriKE) provided herein include, but are not limited to, an anti-CD16a scFv, an anti-CD16a VH domain, an anti-NKG2A scFv, an anti-NKG2A VH domain, an anti-NKG2D scFv, an anti-NKG2D VH domain, an anti-NKp30 scFv, an anti-NKp30 VH domain, an anti-NKp44 scFv, an anti-NKp44 VH domain, an anti-NKp46 scFv, an anti-NKp46 VH domain, an anti-CRTAM scFv, and an anti-CRTAM VH domain.

[0056] In some cases, the cell engagers (e.g., BiTEs) provided herein may be designed to include a linker located between each antigen-binding domain. The cell engagers described herein may be designed using any suitable linker. Examples of linkers that can be used to generate the cell engagers described herein include, but are not limited to, the linker sequences described in Example 10. The cell engagers described herein may be designed to include a linker of any suitable length. For example, cell engagers provided herein can be designed to include a linker having a length of about 3 to about 100 (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17) amino acid residues. In some cases, the hinge of a CAR described herein can be used as a linker to generate the cell engagers described herein. For example, any one of the sequences described in Example 12 may be used as a linker in the cell engagers described herein.

[0057] In some cases, the cell engagers described herein may be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 10 or Example 12. In some cases, the cell engagers described herein may be designed to include a linker that includes, consists essentially of, or consists of one of the amino acid sequences set forth in Example 10 or Example 12, including those with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, in one of the amino acid sequences set forth in Example 10 or Example 12. In some cases, the cell engagers provided herein may be designed to include a linker that consists essentially of, or consists of one of the amino acid sequences set forth in Example 10 or Example 12, including those with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0058] In some cases, a cell engager (e.g., a BiTE) that targets a BAFF-R polypeptide can be designed to include heavy chain variable domains comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as that shown in Example 10, followed by light chain variable domains comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker such as the hinge / linker shown in Example 10 or Example 12, followed by an scFv having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell (e.g., an anti-human CD3 scFv).

[0059] In some cases, a cell engager (e.g., a BiTE) that targets a BAFF-R polypeptide can be designed to include light chain variable domains comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker such as that shown in Example 10, followed by heavy chain variable domains comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO:30), followed by an scFv (e.g., an anti-human CD3 scFv) having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell.

[0060] In some cases, a cell engager (e.g., a BiTE) that targets a BAFF-R polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker such as that shown in Example 10, followed by a light chain variable domain comprising SEQ ID NO:16, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO:30), followed by an scFv (e.g., an anti-human CD3 scFv) having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell.

[0061] In some cases, a cell engager (e.g., a BiTE) that targets a BAFF-R polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO: 16, followed by a linker such as that shown in Example 10, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO: 30), followed by an scFv (e.g., an anti-human CD3 scFv) having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell.

[0062] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a BAFF-R polypeptide can be designed to include heavy chain variable domains comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as that shown in Example 10, followed by a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO:30), followed by an scFv having one or more antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv in the case of BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv in the case of TriKE).

[0063] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a BAFF-R polypeptide can be designed to include light chain variable domains comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, followed by a linker such as that shown in Example 10, followed by a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO:30), followed by an scFv having one or more antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv in the case of BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv in the case of TriKE).

[0064] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a BAFF-R polypeptide can be designed to include a heavy chain variable domain comprising SEQ ID NO:8, followed by a linker such as that shown in Example 10, followed by a light chain variable domain comprising SEQ ID NO:16, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO:30), followed by an scFv having one or more antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv in the case of BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv in the case of TriKE).

[0065] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a BAFF-R polypeptide can be designed to include a light chain variable domain comprising SEQ ID NO: 16, followed by a linker such as that shown in Example 10, followed by a heavy chain variable domain comprising SEQ ID NO: 8, followed by a linker such as the hinge / linker shown in Example 10 or Example 12 (e.g., SEQ ID NO: 30), followed by an scFv having one or more antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv in the case of BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv in the case of TriKE).

[0066] In some cases, a cell engager (e.g., BiTE) that targets a BAFF-R polypeptide can be designed to comprise: (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains); (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, a constant domain (e.g., a kappa or lambda constant domain), and an IgG (e.g., IgG1) configuration having an antigen-binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell.

[0067] In some cases, a cell engager (e.g., BiTE) that targets a BAFF-R polypeptide can be designed to comprise (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO:8, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains), and (b) an IgG (e.g., IgG1) configuration comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO:16, a constant domain (e.g., a kappa or lambda constant domain), and an antigen-binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell.

[0068] In some cases, a cell engager (e.g., BiKE) targeting a BAFF-R polypeptide can be designed to comprise an IgG (e.g., IgG1) configuration having (a) a heavy chain comprising, consisting essentially of, or consisting of heavy chain variable domains, Ig hinges, and constant domains (e.g., CH1, CH2, and CH3 domains) comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and (b) a light chain comprising, consisting essentially of, or consisting of light chain variable domains, constant domains (e.g., kappa or lambda constant domains) comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and an antigen-binding domain capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0069] In some cases, a cell engager (e.g., BiKE) that targets a BAFF-R polypeptide can be designed to comprise: (a) a heavy chain comprising, consisting essentially of, or consisting of a heavy chain variable domain comprising SEQ ID NO: 8, an Ig hinge, and a constant domain (e.g., CH1, CH2, and CH3 domains); (b) a light chain comprising, consisting essentially of, or consisting of a light chain variable domain comprising SEQ ID NO: 16, a constant domain (e.g., a kappa or lambda constant domain), and an IgG (e.g., IgG1) configuration having an antigen-binding domain capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv or an anti-human NKG2A scFv).

[0070] In one embodiment, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) comprises: (i) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with 1, 2, 3, or 4 amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with 1, 2, 3, or 4 amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3. (ii) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 9 (or a variant of SEQ ID NO: 9 with 1, 2, 3, or 4 amino acid modifications), and / or (iii) a light chain variable domain having a CDR2 having the amino acid sequence set forth in SEQ ID NO: 10 (or a variant of SEQ ID NO: 10 with 1, 2, 3, or 4 amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 11 (or a variant of SEQ ID NO: 11 with 1, 2, 3, or 4 amino acid modifications). Examples of such antigen-binding fragments having these CDRs and the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) include, but are not limited to, the variable domain shown in Example 3 and the Fab shown in Example 4.

[0071] In some cases, the binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein are capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and comprise (a) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with 1, 2, 3, or 4 amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with 1, 2, 3, or 4 amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (or a variant of SEQ ID NO: 3 with 1, 2, 3, or 4 amino acid modifications). and / or (b) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 9 (or a variant of SEQ ID NO: 9 with 1, 2, 3, or 4 amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 10 (or a variant of SEQ ID NO: 10 with 1, 2, 3, or 4 amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 11 (or a variant of SEQ ID NO: 11 with 1, 2, 3, or 4 amino acid modifications), may comprise any suitable framework region.For example, such a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) may have (a) a framework region 1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:5 (or a variant of SEQ ID NO:5 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:6 (or a variant of SEQ ID NO:6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications), 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 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications). and / or (b) a light chain variable domain comprising framework region 4 having the amino acid sequence set forth in SEQ ID NO: 15 (or a variant of SEQ ID NO: 15 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), framework region 2 having the amino acid sequence set forth in SEQ ID NO: 16 (or a variant of SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), framework region 3 having the amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications).

[0072] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) having any of the CDRs shown in Example 3 can be designed to include the framework regions shown in Example 3, or can be designed to include one or more framework regions from another antibody, antibody fragment, or antibody domain. For example, a Fab can be designed to include the six CDRs shown in Example 3 and the framework regions shown in Example 3, but one or more of the framework regions can be replaced with the framework regions shown in Example 8 or Example 9. In some cases, an scFv can be designed to include the six CDRs shown in Example 3 and the framework regions shown in Example 3. In some cases, an scFv can be designed to include the six CDRs shown in Example 3 and the framework regions shown in Example 3, but one or more of the framework regions can be replaced with the framework regions shown in Example 8 or Example 9.

[0073] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) may comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:16. For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO:16. In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein may comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:8, and / or (b) a light chain variable domain comprising an amino acid sequence having 100% identity to the amino acid sequence set forth in SEQ ID NO:16.

[0074] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) may comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 8 (with the proviso that the heavy chain variable domain comprises the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3), and / or (b) a light chain variable domain comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 16 (with the proviso that the light chain variable domain comprises the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11). For example, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein can comprise (a) a heavy chain variable domain comprising an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 8, with the proviso that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3, and / or (b) a light chain variable domain comprising an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 16, with the proviso that the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, and 11.

[0075] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) may comprise (a) a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:8, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) to the amino acids set forth in SEQ ID NO:8, and / or (b) a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:16, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) to the amino acids set forth in SEQ ID NO:16. For example, the antibodies or antigen-binding fragments provided herein may have the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and may comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (with the proviso that the heavy chain variable domain comprises the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3), and may comprise a light chain variable domain having the amino acid sequence set forth in SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (with the proviso that the light chain variable domain comprises the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11).

[0076] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) may comprise: (a) a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 3; and / or (b) a light chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 9; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 11. As used herein, a "CDR1 essentially consisting of the amino acid sequence set forth in SEQ ID NO: 1" refers to a CDR1 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 1, a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 1, and / or a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 1, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0077] As used herein, a "CDR2 essentially consisting of the amino acid sequence set forth in SEQ ID NO: 2" refers to a CDR2 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 2, a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 2, and / or a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 2, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0078] As used herein, a "CDR3 essentially consisting of the amino acid sequence set forth in SEQ ID NO: 3" refers to a CDR3 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 3, a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 3, and / or a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 3, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0079] As used herein, a "CDR1 essentially consisting of the amino acid sequence set forth in SEQ ID NO: 9" refers to a CDR1 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 9, a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 9, and / or a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 9, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0080] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10" refers to a CDR2 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 10, a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 10, and / or a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 10, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0081] As used herein, a "CDR3 essentially consisting of the amino acid sequence set forth in SEQ ID NO: 11" refers to a CDR3 having 0, 1, or 2 amino acid substitutions within SEQ ID NO: 11, a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately preceding SEQ ID NO: 11, and / or a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid residues immediately following SEQ ID NO: 11, provided that the binding agent (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains the basic ability to bind to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide).

[0082] When designing a single-chain antibody (e.g., scFv) having a heavy chain variable domain and a light chain variable domain, the two regions can be directly linked, or can be linked using any suitable linker sequence. For example, a heavy chain variable domain having the CDRs of SEQ ID NOS: 1 to 3 can be directly linked via a linker sequence to a light chain variable domain having the CDRs of SEQ ID NOS: 9 to 11, respectively. Examples of linker sequences that can be used to link a heavy chain variable domain and a light chain variable domain to create an scFv include, but are not limited to, the linkers described in Example 10.

[0083] As indicated herein, the amino acid sequences described herein may contain amino acid modifications (e.g., the specified number of amino acid modifications). Such amino acid modifications may include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof. In some cases, amino acid modifications may be made to improve binding and / or contact with an antigen and / or to improve the functional activity of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC). In some cases, amino acid substitutions within the specified sequence identifiers may be conservative amino acid substitutions. For example, conservative amino acid substitutions may be made by replacing one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0084] In some cases, the amino acid substitutions within the linked sequence identifiers may be non-conservative amino acid substitutions. Non-conservative amino acid substitutions may be made by substituting one amino acid residue with another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, but are not limited to, (a) substituting a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine), (b) substituting cysteine ​​or proline with other residues, (c) substituting a residue with a basic side chain (e.g., lysine, arginine, or histidine) with a residue with an acidic side chain (e.g., aspartic acid, glutamic acid), and (d) substituting a residue with a bulky side chain (e.g., phenylalanine) with glycine or other residues with small side chains.

[0085] Methods for generating amino acid sequence variants (e.g., amino acid sequences containing one or more modifications with respect to clearly defined sequence identifiers) can include site-directed or random mutagenesis (e.g., by PCR) of nucleic acids encoding antibodies or fragments thereof. See, e.g., Zoller, Curr. Opin. Biotechnol. 3:348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially derivatized amino acids) can be used to generate the amino acid sequence variants provided herein.

[0086] Binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be produced using any suitable method. For example, binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be produced in recombinant host cells. For example, nucleic acids encoding binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be constructed, introduced into expression vectors, and expressed in suitable host cells. Example 5 is a sequence listing of nucleic acid sequences encoding exemplary binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains) described herein. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be recombinantly produced in prokaryotic hosts such as Escherichia coli, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei. The binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) provided herein can be recombinantly produced in eukaryotic hosts such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), filamentous fungi of the genera Trichoderma (e.g., Trichoderma reesei) and Aspergillus (e.g., Aspergillus niger and Aspergillus oryzae), protozoans such as Leishmania tarentolae, insect cells, or mammalian cells (e.g., mammalian cell lines such as Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, or the human embryonic kidney cell line HEK293). See, for example, Frenzel et al. (Front Immunol., 4:217 (2013)).

[0087] In some cases, the antigen-binding fragments or antibody domains described herein can be produced by proteolytic digestion of whole antibodies. For example, antigen-binding fragments can be obtained by treating antibodies with enzymes such as papain or pepsin. Papain digestion of whole antibodies can be used to produce F(ab)2 or Fab fragments, and pepsin digestion can be used to produce F(ab')2 or Fab' fragments.

[0088] In some cases, the binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein can be substantially pure. The term "substantially pure," as used herein with respect to a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC), means that the binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) is substantially free of other naturally occurring polypeptides, lipids, carbohydrates, and nucleic acids. Thus, a substantially pure binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein is any binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) that has been removed from its natural environment and is at least 60 percent pure. The substantially pure binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.

[0089] Also provided herein are bispecific binding agents (e.g., bispecific antibodies, bispecific antigen-binding fragments, and / or bispecific antibody domains) that bind to two different epitopes, at least one of which is an epitope of a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). In some cases, the bispecific binding agents provided herein can be designed to bind to two different epitopes of the same BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). In some cases, the bispecific binding agents provided herein can bind to an epitope on a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) and a different polypeptide (e.g., a CD3 polypeptide). Bispecific binding agents can be produced by chemically conjugating two different binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains). Bispecific binding agents can also be produced by fusing two antibody-producing cells (e.g., hybridomas) to create a hybrid cell line that produces two different heavy chains and two different light chains within the same cell, resulting in, for example, a bispecific IgG molecule. See Brinkmann and Kontermann, MAbs.,9(2):182-212 (2017).

[0090] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) may be fused or conjugated (e.g., covalently or non-covalently linked) to another polypeptide or other moiety to provide a fusion protein or conjugate. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) may be conjugated (e.g., covalently linked) to a polymer (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), and / or polyglutamic acid (PGA) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), 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 suitable method can be used to conjugate (e.g., covalently or non-covalently) another polypeptide or other moiety to a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein. For example, another polypeptide or other moiety can be conjugated to a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein using the methods described in U.S. Pat. No. 8,021,661.

[0091] In some cases, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be modified with a moiety that improves its stability and / or retention in the circulation, e.g., in blood, serum, or other tissues, by at least 1.5-fold, 2-fold, 5-fold, 10-fold, or 50-fold. For example, a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be attached (e.g., covalently or noncovalently) to a polymer, such as a substantially non-antigenic polymer. In some cases, the polymer can be in the form of a polymeric nanoparticle. Examples of substantially non-antigenic polymers that can be used as described herein include, but are not limited to, polyalkylene oxide and polyethylene oxide. In some cases, the polymers used herein can have any suitable molecular weight. For example, polymers having an average molecular weight of about 200 daltons to about 35,000 daltons (e.g., about 1,000 daltons to about 15,000 daltons, or about 2,000 daltons to about 12,500 daltons) can be used. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be 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, but are not limited to, hydrophilic polyvinyl polymers, polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycol, polyoxyethylenated polyols, poly(lactic-co-glycolic acid) (PLGA), and copolymers and / or block copolymers thereof, provided that the water solubility of the copolymers or block copolymers is maintained.

[0092] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated (e.g., covalently or non-covalently bound) to or contained within nanoparticles. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated to or contained within nanoparticles with carbodiimide chemistry. For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated to or contained within nanoparticles with maleimide chemistry. In some cases, nanoparticles conjugated to or containing the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can also include chitosan, BSA, and / or biotin.

[0093] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) may be attached (e.g., covalently or non-covalently) 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, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) may be covalently attached to polyoxyethylene.

[0094] The present specification also provides ADCs. As used herein, the term "ADC" refers to a conjugate comprising (a) an antigen-binding domain and (b) at least one drug directly or indirectly covalently bound to the antigen-binding domain. In some cases, the ADCs described herein may comprise (a) an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide), and (b) at least one drug directly or indirectly covalently bound to the antigen-binding domain. Any suitable binding agent (e.g., an antibody, antigen-binding fragment, and / or antibody domain) capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) described herein may be used as the antigen-binding domain for generating the ADCs described herein. For example, any of the binding agents described in Example 3 and Example 4 may be used to generate an ADC capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). Examples of drugs that can be used to generate the ADCs described herein include, but are not limited to, auristatins (e.g., monomethylauristatin E (MMAE)), mertansine (DM-1), pyrrolobenzodiazepine (PBD) dimers, tubulysins, duocarmycins, and calicheamicins. Any suitable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) to form the ADCs described herein. For example, a cleavable or non-cleavable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) to form the ADCs described herein.Examples of ADC linkers that can be used to covalently attach one or more agents to an antigen-binding domain capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) to form the ADCs provided herein include, but are not limited to, ADC disulfide linkers, ADC hydrazone linkers, ADC peptide linkers, ADC thioether linkers, and ADC PEG-containing linkers.

[0095] The present specification also provides nucleic acid molecules (e.g., isolated nucleic acid molecules) having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein. For example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a heavy chain variable domain, such as the heavy chain variable domain described in Example 3. In another example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a light chain variable domain, such as the light chain variable domain described in Example 3. In some cases, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding both (a) a heavy chain variable domain and (b) a light chain variable domain, regardless of whether they encode the linker polypeptide described in Example 10. The nucleic acids (e.g., isolated nucleic acid molecules) provided herein can be single-stranded or double-stranded nucleic acids of any suitable type (e.g., DNA, RNA, or DNA / RNA hybrid).

[0096] The present specification also provides vectors (e.g., plasmid vectors or viral vectors) comprising one or more nucleic acids described herein. Examples of plasmid vectors that can be designed to comprise one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding agents described herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, phagemids. Examples of viral vectors that can be designed to comprise one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, retroviral vectors, parvovirus-based vectors (e.g., adenovirus-based vectors and adeno-associated virus (AAV)-based vectors), lentiviral vectors (e.g., herpes simplex (HSV)-based vectors), poxvirus vectors (e.g., vaccinia virus-based vectors and fowlpox virus-based vectors), and hybrid or chimeric viral vectors. For example, viral vectors having an adenoviral backbone containing 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 containing AAV components, such as those described elsewhere (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996)), can be engineered to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein.

[0097] In some cases, the vectors provided herein (e.g., plasmid vectors or viral vectors) can comprise a nucleic acid sequence encoding an scFv or antibody domain (e.g., a VH domain) provided herein. In some cases, the vectors provided herein (e.g., plasmid vectors or viral vectors) can comprise a nucleic acid sequence encoding a CAR provided herein. In some cases, the vectors provided herein (e.g., plasmid vectors or viral vectors) can comprise a nucleic acid sequence encoding a cell engager provided herein.

[0098] The vectors provided herein (e.g., plasmid vectors or viral vectors provided herein) can include any suitable promoter and other regulatory sequences (e.g., initiation and termination codons for transcription and translation) operably linked to a nucleic acid sequence encoding at least a portion of a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager). In some cases, the promoter used to drive expression can be a constitutive promoter or a regulatable promoter. Examples of regulatable promoters that can be used as described herein include, but are not limited to, inducible promoters, repressible promoters, and tissue-specific promoters. Examples of viral promoters that can be used as described herein include, but are not limited to, adenovirus promoters, vaccinia virus promoters, CMV promoters (e.g., immediate-early CMV promoters), and AAV promoters.

[0099] Examples of vectors comprising nucleic acid sequences encoding scFvs or antibody domains (e.g., VH domains) described herein include, but are not limited to, those depicted in FIG. 13.

[0100] Any suitable method can be used to generate nucleic acid molecules (or vectors, such as plasmid vectors or viral vectors) having a nucleic acid sequence encoding at least a portion of the binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) described herein. For example, molecular cloning techniques can be used to generate nucleic acid molecules (or vectors, such as plasmid vectors or viral vectors) having a nucleic acid sequence encoding at least a portion of the binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) provided herein, as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates, and John Wiley & Sons, New York, NY (1994)).

[0101] Also provided herein are host cells comprising a nucleic acid provided herein (e.g., a nucleic acid having a nucleic acid sequence encoding at least a portion of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein). Host cells that can be engineered to contain one or more nucleic acids described herein can be prokaryotic or eukaryotic. Examples of prokaryotic cells that can be engineered to contain a nucleic acid described herein include, but are not limited to, Escherichia coli (e.g., Tb-1, TG-1, DH5α, XL-Blue MRF (Stratagene), SA2821, or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens, or Pseudomonas (e.g., P. aeruginosa) cells. Examples of eukaryotic cells that can be engineered to contain a nucleic acid described herein include, but are not limited to, insect cells (e.g., Sf9 cells 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, 293FT cells, and MDCK cells can be engineered to contain a nucleic acid described herein. Any suitable 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 carrying a nucleic acid sequence encoding at least a portion of a binding agent provided herein) into a host cell.For example, the nucleic acids provided herein can be introduced into host cells using calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high-velocity bombardment with DNA-coated microparticles, direct microinjection into single cells, electroporation, or a combination thereof (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); Neumann et al., EMBO J., 1:841 (1982)).

[0102] In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be engineered to express one or more nucleic acids encoding a CAR described herein. For example, a population of T cells can be infected with a viral vector designed to express a nucleic acid encoding a CAR described herein (e.g., a CAR capable of binding to a BAFF-R polypeptide).

[0103] In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be engineered to express one or more nucleic acids encoding a cell engager described herein. For example, a population of T cells can be infected with a viral vector engineered to express a nucleic acid encoding a cell engager described herein (e.g., a cell engager capable of binding to a BAFF-R polypeptide).

[0104] In some cases, a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein can be produced using a method including: (a) introducing a nucleic acid encoding a polypeptide into a host cell; (b) culturing the host cell in a culture medium under conditions sufficient to express the polypeptide; (c) recovering the polypeptide from the cell or culture medium; and (d) purifying the polypeptide (e.g., to at least 50, 60, 70, 80, 90, 95, 97, 98, or 99 percent purity).

[0105] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) with cancer (e.g., one or more B-cell cancers) to treat the mammal. In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding the antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express the antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express the antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or increase the survival rate of a mammal suffering from cancer. For example, the binding agents described herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs) capable of binding to a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human).In some cases, the pharmaceutical compositions described herein may contain a pharmaceutically acceptable carrier such as a buffer, salt, surfactant, sugar, tonicity adjuster, or a combination thereof, as described elsewhere (Gervasi et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharmaceutically acceptable carriers that can be used to prepare the pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, surfactants (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or sugars (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or a combination thereof. For example, a pharmaceutical composition designed to include a binding agent provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC (or a nucleic acid, vector, or host cell provided herein)) can be formulated to include a buffer (e.g., acetate, citrate, histidine, succinate, phosphate, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar such as sucrose. Other ingredients that may be included within the pharmaceutical compositions provided herein include, but are not limited to, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine or ethylenediaminetetraacetic acid (EDTA), anticancer drugs such as enzalutamide, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosine, bortezomib, carfilzomib, batimastat, ganetespib, obatoclax, navitoclax, taxol, paclitaxel or bevacizumab, or combinations thereof.For example, the pharmaceutical compositions provided herein can comprise one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells engineered to express a CAR capable of binding to a BAFF-R polypeptide, one or more cell engagers, and / or one or more ADCs) in combination with an anti-PD-1 antibody or a PD-1 inhibitor (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-40 14, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarimab, INCMGA00012, AMP-224, or AMP-514), anti-PD-L1 antibodies or PD-L1 inhibitors (e.g., avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189), and / or anti-CTLA-4 antibodies (e.g., ipilimumab).

[0106] In some cases, when a pharmaceutical composition is formulated to include one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells engineered to express a CAR capable of binding to a BAFF-R polypeptide, one or more cell engagers, and / or one or more ADCs), any suitable concentration of the binding agent can be used. For example, the pharmaceutical compositions provided herein may contain about 1 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR +In another example, the pharmaceutical compositions provided herein can be formulated to be solid or semi-solid containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a binding agent (e.g., antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein. In some cases, pharmaceutical compositions comprising a binding agent (e.g., an antibody, antigen-binding fragment, and / or antibody domain) provided herein may be used to administer a pharmaceutical composition containing a binding agent having a titer of about 1×10 5 ~Approx. 1×10 12 (For example, about 1 × 10 5 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 8 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 9 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 11 , or approximately 1 × 10 7 ~Approx. 1×10 10 In some cases, the pharmaceutical composition comprising one or more cells engineered to express a CAR capable of binding to a BAFF-R polypeptide provided herein may contain from about 25 million to about 400 million anti-BAFF-R CARs. +Cells (e.g., about 25 million to about 350 million, about 25 million to about 300 million, about 25 million to about 250 million, about 25 million to about 200 million, about 25 million to about 150 million, about 25 million to about 100 million, about 25 million to about 50 million, about 50 million to about 400 million, about 100 million to about 400 million, about 150 million to about 400 million, about 200 million to about 400 million, about 200 million) 50 million to about 400 million, about 300 million to about 400 million, about 350 million to about 400 million, about 50 million to about 350 million, about 100 million to about 300 million, about 150 million to about 250 million, about 50 million to about 100 million, about 100 million to about 150 million, about 150 million to about 200 million, about 250 million to about 300 million, or about 300 million to about 350 million anti-BAFF-R CARs +In some cases, when pharmaceutical compositions are formulated to include one or more nucleic acids (e.g., vectors such as viral vectors) encoding at least a portion of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein, any suitable concentration of nucleic acid may be used. For example, the pharmaceutical compositions provided herein can be formulated to be liquids containing about 0.5 mg to about 500 mg of a nucleic acid provided herein per mL (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg). In another example, the pharmaceutical compositions provided herein can be formulated to be solid or semi-solid containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg).

[0107] In some cases, pharmaceutical compositions designed to include a binding agent described herein (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) can be formulated to include one or more agents capable of reducing aggregation of the binding agent upon formulation. Examples of such agents that can be used as described herein include, but are not limited to, methionine, arginine, lysine, aspartic acid, glycine, glutamic acid, and combinations thereof. In some cases, one or more of these amino acids can be included in the formulation at a concentration of about 0.5 mM to about 145 mM (e.g., about 1 mM to about 145 mM, about 10 mM to about 145 mM, about 100 mM to about 145 mM, about 0.5 mM to about 125 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 75 mM, or about 10 mM to about 100 mM).

[0108] The pharmaceutical compositions provided herein can be in any suitable form. For example, the pharmaceutical compositions provided herein can be designed to be liquid, semi-solid, or solid. In some cases, the pharmaceutical compositions provided herein can be a liquid solution (e.g., an injection solution and / or an infusion solution), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository. In some cases, the pharmaceutical compositions provided herein can be lyophilized. In some cases, the pharmaceutical compositions provided herein (e.g., a pharmaceutical composition comprising one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs)) can be formulated with a carrier or coating designed to protect against rapid release. For example, the pharmaceutical compositions provided herein may be formulated as controlled- or modified-release formulations, as described elsewhere (U.S. Patent Application Publication Nos. 2019 / 0241667, 2019 / 0233522, and 2019 / 0233498).

[0109] The present specification also provides compositions (e.g., pharmaceutical compositions provided herein) comprising one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells (e.g., CARs) provided herein). + Also provided are methods for administering one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or nucleic acids, vectors, and / or host cells (e.g., CARs) provided herein) to a mammal (e.g., a human). + A composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR) provided herein) can be administered to a mammal (e.g., a human) with cancer (e.g., one or more B-cell cancers) to treat the mammal. In some cases, one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR) provided herein) can be administered to a mammal (e.g., a human) with cancer (e.g., one or more B-cell cancers) to treat the mammal. + Compositions comprising the IL-16-18 (e.g., IL-16-18 cells) (e.g., pharmaceutical compositions provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or improve the survival rate of a mammal afflicted with cancer.

[0110] In some cases, one or more binding agents described herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, and / or host cells described herein (e.g., CAR +a composition (e.g., a pharmaceutical composition described herein) comprising a cancer (e.g., BAFF-R cells) + The compositions can be administered to a mammal (e.g., a human) having cancer (e.g., cancer) to reduce or eliminate one or more symptoms of the cancer. Examples of symptoms of cancer (e.g., B-cell cancer) that can be reduced using a composition comprising one or more binding agents described herein include, but are not limited to, bleeding gums, bone pain, fever, frequent infections, frequent nosebleeds, severe nosebleeds, a lump (e.g., a lump due to swollen lymph nodes in the neck, armpit, abdomen, and / or groin), pale skin, shortness of breath, weakness, fatigue, and decreased energy.

[0111] Any suitable mammal having cancer can be treated with one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR + Examples of mammals that can be treated as described herein include, but are not limited to, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human with cancer can be treated with one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein). + The subject may be treated with a composition (e.g., a pharmaceutical composition provided herein) comprising the inflammatory cytokine agonist (IL-1), ...2), inflammatory cytokine agonist (IL-3), inflammatory cytokine agonist (IL-4), inflammatory cytokine agonist (IL-5), inflammatory cytokine agonist (IL-6), inflammatory cytokine agonist (IL-7), inflammatory cytokine agonist (IL-8), inflammatory cytokine agonist

[0112] Any suitable cancer can be treated with one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or with a nucleic acid, vector, or host cell (e.g., CAR + For example, a mammal (e.g., a human) having cancer can be treated by administering to the mammal a composition (e.g., a pharmaceutical composition) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) described herein. Cancers that can be treated as described herein can be primary or metastatic cancers. In some cases, cancers that can be treated as described herein can be refractory or recurrent cancers. For example, cancers that can be treated as described herein can be antigen-negative (e.g., CD19-negative) recurrent cancers. In some cases, cancers that can be treated as described herein can include one or more solid tumors. In some cases, cancers that can be treated as described herein can be hematological cancers. For example, cancers that can be treated as described herein can be B-cell cancers (e.g., cancers resulting from malignant transformation of B cells or B-cell precursors). For example, a cancer treatable as described herein can be a plasma cell cancer (e.g., a cancer resulting from the malignant transformation of plasma cells). Examples of cancer treatable as described herein include, but are not limited to, CLL, acute lymphoblastic leukemia (ALL), hairy cell leukemia, follicular lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and intravascular large B-cell lymphoma. In some cases, BAFF-R is a vasopressin-resistant leukemia (PBMC). + Cancer (e.g., BAFF-R +A composition (e.g., a pharmaceutical composition) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein can be administered to a mammal (e.g., a human) with a cancer (e.g., B-cell cancer) to treat the mammal (e.g., reduce the number of cancer cells in the mammal).

[0113] Any suitable method can be used to administer the compositions (e.g., pharmaceutical compositions) provided herein to a mammal (e.g., a human). For example, a composition provided herein (e.g., a pharmaceutical composition comprising one or more binding agents provided herein, e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs provided herein) can be administered to a mammal (e.g., a human) intravenously (e.g., by intravenous injection or infusion), subcutaneously (e.g., by subcutaneous injection), intraperitoneally (e.g., by intraperitoneal injection), orally, by inhalation, or intramuscularly (e.g., by intramuscular injection). In some cases, the route and / or method of administration of a composition (e.g., a pharmaceutical composition provided herein) can be tailored to the mammal being treated.

[0114] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR +An effective amount of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be an amount that reduces the number of cancer cells in a mammal having cancer (e.g., one or more B-cell cancers) without causing significant harm to the mammal. In some cases, one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be administered to a mammal having cancer (e.g., one or more B-cell cancers) without causing significant harm to the mammal. +An effective amount of a composition (e.g., a pharmaceutical composition provided herein) comprising a B-cell cancer (e.g., one or more B-cell cancers) can be an amount that extends survival time of a mammal having cancer (e.g., one or more B-cell cancers) compared to a control mammal having a comparable cancer that is not treated with the composition. For example, an effective amount of a binding agent (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be from about 0.001 mg / kg to about 100 mg / kg (e.g., from about 0.001 mg / kg to about 90 mg / kg, from about 0.001 mg / kg to about 80 mg / kg, from about 0.001 mg / kg to about 70 mg / kg, from about 0.001 mg / kg to about 60 mg / kg). , about 0.001mg / kg to about 50mg / kg, about 0.001mg / kg to about 40mg / kg, about 0.001mg / kg to about 30mg / kg, about 0.005mg / kg to about 100mg / kg, about 0. 01mg / kg~about 100mg / kg, about 0.05mg / kg~about 100mg / kg, about 0.1mg / kg~about 100mg / kg, about 0.5mg / kg~about 100mg / kg, about 1mg / kg~about 1 00mg / kg, about 5mg / kg to about 100mg / kg, about 0.01mg / kg to about 25mg / kg, about 0.1mg / kg to about 30mg / kg, about 0.15mg / kg to about 25mg / kg, about 0. 2mg / kg~about 20mg / kg, about 0.5mg / kg~about 20mg / kg, about 1mg / kg~about 30mg / kg, about 1mg / kg~about 25mg / kg, about 1mg / kg~about 20mg / kg, about 2m In another example, an effective amount of a cell (e.g., T cell) expressing a binding agent (e.g., a CAR) provided herein can be about 25 million anti-BAFF-R CARs. + Approximately 400 million anti-BAFF-R CARs were isolated from the cells. +Cells (e.g., about 25 million to about 350 million, about 25 million to about 300 million, about 25 million to about 250 million, about 25 million to about 200 million, about 25 million to about 150 million, about 25 million to about 100 million, about 25 million to about 50 million, about 50 million to about 400 million, about 100 million to about 400 million, about 150 million to about 400 million, about 200 million to about 400 million, about 250 million to about 400 million, about 300 million to about 400 million, about 350 million to about 400 million, about 50 million to about 350 million, about 100 million to about 300 million, about 150 million to about 250 million, about 50 million to about 100 million, about 100 million to about 150 million, about 150 million to about 200 million, about 250 million to about 300 million, or about 300 million to about 350 million anti-BAFF-R CARs + The effective amount may be constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. The actual effective amount used for a particular application may be influenced by a variety of factors. For example, when treating a mammal with cancer (e.g., one or more B-cell cancers), the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of coadministration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require that the actual effective amount of the compositions described herein (e.g., pharmaceutical compositions comprising one or more binding agents described herein) administered be increased or decreased.

[0115] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR +An effective administration frequency of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a frequency that reduces the number of cancer cells in a mammal having cancer (e.g., one or more B-cell cancers) without causing significant toxicity to the mammal. In some cases, administration of one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a frequency that reduces the number of cancer cells in a mammal having cancer (e.g., one or more B-cell cancers) without causing significant toxicity to the mammal. + An effective administration frequency of a composition (e.g., a pharmaceutical composition provided herein) comprising a B-cell cancer (e.g., one or more B-cell cancers) can be a frequency that extends the survival of a mammal having cancer (e.g., one or more B-cell cancers) compared to a control mammal having a comparable cancer and not treated with the composition. For example, an effective administration frequency of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding agents provided herein, can be from about twice daily to about once per week (e.g., about once daily). In some cases, the administration frequency of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding agents provided herein, can be daily. The administration frequency of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition comprising one or more binding agents provided herein, can be constant or variable over the course of treatment. Various factors can affect the actual effective administration frequency in a particular application. For example, the actual effective dosing frequency of the compositions provided herein (e.g., pharmaceutical compositions comprising one or more binding agents provided herein) may need to be increased or decreased depending on the severity of the cancer (e.g., one or more B-cell cancers), the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the attending physician.

[0116] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR + The effective period of administration of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a period that reduces the number of cancer cells in the mammal without causing significant toxicity to the mammal. In some cases, administration of one or more binding agents (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a period that reduces the number of cancer cells in the mammal without causing significant toxicity to the mammal. + The effective administration period of a composition (e.g., a pharmaceutical composition provided herein) comprising a cancer (e.g., one or more B-cell cancers) can be a period that extends the survival of a mammal having cancer (e.g., one or more B-cell cancers) compared to a control mammal having a comparable cancer that is not treated with the composition. For example, the effective administration period of a pharmaceutical composition provided herein, e.g., a pharmaceutical composition comprising one or more binding agents provided herein, can vary from several weeks to several months (e.g., 4 to 12 weeks) from a single administration point. The actual effective administration period used for a particular application can be influenced by several factors. For example, the severity of the cancer (e.g., one or more B-cell cancers), the route of administration, the age and general health of the mammal, the use of excipients, the possibility of coadministration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the attending physician may necessitate extending or shortening the actual effective administration period of a composition provided herein (e.g., a pharmaceutical composition comprising one or more binding agents provided herein).

[0117] In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used to detect the presence or absence of a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) in vitro, in situ, or in vivo (e.g., in vivo imaging in a mammal such as a human). For example, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be designed to include a label (e.g., a covalently attached radioactive, enzymatic, peptide, colorimetric, or fluorescent label). The labeled binding agent can be used to detect the presence or absence of a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) in a biological sample in vitro. Examples of biological samples that can be evaluated using the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) include, but are not limited to, serum samples, plasma samples, tissue samples, biopsy samples, cell line samples, and tissue culture samples. In some cases, biological samples that can be evaluated as described herein can include mammalian body tissues and / or cells, such as leukocytes, ovarian tissue or cells, prostate tissue or cells, cardiac tissue or cells, placental tissue or cells, pancreatic 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, endometrial tissue or cells, colon tissue or cells, colorectal tissue or cells, cervical tissue or cells, stomach tissue or cells, or umbilical cord tissue or cells, which may express a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide). In some cases, binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be immobilized, for example, on a support, and retention of a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) on the support from the biological sample can be detected, and / or vice versa.In some cases, the binding agents provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) may be used in applications such as fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and / or cell sorting (e.g., fluorescence-activated cell sorting).

[0118] In some cases, binding agents described herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) comprising a label described herein (e.g., a covalently attached radiolabel) can be used to detect the presence or absence of a BAFF-R polypeptide (e.g., a human BAFF-R polypeptide) in a mammal (e.g., a human). For example, a binding agent described herein (e.g., an antibody, antigen-binding fragment, and / or antibody domain) labeled (e.g., covalently labeled) with a radiolabel or an MRI-detectable label can be administered to a mammal (e.g., a human), and the mammal can be evaluated using a means for detecting the detectable label. In some cases, the mammal can be scanned to evaluate the location of a labeled binding agent described herein in the mammal. For example, the mammal can be imaged using NMR or other tomography techniques.

[0119] Examples of labels that can be attached (e.g., covalently or non-covalently) to the binding agents (e.g., antibodies, antigen-binding fragments, and / or antibody domains) described herein include: 131 I, 111 In, 123 I, 99m Tc, 32 P, 33 P, 125 I, 3 H, 14 C, and 188 These include, but are not limited to, radioactive labels such as Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron-emitting isotopes detectable by positron emission tomography ("PET") scanners, chemiluminescent materials such as luciferin, and enzymatic markers such as peroxidase or phosphatase. In some cases, short-range radiation emitters may be used, such as isotopes detectable by short-range detector probes.

[0120] In some cases, one or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or compositions comprising the nucleic acids, vectors, or host cells provided herein (e.g., CAR + An effective amount of a CAR (e.g., a pharmaceutical composition provided herein) can be used to treat a mammal having a disease or disorder other than cancer (e.g., in adoptive T cell therapy such as CAR-T cell therapy). For example, one or more T cells (e.g., CAR-T cells) that express (e.g., have been modified to express) a CAR described herein (e.g., a CAR capable of binding to a BAFF-R polypeptide) can be used to treat one or more autoimmune indications. Examples of autoimmune indications that can be treated by administering one or more T cells (e.g., CAR-T cells) that express (e.g., have been modified to express) a CAR described herein (e.g., a CAR capable of binding to a BAFF-R polypeptide) include, but are not limited to, organ transplant rejection, autoimmune diseases (e.g., systemic lupus erythematosus and rheumatoid arthritis), HLA sensitization, vasculitis, and multiple sclerosis.

[0121] One or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or compositions comprising the nucleic acids, vectors, or host cells provided herein (e.g., CAR +When the therapeutic agent (e.g., a pharmaceutical composition provided herein) is used to treat organ transplant rejection and / or HLA sensitization, a composition comprising one or more binding agents provided herein can be administered to a mammal that has received an organ transplant or is preparing to receive an organ transplant (e.g., an allograft or an autograft). For example, a composition comprising one or more binding agents provided herein can be administered to a mammal that has received an organ transplant to reduce or eliminate one or more symptoms of organ transplant rejection in the mammal. Examples of symptoms of organ transplant rejection include, but are not limited to, organ dysfunction, pain and swelling around the transplanted organ, flu-like symptoms, and unexplained changes in blood pressure. For example, a composition comprising one or more binding agents provided herein can be administered to a mammal that is scheduled to receive an organ transplant to delay or prevent organ transplant rejection in the mammal.

[0122] One or more binding agents provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs (or nucleic acids, vectors, or host cells provided herein (e.g., CAR + Compositions comprising one or more binding agents (e.g., pharmaceutical compositions provided herein) can be used to treat mammals (e.g., humans) that have undergone any type of organ transplant. For example, compositions comprising one or more binding agents provided herein can be used to delay or prevent organ transplant rejection in any type of organ transplant. Examples of transplantable organs and organs that can be treated as described herein (e.g., to delay or prevent organ transplant rejection) include, but are not limited to, kidney, lung, liver, heart, pancreas, and bone marrow. The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0123] Example 1: BAFF-R-targeted CAR-T cell therapy in B-cell lymphoma tumors This example describes the design and characterization of molecules capable of binding to BAFF-R polypeptides.

[0124] Materials and Methods cell line Nalm-6, MEC-1, and Z138 cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Jurkat and 293FT cell lines were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and American Type Culture Collection (Manassas, VA, USA), respectively. Cells were maintained in either 90% RPMI 1640, Iscove's MDM, or 90% Dulbecco's Modified Eagle Medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher). Cell lines were authenticated by flow cytometry. BAFF-R knockout in Nalm-6 (BAFF-R KO Nalm-6) and CD19 knockout in Nalm-6 (CD19 KO Nalm-6) were generated, and luciferase-expressing human cell lines were generated for in vivo experiments.

[0125] PBMCs from healthy volunteer donors were isolated by leukapheresis using a leukapheresis system (LRS) cone. To generate CAR-T cells, naive and memory T cell (Tn / mem) populations were isolated from PBMCs in three steps based on the absence of CD14 / CD25 and expression of CD62L on these cells. Negative selection for CD14 and CD25 and positive selection for CD62L were performed using CD14, CD25, and CD62L microbeads according to the manufacturer's protocol (Miltenyi Biotec, Germany).

[0126] For experiments in which the target cell population was B cells, 50 ml of blood was collected from adult subjects diagnosed with CLL at the time of clinically indicated blood draw. Patient and disease characteristics of these subjects were recorded. Tumor cell content in each sample ranged from 80 to 98% by leukapheresis or blood. The minor T cell population present in these tumor samples was removed to avoid confounding results in the BAFF-R CAR-T cell test. B cells were isolated using the EasySep™ Direct Human B-cell Isolation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's protocol. PBMCs and enriched B cells were incubated with anti-BAFFR-AF647 / anti-CD3-BV605 antibodies for 30 minutes at 4°C. Cells were then washed as before and resuspended in 400 μL of FACS for analysis on an Attune flow cytometer (Thermo Fisher). FMO was set as a negative control.

[0127] Development of BAFF-R antibodies We generated monoclonal antibodies targeting human BAFF-R. To enable overexpression of BAFF-R, cDNA encoding human BAFF-R (Origene, Rockville, MD, USA) was cloned into a lentiviral gene delivery system (pCDH cDNA Cloning and Expression Lentivectors, System Biosciences, Palo Alto, CA, USA). NIH / 3T3 or 293FT cells were infected with pCDH-BAFF-R lentivirus and selected with 1 μg / mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for one week. Single-cell clones were established from the selected BAFF-R-positive NIH / 3T3 or 293FT cells, and the BAFF-R-expressing NIH / 3T3 cell clones were used as immunogens for BAFF-R monoclonal antibody development. BAFF-R-positive 293FT cells were used for screening monoclonal antibody clones.

[0128] Crude supernatants were screened in multiples using five 5-fold serial dilutions from hybridoma clones and incubated with BAFF-R-293FT cells for 30 minutes at 4°C. After washing twice with FACS (2% bovine serum albumin in PBS), the cells were incubated with R-PE-conjugated goat anti-mouse IgG for 30 minutes at 4°C. The cells were then washed as above and resuspended in 400 μL of FACS for analysis on an Attune flow cytometer (Thermo Fisher). Mean fluorescence intensity values ​​for the R-PE channel were collected. Undiluted supernatant was incubated with 293FT cells as a negative control. Representative data are included to illustrate the process.

[0129] A lead clone was identified and further purified. Purified monoclonal antibody C21 was incubated with BAFF-R-293FT cells and 293FT cells for 30 minutes at 4°C. After washing twice with FACS (PBS containing 2% bovine serum albumin), the cells were incubated with R-PE-conjugated goat anti-mouse IgG for 30 minutes at 4°C. The cells were then washed as before and resuspended in 400 μL of FACS for analysis on an Attune flow cytometer (Thermo Fisher Scientific). BAFF-R-293FT cells were incubated with R-PE-conjugated anti-mouse IgG as a negative control. Anti-BAFF-R monoclonal antibody production was monitored using R-phycoerythrin-conjugated goat anti-mouse IgG (BD Bioscience, San Jose, CA).

[0130] Antibodies and flow cytometry Productive lentiviral transduction was confirmed by surface expression of truncated epidermal growth factor receptor (tEGFR) by incubating T cells with anti-EGFR-BV421 (clone AY13, BioLegend, San Diego, CA, USA) for 30 min at 4°C. Anti-CD3-BV605 (clone SK7, BD Biosciences) was also used for T cell identification. Functional activity of live T cells (Sytox Blue, Thermo Fisher, or propidium iodide, Thermo Fisher) was imaged using antibodies and flow cytometry. Non-transduced T cells were washed with buffer and incubated with anti-CD107a APC (clone H4A3; BD Biosciences, San Jose, CA, USA), anti-CD4 PE (phycoerythrin clone SK1, BD Biosciences), and anti-CD8 APC-Cy7 (clone SK1, BD Biosciences) for 30 min at 4°C. Gating was performed for either CD4 or CD8. To assess antigen-induced activation and degranulation of CAR-T cells, T cells were incubated with anti-CD107a APC, anti-CD4 PE, anti-CD8 APC-Cy7, anti-EGFR BV421, and antigen-specific antibodies (anti-CD19 APC (clone HIB19, BD Biosciences) or anti-BAFF-R Alexa Fluor 647 (clone 11C1, BD Biosciences)) for 30 minutes at 4°C. Activated CAR-T cells in the degranulation assay were CD4 + EGFR + CD107a + or CD8 + EGFR + CD107a + Data were acquired on a BD Fortessa or MACSQuant Analyzer 10 (Miltenyi Biotech) and analyzed using FlowJo™ Version 10 software.

[0131] CAR-T cell production We generated a second-generation BAFF-R-CAR consisting of the BAFF-R scFv (described herein), the CD28 transmembrane domain, the CD28 and CD3ζ intracellular signaling domains, and tEGFR (described herein) incorporated as a marker and suicide switch. The CAR cDNA was cloned into the pHIV.7 lentiviral vector. A CD19-CAR was also generated using a similar method, substituting the BAFF-R scFv with the CD19 scFv (Qin et al., Science Translational Medicine, 11(511) 2019). Lentivirus was produced in 293FT cells, concentrated, and titered in Jurkat cells. Tn / mem cells were isolated from healthy donor PBMCs and divided into two aliquots. One aliquot was activated and expanded as untransduced (non-CAR) T cells, while the other was used for CAR-T cell generation. These T cells were activated for 24 hours using human T-activator CD3 / CD28 beads (Life Technologies) and then transduced with CAR-encoding lentivirus at a multiplicity of infection (MOI) of 1. CAR-T cells were activated for an additional 7 days by CD3 / CD28 bead stimulation, after which the beads were removed and the CAR-T cells were expanded for an additional 7 days. Non-transduced T cells from the same donor were also expanded according to the CAR-T cell protocol and used as a control. CAR-T cell proliferation was monitored by trypan blue exclusion and viable cell count using a Bio-Rad TC20™ automated cell counter (Bio-Rad Laboratories, Inc., CA, USA). Each batch of CAR-T cells for the research study was assessed for cell quality by fold expansion (>25) and viability (>70% as measured by trypan blue staining), and for CAR-T cell specific properties by identity (>70% as measured by flow cytometry for CD3-positive cells) and potency (>10% as measured by flow cytometry for EGFR-positive T cells).Clinical-grade batches underwent additional safety testing for adventitious viral agents, including lentiviral copy number determination (<5 copies by woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) real-time qPCR and <2.5 copies / 50ng DNA of VSVG (encoding the envelope gene sequence) as detected by qPCR assay).

[0132] In vitro functional assays To determine functional efficacy, CAR-T cells were incubated with target cells at the E:T ratio (as indicated in the figure legend) for 6 hours with CD107a-APC antibody as a degranulation marker. CD107a (also known as lysosome-associated membrane protein-1 (LAMP-1)) is a protein lining secretory cytolytic granules and becomes accessible to the surface upon migration of T cell granules to the plasma membrane in response to immune response signals. CAR-T cells and target cells were co-cultured at an E:T ratio of 4:1 for 72 hours unless otherwise noted. After 72 hours, supernatants were collected and granzyme B was measured using ELISA (U-PLEX Human Granzyme B Assay ELISA kit, Meso Scale Diagnostics, Rockville, MD, USA).

[0133] In vivo modeling Breeding pairs of NOD scid gamma (NSG) mice were purchased from The Jackson Laboratory (stock number 005557) to establish an NSG breeding colony. Mice were housed in a pathogen-free animal facility. NSG mice (8–12 weeks old) were intravenously (IV) injected with luciferase-expressing tumor cell lines and randomly assigned to experimental groups, where they received a single IV injection of CAR-T cells. The number of animals per experimental group is indicated in the figure legend. Tumor burden was quantified by bioluminescence signal intensity after subcutaneous injection of D-luciferin (150 μg luciferin per gram of mouse body weight) into isoflurane-anesthetized mice 10 minutes before imaging with IVIS Imaging (Perkin Elmer, Waltham, MA). Body weight was monitored, and survival data are presented in Kaplan-Meier plots. Animals with excessive tumor burden were humanely euthanized. In some cases, animals exhibited weight loss, lethargy, and / or poor appearance despite minimal or no tumor burden, necessitating humane euthanasia. Necropsy findings suggested graft-versus-host disease. Experiments using mice were performed using all-male or all-female mice. Experiments using cell lines were repeated at least twice.

[0134] statistical analysis Statistical analysis (mean and standard deviation) was performed using Prism Version software (GraphPad, San Diego, USA). Typical comparisons were made between non-CAR and antigen-specific CAR according to the following rules: *, P<0.05, **, P<0.01, ***, P<0.001. For granzyme B release data, an unpaired t-test was performed, and for animal experiments, a log-rank test was performed. Statistical comparisons were indicated by displaying bars above pairs of data sets.

[0135] result Generation of anti-BAFF-R MC10029 CAR An NIH / 3T3 cell line expressing BAFF-R was generated and used to immunize mice (Figure 1A). After successful immunization and hybridoma generation, the supernatants of candidate hybridoma clones were screened against BAFF-R-expressing 293FT cells (Figure 1B). Putative clones were screened by serial dilutions of the raw supernatants in increasing concentrations of BAFF-R-expressing 293FT cells while monitoring mouse IgG by flow cytometry (Figures 1C and 1D). Next, a lead BAFF-R monoclonal antibody was purified (Figure 1E). The lead clone, C21, was sequenced, and the sequences of the heavy and light chain variable regions were identified for application in CAR design.

[0136] Second-generation BAFF-R CARs were generated using a clinical vector (Dong et al., Cancer Immunology, Immunotherapy, 69(10), 2139-2145, 2020). The BAFF-R CAR construct contains a single-chain variable fragment of a monoclonal antibody against BAFF-R, as well as a hinge, CD28 transmembrane domain, CD28 signaling domain, CD3ζ signaling domain, and tEGFR in tandem (Figure 2A). Research-grade BAFF-R CAR-T cells were generated with quality control measures for cell quality, CAR-T cell specificity, and CAR-T cell proliferation (Figures 2B, 2C, and 2D). This CAR T cell generation allowed for comparative production and ensured the quality of CAR-T cells, particularly for experimental evaluation in in vivo experiments.

[0137] MC10029 CAR-T cells demonstrate antigen-specific cytotoxicity against ALL BAFF-R expression was confirmed in the cell lines used for functional analysis of MC10029 CAR T cells (Figure 3A). Nalm-6 expressed both BAFF-R and CD19 (Figure 3B). After incubation with wild-type or BAFF-R KO Nalm-6 cell lines, BAFF-R CAR T cells demonstrated antigen-specific cytotoxicity. Antigen-specific cytotoxicity was measured by CD107a degranulation assay and gated for CD4 or CD8 to highlight the activity of these specific MC10029 CAR T cell populations (Figures 4A and 4B, respectively). Granzyme B released into the supernatant was observed when MC10029 CAR T cells were incubated with wild-type Nalm-6, but not with untransduced T cell controls or BAFF-R KO Nalm-6 cells (Figure 4C). The antigen-specific cytotoxicity of MC10029 CAR T cells was further examined by incubating CAR T cells with GFP-labeled target cells. Cytolysis, as determined by the disappearance of GFP-labeled target cells, was observed only when MC10029 CAR-T cells were incubated with GFP-labeled WT Nalm-6 (Figure 4D).

[0138] MC10029 CAR-T cells demonstrate in vivo antitumor activity targeting ALL The therapeutic efficacy of BAFF-R CAR T cells was assessed by injecting luciferase-labeled human Nalm-6 tumor cells (0.25x10 6 The study was tested in the NSG mouse model, where mice were administered 10x10 transduced T cells. Mice were randomly assigned to groups of five, and each group received either vehicle (PBS), untransduced T cells (10x10 6 cells), or BAFF-R CAR T cells (2 x 10 6NSG mice were administered a single dose of either MC10029 or MC10029 CAR T cells. Bioluminescence imaging of NSG mice after intravenous tumor challenge and CAR T cell administration was used to monitor tumor response over time (Figure 5A). Tumor presence and long-term survival after these three treatments were assessed (Figure 5B). Kaplan-Meier plots show these data as percentage survival versus time after tumor challenge. PBS-treated mice exhibited tumor growth and death within 42 days. Non-transduced T cell-treated mice also exhibited tumor progression and subsequent death. MC10029 CAR T cell-treated mice demonstrated significant clearance of Nalm-6 tumor cells and demonstrated statistically significant survival rates up to 120 days, the end of the study.

[0139] MC10029 CAR-T cells maintain efficacy in a CD19 antigen-deficient model To mimic relapsed / refractory disease, a Nalm-6-based model engineered to lack CD19 was created. MC10029 CAR-T cells, particularly CD8 CAR-T cells targeting the CD19 KO Nalm-6 cell line, retained antigen-specific cytotoxicity, whereas untransduced T cells and CD19 CAR-T cells showed only background activity targeting the CD19 KO Nalm-6 cell line (Figure 6A). This activity profile was confirmed by measuring granzyme B, which demonstrated potent activity of MC10029 CAR T cells compared with untransduced T cells and CD19 CAR T cells (Figure 6B). CAR T cell efficacy was assessed by transfecting NSG mice with luciferase-labeled human CD19-deficient Nalm-6 tumor cells (0.25x10). 6 Seven days after tumor challenge, mice (5 per treatment group) were injected with PBS (vehicle), untransduced T cells (10x10) from the same donor, or 6 cells), MC10029 CAR T cells (2x10 6 cells), or CD19 CAR T cells (2x10 6NSG mice received one of four treatments: CAR T cells (non-transduced T cells), BAFF-R T cells, or PBS. Bioluminescence imaging of NSG mice after intravenous tumor challenge and treatment with either CAR T cells, non-transduced T cells, or PBS was used to track tumor changes along the provided timeline (Figure 6C). Control groups receiving PBS or non-transduced T cells were euthanized around day 60 due to excessive tumor burden. Eighty percent of mice in the CD19 CAR T cell treatment group exhibited tumor burdens similar to those in the control group, and one mouse survived until the end of the experiment at day 84. In the MC10029 CAR T cell treatment group, tumors were eradicated, and all mice survived throughout the 84-day experimental period. A Kaplan-Meier plot shows these data as percent survival versus time after tumor challenge (Figure 6D). Taken together, these results suggest that BAFF-R CAR T cells are effective in treating CD19 antigen escape cases.

[0140] MC10029 CAR-T cells exhibit antigen-specific cytotoxicity against lymphoma The non-Hodgkin's lymphoma (NHL) cell line Z138, which expresses both BAFF-R and CD19 (Figure 3A), was used to test the cytotoxicity of MC10029 CAR T cells against this lymphoma cell line. The antigen-specific cytotoxicity of MC10029 CAR T cells against Z138 was confirmed using a CD107a degranulation assay gated on CD8 (Figure 7A). When MC10029 CAR T cells were incubated with Z138, released granzymes were observed in the supernatant, but not in the untransduced T cell control (Figure 7B). The efficacy of MC10029 CAR T cells was evaluated by transfecting NSG mice with luciferase-labeled human Z138 tumor cells (0.5x10 6 Seven days after tumor challenge and after tumor implantation, mice were injected with PBS (vehicle), untransduced T cells (10x10) from the same donor, or 6 cells), or MC10029 CAR T cells (2x10 6NSG mice received one of three treatments: intravenous tumor challenge and CAR T cell treatment. Tumor response to treatment was monitored using bioluminescence imaging following intravenous tumor challenge and CAR T cell treatment along the timeline shown (Figure 7C). Control groups receiving PBS or untransduced T cells were euthanized within 56 days due to excessive tumor burden. In the MC10029 CAR T cell-treated group, all mice eradicated their tumors and survived throughout the 106-day experimental period. A Kaplan-Meier plot shows these data as percent survival versus time after tumor challenge (Figure 7D). MC10029 CAR T cells recognized and killed Z138 tumor cells in in vitro and in vivo models.

[0141] MC10029 CAR-T cells demonstrate antigen-specific cytotoxicity against CLL The CLL cell line, MEC-1, expressed both BAFF-R and CD19 (Figure 3A). The effect of incubating MC10029 CAR T cells with MEC-1 was examined in both in vitro and in vivo models. The antigen-specific cytotoxicity of MC10029 CAR T cells against MEC-1 was confirmed using a CD107a degranulation assay gated on CD8 (Figure 8A). Granzyme B released into the supernatant was observed when MC10029 CAR T cells were incubated with MEC-1, but not in the untransduced T cell control (Figure 8B). The efficacy of MC10029 CAR T cells was assessed by transfecting NSG mice with luciferase-labeled human MEC-1 tumor cells (1.0x10 6 Ten days after tumor challenge and after tumor implantation, mice were treated with PBS (vehicle), untransduced T cells (10x10) from the same donor, or 6 cells), or MC10029 CAR T cells (2x10 6NSG mice received either 1 or 2 treatments (Figure 8C). Bioluminescence imaging of NSG mice after intravenous tumor challenge and treatment was used to monitor tumor progression along the indicated timeline (Figure 8C), and survival rates were plotted (Figure 8D). The PBS group was euthanized within 37 days due to excessive tumor burden. The non-transduced T cell-treated group experienced steady tumor progression throughout the experimental period, with mice gradually dying due to tumor burden. The MC10029 CAR T cell-treated group experienced tumor regression within 1 month, with an 80% survival rate at 130 days. MC10029 CAR T cells recognized and killed MEC-1 tumor cells in in vitro and in vivo models, suggesting their potential for treating CLL.

[0142] MC10029 CAR-T cells targeted primary human CLL cells MC10029 CAR-T cells were tested against primary B cells isolated from CLL patients. The six subjects selected were three men and three women, aged between 56 and 83 years (see Table 1). [Table 1]

[0143] B cells were enriched from each subject's PBMCs and confirmed to express BAFF-R (Figure 9A). Two batches of MC10029 CAR T cells and untransduced T cells were generated from two healthy donors and incubated with six human primary CLL tumor cells. Gating by EGFR and CD8 revealed activated CD8 T cells after incubation with isolated primary CLL tumor cells. +The percentage of activated MC10029 CAR T cells (Figure 9B, top) was determined. The percentage of activated MC10029 CAR T cells was comparable among B cells isolated from all subjects. Non-transduced T cells generated from two healthy donors showed background activity against B cells, which was also comparable among the collection of CLL tumor cells (Figure 9B, bottom). Complementary cytotoxic activity was determined by measuring the release of granzyme B into the medium of MC10029 CAR T cells or non-transduced T cells incubated with CLL tumor cells for 72 hours. Incubation with CLL tumor cells resulted in statistically significant release of granzyme B from MC10029 CAR T cells compared to non-transduced T cells (Figure 9C).

[0144] Control studies were performed to ensure the quality of both the B cells used and the generated CAR-T cells. To confirm that the activity of MC10029 CAR T cells targeted primary CLL tumor cells, tumor cells, i.e., CLL B cells, were enriched from PBMCs. Using flow cytometry, CD3+ cell populations collected from six CLL patients were characterized in the original PBMC samples (Figure 10, top panel) and after B cell enrichment (Figure 10, bottom panel), confirming the depletion of endogenous T cells.

[0145] Figures 11A-11C show the characterization of MC10029 CAR-T cells, which were used to target primary CLL tumor cells and elicit the responses shown in Figures 9B-9C. MC10029 CAR-T cells were generated from two healthy donors. MC10029 CAR-T cells and two batches of non-CAR-T cells generated from T cells isolated from two healthy donors were shown to be identical as measured by viability, identity, and potency (Figures 11A-11C), allowing for comparison of the two MC10029 CAR-T cell batches in in vitro assays.

[0146] Taken together, these results demonstrate that binding agents having two sets of three CDRs described herein (e.g., SEQ ID NOS: 1-3 and 9-11) can bind to BAFF-R polypeptides. Furthermore, as shown herein, these binding agents can be incorporated into molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers (e.g., BiTEs), or ADCs (e.g., antibody radioconjugates)) that can be used to treat mammals (e.g., humans) with cancer (e.g., B-cell cancers).

[0147] Example 2 Exemplary BAFF-R Polypeptides This example shows an exemplary human BAFF-R sequence. Exemplary Human BAFF-R Polypeptides TIFF2026508306000002.tif27161

[0148] Nucleic acid encoding SEQ ID NO: 42 TIFF2026508306000003.tif72160

[0149] Example 3 Exemplary variable domains capable of binding to BAFF-R polypeptides This example shows the amino acid sequences of the heavy and light chain variable domains of the antibody designated clone #1 (also called C21), along with their respective CDR and framework sequences and diagrams of their structures.

[0150] Anti-BAFF-R clone #1 (C21) heavy chain Heavy chain variable domain TIFF2026508306000004.tif22159 Framework region 1 of the heavy chain variable domain TIFF2026508306000005.tif7162 CDR1 of the heavy chain variable domain TIFF2026508306000006.tif7162 Framework region 2 of the heavy chain variable domain TIFF2026508306000007.tif8161 CDR2 of the heavy chain variable domain TIFF2026508306000008.tif8161 Framework region 3 of the heavy chain variable domain TIFF2026508306000009.tif8161 CDR3 of the heavy chain variable domain TIFF2026508306000010.tif8160 Framework region 4 of the heavy chain variable domain TIFF2026508306000011.tif7160

[0151] Anti-BAFF-R clone #1 (C21) light chain Light chain variable domain TIFF2026508306000012.tif22161 Framework region 1 of the light chain variable domain TIFF2026508306000013.tif8160 CDR1 of the light chain variable domain TIFF2026508306000014.tif8160 Framework region 2 of the light chain variable domain TIFF2026508306000015.tif8160 CDR2 of the light chain variable domain TIFF2026508306000016.tif8160 Framework region 3 of the light chain variable domain TIFF2026508306000017.tif8161 CDR3 of the light chain variable domain TIFF2026508306000018.tif8161 Framework region 4 of the light chain variable domain TIFF2026508306000019.tif8161

[0152] Example 4: Examples of exemplary Fabs with the ability to bind to BAFF-R polypeptides This example shows the amino acid sequences of the heavy and light chain variable domains of the antibody designated clone #1 (also called C21), as well as the respective constant domains, with their structures illustrated.

[0153] Anti-BAFF-R clone #1 (C21) heavy chain Heavy chain variable domain (without the first constant domain) TIFF2026508306000020.tif21161 Human IgG1 heavy chain constant domain 1 (CH1) TIFF2026508306000021.tif14161

[0154] Anti-BAFF-R clone #1 (C21) light chain Light chain variable domain (no kappa / lambda constant domains) TIFF2026508306000022.tif21162

[0155] Example 5: Nucleic acids encoding exemplary BAFF-R binding molecules This example presents the nucleic acid sequences encoding the indicated chains / domains of clone #1 (C21).

[0156] Clone #1 Nucleic acid encoding SEQ ID NO:8 (clone #1; heavy chain) TIFF2026508306000023.tif47160 Nucleic acid encoding SEQ ID NO: 16 (clone #1; light chain) TIFF2026508306000024.tif47160

[0157] Example 6: Exemplary IgGs with the ability to bind to BAFF-R polypeptides This example shows an exemplary structure of an IgG and provides the amino acid and nucleic acid sequences of exemplary hinge, CH2, and CH3 regions / domains. Exemplary Ig (e.g., IgG1) Structures Heavy chain: heavy chain variable domain + CH1 + hinge + CH2 + CH3 Light chain: Light chain variable domain + constant light chain (kappa or lambda)

[0158] Human immunoglobulin G1 hinge region: TIFF2026508306000025.tif28162 Human immunoglobulin G1 heavy chain constant domains 2 and 3 (CH2-CH3) TIFF2026508306000026.tif127161

[0159] Example 7: Exemplary scFvs with the ability to bind to BAFF-R polypeptides This example shows the structure of an exemplary scFv, as well as the amino acid sequences of an exemplary heavy chain variable domain and an exemplary light chain variable domain of an exemplary scFv. The respective CDR and framework sequences are also depicted. Exemplary linker amino acid sequences, such as those shown in Example 10, can be used to link the heavy chain variable domain and the light chain variable domain to form an scFv. The structure of an exemplary scFv is shown, including the amino acid and nucleic acid sequences of the linker, CDR, and framework sequences depicted.

[0160] Exemplary scFv structures: Heavy chain variable domain / region + linker + light chain variable domain / region Exemplary scFv structures: Light chain variable domain / region + linker + heavy chain variable domain / region Exemplary scFv structures: Framework region (FR) 1 + CDR1 + FR2 + CDR2 + FR3 + CDR3 + FR4

[0161] Exemplary variable heavy chain region FR1 sequences: TIFF2026508306000027.tif8160 Exemplary variable heavy chain region FR2 sequences: TIFF2026508306000028.tif8160 Exemplary variable heavy chain region FR3 sequences: TIFF2026508306000029.tif8160 Exemplary variable heavy chain region FR4 sequences: TIFF2026508306000030.tif8160

[0162] Exemplary scFv light chain variable domain structures: FR1+CDR1+FR2+CDR2+FR3+CDR3+FR4 Exemplary variable light chain region FR1 sequence: TIFF2026508306000031.tif8160 Exemplary variable light chain region FR2 sequences: TIFF2026508306000032.tif8161 Exemplary variable light chain region FR3 sequences: TIFF2026508306000033.tif8161 Exemplary variable light chain region FR4 sequences: TIFF2026508306000034.tif8161

[0163] Exemplary scFv structures: Clone #1 CDR Variable light chain region + Linkers (e.g., (G 4 S) 5 Linker) +Clone #1 CDR The variable heavy chain region TIFF2026508306000035.tif141161

[0164] Exemplary scFv structures: Clone #1 CDR Variable light chain region + Linkers (e.g., (G 4 S) 3 Linker) +Clone #1 CDR The variable heavy chain region TIFF2026508306000036.tif140161

[0165] Exemplary scFv structures: Clone #1 CDR Variable heavy chain region + Linkers (e.g., (G 4 S) 5 Linker) +Clone #1 CDR The variable light chain region TIFF2026508306000037.tif21161TIFF2026508306000038.tif122160

[0166] Exemplary scFv structures: Clone #1 CDR Variable heavy chain region + Linkers (e.g., (G 4 S) 3 Linker) +Clone #1 CDR The variable light chain region TIFF2026508306000039.tif76159TIFF2026508306000040.tif70161

[0167] Example 8: Exemplary sequences of heavy chain framework regions This example provides amino acid sequences of exemplary framework regions that can be used in heavy chain variable domains / regions (eg, heavy chain variable domains / regions that can be used to form scFvs).

[0168] Exemplary variable heavy chain region FR1 sequences: TIFF2026508306000041.tif7161 Exemplary variable heavy chain region FR2 sequences: TIFF2026508306000042.tif7161 Exemplary variable heavy chain region FR3 sequences: TIFF2026508306000043.tif7161 Exemplary variable heavy chain region FR4 sequences: TIFF2026508306000044.tif7161

[0169] Example 9: Exemplary sequences of light chain framework regions This example provides amino acid sequences of exemplary framework regions that can be used in light chain variable domains / regions (eg, light chain variable domains / regions that can be used to form scFvs).

[0170] Exemplary variable light chain region FR1 sequence: TIFF2026508306000045.tif8161 Exemplary variable light chain region FR2 sequences: TIFF2026508306000046.tif8161 Exemplary variable light chain region FR3 sequences: TIFF2026508306000047.tif8161 Exemplary variable light chain region FR4 sequences: TIFF2026508306000048.tif8161

[0171] Example 10: Exemplary Linkers This example provides examples of linker amino acid sequences that can be used to link heavy and light chain variable domains to form scFvs. These linker sequences can also be used to generate CARs and cell engagers. TIFF2026508306000049.tif14161 Nucleic acid encoding SEQ ID NO: 25 TIFF2026508306000050.tif14161

[0172] Example 11: Exemplary signal peptides This example shows the amino acid sequences of exemplary signal peptides that can be used in designing CARs. Exemplary signal peptides from human GMCSF: TIFF2026508306000051.tif9161 Nucleic acid encoding SEQ ID NO: 28: TIFF2026508306000052.tif13160

[0173] Example 12: Exemplary Hinge This example shows exemplary hinge amino acid sequences that can be used in designing CARs. Exemplary human IgG4-derived hinge: TIFF2026508306000053.tif33161 Nucleic acid encoding SEQ ID NO: 30: TIFF2026508306000054.tif95161

[0174] Example 13: Exemplary transmembrane domains This example shows the amino acid sequences of exemplary transmembrane domains that can be used to design CARs. Exemplary human CD28 transmembrane domains: TIFF2026508306000055.tif8161 Nucleic acid encoding SEQ ID NO: 32: TIFF2026508306000056.tif14161

[0175] Example 14: Exemplary Intracellular Signaling Domains This example shows the amino acid sequences of exemplary intracellular signaling domains that can be used to design CARs. Exemplary human CD28 intracellular signaling domain (CD28QQ): TIFF2026508306000057.tif10161 Nucleic acid encoding SEQ ID NO: 34: TIFF2026508306000058.tif20162

[0176] Exemplary human CD3ζ intracellular signaling domain: TIFF2026508306000059.tif20162 Nucleic acid encoding SEQ ID NO: 36: TIFF2026508306000060.tif20162TIFF2026508306000061.tif28160

[0177] Example 15: Exemplary detection markers This example shows the amino acid sequences of exemplary detection markers that can be used in designing CARs. Exemplary tEGFR Polypeptides: TIFF2026508306000062.tif48161

[0178] Nucleic acid encoding SEQ ID NO: 38: TIFF2026508306000063.tif88161TIFF2026508306000064.tif54161

[0179] Example 16: Exemplary CARs with the ability to bind to BAFF-R polypeptides This example shows the amino acid sequence of a CAR (CAR #1) designed to contain an scFv generated using the CDRs of clone #1 Fab, and the nucleic acid sequence encoding the CAR. The various components of this CAR (e.g., domains and linkers) are shown. CAR designed using CDRs from clone #1: signal peptide +scFv of Example 7+ Hinge / Linker +CD28 transmembrane domain+ CD28 intracellular signaling domain +CD3ζ Intracellular Signaling Domain+ T2A +tEGFR TIFF2026508306000065.tif60161TIFF2026508306000066.tif76160

[0180] Nucleic acid encoding SEQ ID NO: 40: TIFF2026508306000067.tif128161TIFF2026508306000068.tif215160TIFF2026508306000069.tif76160

[0181] Example 17: BAFF-R CAR-T therapy targeting B-cell lymphoid malignancies The results of this example restate and extend at least some of the results presented in other examples.

[0182] Materials and Methods cell line Nalm-6, MEC-1, and Z-138 cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Jurkat and 293FT cell lines were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and American Type Culture Collection (Manassas, VA, USA), respectively. Cells were maintained in either 90% RPMI 1640, Iscove's MDM, or 90% Dulbecco's Modified Eagle Medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher). Cell lines were authenticated by flow cytometry. The following antigen knockout cell lines were generated: BAFF-R KO Nalm-6, CD19 KO Nalm-6, CD19 KO Z-138, and CD19 KO MEC-1. Luciferase-expressing human cell lines were generated for in vivo experiments as described in Qin et al., Science Translational Medicine, 11(511)2019.

[0183] Isolation of PBMC and Tn / mem from blood samples of healthy donors Peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were isolated by leukapheresis using a leukapheresis system (LRS) cone as described in Dietz et al., Transfusion 46:2083-2089 (2006). To generate CAR-T cells, naive and memory T cell (Tn / mem) populations were isolated from PBMCs using CD14, CD25, and CD62L microbeads in a three-step procedure involving negative selection for CD14 and CD25 and positive selection for CD62L according to the manufacturer's protocol (Miltenyi Biotech, Germany).

[0184] Isolation of T and B cells from blood samples of CLL subjects Peripheral blood samples from CLL subjects were collected according to the biorepository protocol. All subjects provided written informed consent. 50 mL of blood was collected from subjects diagnosed with CLL for primary cell analysis. For CAR-T cell generation from CLL patients, T cells were isolated using a pan T cell isolation kit (Miltenyi Biotec, Germany) according to the accompanying instructions. B cells were isolated using the EasySep™ Direct Human B-cell Isolation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's protocol.

[0185] Generation of CAR-T cells Second-generation BAFF-R-CARs were designed as described in the Results section. The CAR cDNA was cloned into the pHIV.7 lentiviral vector. CD19-CARs were also constructed in the same manner, replacing the BAFF-R antibody single-chain variable fragment (scFv) with the CD19 antibody scFv from the clinically tested CD19-CAR (Qin et al., Science Translational Medicine, 11(511)2019). Lentivirus was produced in 293FT cells, concentrated, and titered in Jurkat cells. Tn / mem or target T cells were isolated and activated with Human T-Activator CD3 / CD28 beads (Life Technologies) for 24 hours before transduction with CAR-encoding lentivirus at a multiplicity of infection (MOI) of 1. Protamine sulfate was used as a transduction promoter for lentiviral CAR-T cell generation. To maximize CAR-T cell efficacy while minimizing residual lentiviral effects (WPRE and VSVG) in CAR-T cells, a range of MOIs (MOI) from 0.1 to 10 was tested to determine the optimal MOI. CAR-T cells were further activated by CD3 / CD28 bead stimulation for 7 days, after which the beads were removed and the CAR-T cells were expanded for an additional 7 days. Non-transduced CAR-T cells from the same donor were expanded according to the CAR-T cell protocol and used as a control.

[0186] In vitro functional assays Degranulation assay CAR-T cells were incubated with target cells at an effector-to-control (E:T) ratio of 2:1 in complete RPMI 1640 medium containing GolgiStop™ Protein Trafficking Inhibitor Reagent (BD Biosciences) and CD107a APC antibody (BD Biosciences) for 6 hours. Cells were then stained with anti-CD3 BV605 (BD Biosciences), anti-CD4 PE-Cy7 (BD Biosciences), anti-CD8 APC-Cy7 (BD Biosciences), and anti-EGFR BV421 (BD Biosciences). Samples were evaluated on an Attune flow cytometer (Thermo Fisher Scientific) or a Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo™ Version 10 software. Non-CAR T cells from the same patient were used as a negative control.

[0187] Granule release assay CAR-T cells and target cells were co-cultured at an E:T ratio of 4:1 for 72 hours, after which time the supernatants were collected. Analytes were quantified using a custom-made U-PLEX Human ELISA kit from Meso Scale Diagnostics (Rockville, MD, USA) according to the manufacturer's instructions.

[0188] Direct killing assay To assess the cytolytic function of CAR-T cells against tumor cells, CAR-T cells were co-cultured with GFP-positive target cells at an E:T ratio of 20:1 for 24 hours. Viability staining with Sytox Blue (Thermo Fisher) was used to identify the percentage of viable GFP-positive tumor cells. Samples were analyzed using an Attune flow cytometer.

[0189] In vivo modeling Breeding pairs of NOD scid gamma (NSG) mice were purchased from The Jackson Laboratory (stock number 005557) to establish breeding colonies. Mice (8–12 weeks old) were intravenously (IV) injected with luciferase-expressing human tumor cell lines (optimized in separate experiments) and randomly assigned to experimental groups (5 mice per group). The non-CAR T cell group typically received 10 × 10 total T cells. 6 10 × 10 total T cells in the CAR-T cell group 6 2 x 10 pieces 6 Mice were treated with a single IV treatment dose of 100 CAR-T cells. Tumor burden was quantified weekly by bioluminescence signal intensity in isoflurane-anesthetized mice injected subcutaneously with D-luciferin (150 μg luciferin per gram of mouse body weight) 10 minutes prior to IVISR imaging (PerkinElmer, Waltham, MA). Survival data were reported and presented as Kaplan-Meier plots.

[0190] statistical analysis All statistical analyses were performed using GraphPad Prism software (San Diego, CA). Data are reported as mean ± SD and analyzed by Student's t-test. Granule protein release data were compared using unpaired t-tests, and for animal studies, a log-rank test was performed. Typical comparisons were made between non-CAR T cells and antigen-specific CAR T cells and followed the following rules: *, p<0.05; **, p<0.01; ***, p<0.001.

[0191] result Generation of a novel anti-BAFF-R MC10029 CAR The MD Anderson Cancer Center Monoclonal Antibody Core Facility undertook the generation of anti-BAFFR monoclonal antibodies (mAbs) using BAFF-R-expressing NIH / 3T3 cells as the immunogen (Figure 1A). To identify lead antibody-producing clones, candidate hybridoma clones were screened against BAFF-R-expressing 293FT cells (Figure 1B). Notably, hybridoma clone 21 (C21) supernatant showed a dose-dependent and antigen-specific binding pattern (Figures 1C and 1D). This antigen-specific binding was further confirmed with antibodies purified from C21 hybridoma supernatant (Figure 1E). Following confirmation of the lead hybridoma clone, the cDNA sequences of the heavy and light chain variable regions of C21 mAb were identified for the construction of the MC10029 CAR.

[0192] The second-generation BAFF-R CAR (MC10029 CAR) was generated using a clinically approved lentiviral vector (Dong et al., Cancer Immunology, Immunotherapy, 69(10), 2139-2145, 2020). This construct contains a novel BAFF-R antibody scFv that sequentially combines an IgG4 hinge, CD28 transmembrane domain, CD28 costimulatory domain, CD3ζ, and tEGFR (Figure 2A). The selection of the CD28 costimulatory domain was empirically determined by comparing the antigen-specific cytotoxicity of BAFF-R CAR constructs containing either CD28 or 4-1BB (Figures 17A-17C). Research-grade MC10029 CAR-T cells were reproducibly generated, meeting specific requirements for cell quality, CAR-T cell-specific properties, and CAR-T cell expansion fold (Figures 2B-2E). This ensured the quality of the experimental CAR-T cells.

[0193] MC10029 CAR-T cells demonstrate antigen-specific cytotoxicity against acute lymphoblastic leukemia (ALL) in both in vitro and in vivo models. BAFF-R expression was confirmed in the cell lines used for functional analysis of MC10029 CAR-T cells (Figure 18A). After confirming BAFF-R expression in the ALL cell line Nalm-6, MC10029 CAR-T cells exhibited antigen-specific cytotoxicity against wild-type (WT) Nalm-6 cells but not against BAFF-R KO Nalm-6 cells (Figure 1A, Figure 20A, Figure 4B, Figure 20B). CD107a degranulation assays were gated on the CD4 CAR-T cell population. Granzyme B release was observed only when MC10029 CAR-T cells were incubated with WT Nalm-6 cells (Figure 4C). When MC10029 CAR-T cells were incubated with GFP-labeled WT Nalm-6 cells, cytolysis, as determined by the disappearance of target cells engineered to express green fluorescent protein (GFP), was observed in BAFF-R KO Nalm-6 cells, but not in BAFF-R KO Nalm-6 cells, again confirming antigen-specific cytotoxicity (Figure 4D).

[0194] The therapeutic efficacy of MC10029 CAR-T cells was evaluated in NSG mice injected with Nalm-6 tumor cells. Tumor changes were monitored over time by bioluminescence imaging (Figure 5A), and long-term survival was monitored by Kaplan-Meier plots (Figure 5B). PBS-treated mice demonstrated tumor growth and death within 42 days, while tumor progression was observed in the non-CAR-T cell treated group. Mice injected with MC10029 CAR-T cells showed a significant reduction in Nalm-6 tumor presence and a statistically significant increase in survival up to 120 days.

[0195] MC10029 CAR-T cells were also effective in a CD19 antigen-deficient model To mimic antigen-evasive disease, we developed a CD19-deficient Nalm-6-based model. MC10029 CAR-T cells maintained antigen-specific cytotoxicity against the CD19 KO Nalm-6 cell line, whereas only background activity was observed with non-CAR-T cells and CD19 CAR-T cells (Figures 6A and 20C). The potent activity of MC10029 CAR-T cells against CD19-deficient tumor cells was confirmed by measuring granzyme B (Figure 6B). The antitumor activity of MC10029 CAR-T cells against CD19-deficient tumors was consistently reproduced in two additional CD19-deficient B-cell tumor models: CD19 KO Z-138 and CD19 KO MEC-1 (Figures 18B–18F). This confirmation further supported the initial observations made in the CD19 KO Nalm-6 model. NSG mice injected with CD19 KO Nalm-6 tumor cells were then examined after receiving one of four treatments: PBS, non-CAR-T cells, MC10029 CAR-T cells, or CD19 CAR-T cells. Bioluminescence imaging demonstrated tumor progression (Figure 6C), and Kaplan-Meier curves plotted survival rates (Figure 6D). The PBS and non-CAR-T cell control groups were euthanized at approximately 60 days due to excessive tumor burden. Eighty percent of the mice in the CD19 CAR-T cell treatment group developed tumor burden similar to that of controls, and one mouse survived to the end of the 84-day experiment. The MC10029 CAR-T cell treatment group demonstrated a significant reduction in tumor volume and statistically significant survival.

[0196] MC10029 CAR-T cells demonstrate antigen-specific cytotoxicity against lymphoma in both in vitro and in vivo models Z-138 is a non-Hodgkin's lymphoma (NHL) cell line that expresses BAFF-R (Figure 18B). The antigen-specific cytotoxicity of MC10029 CAR-T cells against Z-138 was confirmed by CD107a degranulation assay (Figures 7A and 20D) and granzyme B release measurement (Figure 7B). The therapeutic efficacy of MC10029 CAR-T cells was evaluated by administering one of three treatments to NSG mice injected with Z-138 tumor cells. Bioluminescence imaging of the mice showed tumor progression (Figure 3c), and survival rates were also plotted (Figure 7D). Due to excessive tumor burden, the control groups (PBS group and non-CAR-T cell group) were euthanized within 56 days. The MC10029 CAR-T cell-treated group showed a significant reduction in tumor presence and a statistically significant survival rate.

[0197] MC10029 CAR-T cells are a promising treatment for chronic lymphocytic leukemia (CLL) CLL remains a major unmet need for CAR-T cell therapy for B-cell hematopoietic malignancies. While CD19 CAR-T cell therapy has been studied in CLL patients, complete remissions have not been comparable to those reported in diseases such as ALL, FL, and MCL (Wang et al., N. Engl. J. Med., 382:1331-1342 (2020); Jacobson et al., Lancet Oncol., 23:91-103 (2022); Fowler et al., Nat. Med., 28:325-332 (2022); and Fraietta et al., Nat. Med., 24:563-571 (2018)). Given the high expression of BAFF-R in CLL, the potential of MC10029 CAR-T therapy targeting BAFF-R was explored as an alternative treatment option for CLL patients. The CLL cell line, MEC-1, was first utilized to examine the cytotoxicity of MC10029 CAR-T cells against CLL. As shown in Figure 8B and Figure 20E, the efficacy of MC10029 CAR-T cells against MEC-1 cells was confirmed by granule degranulation. Furthermore, granzyme B released in response to tumor cells provides further evidence supporting the efficacy of MC10029 CAR-T cells in targeting MEC-1 cells and inducing cytotoxic effects (Figure 8B).

[0198] Next, we evaluated MC10029 CAR-T cells against primary B cells isolated from CLL patients. The subjects selected were three men and three women, ages 56 to 83 (Figure 19A, identifiers 1–6). B cells were enriched from each subject's PBMCs and confirmed to express BAFF-R (Figure 9A). B cell enrichment effectively removed endogenous T cells (Figure 19B). In initial studies, two batches of MC10029 CAR-T cells and non-CAR-T cells were generated from T cells isolated from two healthy donors. These batches demonstrated identical viability, identity, and potency (Figures 11A–11C).

[0199] CD8 +Activation of MC10029 CAR-T cells was comparable among the six primary CLL tumor cells after incubation (Figure 9B, top row and Figure 19C). Furthermore, incubation of MC10029 CAR-T cells with CLL tumor cells resulted in the release of granzyme B (Figure 9C). Four of the CLL subjects provided sufficient B cells to assess the release of additional granule proteins. Incubation of MC10029 CAR-T cells with primary CLL tumor cells resulted in the release of statistically significant amounts of granzyme A, perforin, and IFN-γ (Figure 19D).

[0200] To more realistically model the efficacy of MC10029 CAR-T cells as a clinical therapeutic, three additional CLL patients were identified (Figure 19A, identifiers 7–9). Patient-derived MC10029 CAR-T cells were generated and incubated with autologous B cells. MC10029 CAR-T cells generated from the three CLL patients were characterized by monitoring fold expansion (>25; Figure 15A), identity (>80% CD3+ cells; Figure 15B, top row), and potency (>10% EGFR+ cells; Figure 15B, bottom row). These MC10029 CAR-T cells and corresponding non-CAR-T cells (negative control) were incubated with corresponding autologous B cells or MEC-1 cells (positive control). The cytotoxicity of patient-derived MC10029 CAR-T cells against autologous tumor cells was confirmed by CD8+ / CD107a degranulation assay (Figure 15C). Nonspecific activity is persistently observed when patient-derived non-CAR-T cells are incubated with target cells, particularly autologous B cells.

[0201] MC10029 CAR-T cell therapy: Readiness for clinical application As part of preparation for an IND application and a Phase 1a / 1b clinical trial, an engineering run was conducted to obtain three production batches of CAR-T cells using MC10029-expressing lentiviral vectors manufactured under GMP conditions. A series of assays used as process quality control (QC) and final product QC assays were established and validated. These assays were performed on the production batches generated in the laboratory (Figures 16A and 16B). Production batches of MC10029 CAR-T cells were assessed for cell quality by fold expansion and viability (>70% as measured by a Muse Cell Analyzer), identity (>70% as measured by flow cytometry in CD3+ cells) and potency (>10% as measured by flow cytometry in EGFR+ T cells) for CAR-T cell specific characterization; and safety from adventitious viral agents by measuring lentiviral copy number by two methods: <5 copies of woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) per cell as detected by a real-time qPCR assay, and <5 copies of vesicular stomatitis virus G glycoprotein (VSVG; encoding the envelope gene sequence) per 50 ng DNA. All three production batches of MC10029 CAR-T cells met the criteria required for commercial qualification (Figure 16A). Furthermore, the antigen-specific cytotoxicity of these MC10029 CAR-T cells was confirmed using our standard degranulation assay (Figure 16B, Figure 20F). The successful quality assessment of these batches confirms their suitability for further clinical development.

[0202] Taken together, these results demonstrate that binding agents having two sets of three CDRs described herein (e.g., SEQ ID NOS: 1-3 and 9-11) can bind to BAFF-R polypeptides. Furthermore, as demonstrated herein, these binding agents can be engineered into molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers (e.g., BiTEs), or ADCs (e.g., antibody radioconjugates)) that can be used to treat mammals (e.g., humans) with cancer (e.g., B-cell cancer).

[0203] Example 18: BAFF-R CAR-T Therapy for Sensitized Kidney Transplant Patients The demand for organ transplants, particularly in patients with end-stage renal disease (ESKD), far exceeds the current organ supply. This challenging situation is further exacerbated by the presence of high levels of pre-transplant anti-HLA antibodies, clinically known as sensitization (Heidt et al., Expert Rev. Clin. Immunol., 14:673-679 (2018)), which significantly complicates the matching process. This can result in extremely long wait times on transplant waiting lists and potentially catastrophic organ failure before receiving a life-saving transplant (Sapir-Pichhadze et al., J. Am. Soc. Nephrol., 27:570-578 (2016)).

[0204] It has been widely reported that anti-HLA donor-specific antibodies play an important role in kidney transplant rejection and associated adverse outcomes. Several approaches have been used to desensitize these patients, but none of these treatments have shown durable clinical benefits.

[0205] This example describes how MC10029 CAR T cells can be used to target B cells in kidney transplant patients, desensitizing them and minimizing the risk of organ transplant rejection.

[0206] Materials and Methods cell line The Nalm-6 cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany), and the BAFF-R knockout mutant (BAFF-R KO Nalm-6) was generated as described in Qin et al., Science Translational Medicine, 11(511) 2019. 293FT cells and Jurkat cells were obtained from ATCC. Prior to cryopreservation, cell lines were authenticated for the antigens of interest using flow cytometry.

[0207] human blood samples Peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were isolated by leukapheresis using a leukapheresis system (LRS) cone at the Department of Transfusion Medicine, Mayo Clinic, Rochester, Minnesota, according to current regulatory requirements and the method described in Dietz et al., Transfusion 46:2083-2089 (2006).

[0208] Patient blood samples were collected as part of a biorepository protocol. All patients provided written informed consent. Consenting patients underwent two blood draws, at least 2 weeks apart, yielding approximately 40 mL of blood. Disease characteristics of these patients were recorded.

[0209] Isolation of PBMCs from patient blood samples Peripheral blood collected from subjects was diluted 1:1 (v / v) with PBS, and the diluted blood sample (20 mL) was layered on 10 mL of Ficoll (Sigma). The gradient was centrifuged at 2000 rpm for 20 minutes at room temperature. The PBMC layer was then carefully separated and isolated. A cell counter (TC 20 Automated Cell Counter, Bio-Rad) was used to assess the number and viability of PBMCs.

[0210] Generation of patient-derived MC10029 CAR-T cells All patients consented to two blood draws, usually 2 weeks apart. Each draw yielded 12–20 mL of blood. The first blood draw was used to generate patient-derived CAR-T cells. The collected PBMCs were subjected to T cell isolation using a pan T cell isolation kit (Miltenyi Biotec, Germany). Briefly, PBMCs were first labeled with a biotin-antibody cocktail, followed by a microbead cocktail in FACS buffer. The labeled cells were applied to an LD column and subjected to magnetic cell separation. The unlabeled T cells that passed through the column were collected for CAR-T cell production.

[0211] A second-generation BAFF-R-CAR (MC10029) was generated, consisting of a novel BAFF-R antibody scFv, an IgG4 transmembrane domain, CD28, and a CD3ζ intracellular signaling domain. Furthermore, this CAR construct was engineered to contain a safety switch, truncated human epidermal growth factor receptor (tEGFR), allowing CAR T cell depletion after administration of the FDA-approved anti-EGFR mAb, cetuximab. The CAR cDNA was cloned into the pHIV.7 lentiviral vector. Lentivirus was produced in 293FT cells, concentrated, and titered in Jurkat cells. T cells isolated from the patient's PBMCs were divided into two aliquots. One aliquot was expanded as untransduced (non-CAR) T cells and used as an alloreactive control, while the remaining cells were used for CAR T cell production. To generate patient-derived CAR T cells, T cells were activated with Human T-Activator CD3 / CD28 beads (Life Technologies) for 24 hours and then transduced with CAR lentivirus at a multiplicity of infection (MOI) of 1. CAR T cells were activated with CD3 / CD28 bead stimulation for an additional 6 days, after which the beads were removed and the CAR T cells were expanded for an additional 7 days. Non-CAR T cells were untransduced T cells from the same donor and expanded according to the CAR T cell protocol. Each batch of CAR T cells was assessed for cell quality by fold expansion and viability (determined by trypan blue staining), and CAR T cell specificity was assessed by flow cytometry for identity (CD3-positive cells) and potency (EGFR-positive T cells). Characterized patient-derived MC10029 CAR T cells were cryopreserved for subsequent functional assays.

[0212] Isolation of autologous B cells for CAR T cell functional assays When the patient was available for a second blood draw, autologous B cells were isolated from PBMCs using the EasySep™ Human B Cell Isolation Kit (STEMCELL technologies, Canada). Briefly, the isolation cocktail was added to PBMCs, followed by incubation with RapidSpheres™. The tubes were then placed on a magnet for negative selection. The isolated B cells were stained and characterized with anti-CD3 BV605 (BD Biosciences), anti-CD20 BUV395 (BD Biosciences), and anti-BAFF-R-AF647 (BD Biosciences). These newly isolated autologous B cells were used as target cells to stimulate cryopreserved MC10029 CAR T cells in T cell functionality assays. At least 2 million enriched B cells were required for degranulation and granule release assays. Of the 10 enrolled patients, we successfully harvested 1.5 million to 2.65 million B cells from seven patients (six kidney transplant patients and one lung transplant patient) and performed at least one CAR T cell functional assay. The remaining three kidney transplant patients (patients 7–9) had difficulty obtaining sufficient autologous B cells (fewer than 0.5 million). Therefore, we tested the antigen-specific function of cryopreserved CAR T cells using target cell lines (Nalm-6 and BAFF-R KO Nalm-6).

[0213] Degranulation assay CAR T cells were incubated with target cells at a 2:1 effector-to-target (E:T) ratio in complete RPMI 1640 medium containing GolgiStop protein trafficking inhibitor reagent (BD Bioscience) and CD107a APC antibody (BD Biosciences) for 6 hours. Cells were then stained with anti-CD3 BV605 (BD Biosciences), anti-CD4 PE-Cy7 (BD Biosciences), anti-CD8 APC-Cy7 (BD Biosciences), and anti-EGFR BV421 (BD Biosciences). Samples were run on an Attune flow cytometer (Thermo Fisher Scientific) or a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo™ Version 10 software. Non-CAR T cells from the same patient served as a negative control.

[0214] Granule release assay CAR T cells and target cells were co-cultured at an E:T ratio of 4:1 for 72 hours. After incubation, supernatants were collected and granule release was assessed. Levels of granule proteins involved in cytotoxicity, such as granzyme B, granzyme A, and perforin, were quantified using a customized U-PLEX Human ELISA kit from Meso Scale Diagnostics (Rockville, MD, USA) according to the manufacturer's instructions. This multiplex kit enabled simultaneous measurement of multiple secreted proteins related to CAR-T cell function.

[0215] Direct killing assay using B cells as target cells A direct killing assay using B cells as target cells was established using MC10029 CAR-T cells derived from a healthy donor. This established assay was then applied to patient-derived MC10029 CAR-T cells. MC10029 CAR-T cells were co-incubated with B cells at an E:T ratio of 10:1 for 1 hour. Cells were then stained with anti-CD20 BUV395 (BD Biosciences) and Sytox Blue (Thermo Fisher). Samples were evaluated using a Fortessa flow cytometer (BD Biosciences), and the resulting data were analyzed using FlowJo™ version 10 software. Non-CAR-T cells from the same donor were used as a negative control.

[0216] statistical analysis All statistical analyses were performed using GraphPad Prism software. Data are reported as mean ± standard error of mean (SEM) and analyzed using Student's t-test.

[0217] result Study design The primary objective of this study was to investigate the cytotoxicity of patient-derived MC10029 CAR T cells against autologous B cells. Inclusion criteria were adult patients aged 18 years or older, with a high sensitization level of calculated panel reactive antibodies (cPRA) of 98% or higher, and who agreed to provide informed consent, clinical data, and blood samples. Patients who underwent desensitization therapy were excluded from this study. A total of 10 eligible patients participated in this study, including nine kidney transplant patients and one lung transplant patient. Patient and disease information is shown in Tables 2 and 3. [Table 2] TIFF2026508306000071.tif254159 [Table 3]

[0218] Targeting BAFF-R on patient B cells with MC10029 CAR T cells A protocol for generating patient-derived CAR T cells from limited blood samples (12–20 mL) (Figure 22) was developed and optimized for isolating enriched autologous B cells from the same patients, which were used as target cells in CAR T cell functional experiments (Table 2 and Figure 23). CAR T cells were generated for all 10 enrolled patients (Table 2 and Table 3). Seven of the 10 patients, including the patient who underwent lung transplantation, provided sufficient autologous B cells to evaluate the cytotoxicity of patient-derived CAR T cells against autologous B cells (Table 2 and Figure 24, respectively). For the remaining three patients from whom sufficient B cells could not be isolated for autologous experiments, target cell lines (Nalm-6 and BAFF-R KO Nalm-6) were used to verify the antigen-specific functionality of CAR T cells (Figure 25).

[0219] Autologous CD20-positive B cells from sensitized patients were enriched from blood samples and used as target cells. Enriched B cells from healthy donors served as a control. This enrichment process efficiently removed CD3-positive T cells, reducing the nonspecific background that these T cells might introduce in subsequent CAR T cell functional assays (Figure 21A). Notably, B cells from sensitized patients displayed BAFF-R expression levels comparable to those found in B cells from healthy donors (Figure 21A), suggesting the possibility of targeting BAFF-R for B cell elimination in desensitization strategies. The cytotoxicity of patient-derived MC10029 CAR T cells against autologous B cells was assessed by assessing degranulation activity and further confirmed by measuring granzyme B release. Figure 21 shows representative patient data (patient 1 in Table 2), demonstrating readily detectable degranulation activity of MC10029 CAR T cells in response to autologous B cells (Figure 21B). Furthermore, upon encountering autologous B cells, CAR T cells significantly released cytotoxic granzyme B (Figure 21C). Non-CAR T cells from the same patient served as a control in both the degranulation assay and the granzyme B release experiment, representing the baseline level of T cell activation observed in patients. Importantly, CAR T cell function was shown to be antigen-specific, as evidenced by the CAR T cells' activation only against Nalm-6 cells expressing endogenous BAFF-R, but not against a genetically engineered mutant lacking BAFF-R expression (Figures 21B-21C). These data validate the autologous experimental system and support the use of MC10029 CAR T cell therapy as a desensitization strategy.

[0220] Evaluation of patient-derived MC10029 CAR T cells against autologous B cells Seven of the 10 enrolled patients provided sufficient autologous B cells to assess the cytotoxicity of patient-derived CAR T cells against autologous B cells. These included six kidney transplant patients (patients 1–6 in Table 2) and one lung transplant patient (patient 10 in Figure 24). Functional analysis data for the six kidney transplant patients are shown in Table 2. CAR T cells from these patients consistently demonstrated degranulation activity against autologous B cells, as evidenced by the release of cytotoxic proteins (granzyme A, granzyme B, and perforin). To account for variability in baseline levels of T cell activation between patients, we calculated the fold change between MC10029 CAR T cells and non-CAR T cells and normalized the data for inter-patient comparisons (Table 2). Furthermore, CD107a degranulation data also provided compelling evidence of the cytotoxicity of patient-specific CAR T cells against autologous B cells. Consistent positive staining of CD107a on the surface of these CAR T cells indicated robust degranulation activity in response to autologous B cells (Table 2). Similar to observations in kidney transplant patients, MC10029 CAR T cells from lung transplant patients elicited cytotoxicity against autologous B cells, as evidenced by both degranulation and release of granzyme B (Figure 24). Functionality of patient-derived MC10029 CAR T cells was confirmed as antigen-specific upon interaction with BAFF-R-positive Nalm-6 cells, which demonstrated T cell degranulation. However, no response was observed when these CAR T cells interacted with a mutant Nalm-6 cell line genetically engineered to lack BAFF-R expression (Figures 23 and 24).

[0221] A direct killing assay using B cells as target cells was first established using MC10029 CAR T cells derived from a healthy donor. After CAR-T cell introduction, a significant decrease in the percentage of viable B cells was observed in the MC10029 CAR-T group compared to the non-CAR-T group. The effect of MC10029 CAR T cells derived from a representative sensitized kidney transplant patient (Patient 5) on direct B cell killing was evaluated using healthy donor-derived B cells as surrogate target cells. A significant decrease in viable CD20-positive B cells was observed (Figure 26).

[0222] Of the 10 enrolled patients, three patient samples (patients 7-9) did not yield enough B cells for autologous experiments. However, we successfully generated patient-specific MC10029 CAR T cells for these patients (Table 3). Using a set of BAFF-R-positive and BAFF-R-negative Nalm-6 cells as target cells, we demonstrated the antigen-specific function of these CAR T cells (representative data for patient 7 in Figure 25). The function was comparable to that observed in patients 1-6.

[0223] Taken together, these results demonstrate that MC10029 CAR T cells may be used to treat organ transplant rejection. For example, MC10029 CAR T cells generated from T cells from a sensitized patient exhibited cytotoxicity against autologous B cells and may therefore be used to desensitize a mammal undergoing an organ transplant (e.g., a kidney transplant) to the transplanted organ, thereby reducing or eliminating the risk of organ transplant rejection in that mammal.

[0224] Example 19: Cancer Treatment Cells (e.g., T cells) engineered to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R polypeptide are administered to a human identified with a B-cell cancer. Cells (e.g., T cells) modified to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R polypeptide are administered by intravenous injection. After administration of cells (e.g., T cells) modified to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R polypeptide, the number of cancer cells (e.g., cancer cells expressing a BAFF-R polypeptide) in the human body is reduced. Following administration of cells (e.g., T cells) modified to express one or more binding agents (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a BAFF-R polypeptide, one or more tumors (e.g., tumors expressing a BAFF-R polypeptide) in a human body are reduced in size.

[0225] Example 20: Generation of T cells expressing one or more binding agents capable of binding to a BAFF-R polypeptide T cells are obtained from a human identified as having a B-cell cancer. A nucleic acid designed to express one or more binding agents (e.g., one or more CARs) capable of binding to a BAFF-R polypeptide described herein is introduced into the T cells by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection, thereby causing the T cells to express the binding agents capable of binding to a BAFF-R polypeptide. The T cells modified to express one or more binding agents (e.g., one or more CARs) capable of binding to a BAFF-R polypeptide are re-administered to the human, for example, via intravenous injection. After administration of cells (e.g., T cells) modified to express one or more binding agents (e.g., one or more CARs) capable of binding to a BAFF-R polypeptide, the number of cancer cells (e.g., cancer cells expressing a BAFF-R polypeptide) in the human is reduced. Following administration of cells (e.g., T cells) modified to express one or more binding agents (e.g., one or more CARs) capable of binding to a BAFF-R polypeptide, one or more tumors (e.g., tumors expressing a BAFF-R polypeptide) in a human are reduced in size.

[0226] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 2 (or SEQ ID NO: 2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 3 (or SEQ ID NO: 3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO: 10 (or SEQ ID NO: 10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO: 11 (or SEQ ID NO: 11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids).

2. The antibody of claim 1, having the ability to bind to sequence number 42.

3. The antibody of any one of claims 1 to 2, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

8.

4. The antibody of any one of claims 1 to 2, wherein the light chain variable domain or region comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

16.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

5. The antibody according to any one of claims 1 to 5, wherein the antibody is an scFv antibody.

6. An antigen-binding fragment comprising a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:2 (or SEQ ID NO:2 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:3 (or SEQ ID NO:3 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and a light chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), SEQ ID NO:10 (or SEQ ID NO:10 with the addition, deletion, or substitution of 1, 2, or 3 amino acids), and SEQ ID NO:11 (or SEQ ID NO:11 with the addition, deletion, or substitution of 1, 2, or 3 amino acids).

7. The antigen-binding fragment of claim 6, wherein the antigen-binding fragment has the ability to bind to SEQ ID NO:

42.

8. The antigen-binding fragment of any one of claims 6 to 7, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 85% identity with the amino acid sequence set forth in SEQ ID NO:

8.

9. The antigen-binding fragment of any one of claims 6 to 7, wherein the light chain variable domain or region comprises an amino acid sequence having at least 85% identity with the amino acid sequence set forth in SEQ ID NO:

16.

10. The antigen-binding fragment of any one of claims 6 to 9, wherein the antigen-binding fragment is monoclonal.

11. The antigen-binding fragment of any one of claims 6 to 10, wherein the antigen-binding fragment is a Fab.

12. A chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 1 to 11.

13. 13. The chimeric antigen receptor of claim 12, wherein the antigen-binding domain comprises an scFv capable of binding to a B-cell activating factor receptor (BAFF-R) polypeptide.

14. The chimeric antigen receptor of any one of claims 12 to 13, wherein the hinge comprises the amino acid sequence shown in SEQ ID NO:

30.

15. The chimeric antigen receptor according to any one of claims 12 to 14, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

32.

16. The chimeric antigen receptor of any one of claims 12 to 15, wherein the chimeric antigen receptor comprises one or more signaling domains set forth in any one of SEQ ID NOs: 34 and 36.

17. A cell comprising the chimeric antigen receptor of any one of claims 12 to 16.

18. 18. The cell of claim 17, wherein the cell is a T cell, a stem cell, or a NK cell.

19. A cell engager comprising a first antigen-binding domain comprising the antibody or antigen-binding fragment of any one of claims 1 to 11, a linker, and a second antigen-binding domain.

20. 20. The cell engager of claim 19, wherein the first antigen-binding domain comprises an scFv capable of binding to a BAFF-R polypeptide.

21. 21. The cell engager of claim 20, wherein the first antigen-binding domain is an IgG capable of binding to a BAFF-R polypeptide.

22. 22. The cell engager of any one of claims 19 to 21, wherein the linker comprises a linker set forth in any one of SEQ ID NOs: 25, 26 and 30.

23. The cell engager of any one of claims 19 to 22, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a T cell.

24. 24. The cell engager of claim 23, wherein the polypeptide expressed on the surface of the T cell is a CD3 polypeptide.

25. The cell engager of any one of claims 19 to 22, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a natural killer cell.

26. 26. The cell engager of claim 25, wherein the polypeptide expressed on the surface of the NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44, NKp46, or CRTAM polypeptide.

27. 27. The cell engager of any one of claims 19 to 26, wherein the cell engager comprises a third antigen binding domain.

28. 28. The cell engager of claim 27, wherein the third antigen-binding domain binds to a polypeptide expressed on the surface of a natural killer cell.

29. 29. The cell engager of claim 28, wherein the polypeptide expressed on the surface of a natural killer cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44, NKp46, or CRTAM polypeptide.

30. A nucleic acid comprising a nucleic acid sequence encoding at least a portion of the antibody or antigen-binding fragment of any one of claims 1 to 11.

31. 31. The nucleic acid of claim 30, wherein the nucleic acid sequence encodes the heavy chain variable domain or region of claim 1.

32. 32. The nucleic acid of any one of claims 30 to 31, wherein the nucleic acid sequence encodes a light chain variable domain or region according to claim 1.

33. The nucleic acid according to any one of claims 30 to 31, wherein the nucleic acid is a viral vector.

34. The nucleic acid according to any one of claims 30 to 31, wherein the nucleic acid is a phagemid.

35. A nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 12 to 16 or the cell engager of any one of claims 19 to 29.

36. 36. The nucleic acid of claim 35, wherein the nucleic acid is a viral vector.

37. 36. The nucleic acid of claim 35, wherein the nucleic acid is a phagemid.

38. A host cell comprising a nucleic acid according to any one of claims 30 to 37.

39. A host cell expressing the chimeric antigen receptor of any one of claims 12 to 16 or the cell engager of any one of claims 19 to 29.

40. The host cell according to any one of claims 38 to 39, wherein the host cell is a T cell, a stem cell, or a NK cell.

41. 12. An antibody-drug conjugate (ADC) comprising an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 1 to 11.

42. 42. The ADC of claim 41, wherein the antigen-binding domain comprises an scFv capable of binding to a BAFF-R polypeptide.

43. 42. The ADC of claim 41, wherein the antigen-binding domain is an IgG capable of binding to a BAFF-R polypeptide.

44. 44. The ADC of any one of claims 41 to 43, wherein the drug is selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

45. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 11.

46. The composition of claim 45, comprising an antibody of any one of claims 1 to 5.

47. The composition of claim 45, comprising an antigen-binding fragment of any one of claims 6 to 11.

48. A composition comprising the cell engager of any one of claims 19 to 29.

49. A composition comprising the cells of any one of claims 17, 18, and 38 to 40.

50. A composition comprising the ADC of any one of claims 41 to 44.

51. 51. The composition of any one of claims 45 to 50, comprising a checkpoint inhibitor.

52. 52. The composition of claim 51, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

53. 53. A method of treating a mammal with cancer, comprising administering to the mammal a composition according to any one of claims 45 to 52.

54. 54. The method of claim 53, wherein the mammal is a human.

55. Cancer is BAFF-R ＋ The method according to any one of claims 53 to 54, wherein the treatment is cancer.

56. BAFF-R ＋ 56. The method of claim 55, wherein the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, follicular lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and intravascular large B-cell lymphoma.

57. 57. The method of any one of claims 53 to 56, wherein the number of cancer cells in the mammal is reduced after the administering step.

58. 1. A method of treating a mammal having cancer, comprising the steps of: (a) administering to a mammal a composition according to any one of claims 45 to 50; and (b) administering to the mammal a composition comprising a checkpoint inhibitor. A method comprising:

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

60. Cancer is BAFF-R ＋ The method of any one of claims 58 to 59, wherein the treatment is cancer.

61. BAFF-R ＋ 61. The method of claim 60, wherein the cancer is selected from the group consisting of CLL, ALL, hairy cell leukemia, follicular lymphoma, NHL, Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, DLBCL, and intravascular large B-cell lymphoma.

62. 62. The method of any one of claims 58 to 61, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

63. 63. The method of any one of claims 58 to 62, wherein after administering steps (a) and (b), the number of cancer cells in the mammal is reduced.

64. A method for binding a binding molecule to a BAFF-R polypeptide, the method comprising contacting the BAFF-R polypeptide with the antibody or antigen-binding fragment of any one of claims 1-11.

65. 65. The method of claim 64, wherein the contacting is performed in vitro.

66. 65. The method of claim 64, wherein the contacting is performed in vivo.

67. 67. The method of claim 66, wherein the contacting is performed in a mammal by administering the antibody or antigen-binding fragment to the mammal.

68. 68. The method of claim 67, wherein the mammal is a human.

69. 19. A method of binding a binding molecule to a BAFF-R polypeptide, comprising contacting the BAFF-R polypeptide with a chimeric antigen receptor of any one of claims 12-16, a cell engager of any one of claims 19-29, or an ADC of any one of claims 41-44.

70. 70. The method of claim 69, wherein the contacting is performed in vitro.

71. 70. The method of claim 69, wherein the contacting is performed in vivo.

72. 72. The method of claim 71, wherein the contacting is carried out in the mammal by administering the chimeric antigen receptor, cell engager, or ADC to the mammal.

73. 73. The method of claim 72, wherein the mammal is a human.

74. 53. A method for treating a mammal having a transplanted organ, comprising administering to the mammal a composition according to any one of claims 45 to 52.

75. 75. The method of claim 74, wherein the mammal is a human.

76. 76. The method of any one of claims 74 to 75, wherein the transplant organ is selected from the group consisting of kidney, lung, liver, heart, pancreas, and bone marrow.

77. 53. A method of delaying or preventing organ transplant rejection in a mammal that has undergone or is preparing to receive an organ transplant, the method comprising administering to the mammal a composition according to any one of claims 45 to 52.

78. 78. The method of claim 77, wherein the mammal is a human.

79. 79. The method of any one of claims 77 to 78, wherein the organ transplant is selected from the group consisting of kidney transplant, lung transplant, liver transplant, heart transplant, pancreas transplant, and bone marrow transplant.

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