Induced NK cells responding to CD3 / TAA bispecific antibodies

Abstract

In certain aspects, provided herein are compositions and methods for treating cancer. The methods of the present disclosure include administering to a subject in need thereof NK cells expressing a CAR in combination with an antigen binding molecule that binds to a tumor antigen, wherein the CAR-NK cells target tumor cells via binding to the antigen binding molecule.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 446,428, filed February 17, 2023, and U.S. Provisional Patent Application No. 63 / 458,765, filed April 12, 2023, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on February 16, 2024, is named RPB-02825_SL.xml and is 1,084,637 bytes in size. [Background technology]

[0003] Cancer is the second leading cause of death in the United States. Current treatments for many cancers fail in certain patient populations or produce adverse side effects that significantly impact patients' quality of life. Adoptive immunotherapy, which involves the transplantation of ex vivo generated antigen-specific immune cells (e.g., T cells or NK cells), is a promising strategy for treating cancer. Immune cells used in adoptive immunotherapy can be generated, for example, by redirecting immune cells through genetic engineering (e.g., by engineering them to express chimeric antigen receptors, or "CARs"). CAR-NK cells offer a better safety profile than CAR-T cells, minimal cytokine release, and mild graft-versus-host disease. However, engineering unique CAR-NK cells specific for every potential cancer antigen is a laborious and time-consuming process. Thus, there is a need in the art for improved adoptive immunotherapy approaches for cancer treatment. Summary of the Invention

[0004] The present disclosure is based, in part, on the discovery that co-administration of a cancer antigen-binding molecule (e.g., a cancer antigen-specific antibody) with NK cells expressing a CAR containing a binding domain specific for the cancer antigen-binding molecule induces cytotoxicity in tumor cells expressing that cancer antigen. Thus, in certain embodiments, provided herein are CAR-NK cells that can be used as off-the-shelf therapeutics and can target a wide range of different cancers by co-administering a cancer antigen-specific antibody.

[0005] Thus, in some embodiments, provided herein is a chimeric antigen receptor (CAR) polypeptide comprising: (a) an extracellular domain comprising: (i) the extracellular domain of CD3, or a fragment thereof; (ii) an antigen-binding domain specific for the idiotype of an anti-CD3 antibody; or (iii) an antigen-binding domain specific for an Fc domain; (b) a hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0006] In some embodiments, the extracellular domain comprises the extracellular domain of CD3 or a fragment thereof. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an epitope recognized by an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises at least 10 contiguous amino acids of SEQ ID NO: 1959. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1959. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 1959.

[0007] In some embodiments, the extracellular domain comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises the CDR sequences of the heavy and light chains of an scFv listed in Table 1. In some embodiments, the antigen-binding domain comprises the variable region sequences of the heavy and light chains of one of the scFvs listed in Table 1. In some embodiments, the antigen-binding domain comprises the amino acid sequence of an scFv listed in Table 1.

[0008] In some embodiments, the extracellular domain comprises an antigen-binding domain specific for an Fc domain. In some embodiments, the Fc domain is selected from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain. In some embodiments, the Fc domain is an IgG3 Fc domain. In some embodiments, the Fc domain comprises the Fc amino acid sequence shown in Figure 3.

[0009] In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv).

[0010] In some embodiments, the hinge domain is a CD28 or CD8 hinge domain. In some embodiments, the hinge domain comprises an amino acid sequence selected from SEQ ID NOs: 1-5. In some embodiments, the transmembrane domain is an NKG2D transmembrane domain, an NKG2D reverse transmembrane domain, a CD28 transmembrane domain, a CD8 transmembrane domain, a CD16 transmembrane domain, or an FcgR1 (CD64) transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence selected from SEQ ID NOs: 6-13. In some embodiments, the intracellular signaling comprises any combination of an FcgR1 intracellular signaling domain, a CD3z intracellular signaling domain, a 4-1BB intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16 intracellular signaling domain, a CD64 intracellular signaling domain, or a CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain is an FcgR1 intracellular signaling domain, a 4-1BB-CD3z intracellular signaling domain, a 2B4-CD3z intracellular signaling domain, a CD16 intracellular signaling domain, a CD64 intracellular signaling domain, or a CD28-CD3z intracellular signaling domain.

[0011] In some aspects, provided herein are vectors comprising the nucleic acids described herein. In some embodiments, the vectors are expression vectors. In some embodiments, the vectors are viral vectors. In some embodiments, the viral vectors are lentiviral vectors.

[0012] In some aspects, provided herein are natural killer (NK) cells comprising a nucleic acid described herein. In some aspects, provided herein are natural killer (NK) cells expressing a CAR polypeptide described herein. In some embodiments, the cells are primary NK cells or induced NK cells differentiated from induced pluripotent stem cells (iPSCs). In some aspects, provided herein are immune cells (e.g., phagocytes) comprising a nucleic acid described herein or expressing a CAR polypeptide described herein.

[0013] In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject (A) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain; and (B) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to the extracellular domain. In certain embodiments, the method comprises administering to the subject (A) natural killer (NK) cells expressing a CAR polypeptide comprising a CD3 extracellular domain or a fragment thereof; and (B) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to the CD3 extracellular domain or a fragment thereof and a tumor antigen-binding domain that specifically binds to a tumor antigen.

[0014] In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject (A) an antigen binding molecule that binds to a tumor antigen; and (B) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the antigen binding molecule. In some embodiments, the method comprises administering to the subject (A) a multispecific antigen binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen; and (B) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule.

[0015] In some aspects, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject (a) an antigen binding molecule that binds to a tumor antigen and comprises an Fc domain; and (b) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the Fc domain. In some embodiments, the methods comprise administering to the subject (A) a multispecific antigen binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; and (B) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the Fc domain, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen binding molecule.

[0016] In some embodiments, the antigen binding molecule (e.g., a multispecific antigen binding molecule) and the NK cells are administered simultaneously or sequentially. In some embodiments, the antigen binding molecule (e.g., a multispecific antigen binding molecule) and the NK cells are premixed and administered simultaneously to the subject. In some embodiments, the subject is lymphopenic, and the antigen binding molecule (e.g., a multispecific antigen binding molecule) and the NK cells are premixed and administered simultaneously to the subject. In some embodiments, the NK cells or premixed NK cells and antigen binding molecule (e.g., premixed NK cells and a multispecific antigen binding molecule) are administered after at least one administration of the antigen binding molecule (e.g., a multispecific antigen binding molecule).

[0017] In some embodiments, the antigen-binding molecule (e.g., a multispecific antigen-binding molecule) is a bispecific antigen-binding molecule. In some embodiments, tumor antigens include, but are not limited to, CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, BCMA, CA19.9, MSLN, CD22, SLC3A2-APIS, CLDN18.2, and CEACAM5. In some embodiments, the antigen-binding molecule (e.g., a multispecific antigen-binding molecule) is a multispecific antibody or antigen-binding fragment thereof. In some embodiments, the multispecific antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody.

[0018] In some embodiments, the antigen binding molecule (e.g., a multispecific antigen binding molecule) is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody. In some embodiments, the antigen binding molecule (e.g., a multispecific antigen binding molecule) is a multispecific antigen binding molecule listed in Table 6.

[0019] In some aspects, provided herein is a pharmaceutical composition comprising: (A) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain comprising a CD3 extracellular domain or a fragment thereof; and (B) a multispecific antigen binding molecule comprising a CD3 binding domain that specifically binds to the CD3 extracellular domain or a fragment thereof and a tumor antigen binding domain that specifically binds to a tumor antigen.

[0020] In some aspects, provided herein is a pharmaceutical composition comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen; and (B) a natural killer (NK) cell expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule.

[0021] In some aspects, provided herein is a pharmaceutical composition comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; and (B) a natural killer (NK) cell expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the Fc domain, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen-binding molecule.

[0022] In some embodiments, provided herein is a cell bank comprising NK cells expressing a CAR described herein. [Brief explanation of the drawings]

[0023] [Figure 1] A shows NFAT activity in Jurkat / NFAT-Luc cl.3C7 cells, and B shows NFAT activity in Jurkat / NFAT-Luc / ahFc-CD28-CD3 cells. Both cells were incubated with titrated isotype control (REGN1932, gray squares) or single-arm anti-CD20 antibody (REGN2959, black) and target cells lacking CD20 expression (Jurkat, open symbols / dashed lines) or CD20-positive target cells (Ramos.2G6.4C10, black symbols / solid lines). After 5 hours, NFAT activity was assessed by luminescence reading. [Figure 2] Cytotoxicity of KHYG / ahFc-CD28-CD3 cells. KHYG / ahFc-CD28-CD3 cells were incubated with titrated isotype control (REGN1932, gray squares, gray dashed line) or single-arm anti-CD20 antibody (REGN2959, black circles, black solid line) in the presence of a fixed amount of Ramos / GFP target cells. After 4 hours, tag release was detected using an extracellular detection system. [Figure 3]Sequence alignment between hIgG3 and IgG4 Stealth* constant regions. hIgG3, REGN2280, and REGN7075 contain 100% sequence identity in the CH3 region, including the star mutation: FSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 14). The figure discloses SEQ ID NOs: 91-94, respectively, in order of appearance. [Figure 4A]Single-chain variable fragments (scFv) derived from anti-human CD3 idiotypic monoclonal antibodies (mAbs) that blocked anti-hCD3 mAb binding to immobilized hCD3ε / δ (epsilon / delta heterodimer) are shown. 1PN29950_2HCLC (open circles) and PN29950_2LCHC (open squares) blocked binding of 20.0 pM REGN18409 (Figure 4A, anti-hCD3, 7221G) or REGN18411 (Figure 4B, anti-hCD3, 7221G20) to immobilized hCD3ε / δ. PN77570_HCLC (open circles) and PN77570_LCHC (open squares) blocked binding of 20.0 pM REGN2533 (anti-hCD3, 9F7) to immobilized hCD3ε / δ. The inset graphs show the dose-dependent binding of REGN18409 (Figure 4A), REGN18411 (Figure 4B), and REGN2533 (Figure 4C) (black inverted triangles) to immobilized hCD3ε / δ, with EC50 values ​​of 7.5 pM, 11.3 pM, and 8.1 pM, respectively. The parental bivalent mAb REGN5766 (black circles) inhibits the binding of 20.0 pM REGN18409 (Figure 4A) or REGN18411 (Figure 4B) to hCD3ε / δ with IC50 values ​​of 42.0 pM and 40.0 pM, respectively. The parental bivalent mAb REGN2984 (black circles) inhibits the binding of 20.0 pM REGN2533 to hCD3ε / δ with an IC50 value of 32.0 pM. The isotype controls, mIgG1 (Figures 4A and 4B) and mIgG2a (Figure 4C) (southern biotech #0102-01 and 0103-01, respectively; filled triangles), and the negative scFv control (Figures 4A, 4B, and 4C) (REGN4393; filled squares) showed no inhibition under identical assay conditions. The x-axis is Log10 molar concentration for the inset graphs, and dilution factor for the first graphs (derived from Log10 dilution factor). The y-axis for all graphs indicates absorbance at 450 nm. 1PN annotates the root protein number. 2HCLC or 2LCHC corresponds to the orientation of the scFv heavy chain, linker, and light chain. [Figure 4B]Single-chain variable fragments (scFv) derived from anti-human CD3 idiotypic monoclonal antibodies (mAbs) that blocked anti-hCD3 mAb binding to immobilized hCD3ε / δ (epsilon / delta heterodimer) are shown. 1PN29950_2HCLC (open circles) and PN29950_2LCHC (open squares) blocked binding of 20.0 pM REGN18409 (Figure 4A, anti-hCD3, 7221G) or REGN18411 (Figure 4B, anti-hCD3, 7221G20) to immobilized hCD3ε / δ. PN77570_HCLC (open circles) and PN77570_LCHC (open squares) blocked binding of 20.0 pM REGN2533 (anti-hCD3, 9F7) to immobilized hCD3ε / δ. The inset graphs show the dose-dependent binding of REGN18409 (Figure 4A), REGN18411 (Figure 4B), and REGN2533 (Figure 4C) (black inverted triangles) to immobilized hCD3ε / δ, with EC50 values ​​of 7.5 pM, 11.3 pM, and 8.1 pM, respectively. The parental bivalent mAb REGN5766 (black circles) inhibits the binding of 20.0 pM REGN18409 (Figure 4A) or REGN18411 (Figure 4B) to hCD3ε / δ with IC50 values ​​of 42.0 pM and 40.0 pM, respectively. The parental bivalent mAb REGN2984 (black circles) inhibits the binding of 20.0 pM REGN2533 to hCD3ε / δ with an IC50 value of 32.0 pM. The isotype controls, mIgG1 (Figures 4A and 4B) and mIgG2a (Figure 4C) (southern biotech #0102-01 and 0103-01, respectively; filled triangles), and the negative scFv control (Figures 4A, 4B, and 4C) (REGN4393; filled squares) showed no inhibition under identical assay conditions. The x-axis is Log10 molar concentration for the inset graphs, and dilution factor for the first graphs (derived from Log10 dilution factor). The y-axis for all graphs indicates absorbance at 450 nm. 1PN annotates the root protein number. 2HCLC or 2LCHC corresponds to the orientation of the scFv heavy chain, linker, and light chain. [Figure 4C]Single-chain variable fragments (scFv) derived from anti-human CD3 idiotypic monoclonal antibodies (mAbs) that blocked anti-hCD3 mAb binding to immobilized hCD3ε / δ (epsilon / delta heterodimer) are shown. 1PN29950_2HCLC (open circles) and PN29950_2LCHC (open squares) blocked binding of 20.0 pM REGN18409 (Figure 4A, anti-hCD3, 7221G) or REGN18411 (Figure 4B, anti-hCD3, 7221G20) to immobilized hCD3ε / δ. PN77570_HCLC (open circles) and PN77570_LCHC (open squares) blocked binding of 20.0 pM REGN2533 (anti-hCD3, 9F7) to immobilized hCD3ε / δ. The inset graphs show the dose-dependent binding of REGN18409 (Figure 4A), REGN18411 (Figure 4B), and REGN2533 (Figure 4C) (black inverted triangles) to immobilized hCD3ε / δ, with EC50 values ​​of 7.5 pM, 11.3 pM, and 8.1 pM, respectively. The parental bivalent mAb REGN5766 (black circles) inhibits the binding of 20.0 pM REGN18409 (Figure 4A) or REGN18411 (Figure 4B) to hCD3ε / δ with IC50 values ​​of 42.0 pM and 40.0 pM, respectively. The parental bivalent mAb REGN2984 (black circles) inhibits the binding of 20.0 pM REGN2533 to hCD3ε / δ with an IC50 value of 32.0 pM. The isotype controls, mIgG1 (Figures 4A and 4B) and mIgG2a (Figure 4C) (southern biotech #0102-01 and 0103-01, respectively; filled triangles), and the negative scFv control (Figures 4A, 4B, and 4C) (REGN4393; filled squares) showed no inhibition under identical assay conditions. The x-axis is Log10 molar concentration for the inset graphs, and dilution factor for the first graphs (derived from Log10 dilution factor). The y-axis for all graphs indicates absorbance at 450 nm. 1PN annotates the root protein number. 2HCLC or 2LCHC corresponds to the orientation of the scFv heavy chain, linker, and light chain. [Figure 5] Binding of antibodies to KHYG-1 / NFAT-Luc / CAR1 cells is shown. [Figure 6]Binding of antibodies to KHYG-1 / NFAT-Luc / CAR6 and CAR15 cells is shown. [Figure 7] Reporter activation of KHYG-1 / NFAT-Luc / CAR1 cells is shown. [Figure 8] Reporter activation in KHYG-1 / NFAT-Luc / CAR6 and CAR15 cells is shown. [Figure 9] 1 shows the cytotoxic activation of KHYG-1 / NFAT-Luc / CAR1 cells. [Figure 10] Cytotoxic activation of KHYG-1 / NFAT-Luc / CAR6 cells and CAR15 cells is shown. [Figure 11] Cytotoxicity and cytokine release from CBNK / CAR6 cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] General Provided herein are methods and compositions for the treatment of cancer. As disclosed herein, administration of NK cells expressing a CAR comprising an antigen-binding domain specific for the idiotype of an anti-CD3 antibody or an antigen-binding domain specific for the Fc domain is performed in combination with a bispecific antibody that binds to CD3 and tumor antigen (TAA)-induced cytotoxicity in tumor cells expressing the specific tumor antigen.

[0025] Thus, in some embodiments, provided herein is a chimeric immunoreceptor (CAR) comprising an extracellular domain comprising a CD3 extracellular domain, an antigen-binding domain specific for the idiotype of an anti-CD3 antibody, or an antigen-binding domain specific for an Fc domain.

[0026] In some aspects, provided herein are methods of treating cancer using NK cells (e.g., induced NK cells) expressing a CAR described herein in combination with an antigen binding molecule that binds to a tumor antigen, wherein the CAR-NK cells then bind to the antigen binding molecule that targets cancer cells expressing the tumor antigen to induce anti-tumor activity (e.g., cytotoxicity).

[0027] In some embodiments, the method comprises administering to a subject (A) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain (e.g., a CD3 extracellular domain or a fragment thereof) together with (B) a multispecific antigen binding molecule comprising a first antigen binding domain that binds to a tumor antigen and a second antigen binding domain that binds to the extracellular domain (e.g., a CD3 extracellular domain or a fragment thereof).

[0028] In some embodiments, the method may include administering to a subject: (A) an antigen-binding molecule that binds to a tumor antigen (e.g., a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen); and (B) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the antigen-binding molecule (e.g., an extracellular domain comprising an antigen-binding domain specific for the idiotype of the CD3 multispecific antigen-binding molecule).

[0029] In some embodiments, the method may include administering to a subject: (a) an antigen binding molecule that binds to a tumor antigen and comprises an Fc domain (e.g., a multispecific antigen binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain); and (b) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the Fc domain.

[0030] In some aspects, provided herein is a pharmaceutical composition comprising a CAR-NK cell described herein and an antigen binding molecule described herein, wherein the CAR-NK cell binds to the antigen binding molecule. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0031] Compared with CAR-T cells, CAR-NK cells offer a better safety profile, minimal cytokine release, and reduced graft-versus-host disease. Instead of engineering NK cells each time to express a CAR specific for a particular tumor antigen, the CAR-NK cells disclosed herein can be combined with various tumor antigen-binding molecules to serve as readily available "universal" CAR-NK cells capable of targeting various tumor types. For example, NK cells expressing a CAR containing an antigen-binding domain specific for the CD3 extracellular domain or the idiotype of an anti-CD3 antibody can be combined with various CD3 bispecific antibodies known in the art. NK cells expressing a CAR containing an antigen-binding domain specific for an Fc domain can be combined with various CD3 bispecific antibodies with an Fc domain or any other antibody that binds to a tumor antigen and contains an Fc domain.

[0032] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0033] As used herein, the term "about," when used in reference to a particular stated numerical value, means that the value may vary by 1% or less from the stated value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0034] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] As used herein, the terms "administering" or "administration" mean providing a pharmaceutical agent or composition to a subject, and include, but are not limited to, administration by a medical professional and self-administration. Such agents can contain, for example, CAR T cells provided herein.

[0036] As used herein, the term "antibody" may refer to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain comprises a light chain variable region (abbreviated herein as V) and a heavy chain constant region. L V H Area and V L The regions can be further subdivided into hypervariable regions called "complementarity-determining regions" (CDRs), which are separated by more conserved regions called "framework regions" (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The term "antibody" encompasses, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), single-chain antibodies, and antigen-binding antibody fragments.

[0037] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), e.g., CDR3 peptides) or FR3-CDR3-FR4-constrained peptides). Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the scope of the term "antigen-binding fragment" as used herein.

[0038] "Cancer" broadly refers to the uncontrolled, abnormal growth of a host's own cells, resulting in invasion of surrounding tissues and potentially tissues distal to the initial site of abnormal cell growth within the host. Major types include carcinomas, which are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas, which are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of hematopoietic tissues (e.g., bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells; and central nervous system cancers, including cancers originating from brain and spinal cord tissue. "Cancer(s)" and "neoplasm(s)" are used interchangeably herein. As used herein, "cancer" refers to all types of cancers or neoplasms or malignant tumors, including leukemias, carcinomas, and sarcomas, whether new or recurrent. Specific examples of cancers are carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Non-limiting examples of cancers include new or recurrent cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, and medulloblastoma. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a metastasis.

[0039] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen), with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cell immune activity. Generally, CARs consist of an extracellular single-chain antigen-binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex ζ chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0040] As used herein, the phrase "co-administration" or "co-administered" refers to any form of administration of two or more different therapeutic agents, such that a second agent is administered while a previously administered therapeutic agent is still active in the body (e.g., the two agents are active in a subject simultaneously, which may involve a synergistic effect of the two agents). For example, the different therapeutic agents can be administered in the same formulation, or in separate formulations, either simultaneously or sequentially. In certain embodiments, the different therapeutic agents can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or within 1 week of each other. Thus, subjects receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0041] A "costimulatory domain" or "costimulatory molecule" refers to the corresponding binding partner on an immune cell (e.g., a B cell, a T cell, a NK cell, or a myeloid cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. The costimulatory domain may be a human costimulatory domain. Exemplary costimulatory molecules include CD28, CD27, 4-1BB (CD137), OX40, CD30, CD40, ICOS, CD2, LIGHT, CD244 (2B4), and NKG2C.

[0042] A "costimulatory ligand" refers to a molecule present on an antigen-presenting cell that specifically binds to a corresponding costimulatory molecule on an immune cell (e.g., B cell, T cell, NK cell, or myeloid cell), thereby providing a signal that mediates immune cell (e.g., B cell, T cell, NK cell, or myeloid cell) responses, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOSLG), intercellular adhesion molecule (ICAM), CD30L, CD40L, CD70, MICA, MICB, and HVEM.

[0043] A "costimulatory signal" refers to a signal that, in combination with a primary signal, causes an immune cell (e.g., a B cell, T cell, NK cell, or myeloid cell) to proliferate and / or up-regulate or down-regulate key molecules.

[0044] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen can have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues of a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl moieties on an antigen.

[0045] A "genetic construct" refers to a nucleic acid, such as a vector, plasmid, or viral genome, that contains a "coding sequence" for a polypeptide or that is otherwise capable of being transcribed into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), transfected into a cell, e.g., a mammalian cell, and causing expression of the coding sequence in cells transfected with the construct. A genetic construct may include one or more regulatory elements operably linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0046] The terms "ligand binding domain" and "antigen binding domain" are used interchangeably herein and refer to the portion of a chimeric antigen receptor that specifically binds to a given antigen.

[0047] The term "linker" is art-recognized and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. A linker may consist of a single linking molecule or may include a linking molecule and a spacer molecule whose purpose is to separate the linking molecule and the compounds by a specific distance.

[0048] The term "operably linked" refers to the functional relationship of one nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operably linking DNA to a transcriptional regulatory element refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0049] As used herein, the phrase "pharmaceutically acceptable" refers to agents, compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0050] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the movement or transport of a drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0051] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to natural or synthetic molecules, or some combination thereof, containing a single nucleotide, or two or more nucleotides linked at the 3' position of one nucleotide to the 5' end of another nucleotide via a phosphate group. Polymeric forms of nucleotides can be of any length and can contain deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and perform any function. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all of the nucleic acid sequences provided herein, U nucleotides can be substituted for T nucleotides. A polynucleotide is not necessarily associated with the cell in which the nucleic acid is found in nature and / or is not necessarily operably linked to the polynucleotide with which it is linked in nature.

[0052] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0053] As used herein, the "signal transduction domain" or "signal transduction domain" of a CAR is responsible for intracellular signal transduction after the extracellular ligand-binding domain binds to a target, thereby resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "signal transduction domain" refers to a portion of a protein that transduces an effector function signal and induces the cell to perform its intrinsic function. Examples of signal transduction domains for use in CARs can be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after antigen receptor ligation, as well as any derivatives or variants of these sequences, and any synthetic sequence with the same functional capabilities. In some cases, the signal transduction domain includes two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may include a signaling motif known as an ITAM, an immunoreceptor tyrosine-based activation motif. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD28, 4-1BB, CD79b, and CD66d.

[0054] As used herein, "spacer" refers to a peptide that connects proteins (e.g., in a fusion protein). Generally, spacers have no particular biological activity other than connecting proteins together or maintaining some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to affect some property of the molecule, such as molecular folding, net charge, or hydrophobicity.

[0055] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known sequence identity algorithm, such as, for example, FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0056] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity, when optimally aligned, for example, using default gap weights with the GAP or BESTFIT programs. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992), Science, 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0057] As used herein, the term "specifically binds" or "specific binding," when referring to a polypeptide, refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological products. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its particular "target" if it does not bind in significant amounts to other proteins present in the sample or to other proteins with which the ligand or antibody may come into contact in an organism (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule will bind to that second molecule in a manner that is approximately 10 times more specific than the first molecule. 5 M -1 More than (e.g., 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 For example, in terms of the ability of a PIg-specific CAR to bind a peptide presented on MHC (e.g., class I MHC or class II MHC), the CAR typically binds specifically to that peptide / MHC with an affinity of at least about 10 M or less, KD, and binds to a given antigen / binding partner with an affinity (expressed as a KD) that is at least 10-fold, at least 100-fold, or at least 1000-fold less than the affinity with which it binds to a nonspecific and unrelated peptide / MHC complex (e.g., one containing a BSA peptide or a casein peptide).

[0058] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.

[0059] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a clinical condition in a treated individual. Desirable effects of treatment include a reduction in the rate of progression, an improvement or alleviation of the pathological condition, and an improvement in the remission or prognosis of a particular disease, disorder, or condition. For example, an individual is considered to be successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0060] The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (e.g., degenerate variants), substitutions within the wobble positions of each codon encoding an amino acid (e.g., in DNA and RNA), amino acids added to the C-terminus of a peptide, or peptides with 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0061] The term "vector" refers to a means by which nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, to which nucleic acid can be linked and which may or may not be capable of autonomous replication or which may or may not be capable of integration into a host cell chromosome. Such vectors may include any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct (e.g., linked to transcriptional control elements) in a form suitable for expression by a cell.

[0062] Chimeric antigen receptor (CAR) A CAR is a receptor comprising a targeting moiety linked to one or more signaling and / or costimulatory domains within a single fusion molecule. In some aspects, the binding portion of the CAR comprises an extracellular domain comprising (i) the extracellular domain of CD3 or a fragment thereof; (ii) an antigen-binding domain specific for the idiotype of an anti-CD3 antibody; or (iii) an antigen-binding domain specific for an Fc domain. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising the variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. In certain embodiments, the CAR further comprises a transmembrane domain, a transmembrane domain, and an intracellular signaling domain.

[0063] CD3 extracellular domain In some embodiments, provided herein is a CAR comprising an extracellular domain comprising the extracellular domain of CD3 or a fragment thereof.

[0064] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence set forth in SEQ ID NO: 116 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-delta comprises the amino acid sequence set forth in SEQ ID NO: 117 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 118 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; and CD3-gamma comprises the amino acid sequence set forth in SEQ ID NO: 119 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless expressly identified as being from a non-human species. Thus, the term "CD3" refers to human CD3 unless it is specified to be from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0065] As used herein, the phrase "cell surface-expressed CD3" refers to one or more CD3 proteins expressed on the surface of a cell in vitro or in vivo such that at least a portion of the CD3 protein is exposed on the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. Cell surface-expressed CD3 includes CD3 protein contained within the context of a functional T cell receptor on the cell membrane. Cell surface-expressed CD3 includes CD3 protein expressed as part of a homodimer or heterodimer (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers) on the surface of a cell. Cell surface-expressed CD3 also includes CD3 chains expressed alone, without other CD3 chain types, on the surface of a cell (e.g., CD3-epsilon, CD3-delta, or CD3-gamma). Cell surface-expressed CD3 can comprise or consist of a CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, the cell surface expressed CD3 can comprise or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0066] In some embodiments, the CD3 extracellular domain or fragment thereof is an extracellular domain of human CD3 or a fragment thereof, such as the extracellular domain of human CD3ε or a fragment thereof, the extracellular domain of human CD3δ or a fragment thereof, the extracellular domain of human CD3γ or a fragment thereof, or the extracellular domain of human CD3ζ or a fragment thereof. In some embodiments, the extracellular domain of human CD3ε is set forth in SEQ ID NO: 33, the extracellular domain of human CD3δ is set forth in SEQ ID NO: 34, and the extracellular domain of human CD3γ is set forth in SEQ ID NO: 35 of WO2016 / 085889, which is incorporated herein by reference in its entirety.

[0067] In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 113 consecutive amino acids of SEQ ID NO: 33 of WO2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below). In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33 of WO2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below). In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 33 of WO2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below). [Table 1]

[0068] In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 87 contiguous amino acids of SEQ ID NO: 34 of WO2016 / 085889. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34 of WO2016 / 085889. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 34 of WO2016 / 085889.

[0069] In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 97 contiguous amino acids of SEQ ID NO: 35 of WO2016 / 085889. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35 of WO2016 / 085889. In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 35 of WO2016 / 085889.

[0070] In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an epitope recognized by an anti-CD3 antibody. As used herein, "antibodies that bind CD3" or "anti-CD3 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments disclosed herein may bind to soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 protein as well as recombinant CD3 protein variants, e.g., monomeric and dimeric CD3 constructs that lack the transmembrane domain or are otherwise not associated with the cell membrane. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6.

[0071] Idiotype-specific antigen-binding domain of anti-CD3 antibodies In certain embodiments, the binding domain and / or extracellular domain of a CAR provided herein provides a CAR with the ability to bind to a target antigen of interest. A binding domain (e.g., a ligand-binding domain or an antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or a tumor protein, or a component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule of interest. For example, as further described herein, a binding domain can be the variable regions of an antibody light and heavy chain, and the light and heavy chain variable regions can be combined in a single chain in either orientation (e.g., V L -V H or V H -V L ) can be linked together. Various assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance (e.g., using BIACORE analysis). Exemplary methods for generating anti-idiotypic antibodies are described in U.S. Pat. No. 10,150,817 B2 and Example 1 of WO2017 / 162587A1, each of which is incorporated by reference in its entirety.

[0072] In some embodiments, the binding domain and / or extracellular domain of a CAR provided herein comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In particular embodiments, the anti-CD3 antibody is the anti-CD3 antibody designated as CH2527 in WO2017 / 162587A1. For example, in some embodiments, the anti-CD3 antibody comprises the CDR H1, CDR H2, and CDR H3 sequences of SEQ ID NOs: 11, 12, and 13, respectively, as disclosed in WO2017 / 162587, which is incorporated by reference in its entirety. In some embodiments, the anti-CD3 antibody comprises the CDR H1, CDR H2, and CDR H3 sequences of SEQ ID NOs: 44, 45, and 46, respectively, as disclosed in WO2017 / 162587, which is incorporated by reference in its entirety. In some embodiments, the anti-CD3 antibody comprises the variable heavy chain (VH) sequence of SEQ ID NO: 43, as disclosed in WO2017 / 162587, which is incorporated by reference in its entirety.

[0073] In some embodiments, the antigen-binding domain described herein is a single-chain variable fragment (scFv) specific for the idiotype of an anti-CD3 antibody, which may be a murine, human, or humanized scFv. In some embodiments, the single-chain variable fragment (scFv) specific for the idiotype of an anti-CD3 antibody is the scFv designated 4.15.64 or 4.32.63, as disclosed in WO2017 / 162587, which is incorporated herein by reference in its entirety.

[0074] In some embodiments, the antigen binding domain comprises the heavy and / or light chain CDR sequences of an scFv listed in Table 1. In some embodiments, the antigen binding domain comprises the heavy and / or light chain variable region sequences of an scFv listed in Table 1. In some embodiments, the heavy chain variable regions disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the heavy chain variable regions of an scFv listed in Table 1. In some embodiments, the light chain variable regions disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the light chain variable regions of the scFvs listed in Table 1. In some embodiments, the scFvs described herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the scFvs listed in Table 1. [Table 2-1] [Table 2-2]

[0075] In some embodiments, provided herein is a chimeric antigen receptor (CAR) polypeptide comprising: (a) an extracellular domain comprising an antigen-binding domain specific for the idiotype of an anti-CD28 antibody; (b) a hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. Such a CAR can be used in an anti-CD28 x TAA combination to target tumor cells expressing the TAA.

[0076] Fc domain-specific antigen-binding domain In some embodiments, the extracellular domain comprises an antigen-binding domain specific for an Fc domain. In some embodiments, the Fc domain is selected from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain. In some embodiments, the Fc domain is an IgG3 Fc domain. In some embodiments, the Fc domain comprises the Fc amino acid sequence shown in Figure 3.

[0077] In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the single-chain variable fragment (scFv) specific for the idiotype or Fc domain of an anti-CD3 antibody may be a murine, human, or humanized scFv. Single-chain antibodies may be cloned from V-region genes of hybridomas specific for a desired target. Techniques that can be used to clone variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain, but may also be a non-antibody-derived binding domain. The antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, where the fragment is responsible for binding to the antigen.

[0078] In certain embodiments, the CAR of the present disclosure may include linkers between various domains, added for proper spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the VH or VL binding domains that may be 1 to 10 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1 to 20 or 20 amino acids in length. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Linkers of lengths ranging from 10 to 30 amino acids inclusive, including the numbers described herein, are also encompassed herein.

[0079] In certain embodiments, a linker suitable for the CARs described herein is a flexible linker. Suitable linkers can be readily selected and can be of any different suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0080] Exemplary flexible linkers include glycine polymers (G)n (where n is at least an integer), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can potentially function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more Φ-Ψ space than alanine and is much less restrictive than residues with longer side chains. Those skilled in the art will recognize that CAR designs can include fully or partially flexible linkers, and thus can include not only flexible linkers but also one or more moieties that confer less flexible structure to provide the desired CAR structure.

[0081] Hinge domain, transmembrane domain, and intracellular signaling domain The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that distances the extracellular domain or binding domain (e.g., a CD3 extracellular domain as described herein, or an antigen binding domain specific for the idiotype or Fc domain of an anti-CD3 antibody) from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of a CAR is generally between the transmembrane (TM) domain and the extracellular domain or binding domain (e.g., a CD3 extracellular domain as described herein, or an antigen binding domain specific for the idiotype or Fc domain of an anti-CD3 antibody). In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified.

[0082] In some embodiments, a CAR described herein further comprises a hinge domain. The hinge domain may be selected from the hinge domains in Table 2 below. In specific embodiments, the hinge domain is a CD28 or CD8 hinge domain. In some embodiments, a CAR further comprises a hinge domain comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in Table 2. In specific embodiments, the hinge domain comprises an amino acid sequence selected from SEQ ID NOs: 1-5. [Table 3-1] [Table 3-2]

[0083] In some embodiments, the CAR described herein further comprises a transmembrane domain. A "transmembrane" region or domain is a portion of a CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the extracellular domain or binding domain (e.g., a CD3 extracellular domain as described herein, or an antigen binding domain specific for the idiotype or Fc domain of an anti-CD3 antibody) with its binding partner. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain. Other transmembrane domains that may be employed in some embodiments include those derived from CD8, CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine.

[0084] In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, ICOS, 4-1BB, CD4, Tim4, OX40, CD27, CD2, LFA-1, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, NKG2D, NKp44, NKp46, DAP12, CD16, NKp30, FcRγ, DAP10, 2B4, or DNAM-1. In some embodiments, the transmembrane domain may be selected from the transmembrane domains in Table 3 below. In specific embodiments, the transmembrane domain is an NKG2D transmembrane domain, an NKG2D reverse transmembrane domain, a CD28 transmembrane domain, a CD8 transmembrane domain, a CD16 transmembrane domain, or an FcgR1 (CD64) transmembrane domain. In some embodiments, the CAR further comprises a transmembrane domain comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in Table 3. In some embodiments, the transmembrane domain comprises an amino acid sequence selected from SEQ ID NOs:6-13. [Table 4-1] [Table 4-2]

[0085] In certain embodiments, the CARs provided herein further comprise an intracellular signaling domain. The intracellular signaling domain (also referred to herein as a "signaling domain") comprises a portion of a chimeric antigen receptor protein that is responsible for transmitting the message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell, or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0086] In certain embodiments, the CARs provided herein comprise one or more immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing useful primary cytoplasmic signaling sequences include those derived from TCRζ, FcR gamma, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD28, 4-1BB, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain of a CAR described herein is derived from CD3ζ.

[0087] In certain embodiments, the CARs provided herein further comprise a costimulatory domain. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide secondary signals necessary for efficient activation and function of immune cells (e.g., lymphocytes or NK cells) upon antigen binding. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), CD2, LIGHT, and NKD2C. Accordingly, the present disclosure provides an exemplary costimulatory domain derived from CD28. The inclusion of one or more costimulatory signaling domains may enhance the efficacy and proliferation of T cells expressing the CAR receptor. Also disclosed herein are CAR polypeptides whose cytoplasmic / costimulatory region further comprises a 4-1BB domain (e.g., in addition to the CD28 / zeta domain). The costimulatory region of such CAR polypeptides may comprise the complete 4-1BB domain or a fragment thereof, and / or the complete CD28 / zeta domain or a fragment thereof. The intracellular signaling domain and the costimulatory signaling domain may be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0088] In some embodiments, the intracellular signaling domain is an FcgR1 intracellular signaling domain, a 4-1BB-CD3z intracellular signaling domain, a 2B4-CD3z intracellular signaling domain, a CD16 intracellular signaling domain, a CD64 intracellular signaling domain, or a CD28-CD3z intracellular signaling domain.

[0089] In certain aspects, the CAR polypeptides provided herein comprise at least one cytoplasmic / co-stimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain. Additionally, the hinge / spacer region and / or transmembrane region of the CAR, or the transmembrane region of the CAR, may comprise a CD28 / ζ domain. The CAR may comprise at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 22. The CAR polypeptides disclosed herein may comprise at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NOs: 5, 6, and 22. A CAR polypeptide may comprise all three sequences represented by SEQ ID NOs: 5, 6, and 22. For example, the CAR hinge domain may comprise SEQ ID NO: 5, the transmembrane domain may comprise SEQ ID NO: 6, and the costimulatory domain may comprise SEQ ID NO: 22. [Table 5]

[0090] The CAR polypeptide sequences disclosed herein may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 26-28. The CAR polypeptides disclosed herein may comprise at least an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence set forth in SEQ ID NOs: 26-28. [Table 6]

[0091] Nucleic acids and vectors In certain aspects, also disclosed are nucleic acid and polynucleotide vectors that encode the CAR polypeptides disclosed herein.

[0092] The nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art, for example, by screening libraries of cells expressing the gene, by deriving the gene from a vector known to contain it, or by isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0093] Expression of a nucleic acid encoding a CAR is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0094] In certain embodiments, the polynucleotide encoding a CAR described herein is inserted into a vector. A vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted to effect protein expression and / or polynucleotide cloning. Such vectors are sometimes referred to as "expression vectors." An isolated polynucleotide may be inserted into a vector using any suitable method known in the art, for example, but not limited to, a vector may be digested with an appropriate restriction enzyme and then ligated to an isolated polynucleotide having corresponding restriction ends. Expression vectors are capable of incorporating and expressing heterologous or modified nucleic acid sequences that encode at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and optionally translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions, which are discussed below. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification.

[0095] The expression vector may have necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences such as the CMV, PGK, and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and 3' UTR AAUAAA transcription termination sequences for efficient gene transcription and translation in the corresponding host cells. Other suitable promoters include the constitutive promoter of the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters, including but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, may also be used. In certain embodiments, inducible promoters are also contemplated as part of vectors expressing chimeric antigen receptors. This provides a molecular switch that can turn on or off the expression of a polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0096] Expression vectors may have additional sequences, such as hexa-histidine (SEQ ID NO: 88), c-Myc, and a FLAG tag, that are incorporated into the expressed CAR. Thus, expression vectors may be engineered to contain 5' and 3' untranslated regulatory sequences that may function as enhancer sequences, promoter regions and / or terminator sequences that can facilitate or increase efficient transcription of the nucleic acid(s) of interest contained in the expression vector. Expression vectors may also be engineered for replication and / or expression functions (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors may also include selectable markers for maintaining the vector within host or recipient cells.

[0097] In various embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (such as lambda phage or M13 phage), and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. The coding sequences of the CARs disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0098] In certain embodiments, the nucleic acid encoding the CAR is provided in a viral vector. The viral vector may be derived, for example, from a retrovirus (e.g., a foamy virus) or a lentivirus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain coding sequences for various chimeric proteins described herein in place of non-essential viral genes. The vectors and / or particles can be utilized to transcribe DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0099] In certain embodiments, the viral vector containing the coding sequence of the CAR described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector that contains structural and functional genetic elements that are primarily derived from retroviruses. The term "lentiviral vector" refers to a vector that contains structural and functional genetic elements outside the LTR that are primarily derived from lentiviruses.

[0100] Retroviral vectors used herein can be derived from any known retrovirus (e.g., C-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retrovirus" also includes the lentivirus family of retroviruses and other classes of retroviruses, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, as well as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV).

[0101] As used herein, lentiviral vector refers to a vector derived from lentivirus, a group (or genus) of retroviruses that cause slowly progressive diseases. Viruses in this group include HIV (human immunodeficiency virus; HIV types 1 and 2); Visna-Maedi; Caprine Arthritis-Encephalitis Virus; Equine Infectious Anemia Virus; Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). Preparation of recombinant lentivirus can be achieved using the methods described by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72:8463-8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).

[0102] The retroviral vectors used (i.e., both lentiviral and non-lentiviral) can be constructed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985), Science, 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA, 85:6460-6464; Wilson et al. (1988), Proc. Natl. Acad. Sci. USA, 85:3014-3018; Armentano et al. al.(1990),Proc.Natl.Acad.Sci.USA,87:6141-6145;Huber et al.(1991),Proc.Natl.Acad.Sci.USA,88:8039-8043;Ferry et al. al.(1991),Proc.Natl.Acad.Sci.USA,88:8377-8381;Chowdhury et al.(1991),Science,254:1802-1805;van Beusechem et al.(1992),Proc.Natl.Acad.Sci.USA,89:7640-7644;Kay et al. al.(1992),Human Gene Therapy,3:641-647;Dai et al. al. (1992), Proc. Natl. Acad. Sci. USA, 89:10892-10895; Hwu et al. (1993), J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application No. WO89 / 07136; PCT Application No. WO89 / 02468; PCT Application No. WO89 / 05345; and PCT Application No. WO92 / 07573).

[0103] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for vector generation include genomic RNA and cDNA available from commercial sources such as the Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0104] For the expression of CAR, a vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. Expression vectors may contain various elements for controlling expression, such as, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection marker, and a signal sequence. As described above, these elements can be appropriately selected by those skilled in the art. For example, a promoter sequence can be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, a T7 promoter, a T3 promoter, an SP6 promoter, a β-actin promoter, an EF1a promoter, a CMV promoter, and an SV40 promoter. An enhancer sequence can be selected to enhance transcription of a polynucleotide. A selection marker can be selected to enable selection of host cells into which the vector has been inserted from host cells into which it has not been inserted; for example, the selection marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to enable transport of the expressed polypeptide out of the host cell.

[0105] For cloning polynucleotides, a vector may be introduced into a host cell (isolated host cell) to allow replication of the vector itself and thereby amplify copies of the polynucleotide contained in the vector. Cloning vectors may generally contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements can be appropriately selected by those skilled in the art. For example, an origin of replication can be selected to promote autonomous replication of the vector within the host cell.

[0106] In certain embodiments, the present disclosure provides an isolated host cell comprising a vector provided herein. Host cells comprising a vector may be useful for expressing or cloning a polynucleotide contained within the vector. Suitable host cells include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Prokaryotic cells suitable for this purpose include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typHimurium, Serratia, e.g., Erratia marcescan, and ShIgella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0107] The CAR is introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid may be transiently maintained or stably introduced. Transfection can be performed by various methods known in the art, including, but not limited to, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector via viral infection instead of transfection. In certain embodiments, a retroviral vector is transduced by packaging the vector into a virion before contacting the cell. For example, a nucleic acid encoding a CAR carried by a retroviral vector can transduce cells via infection and proviral integration.

[0108] To assess the expression of a CAR polypeptide or portion thereof, the expression vector introduced into cells can contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0109] Reporter genes are used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes that encode polypeptides that are not present in or expressed by recipient organisms or tissues, and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is measured at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include luciferase, β-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, a construct containing a minimal 5' flanking region that exhibits high levels of reporter gene expression is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to regulate promoter-induced transcription.

[0110] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0111] In cases where a non-viral delivery system is utilized, a representative delivery vehicle is a liposome. In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule bound to both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contained with or complexed with micelles, or otherwise associated with the lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles or in bilayer structures with a "collapsed" structure. They may also simply be interspersed within the solution, occasionally forming aggregates that are not uniform in size or shape. The lipid is a lipid substance that may be a natural lipid or a synthetic lipid. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0112] Induced natural killer (NK) cells In certain aspects, also disclosed herein are natural killer (NK) cells that have been engineered to express the disclosed CAR polypeptides. Natural killer (NK) cells express CD56 receptors that can kill virally infected and transformed cells and constitute an important cell subset of the innate immune system. + CD3 - It is a large granular lymphocyte (Godfrey J, et al. Leuk Lymphoma, 2012, 53:1666-1676). cytotoxic CD8 + Unlike T lymphocytes, NK cells can initiate cytotoxic effects against tumor cells and eradicate MHC-I negative cells without the need for prior sensitization (Narni-Mancinelli E, et al. Int. Immunol. 2011, 23:427-431). NK cells are safer effector cells because they avoid potentially fatal complications of cytokine storm (Morgan RA, et al. Mol. Ther. 2010, 18:843-851), tumor lysis syndrome (Porter DL, et al. N. Engl. J. Med. 2011, 365:725-733), and adverse effects beyond the targeted tumor.

[0113] In some embodiments, the NK cells are harvested from the subject to be treated (i.e., autologous). However, in certain embodiments, NK cell lines or donor effector cells (allogeneic) are used. In some embodiments, the NK cells are induced NK cells differentiated from induced pluripotent stem cells (iPSCs).

[0114] NK cells can be obtained from multiple sources, including peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), bone marrow, umbilical cord blood, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), by methods well known in the art. NK cells can be detected by specific surface markers such as CD16, CD56, and CD8, which do not express CD3 in humans.

[0115] In some embodiments, NK cells may be obtained from blood drawn from a subject using any number of techniques known to those skilled in the art. For example, a starting population of NK cells may be obtained by isolating mononuclear cells using Ficoll density gradient centrifugation. Specific subpopulations of NK cells can be further isolated by positive or negative selection techniques. For example, NK cells can be isolated using a combination of antibodies against surface markers specific to the positively selected cells, e.g., by incubating with antibody-conjugated beads for a time sufficient for positive selection of NK cells. Alternatively, enrichment of the NK cell population can be achieved by negative selection using a combination of antibodies against surface markers specific to the negatively selected cells. For example, cell cultures may be depleted of any cells expressing CD3, CD14, and / or CD19, and may be enriched for CD56. + / CD 3- The cells may be characterized to determine the proportion of NK cells or NK cells.

[0116] In some embodiments, umbilical cord blood (CB) is used to induce NK cells. In some embodiments, NK cells are isolated and expanded by previously described methods for ex vivo expansion of NK cells. For example, CB mononuclear cells may be isolated by Ficoll density gradient centrifugation and cultured in a bioreactor with IL-2 and artificial antigen-presenting cells (aAPCs). After several days, the cell culture is depleted of any cells expressing CD3 and re-cultured for several more days. Cells are again CD3-depleted and characterized to identify CD56 + / CD3 - In another embodiment, umbilical cord CB is used to determine the percentage of CD34 cells or NK cells. + Isolate cells and isolate CD34 + The cells were cultured in medium containing SCF, IL-7, IL-15, and IL-2 to express CD56 + / CD3 - NK cells are induced by differentiating them into cells.

[0117] In some embodiments, NK cells are generated from pluripotent stem cells. Pluripotent stem cells, either human embryonic stem cells (hESCs) or iPSCs, can proliferate indefinitely in an undifferentiated state through self-renewal. Therefore, the ability to routinely derive NK cells from hESCs and iPSCs allows for the production of unlimited numbers of uniform NK cells from a starting pluripotent stem cell population, providing a standardized, off-the-shelf approach. iPSC-derived NK (iNK) cells have also been reported to produce inflammatory cytokines and exert potent cytotoxicity against a range of hematological and solid tumors. hESCs and iPSCs can be genetically engineered to express the CARs described herein using genetic engineering approaches such as transposon and lentiviral delivery, which ensure efficient transgene insertion and stable expression in iPSCs. TALENS and CRISPR / Cas9 may also be used to more precisely knock-in or delete specific genes. The engineered, undifferentiated hESCs or iPSCs may then be used for differentiation into NK cells expressing the CARs described herein. Methods for differentiating hESCs or iPSCs into NK cells are described in Cichocki et al, Sci. Transl. Med. 2020;12(568):eaaz5618; Goldenson et al. Front Immunol. 2022;13:841107; Li et al. Cell Stem Cell, 2018;23:181-192; Maddineni S, et al. J. Immunother. Cancer, 2022;10:e004693, each of which is incorporated by reference in its entirety.

[0118] The present disclosure provides a method for generating NK cells expressing a CAR described herein. In one embodiment, the method comprises transfecting or transducing NK cells isolated from a subject so that the NK cells express one or more CARs described herein. In certain embodiments, NK cells are isolated from an individual and genetically engineered in vitro without further manipulation. Such cells can then be directly readministered to the individual. In a further embodiment, NK cells are expanded in vitro before being genetically engineered to express a CAR. In this regard, NK cells may be cultured before or after being genetically engineered (i.e., transduced or transfected to express a CAR described herein). NK cells may be expanded in the presence of artificial antigen-presenting cells (aAPCs). The expansion culture may further include proliferation-promoting cytokines, such as IL-2, IL-21, and / or IL-18. Cytokines may be replenished in the expansion culture, for example, every 2-3 days. APCs may be added to the culture at least a second time, such as after CAR transduction.

[0119] In certain aspects, also disclosed herein are other immune cells (phagocytes) that have been engineered to express the disclosed CAR polypeptides.

[0120] Binding properties of chimeric antigen receptors As used herein, the term "binding" refers to the binding of a chimeric antigen receptor comprising an extracellular domain described herein to, for example, an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and a tumor antigen). Binding typically refers to the interaction or association between at least two entities or molecular structures, such as the interaction of an antigen-binding domain and an antigen. For example, the binding affinity typically is about 10, as measured by surface plasmon resonance (SPR) technology on a Biacore 3000 instrument using, for example, an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or antiligand). -8 M, about 10 -9 M about 10 -7K below M D Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J. Immunol. Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J. Immunol. Methods. 2005, 302(1-2):68-77).

[0121] Thus, in some embodiments, the chimeric antigen receptor of the present disclosure has a K that is at least 10-fold lower than its affinity for binding to a nonspecific antigen (e.g., BSA, casein). D In accordance with the present disclosure, in some embodiments, the K value is 10-fold or less lower than that of a nonspecific binding partner. D The affinity of a chimeric antigen receptor with a value may be considered as undetectable binding.

[0122] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antigen-binding domain:antigen interaction. D Since there is an inverse correlation between binding affinity and K D The smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, hence a higher K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, hence a lower K D In some contexts, a higher binding affinity (or K ) of a particular molecule (e.g., chimeric antigen receptor) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., chimeric antigen receptor) to another interaction partner molecule (e.g., antigen Y) can refer to a higher binding affinity (or K ) of the molecule (e.g., chimeric antigen receptor) to its interaction partner molecule (e.g., antigen Y). D) can be expressed as a binding ratio determined by dividing the larger KD value (lower, i.e., weaker affinity) by the smaller KD value (higher, i.e., stronger affinity), and may be expressed, for example, as a 5-fold or 10-fold greater binding affinity, as the case may be.

[0123] The term "k" d The term "sec-1 or 1 / s" refers to the dissociation rate constant of a particular antigen-binding domain:antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. The above value is also used to calculate the k off Also called value.

[0124] The term "k" a " (M-1 × sec-1 or 1 / M) refers to the association rate constant of a particular antigen-binding domain:antigen interaction, or the association rate constant of a chimeric antigen receptor.

[0125] The term “K A " (M-1 or 1 / M) refers to the association equilibrium constant of a particular antigen-binding domain:antigen interaction, or the association equilibrium constant of a chimeric antigen receptor. The association equilibrium constant is k a k d is obtained by dividing by

[0126] The terms "EC50" or "EC 50 " refers to the half-maximal effective concentration, which includes the concentration of chimeric antigen receptor that induces a response halfway between baseline and maximum after a given exposure time. EC 50 essentially represents the concentration of chimeric antigen receptor at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of a chimeric antigen receptor of the present disclosure that confers half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen) as measured, for example, by a FACS binding assay. Thus, the EC 50 , i.e., as the 50% effective concentration value increases, decreased or weaker binding is observed.

[0127] In one embodiment, the reduction in binding is measured by the chimeric antigen receptor concentration, EC 50 It can be defined as an increase in

[0128] The present disclosure provides high affinity (e.g., nanomolar or subnanomolar K D The present invention provides a chimeric antigen receptor comprising an antigen-binding domain derived from an antibody that binds to a human antigen at a specific target site (a specific target site).

[0129] According to certain embodiments, the present disclosure provides a compound having a K of less than about 5 nM as measured by surface plasmon resonance. D The present invention provides a chimeric antigen receptor having an antigen-binding domain derived from a corresponding antibody that binds to an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and a tumor antigen) at (e.g., at 25°C) at a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM, as measured by surface plasmon resonance. D The antibody binds to an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and a tumor antigen).

[0130] The present disclosure also provides chimeric antigen receptors having an antigen-binding domain derived from a corresponding antibody that binds to an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and a tumor antigen) with a dissociation half-life (t) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25° C. In certain embodiments, the corresponding antibody binds to an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and a tumor antigen) with a t of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25° C.

[0131] therapeutic antibodies General In certain aspects, the methods and compositions provided herein relate to the use of therapeutic antibodies (e.g., bispecific CD3 binding antibodies).

[0132] As mentioned above, the term "antibody" as used herein encompasses both intact antibody molecules and antigen-binding fragments of intact antibody molecules. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) heavy chain Fab (Fd) fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) domain antibody (dAb) fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the scope of the expression "antigen-binding fragment" as used herein.

[0133] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and typically contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H For antigen-binding fragments containing domains, V H and V L The domains may be positioned in any suitable arrangement relative to each other. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V LAlternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0134] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragments of antibodies disclosed herein include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -CH3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies disclosed herein may be linked to each other and / or to one or more monomeric V H Domain or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the above listed variable and constant domain configurations, in which the domains are non-covalently associated (e.g., by disulfide bond(s)).

[0135] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, wherein each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of the antigen-binding fragments of antibodies disclosed herein using routine techniques available in the art. In certain embodiments provided herein, at least one variable domain of the multispecific antibody is capable of specifically binding to CD3.

[0136] In some embodiments, the antibodies provided herein may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies disclosed herein in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC are well known in the art and can be measured using available assays (see, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998), Proc. Natl. Acad. Sci. (USA), 95:652-656). The constant region of an antibody is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0137] In certain embodiments provided herein, the CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0138] In some embodiments, the antibodies provided herein can be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to encompass all human antibodies that are produced, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from the combination of a recombinant human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving the insertion of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), which results in the V H and V L The amino acid sequence of the region is human germline V H and V L A sequence that is derived from and related to a sequence, but that may not naturally occur within the human antibody germline repertoire in vivo.

[0139] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming an approximately 75-80 kDa molecule (half antibody) composed of covalently linked light and heavy chains. These forms have been very difficult to separate, even after affinity purification.

[0140] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993), Molecular Immunology, 30:105) to the level typically observed using a human IgG1 hinge. In certain embodiments, the present disclosure provides a method for the identification of a second form in the hinge, C H 2, or C H The present invention encompasses antibodies with one or more mutations in three regions, which may be desirable, for example, to improve the yield of a desired antibody form in manufacturing.

[0141] The antibodies disclosed herein may be isolated antibodies. As used herein, "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes disclosed herein. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0142] In certain embodiments, the methods and compositions provided herein comprise a single-armed antibody that binds to a tumor antigen (TAA). As used herein, "single-armed antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain.

[0143] Sequence variants In some embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. In certain embodiments, the present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. H and / or V LAll framework and / or CDR residues within a domain are backmutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residue in a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., KD), improved or enhanced antagonistic or agonistic biological properties (as the case may be), or reduced immunogenicity.

[0144] In some embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, in certain embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein have HCVR, LCVR, and / or CDR amino acid sequences with, e.g., no more than 10, no more than 8, no more than 6, no more than 4, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0145] Fc variants According to certain embodiments provided herein, there are provided antibodies and multispecific antigen-binding molecules comprising an Fc domain comprising one or more mutations that enhance or attenuate binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. In certain embodiments, the present disclosure provides antibodies and multispecific antigen-binding molecules comprising an Fc domain comprising one or more mutations that enhance or attenuate binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C H 3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes at a pH ranging from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0146] In certain embodiments, the present disclosure includes CD3 multispecific antigen binding molecules (e.g., anti-CD3 / anti-MUC16 bispecific antibodies, anti-BCMA x anti-CD3 antibodies, or anti-CD3 / anti-CD20 bispecific antibodies) comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated herein.

[0147] In some embodiments, a CD3xTAA bispecific antibody of the present disclosure comprises an IgG Fc sequence with amino acid substitutions, e.g., using two residues from IgG3. For example, a CD3xTAA bispecific antibody of the present disclosure may comprise an IgG1 Fc sequence with amino acid substitutions H365R and Y366F in the CH3 region. An exemplary Fc sequence is also shown in Figure 3.

[0148] bioequivalence Provided herein are antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but retain the ability to bind to the same antigen or antigens. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit essentially the same biological activity as the described bispecific antigen-binding molecules.

[0149] In certain embodiments, the present disclosure includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules presented herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutically equivalent or pharmaceutically alternative formulations that do not show significant differences in their rate and extent of absorption when administered in either single or repeated doses at the same molar dose under similar experimental conditions. Some antigen-binding proteins are considered equivalent or pharmaceutically alternative formulations if they are equivalent in extent of absorption but not in absorption rate, yet can be considered bioequivalent because such differences in absorption rate are intentional and reflected in the label, and are not critical, for example, to achieving effective body drug concentrations in chronic use and are not medically significant with respect to the particular formulation being studied.

[0150] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically relevant differences in their safety, purity, and potency.

[0151] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference and biologic products one or more times without expected increased risk of adverse effects, e.g., clinically relevant changes in immunogenicity, or decreased efficacy, compared to continued treatment without switching.

[0152] In one embodiment, two antigen binding proteins are bioequivalent if they both act in a condition or conditions of use by a common mechanism or mechanism of action, to the extent such mechanism is known.

[0153] Bioequivalence may be demonstrated by in vivo and in vitro methods. Methods for measuring bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid is measured as a function of time; (b) in vitro studies that correlate with and reasonably predict in vivo human bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein. Biologically equivalent variants of the exemplary bispecific antigen-binding molecules shown herein may be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be removed or replaced with other amino acids to prevent the formation of unnecessary or improper intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules shown herein that contain amino acid changes that alter the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0154] antibody binding As used herein, the term "binding" refers to an interaction or association between at least two entities or molecular structures, e.g., an antibody-antigen interaction, typically in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to any given antigen, e.g., a cell surface protein or fragment thereof.

[0155] For example, binding affinities are typically around 10 when measured, for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 K below M D Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J. Immunol. Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J. Immunol. Methods. 2005, 302(1-2):68-77).

[0156] Thus, the antibodies or antigen-binding proteins provided herein have a K that is at least 10-fold lower than the binding affinity for a non-specific antigen (e.g., BSA, casein). D The antibody binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value. In some embodiments, an antibody affinity corresponding to a K value that is at least 10-fold lower than that of a non-specific antigen may be considered undetectable binding, and such an antibody may be paired with a second antigen-binding arm to create a bispecific antibody as disclosed herein.

[0157] "K D The term "M" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D Since there is an inverse correlation between binding affinity and K D The smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, hence the K DConversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, hence a lower K D In some contexts, a higher binding affinity (or K ) of a particular molecule (e.g., antibody) to an interaction partner molecule (e.g., antigen X) compared to the binding affinity of that molecule (e.g., antibody) to another interaction partner molecule (e.g., antigen Y) can be used. D ) is an even larger K D A smaller K value (lower or weaker affinity) D The binding affinity may be expressed as a binding ratio determined by dividing by (higher or stronger affinity), e.g., 5-fold or 10-fold higher binding affinity in some cases.

[0158] "k d The term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. off Also called value.

[0159] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0160] The term “K A " (M-1 or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d is obtained by dividing by

[0161] "EC50" or "EC 50 The term "50% effective concentration" refers to the concentration of antibody that elicits a response halfway between baseline and maximum after a specific exposure time. 50 The EC essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50The EC value is equal to the concentration of an antibody disclosed herein that confers half-maximal binding to cells expressing CD3 or a tumor-associated antigen (e.g., CD123, STEAP2, CD20, PSMA, SSTR2, CD38, STEAP1, 5T4, ENPP3, MUC16, or BCMA), as determined, for example, by a FACS binding assay. 50 As the 50% effective concentration value increases, decreased or weaker binding is observed.

[0162] In one embodiment, the reduced binding of an antibody (e.g., a CD3 multispecific antibody) is an EC that allows binding to half-maximal amounts of target cells. 50 It can be defined as an increase in antibody concentration.

[0163] In another embodiment, EC 50 The values ​​represent the concentration of antibody (e.g., a CD3 multispecific antibody disclosed herein) that causes half-maximal depletion of target cells by the cytotoxic activity of effector cells (e.g., T cells or NK cells). 50 Alternatively, at 50% effective concentration values, enhanced cytotoxic activity (eg, T cell or NK cell mediated tumor cell killing) is observed.

[0164] In yet another embodiment, EC 50 The values ​​represent the concentration of antibody (e.g., a CD3 multispecific antibody disclosed herein) that elicits half-maximal activation of target cells by activating effector cells (e.g., T cells or NK cells). For example, T cell activation can be measured by a Jurkat NFAT reporter bioassay (e.g., the Jurkat / NFAT-Luc bioassay described in Example 1). Thus, an increase in effector cell activation (e.g., T cell or NK cell activation) is associated with an increase in EC 50 That is, a decrease in the 50% effective concentration value is observed.

[0165] pH-dependent binding In certain embodiments, the present disclosure includes antibodies and multispecific antigen-binding molecules with pH-dependent binding properties. For example, the CD3 multispecific antibodies disclosed herein may exhibit reduced binding to CD3 at acidic pH compared to neutral pH. Alternatively, the CD3 multispecific antibodies disclosed herein may exhibit increased binding to CD3 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or lower. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0166] In some cases, "decreased binding at acidic pH compared to neutral pH" refers to the K of an antibody that binds to an antigen at acidic pH. D K value of antibody binding to antigen at neutral pH D For example, the acidic / neutral K D For purposes disclosed herein, a CD3 multispecific antibody or antigen-binding fragment thereof may be considered to have "reduced binding to CD3 at acidic pH compared to neutral pH" if it exhibits a ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic / neutral K for the antibodies or antigen-binding fragments disclosed herein D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0167] Antibodies with pH-dependent binding properties may be obtained, for example, by screening a population of antibodies that exhibit reduced (or enhanced) binding to a particular antigen at acidic pH compared to neutral pH. Furthermore, antibodies with pH-dependent characteristics may be obtained by modifying the antigen-binding domain at the amino acid level. For example, an antibody with reduced antigen binding at acidic pH compared to neutral pH may be obtained by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues.

[0168] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and a human tumor antigen (e.g., MUC16, BCMA, CD20, etc.)) can be appropriately positioned relative to each other using routine methods to generate the bispecific antigen-binding molecules disclosed herein. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules disclosed herein are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules disclosed herein can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), high-affinity chimeric antibodies against specific antigens (e.g., CD3 and human tumor antigens (e.g., MUC16, BCMA, CD20, etc.)) with human variable regions and mouse constant regions are first isolated. The antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. To generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules disclosed herein, the mouse constant regions are replaced with the desired human constant regions.

[0169] Genetically engineered animals can be used to produce human bispecific antigen-binding molecules. For example, genetically engineered mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, where the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to a mouse κ constant gene at the endogenous mouse κ locus. Such genetically engineered mice can be used to produce fully human bispecific antigen-binding molecules containing two different heavy chains associated with an identical light chain containing variable domains derived from one of two different human light chain variable region gene segments (see US2011 / 0195454). Fully human refers to an antibody or antigen-binding fragment or immunoglobulin domain that contains an amino acid sequence encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain. In some examples, the fully human sequence is derived from a protein endogenous to humans. In other examples, the fully human protein or protein sequence includes a chimeric sequence in which the sequence of each component is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, for example, compared to regions or domains of any wild-type human immunoglobulin.

[0170] CD3 multispecific antigen binding molecule In certain embodiments, the methods and compositions provided herein relate to CD3 antigen binding molecules (i.e., antigen binding molecules comprising at least one antigen binding domain that binds to CD3). In certain embodiments, the CD3 multispecific antigen binding molecules provided herein further comprise an antigen binding domain that binds to a cancer antigen (i.e., an antigen expressed on a cancer cell). In certain embodiments, the CD3 multispecific antigen binding molecules provided herein further comprise an antigen binding domain that binds to a costimulatory receptor (e.g., CD28).

[0171] In some embodiments, the CD3 antibody is any one of the CD3 antibodies listed in Table 6. [Table 7]

[0172] As used herein, the term "multispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. In some embodiments, each antigen-binding domain within a multispecific antigen-binding molecule may comprise at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context disclosed herein, the first antigen-binding domain specifically binds to a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds to a second, different antigen (e.g., a tumor antigen).

[0173] In some embodiments, the CD3 multispecific antigen-binding molecule is a CD3 multispecific antibody. The CD3 multispecific antibodies provided herein may be, for example, bispecific or trispecific. A multispecific antibody may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The bispecific CD3 antibodies provided herein may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, to create a bispecific or multispecific antibody with a second or additional binding specificity, an antibody or fragment thereof may be operably linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other method) to one or more other molecular entities, such as another antibody or antibody fragment.

[0174] In certain embodiments, the present disclosure includes bispecific antibodies in which one immunoglobulin arm binds CD3 and the other immunoglobulin arm is specific for a cancer antigen (also referred to herein as a tumor antigen, or "TAA"). In certain embodiments, the present disclosure includes trispecific antibodies in which a first immunoglobulin arm binds CD3, a second immunoglobulin arm is specific for a tumor antigen, and a third immunoglobulin arm binds an additional T cell antigen (e.g., CD28) or an additional tumor antigen.

[0175] In some embodiments, the CD3-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences as disclosed in WO2014 / 047231 or WO2017 / 053856. In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and, in the context of a bispecific or multispecific antibody, induces killing of cells expressing a tumor-associated antigen. In other embodiments, the CD3-binding arm binds or associates weakly with human and cynomolgus monkey (monkey) CD3, but the binding interaction is not detectable by in vitro assays known in the art.

[0176] In certain embodiments, the multispecific antibody or antigen-binding fragment comprises an antigen-binding arm that binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2 and induces T cell activation.

[0177] In certain embodiments, the multispecific CD3 antigen binding molecule comprises an antigen binding domain specific for a cancer antigen, such as AIM-2, ALDH1A1, alpha-actin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCMA, BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD19, CD20, CD22, CD38, CD45, CD123, CD274, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, CLDN18.2, CEACAM5, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin D2, cyclin D3, cyclin D4, cyclin D5, cyclin D6, cyclin D7, cyclin D8, cyclin D9, cyclin D10, cyclin D11, cyclin D12, cyclin D13, cyclin D14, cyclin D15, cyclin D16, cyclin D17, cyclin D18, cyclin D20, cyclin D21, cyclin D22, cyclin D23, cyclin D24, cyclin D25, cyclin D16, cyclin D26, cyclin D27, cyclin D28, cyclin D30, cyclin D31, cyclin D42, cyclin D19, cyclin D29, cyclin D32, cyclin D43, cyclin D18, cyclin D19, cyclin D20, cyclin D21, cyclin D19, cyclin D D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENPP3, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1, 2, 8, GPRC5D, GAGE-3, 4, 5, 6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KMHN1, LAGE-1, LDLR-fucosyltransferase fusion protein, also known as CCDC110, KMHN1, K-ras, KIF20A, KMHN1, K-ras ... , Lengusin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, Malic enzyme, Mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MSLN, MUC1, MUC5AC, MUC16, Mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosink RasI, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, cecernin1, SIRT2, SLC3A2 -APIS, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, survivin, SSTR2, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-beta RII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, 5T4, and XAGE-1b / GAGED2a.

[0178] In some embodiments, cancer antigens include ADAM17, BCMA, CA-IX, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79b, CD123, CD138, CDH3, CEA, EphA2, EpCAM, ERBB2, ENPP3, EGFR, EGFR-vIII, FLT3, FOLRI, GD-2, glypican-3, gpA33, GPNMB, GPRC5D, HER2, HER3, LMP1, LMP2A, MUC16, mesothelin, PSMA, PSCA, RON, ROR1, ROR2, STEAP1, STEAP2, SSTR2, SSTR5, 5T4, and Trop-2. In some embodiments, the tumor antigen may be CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, BCMA, CA19.9, MSLN, CD22, SLC3A2-APIS, CLDN18.2, or CEACAM5.

[0179] In some embodiments, the tumor antigen may be CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, BCMA, CA19.9, MSLN, CD22, SLC3A2-APIS, CLDN18.2, or CEACAM5.

[0180] In some embodiments, the cancer antigen is CD20, MUC16, BCMA, PSMA, or STEAP2.

[0181] CD20 is a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered a B cell tumor-associated antigen because it is expressed by more than 95% of B cell non-Hodgkin's lymphomas (NHLs) and other B cell malignancies, but is absent from precursor B cells, dendritic cells, and plasma cells. Human CD20 protein has the amino acid sequence set forth in SEQ ID NO: 5 of U.S. Patent Application No. US2020 / 0129617 (the contents of which are incorporated herein by reference in their entirety).

[0182] MUC16 refers to mucin 16. MUC16 is a single transmembrane domain, highly glycosylated integral membrane protein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is set forth in SEQ ID NO: 1899 of U.S. Patent Application No. US2018 / 0118848A1 (the contents of which are incorporated herein by reference in their entirety).

[0183] BCMA refers to B-cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B-cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is set forth in SEQ ID NO: 115 of U.S. Patent Application No. US2020 / 0024356 (the contents of which are incorporated herein by reference in their entirety). It can also be found in GenBank Accession No. NP_001183.2.

[0184] PSMA refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is a non-shedding membrane integral glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. The amino acid sequence of human PSMA is shown in SEQ ID NO: 7 of U.S. Patent Application No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0185] STEAP2 refers to the six-transmembrane epithelial antigen of the prostate. STEAP2 is an important six-transmembrane protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. STEAP2 is a 490-amino acid protein encoded by the STEAP2 gene located in human chromosome region 7q21. The amino acid sequence of human STEAP2 is shown in SEQ ID NO: 9 of U.S. Patent Application No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0186] In some embodiments, the CD3 multispecific antibody may be a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

[0187] In certain embodiments, the present disclosure includes antibodies having the HCVR, LCVR and / or CDR amino acid sequences of the antibodies set forth herein, anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856, anti-CD20 x anti-CD3 antibodies disclosed in WO2014 / 047231, anti-PSMA x anti-CD3 bispecific antibodies disclosed in WO2017 / 023761, anti-MUC16 x anti-CD3 bispecific antibodies disclosed in WO2018 / 067331, anti-STEAP2 x anti-CD3 bispecific antibodies disclosed in WO2018 / 058001, or anti-BCMA x anti-CD3 bispecific antibodies disclosed in WO2020 / 018820, each of which is incorporated herein by reference.

[0188] In certain embodiments, the multispecific antigen-binding molecule is a multispecific antibody or antigen-binding fragment thereof. Each antigen-binding domain of a multispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "A1", and the CDRs of the second antigen-binding domain may be designated with the prefix "A2". Accordingly, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3. In the context of a trispecific antigen-binding molecule (e.g., a trispecific antibody) comprising a first, second, and third antigen-binding domain, the CDRs of the first antigen-binding domain may be designated using the prefix "A1", the CDRs of the second antigen-binding domain may be designated using the prefix "A2", and the CDRs of the third antigen-binding domain may be designated using the prefix "A3". Accordingly, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3, and the CDRs of the third antigen-binding domain may be referred to herein as A3-HCDR1, A3-HCDR2, and A3-HCDR3.

[0189] The bispecific antigen-binding molecule described above or herein may be a bispecific antibody. In some examples, the bispecific antibody comprises a human IgG heavy chain constant region. In some examples, the human IgG heavy chain constant region is of the isotype IgG1. In some examples, the human IgG heavy chain constant region is of the isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0190] The first and second antigen-binding domains may be linked to each other directly or indirectly to form the bispecific antigen-binding molecules disclosed herein. Alternatively, the first and second antigen-binding domains may each be linked to a separate multimerization domain. Association of one multimerization domain with another multimerization domain promotes association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, a "multimerization domain" refers to any macromolecule, protein, polypeptide, peptide, or amino acid capable of associating with a second multimerization domain of the same or similar structure or configuration. For example, the multimerization domain may be a polypeptide containing an immunoglobulin CH3 domain. A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (containing a CH2-CH3 domain), such as the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0191] The bispecific antigen-binding molecules disclosed herein typically comprise two multimerization domains, e.g., two Fc domains, each of which is part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0192] In certain embodiments, the multimerization domain is an Fc fragment containing at least one cysteine ​​residue or an amino acid sequence of 1 to about 200 amino acids in length. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0193] Any bispecific antibody format or technology may be used to create the bispecific antigen-binding molecules disclosed herein. For example, to create a bispecific antigen-binding molecule, an antibody or fragment thereof having a first antigen-binding specificity may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or another method) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity. Certain exemplary bispecific formats that may be used in the context disclosed herein include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-in-hole, common light chain (e.g., common light chain with knob-in-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of the above formats, see, e.g., Klein et al. 2012, mAbs, 4:6, 1-11 and references cited therein).

[0194] In the context of the bispecific antigen-binding molecules provided herein, the multimerization domain, e.g., the Fc domain, may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring Fc domain. In certain embodiments, the present disclosure includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that result in a modified Fc domain with altered (e.g., enhanced or weakened) binding interactions between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C HThe FcRn comprises modifications in three regions, where the modifications increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., E or Q); a modification at positions 250 and 428 (e.g., L or F); a modification at positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or a modification at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); a modification at positions 250 and / or 428; or a modification at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and a 428L modification (e.g., T250Q and M428L); and a 307 and / or a 308 modification (e.g., 308F or 308P).

[0195] In certain embodiments, provided herein is a first C H 3 domain and second Ig C H A bispecific antigen-binding molecule comprising three domains, wherein a first and a second Ig C H In one embodiment, the three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C HThe second CH3 domain may contain a mutation that reduces or eliminates Protein A binding, e.g., an H95R modification (according to IMGT exon numbering; H435R in EU numbering). The second CH3 may further contain a Y96F modification (according to IMGT; Y436F in EU numbering). See, e.g., U.S. Patent No. 8,586,713. H Further modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in EU); N44S, K52N, and V82I for IgG2 antibodies (according to IMGT; N384S, K392N, and V422I in EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU).

[0196] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may combine the Fc sequences of human IgG1, human IgG2, or human IgG4. H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4 H The chimeric Fc domain may comprise part or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules presented herein is a chimeric Fc domain that comprises, from the N-terminus to the C-terminus, [IgG4 C HAnother example of a chimeric Fc domain that may be included in any of the antigen-binding molecules provided herein comprises, from the N-terminus to the C-terminus, [IgG1 C H IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules disclosed herein are described in U.S. Patent Application Publication No. 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these overall structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0197] The multispecific (e.g., bispecific or trispecific) CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. In certain embodiments, the present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. H and / or V LAll framework and / or CDR residues within a domain are backmutated to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies disclosed herein can contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues in the framework and / or CDR regions are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may be characterized for one or more desired properties, such as improved binding specificity, enhanced binding (e.g., as assessed by measuring cell-binding antibody titers or FACS binding), or binding affinity (e.g., K D ), (potentially) improved or enhanced antagonist or agonist biological properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed within the scope of the present disclosure.

[0198] Also provided herein are CD3 multispecific (e.g., bispecific or trispecific) antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. In certain embodiments, the present disclosure includes CD3 multispecific (e.g., bispecific or trispecific) antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0199] Exemplary CD3xMUC16 Antibodies In some embodiments, the methods and compositions provided herein comprise bispecific antibodies, wherein one immunoglobulin arm binds human CD3 and the other immunoglobulin arm is specific for human MUC16. As used herein, the term "MUC16" refers to the human MUC16 protein unless otherwise specified as being from a non-human species (e.g., "mouse MUC16," "simian MUC16," etc.). The human MUC16 protein has the amino acid sequence set forth in SEQ ID NO: 1899 of U.S. Patent Application No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-CD3 / anti-MUC16," or "anti-CD3 x MUC16," or "CD3 x MUC16" bispecific molecules, or other similar terms (e.g., anti-MUC16 / anti-CD3). Such bispecific antigen-binding molecules are constructed with a first antigen-binding arm that binds MUC16 and a second antigen-binding arm that binds CD3. The MUC16-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Table 7 herein. The CD3-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Tables 8-12 herein. The sequences in Tables 7-12 are disclosed in U.S. Patent Application Publication No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety.

[0200] Table 7 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MUC16 antibodies disclosed herein. [Table 8]

[0201] Table 8 provides amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-CD3 antibodies disclosed herein. Methods for making the anti-CD3 antibodies disclosed herein can also be found in U.S. Publication No. 2014 / 0088295. [Table 9-1] [Table 9-2] [Table 9-3]

[0202] Tables 9 and 10 provide amino acid sequence identifiers for the heavy chain variable regions (Table 9) and light chain variable regions (Table 10) of additional anti-CD3 HCVRs and LCVRs useful in the anti-MUC16 x anti-CD3 bispecific antibodies disclosed herein and their corresponding CDRs. [Table 10] [Table 11]

[0203] Table 11 shows the amino acid sequence identifiers for the heavy chain variable regions and CDRs of the engineered anti-CD3 antibodies disclosed herein. The amino acid sequence identifiers for the light chain variable regions and CDRs are also identified below in Table 12. [Table 12] [Table 13]

[0204] In certain exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 comprises heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786, and 1866, and comprises light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 117.

[0205] In certain exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2, and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2, and A1-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790, and 1870; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1736, 1768, 1784, 1792, and 1872; A1-LCDR1 comprises the amino acid sequence of SEQ ID NO: 119; A1-LCDR2 comprises the amino acid sequence TAS; and A1-LCDR3 comprises the amino acid sequence of SEQ ID NO: 312.

[0206] In certain exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 comprises the heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1730 / 117, 1762 / 117, 1778 / 117, 1786 / 117, and 1866 / 117.

[0207] In certain exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2, and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2, and A1-LCDR3). the second antigen-binding domain that specifically binds to human MUC16 comprises three heavy chain complementarity-determining regions (A2-HCDR1, A2-HCDR2, and A2-HCDR3) and three light chain complementarity-determining regions (A2-LCDR1, A2-LCDR2, and A2-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790, and 1870; and A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 173 A1-LCDR1 comprises the amino acid sequence of SEQ ID NO: 119; A1-LCDR2 comprises the amino acid sequence TAS; and A1-LCDR3 comprises the amino acid sequence of SEQ ID NO: 312; A2-HCDR1 comprises the amino acid sequence of SEQ ID NO: 111; A2-HCDR2 comprises the amino acid sequence of SEQ ID NO: 113; A2-HCDR3 comprises the amino acid sequence of SEQ ID NO: 115; A2-LCDR1 comprises the amino acid sequence of SEQ ID NO: 119; A2-LCDR2 comprises the amino acid sequence TAS; and A2-LCDR3 comprises the amino acid sequence of SEQ ID NO: 312.

[0208] Additional bispecific anti-MUC16 x anti-CD3 antibodies are disclosed, for example, in WO2018 / 067331, which is incorporated herein by reference.

[0209] Exemplary CD3xBCMA Antibodies In some embodiments, the methods and compositions provided herein include bispecific antibodies, wherein one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for human BCMA. As used herein, the term "BCMA" refers to human BCMA protein unless otherwise specified as being from a non-human species (e.g., "mouse BCMA," "monkey BCMA," etc.). Human BCMA protein has the amino acid sequence set forth in SEQ ID NO: 115 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-BCMA x anti-CD3," or "anti-CD3 / anti-BCMA," or "anti-CD3 x BCMA," or "CD3 x BCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3). The BCMA-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 13 herein. The CD3-binding arm can comprise any of the amino acid sequences of the HCVR / LCVR or CDRs set forth in Table 14 herein, or any of the amino acid sequences of the anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856. The sequences in Tables 13 and 14 are those disclosed in U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0210] Table 13 provides the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-BCMA antibodies disclosed herein. [Table 14]

[0211] Table 14 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-CD3 antibodies. Other anti-CD3 antibodies for use in preparing bispecific antibodies according to the present disclosure can be found, for example, in WO2014 / 047231. [Table 15]

[0212] In certain exemplary embodiments, an isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 217; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 394. Optionally, the isolated bispecific antigen-binding molecule comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 219, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 221, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 223. Optionally, the isolated bispecific antigen-binding molecule comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 396, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 312. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 217 and an LCVR comprising the amino acid sequence of SEQ ID NO: 394.

[0213] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises a second antigen binding domain comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1610 or SEQ ID NO: 1866; and (b) three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 394. Optionally, the second antigen binding domain comprises (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 740; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 438 or SEQ ID NO: 406; and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 1512 or SEQ ID NO: 1848. In some cases, the second antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 396, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 312. In some cases, the second antigen-binding domain comprises (a) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 740, 438, and 1512, respectively; LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, and SEQ ID NO: 312, respectively; (b) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 740, 406, and 1848, respectively; and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, and SEQ ID NO: 312, respectively. In some cases, the second antigen-binding domain comprises (a) an HCDR comprising the amino acid sequence of SEQ ID NO: 1610 and an LCDR comprising the amino acid sequence of SEQ ID NO: 394, or (b) an HCDR comprising the amino acid sequence of SEQ ID NO: 1866 and an LCDR comprising the amino acid sequence of SEQ ID NO: 394.

[0214] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 219, 221, 223, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, SEQ ID NO: 312, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 740, 438, 1512, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, SEQ ID NO: 312, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCDR comprising the amino acid sequence of SEQ ID NO: 217 and an LCDR comprising the amino acid sequence of SEQ ID NO: 394; and (b) a second antigen-binding domain comprising an HCDR comprising the amino acid sequence of SEQ ID NO: 1610 and an LCDR comprising the amino acid sequence of SEQ ID NO: 394.

[0215] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 219, 221, 223, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, SEQ ID NO: 312, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 740, 406, 1848, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 396, AAS, SEQ ID NO: 312, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 217 and an LCVR comprising the amino acid sequence of SEQ ID NO: 394; and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 1866 and an LCVR comprising the amino acid sequence of SEQ ID NO: 1512 or SEQ ID NO: 394.

[0216] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises (a) a first antigen-binding domain that specifically binds human BCMA and comprises a CDR of an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 165, 179, 191, 203, 217, 227, and 231, and a CDR of an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 173, 187, 197, 211, 225, 394, 229, and 125; and (b) a second antigen-binding domain that specifically binds human CD3. In some cases, the first antigen-binding domain comprises CDRs derived from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 165 / 173, 179 / 187, 191 / 197, 203 / 211, 217 / 225, 227 / 229, 231 / 125, 165 / 394, 179 / 394, 191 / 394, 203 / 394, 217 / 394, 227 / 394, and 231 / 394. In some cases, the first antigen-binding domain has the sequence set forth in SEQ ID NOs: 167-169-171-175-AAS-177, 181-183-185-972-AAS-189, 1740-193-195-199-TAS-201, 205-207-209-213-AAT-215, 219-221-223-396-AAS-1640, 167-169-171- and 219-221-223-396-AAS-312. In some cases, the first antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 165 / 173, 179 / 187, 191 / 197, 203 / 211, 217 / 225, 227 / 229, 231 / 125, 165 / 394, 179 / 394, 191 / 394, 203 / 394, 217 / 394, 227 / 394, and 231 / 394.In some cases, the second antigen-binding domain comprises the CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1610 / 394 and 1866 / 394.

[0217] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule competes for binding to BCMA or binds to the same epitope on BCMA as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 217 / 394, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 1610 / 394 or SEQ ID NO: 1866 / 394.

[0218] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule competes for binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 217 / 394 and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 1610 / 394 or SEQ ID NO: 1866 / 394.

[0219] Further bispecific anti-BCMA x anti-CD3 antibodies are disclosed, for example, in WO2020 / 018820.

[0220] CD3×CD20 antibody In some embodiments, provided herein are bispecific antibodies, wherein one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for human CD20. As used herein, the term "CD20" refers to the human CD20 protein unless specified to be from a non-human species (e.g., "mouse CD20," "monkey CD20," etc.). The human CD20 protein has the amino acid sequence set forth in SEQ ID NO: 1369 of U.S. Patent No. 9,657,102 B2, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-CD3 / anti-CD20," or "anti-CD3xCD20," or "CD3xCD20" bispecific molecules, or other similar terms.

[0221] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from SEQ ID NOs: 1250, 1266, 1282, 1298, 1314, and 1329, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. All sequences disclosed in the section on antigen-binding domains that specifically bind to CD3 or CD20 and corresponding SEQ ID NOs (i.e., the "CD3xCD20 Antibodies" section) are derived from U.S. Pat. No. 9,657,102 B2, the contents of which are incorporated herein by reference in their entirety.

[0222] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322, and 1333, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0223] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises an amino acid sequence pair of an HCVR and an LCVR (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 1250 / 1258, 1266 / 1274, 1282 / 1290, 1298 / 1306, 1314 / 1322, and 1329 / 1333.

[0224] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252, 1268, 1284, 1300, 1316, and 1330, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1254, 1270, 1286, 1302, 1318, and 1331. a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320, and 1332, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320, and 1332, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. a light chain CDR3 (HCDR3) domain; an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity. and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0225] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 1252-1254-1256-1260-1262-1264; 1268-1270-1272-1276-1278-1280; 1284-1286-1288-1292-1294-1296; 1300-1302-1304-1308-1310-1312; 1316-1318-1320-1324-1326-1328; and 1330-1331-1332-1334-1335-1336, respectively.

[0226] In certain embodiments, the second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 1242, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0227] In certain embodiments, the second antigen-binding domain that specifically binds to CD20 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322, and 1333, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0228] In certain embodiments, the second antigen-binding domain that specifically binds to CD20 comprises an amino acid sequence pair of an HCVR and an LCVR (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 1242 / 1258, 1242 / 1274, 1242 / 1290, 1242 / 1306, 1242 / 1322, and 1242 / 1333.

[0229] In certain embodiments, the second antigen-binding domain that specifically binds to CD20 is selected from the group consisting of: a heavy chain CDR1 (HCDR1) domain having the amino acid sequence of SEQ ID NO: 1244, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having the amino acid sequence of SEQ ID NO: 1246, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CDR3 (HCDR3) domain having the amino acid sequence of SEQ ID NO: 1248, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0230] In certain embodiments, the second antigen-binding domain that specifically binds to CD20 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 1244-1246-1248-1260-1262-1264; 1244-1246-1248-1276-1278-1280; 1244-1246-1248-1292-1294-1296; 1244-1246-1248-1308-1310-1312; 1244-1246-1248-1324-1326-1328; and 1244-1246-1248-1334-1335-1336, respectively.

[0231] Additional bispecific anti-CD20 / anti-CD3 antibodies are disclosed, for example, in US Pat. No. 9,657,102, which is incorporated herein by reference in its entirety.

[0232] Other Exemplary CD3 Multispecific Antibodies Additional exemplary CD3 multispecific antibodies that can be used in the compositions and methods disclosed herein include, for example, the bispecific CD3xCD123 antibodies disclosed in U.S. Pat. No. 10,787,521 B2, U.S. Patent Application Publication Nos. 2018 / 0222987 A1, and US2019 / 0241657 A1, and International Application Publication Nos. WO2016 / 036937 A1, WO2017 / 210443 A1, WO2019 / 050521 A1, WO2019 / 210147 A1, WO2019 / 232528 A1, and WO2020 / 092404 A1; The bispecific CD3×STEAP2 antibodies disclosed in WO2018 / 058001A1; WO2014 / 047231A1, WO2015 / 143079A1, WO2016 / 081490A1, WO2017 / 112775A1, WO2017 / 210485A1, WO2018 / 114748A1, WO2018 / 093821A8, WO2018 / 223004A1, WO2018 / 188612A1, WO2019 / 155008A1, WO2019 / 228406A1, WO2020 / 088608A1, WO2020 / 156405A1, and U.S. Pat.The CD3xCD20 antibodies disclosed in Patent Application Publication Nos. US2020 / 0199231A1 and US2020 / 0172627A1; the bispecific CD3xSSTR2 antibodies disclosed in International Application Publication Nos. WO2018 / 005706A1; the bispecific CD3xCD38 antibodies disclosed in International Application Nos. WO2015 / 149077A1 and WO2020 / 018556A1, and U.S. Patent Application Publication Nos. US2018 / 0305465A1 and US2020 / 0102403A1; Olivier Nolan-Stevau (2020), Abstract at Proceedings of the American Association for Cancer Research the bispecific CD3×STEAP1 antibody disclosed in International Application Publication No. WO2013 / 041687A1, U.S. Patent Application Publication No. US2017 / 0342160A1, and U.S. Patent Application Publication No. US20200277397A1; the bispecific CD3×ENPP3 antibody described in International Application Publication No. WO2020 / 180726A1; Bispecific CD3xMUC16 antibodies disclosed in International Application Publication Nos. WO2018 / 067331A9 and WO2019 / 246356A1; International Application Publication Nos. WO2013 / 072406A1, WO2014 / 140248A1, WO2016 / 166629A1, WO2017 / 031104A1, WO2017 / 134134A1, WO2017 / 095267A1, WO2019 / 220369A3, WO2019 / 075359A1, WO2019 / 226761A1, WO2020 / 025596A 1, bispecific CD3×BCMA antibodies disclosed in WO2020 / 191346A1, WO2020018820A1, U.S. Patent Application Publication Nos. US2013 / 0273055A1, and US2019 / 0263920A1; International Application Publication Nos. WO2012 / 055961A1, WO2016 / 048938A1, WO2017 / 087603A1, WO2017 / 096368A1, WO2018 / 188612A1, WO2019 / 237081A1, WO2020 / 048525A1, and WO2020 / 135 335A1, the bispecific CD3×CD19 antibody disclosed in U.S. Patent Application Publication Nos. US2016 / 0326249A1, US2020 / 0283523A1, US2019 / 0284279A1, U.S. Patent Nos. 9,315,567B2, 7,575,923B2, and 7,635,472B2; the bispecific CD3×GPRC5D antibody disclosed in International Patent Application Publication Nos. WO2018 / 017786A3 and WO2019 / 220369A3; the bispecific CD3×GPRC5D antibody disclosed in U.S. Patent Application Publication No. US2017 / 0320947A1; The bispecific CD3xPSMA antibody disclosed in U.S. Patent Application Publication No. US2020 / 0140552A1; the trispecific CD3xCD28xCD38 antibody disclosed in International Application Publication Nos. WO2016 / 086189A2, WO2020 / 088608A1, WO2019191120A1, and WO2016 / 105450A3; the contents of each of the foregoing are incorporated by reference in their entirety.

[0233] In some embodiments, the multispecific (e.g., bispecific or trispecific) antigen-binding molecules described above that specifically bind to CD3 and a tumor antigen have a K of greater than about 40 nM as measured by an in vitro binding affinity assay. D The bispecific antigen-binding molecule may also include an anti-CD3 antigen-binding molecule that binds to CD3 with weak binding affinity, such as exhibiting an EC50 of greater than about 100 nM, as measured by a FACS-based titration assay. The bispecific antigen-binding molecule may also include an anti-CD3 antigen-binding molecule that binds to CD3 and exhibits an EC50 of greater than about 100 nM, as measured by an in vitro binding affinity assay or a FACS-based titration assay, but that does not exhibit measurable or observable binding to CD3, as measured by an in vitro binding affinity assay or a FACS-based titration assay, but retains the ability to activate human PBMC cells and / or induce cytotoxic activity against cell lines expressing tumor antigens.

[0234] Therapeutic Formulations and Administration In some embodiments, provided herein are pharmaceutical compositions comprising NK cells expressing a CAR described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a CD3 multispecific antigen-binding molecule described herein. In some embodiments, provided herein are pharmaceutical compositions in which a CD3 multispecific antigen-binding molecule described herein is co-formulated with NK cells expressing a CAR described elsewhere herein.

[0235] The pharmaceutical compositions provided herein can be formulated with suitable carriers, excipients, and other agents that provide improved transport, delivery, durability, etc. Many suitable formulations can be found in formularies known to all pharmacists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J. Pharm. Sci. Technol. 52:238-311.

[0236] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, and the like.

[0237] Various delivery systems, such as liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions provided herein (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active substances. Administration can be systemic or local.

[0238] In some embodiments, the pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical compositions disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are manufactured pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition is dispensed and the reservoir is emptied, the entire device is discarded.

[0239] Numerous reusable pen-type autoinjector delivery devices have been adapted for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include the AUTOPen™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, and the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVO Pen™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVO Pen JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), and the BD™ pen (Becton Dickinson, Franklin, MA), to name just a few. Lakes, NJ), OPTI Pen™, OPTIPEN, PRO™, OPTIPEN, STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices applicable to subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLExPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name just a few.

[0240] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are described in the review by Langer, 1990, Science, 249:1527-1533.

[0241] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like, which may be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which may be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. Therefore, injectable preparations are preferably filled into appropriate ampoules.

[0242] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for administration of the active ingredient.Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0243] In some aspects, provided herein is a pharmaceutical composition comprising an NK cell (e.g., an induced NK cell) expressing a CAR described herein.

[0244] In certain embodiments, the CAR-NK cell population may be administered alone or in combination with a pharmaceutically or physiologically acceptable carrier, diluent, excipient, and / or other components or cell populations as a pharmaceutical composition. Such compositions may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions disclosed herein may be formulated for intravenous administration.

[0245] Administration of CAR-NK cells may be performed in any convenient manner, including injection, infusion, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may also be injected directly into a tumor or lymph node.

[0246] How to Treat Cancer In certain embodiments, the present disclosure includes a method of treating a subject. In some embodiments, the method may include administering to a subject in need thereof (1) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to the extracellular domain, together (e.g., simultaneously or sequentially). In certain embodiments, the method may include administering to a subject in need thereof (1) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that includes a CD3 extracellular domain or a fragment thereof; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to the CD3 extracellular domain or a fragment thereof, together (e.g., simultaneously or sequentially). In some embodiments, the extracellular domain of CD3 or a fragment thereof comprises an epitope recognized by an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In some embodiments, the extracellular domain of CD3, or a fragment thereof, comprises at least 10 contiguous amino acids of SEQ ID NO: 1959. In some embodiments, the CD3 extracellular domain, or a fragment thereof, comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1959. In certain embodiments, the CD3 extracellular domain, or a fragment thereof, comprises the amino acid sequence of SEQ ID NO: 1959.

[0247] In some embodiments, the method may comprise administering to a subject in need thereof a pharmaceutical composition comprising: (1) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain comprising the CD3 extracellular domain or a fragment thereof; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to the CD3 extracellular domain or a fragment thereof. The therapeutic composition may further comprise a pharmaceutically acceptable carrier or diluent.

[0248] In some embodiments, the method may include administering to a subject in need thereof (1) an antigen binding molecule that binds to a tumor antigen; and (2) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the antigen binding molecule, together (e.g., simultaneously or sequentially).

[0249] In certain embodiments, the method may comprise administering to a subject in need thereof (e.g., simultaneously or sequentially) (1) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds CD3 and a tumor antigen-binding domain that specifically binds a tumor antigen; and (2) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain comprising an antigen-binding domain specific for the idiotype of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises the heavy and light chain CDR sequences of an scFv listed in Table 1. In some embodiments, the antigen-binding domain comprises the heavy and light chain variable region sequences of one of the scFvs listed in Table 1. In some embodiments, the antigen-binding domain comprises the amino acid sequence of an scFv listed in Table 1.

[0250] In some embodiments, the method may comprise administering to a subject in need thereof a pharmaceutical composition comprising: (1) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen; and (2) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule. The therapeutic composition may further comprise a pharmaceutically acceptable carrier or diluent.

[0251] In some embodiments, the method may include administering to a subject in need thereof (e.g., simultaneously or sequentially) (a) an antigen binding molecule that binds to a tumor antigen and includes an Fc domain; and (b) natural killer (NK) cells expressing a CAR polypeptide that includes an extracellular domain that binds to the Fc domain.

[0252] In certain embodiments, the method may comprise administering to a subject in need thereof (e.g., simultaneously or sequentially) (1) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; and (2) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the Fc domain, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen-binding molecule.

[0253] In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises the heavy and light chain CDR sequences of an scFv listed in Table 1. In some embodiments, the antigen-binding domain comprises the heavy and light chain variable region sequences of one of the scFvs listed in Table 1. In some embodiments, the antigen-binding domain comprises the amino acid sequence of an scFv listed in Table 1.

[0254] In some embodiments, the method may include administering to a subject in need thereof a pharmaceutical composition comprising: (1) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; and (2) natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that comprises an antigen-binding domain specific for the Fc domain, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen-binding molecule. The therapeutic composition may further comprise a pharmaceutically acceptable carrier or diluent.

[0255] As used herein, the terms "treat," "treating," and the like refer to alleviating symptoms or eliminating the cause of symptoms, either temporarily or permanently. For example, "treating cancer" can mean slowing or inhibiting tumor growth, reducing tumor cell burden or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, and / or increasing survival of a subject.

[0256] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or indications of cancer and / or having been diagnosed with cancer and in need of treatment. In many embodiments, the term "subject" may be used interchangeably with the term "patient."

[0257] In some embodiments, cancers that may be treated by the methods and compositions provided herein include, but are not limited to, cancers originating from the cervix, anus, vagina, vulva, penis, base of tongue, larynx, tonsils, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, non-melanoma skin cancer (NMSC), cutaneous squamous cell carcinoma (SCC), stomach, testis, tongue, or uterus. In addition, cancers include, specifically, the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; hairy cell carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combined hepatocellular carcinomas and cholangiocarcinomas; trabecular adenomas; adenoid cystic carcinomas; adenocarcinomas with adenomatous polyposis; adenocarcinomas, familial adenomatous polyposis; solid tumors; malignant carcinoid tumors; bronchioloalveolar adenocarcinomas ;papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; ; mucosal epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Adenocarcinoma;Malignant thymoma;Malignant ovarian stromal tumor;Malignant theca tumor;Malignant granulosa cell tumor;Malignant androgen-producing tumor;Sertoli cell tumor;Malignant Leydig cell tumor;Malignant lipid cell tumor;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Hemangioangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Transverse Rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian goiter;choriocarcinoma;malignant mesonephroma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;mesenchymal chondrosarcoma;Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillar astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory nerve tumor; Malignant meningioma; Neurofibrosarcoma; Malignant neurilemmoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma The disease may include, but is not limited to, lymphoma, granulomatous ...

[0258] In some embodiments, cancers that may be treated by the methods and compositions provided herein express a tumor antigen that is targeted by an antigen-binding molecule or multispecific antigen-binding molecule (e.g., a CD3 multispecific antigen-binding molecule). In some embodiments, cancers treated by the methods and compositions provided herein may be tumors with tumor antigen expression as determined by flow cytometry in 20% or more of tumor cells. In particular, the compositions and methods disclosed herein may be used to treat tumors with tumor antigen expression or activity, e.g., CD20, PSMA, MUC16, STEAP2, or BCMA, or CD20 + , PSMA + , MUC16 + , STEAP2 + , or BCMA +They may be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by cell proliferation. The mechanisms by which the treatments disclosed herein are achieved include, for example, killing cells expressing such antigens in the presence of effector cells by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0259] In some embodiments, the CD3 multispecific antigen-binding molecule used in the compositions or methods of the present invention is a bispecific anti-CD3 x anti-PSMA antibody. The compositions or methods are useful for treating PSMA-expressing cancers, including prostate cancer, kidney cancer, bladder cancer, colon cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0260] In some embodiments, the CD3 multispecific antigen-binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-MUC16 antibody. The compositions or methods are useful for treating MUC16-expressing cancers, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma (mass-forming type), adenocarcinoma of the cervix, and adenocarcinoma of the gastrointestinal tract. In some embodiments, the cancer is ovarian cancer.

[0261] In some embodiments, the CD3 multispecific antigen-binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-STEAP2 antibody. The compositions or methods are useful for treating cancers that express STEAP2, including prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer, and ovarian cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0262] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-BCMA antibody. The compositions or methods are useful for treating multiple myeloma or other B-cell or plasma cell cancers, such as BCMA-expressing cancers, including Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. In some embodiments, the cancer is multiple myeloma.

[0263] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-CD20 antibody. The compositions or methods are useful for treating CD20-expressing cancers, including non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom's macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Burkitt's lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0264] In certain embodiments, the methods disclosed herein are used in subjects being treated with a particular anti-cancer agent (e.g., cancer immunotherapy, CAR-T cell therapy, or a CD3 multispecific antigen-binding molecule such as those described herein).

[0265] In some embodiments, for any of the methods disclosed herein, the subject being treated or assessed is a subject being treated with a cancer immunotherapy, e.g., a CD3 multispecific antigen-binding molecule as described herein.

[0266] In some embodiments, the methods provided herein treat, slow, or inhibit tumor growth or induce tumor cell death. In certain embodiments, the methods provided herein promote tumor regression. In certain embodiments, the methods provided herein reduce tumor cell burden or reduce tumor burden. In certain embodiments, the methods provided herein prevent tumor recurrence.

[0267] In certain embodiments, the disclosed natural killer (NK) cells and / or antigen binding molecules (e.g., CD3 multispecific antigen binding molecules) expressing a CAR polypeptide are administered to a patient in combination with (e.g., before, concurrently with, or after) any number of related therapies, including, but not limited to, an additional cancer treatment.

[0268] The additional therapeutically active ingredient(s) may be administered immediately before, during, or immediately after administration of the disclosed natural killer (NK) cells and / or antigen binding molecules (e.g., CD3 multispecific antigen-binding molecules) expressing a CAR polypeptide; (for purposes of this disclosure, such administration regimens will be considered administration of the disclosed natural killer (NK) cells and / or antigen binding molecules (e.g., CD3 multispecific antigen-binding molecules) expressing a CAR polypeptide "in combination with" the additional therapeutically active ingredient).

[0269] As described above, co-administration may be simultaneous, separate, or sequential. In the case of simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0270] Drug administration regimen In certain embodiments, provided herein are methods that include administering to a subject NK cells expressing a CAR described herein at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0271] In certain embodiments, provided herein are methods comprising administering a CD3 multispecific antigen binding molecule to a subject at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0272] In certain embodiments, the methods comprise administering NK cells expressing a CAR described herein in combination with a CD3 multispecific antigen binding molecule at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0273] According to certain embodiments, multiple doses of NK cells expressing a CAR described herein in combination with a CD3 multispecific antigen-binding molecule may be administered to a subject over a defined time course. The methods according to this aspect disclosed herein may comprise sequentially administering to a subject multiple doses of NK cells expressing a CAR described herein in combination with a CD3 multispecific antigen-binding molecule. As used herein, "sequentially administering" means that each dose of CAR-NK cells or antigen-binding molecule is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hour, day, week, or month). In certain embodiments, the present disclosure encompasses methods comprising sequentially administering to a patient a single initial dose of NK cells expressing a CAR described herein, followed by one or more second doses of NK cells expressing a CAR described herein, optionally followed by one or more third doses of NK cells expressing a CAR described herein. In certain embodiments, the present disclosure further comprises sequentially administering to the patient a single initial dose of the CD3 multispecific antigen-binding molecule, followed by one or more second doses of the CD3 multispecific antigen-binding molecule, optionally followed by one or more third doses of the CD3 multispecific antigen-binding molecule.

[0274] The terms "first dose," "second dose," and "third dose" refer to the temporal order of administration of the antigen-binding molecules disclosed herein. Thus, the "first dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the first dose; and the "third dose" is the dose administered after the second dose. The first, second, and third doses may all contain the same amount of a therapeutic agent described herein, but generally may differ from each other in terms of administration frequency. However, in certain embodiments, the amount of antigen-binding molecule contained in the first, second, and / or third dose varies from each other during the course of treatment (e.g., adjusted upward or downward as necessary). In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered at the beginning of a treatment regimen as "loading doses," followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0275] In one exemplary embodiment disclosed herein, each of the second and / or third doses is administered 1 to 26 weeks (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26) or more weeks after the immediately preceding administration. As used herein, the phrase "immediately preceding administration" means a dose of a therapeutic agent administered to a patient prior to administration of the immediately next dose in the sequence, with no intervening administrations in between.

[0276] The methods according to this aspect disclosed herein may include administering to the patient any number of second and / or third doses of a therapeutic agent described herein. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) third doses are administered to the patient.

[0277] In embodiments comprising multiple second doses, each second dose may be administered with the same frequency as the other second doses. For example, each second dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments comprising multiple third doses, each third dose may be administered with the same frequency as the other third doses. For example, each third dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the second and / or third doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted during the course of treatment by a physician according to the needs of an individual patient after clinical testing. [Example]

[0278] Example 1: Evaluation of the ability of anti-CD20 single-armed antibodies to induce target-dependent ahFc-CAR-dependent signaling using Jurkat / NFAT-Luc / ahFc-CD28-CD3z and ahFc-CAR and cytolysis of ahFc-CAR Ramos using KHYG1 / ahFc-CD28-CD3z Testing Procedure: Cell line manipulation Engineering a luciferase-based reporter cell line: The Jurkat E6 cell line, derived from human acute T-cell leukemia, was transduced with an NFAT-response element-driven luciferase reporter construct. Puromycin-resistant cells were maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, and 1 mg / mL puromycin. This cell line was subjected to single-cell sorting, and a single clone was identified and renamed Jurkat / NFAT-Luc cl.3C7 (ACL8722). Jurkat / NFAT-Luc cl.3C7 was transduced with a vector encoding a chimeric construct consisting of the mROR signal sequence, an anti-human Fc scFv portion, a G4S linker (SEQ ID NO: 89), the CD28 hinge, transmembrane domain, and cytoplasmic domain, and the CD3z cytoplasmic domain. Blasticidin-resistant cells were maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 1 mg / mL puromycin, and 10 mg / mL blasticidin. The cell line was renamed Jurkat / NFAT-Luc / ahFc-CD28-CD3z (ACL21770).

[0279] Engineering B cell line reporter cells to assess cytolytic activity: The human B lymphocyte cell line Ramos.2G6.4C10 was transduced with a vector encoding a chimeric construct consisting of enhanced GFP (eGFP) (WP_031943942.1 M1-K239), a GSGGSG linker (SEQ ID NO: 90), and a HiBiT tag (VSGWRLFKKIS) (SEQ ID NO: 1960). + Cells were sorted and maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. The cell line was renamed Ramos / GFP (ACL21777).

[0280] Engineering an NK cell line with cytolytic activity: The human natural killer cell leukemia-derived KHYG-1 cell line was transduced with a vector encoding a chimeric construct consisting of the mROR signal sequence, a scFv portion targeting human Fc, a G4S linker (SEQ ID NO: 89), the CD28 hinge, transmembrane, and cytoplasmic domains, and the CD3z cytoplasmic domain. Blasticidin-resistant cells were maintained in RPMI 1640 medium supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 10 ng / mL IL-2, and 5 mg / mL blasticidin. The cell line was renamed KHYG / ahFc-CD28-CD3z (ACL21772).

[0281] Jurkat / NFAT-Luc / ahFc-CD28-CD3z signaling bioassay: To evaluate the targeting and antibody-dependent agonist activity of the anti-Fc chimeric construct, a cell-based reporter assay was established in which the antibody was co-incubated with target cells and Jurkat / NFAT-Luc cells expressing the anti-hFc CAR construct, which, upon clustering of the CAR construct, results in activation of luciferase expression driven by the nuclear factor of activated T cells (NFAT) response element. The effects of an anti-CD20 antibody (REGN2959: anti-CD20 (10F2) single-armed antibody IgG1) and a non-targeting isotype control (REGN1932: isotype control IgG1) were evaluated in the presence of either cells positive for CD20 expression (Ramos.2G6.4C10) or negative for CD20 expression (Jurkat).

[0282] RPMI1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin was used as the assay medium to prepare cell suspensions and antibody dilutions. The day before screening, all reporter and target cells were cultured at 3 × 10 5 On the day of the assay, Jurkat / NFAT-Luccl.3C7 or Jurkat / NFAT-Luc / ahFc-CD28-CD3z reporter cells were plated in a 96-well white flat-bottom plate at 2.5 × 10 4Reporter cells were plated at 2.5 × 10 cells / well. Anti-CD20 single-arm antibody [REGN2959] or isotype control [REGN1932] was serially diluted (1:4) over a nine-point set range (25 nM–0.38 pM) (Figures 1A and 1B), with the tenth point containing no antibody (represented as 0.10 pM) but at 2.5 × 10 cells / well. 4 100 μL of detection reagent was added to the cells before adding 100 μL of Ramos.2G6.4C10 or Jurkat target cells / well. The plates were incubated at 37°C / 5% CO2 for 5 hours, after which 100 μL of detection reagent was added to the wells to lyse the cells and detect luciferase activity. Light emitted was measured in RLU using a multilabel plate reader, Envision (PerkinElmer).

[0283] Fold induction was calculated using the following formula:

number

[0284] KHYG / ahFc-CD28-CD3z target cell lysis assay: To evaluate antibody-dependent NK cell activation via anti-Fc chimeric constructs, an NK cell line-based cytolysis assay was established in which antibodies were co-incubated with target cells and KHYG cells expressing anti-hFc CAR constructs. Clustering of the CAR constructs leads to cytolysis of target cells. Detection of released tags in the supernatant was used as a surrogate for target lysis.

[0285] RPMI1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin was used as the assay medium to prepare cell suspensions and antibody dilutions. The day before screening, transduced NK cell lines and target cells were incubated at 3 × 10 5 On the day of the assay, KHYG / ahFc-CD28-CD3z cells were plated in a 96-well white flat-bottom plate at 2.5 × 10 4 Reporter cells / well were seeded at 5 x 10 3100 μL of Ramos / GFP was added. A serial dilution (1:4) of an anti-CD20 single-arm antibody [REGN2959] or an isotype control [REGN1932] was added to the cells over a nine-point setpoint range (25 nM–0.38 pM) (Figures 1A and 1B), with the tenth point containing no antibody (represented as 0.10 pM). The plate was incubated at 37°C / 5% CO2 for 5 hours, after which 100 μL of detection reagent was added to detect extracellular tags. Emission light was measured in RLU using a multilabel plate reader, Envision (PerkinElmer).

[0286] The cytotoxicity rate was calculated using the following equation:

number

[0287] The maximum cytotoxicity rate was the most reproducible mean cytotoxicity value across the antibody dose range.

[0288] result: Jurkat / NFAT-Luc / ahFc-CD28-CD3z signaling bioassay: As shown in Figures 1A and 1B, in the presence of Jurkat reporter cells expressing CAR, target-expressing cells (Ramos), and anti-CD20 antibody (REGN2959), a dose-dependent increase in NFAT-driven luciferase reporter expression was detected (maximum signal: 4.54-fold). In contrast, the absence of CAR expression on Jurkat reporter cells, target expression (using Jurkat as target cells), or antibody targeting (using isotype control REGN1932) did not lead to an increase in luciferase reporter expression (maximum signal: 1.33-fold) (Figures 1A and 1B, Table 15).

[0289] Table 15 shows the maximum fold induction of signal across a range of antibody doses from Jurkat or Ramos and Jurkat / NFAT-Luccl.3C7 or Jurkat / NFAT-Luc / ahFc-CD28-CD3 incubated with anti-CD20 (REGN2959) or isotype control (REGN1932). [Table 16]

[0290] KHYG / ahFc-CD28-CD3z target cell lysis assay: As shown in Figure 2, in the presence of the CAR-expressing KHYG-1 NK cell line and target cells (Ramos / GFP), the addition of an anti-CD20 antibody (REGN2959) resulted in a dose-dependent increase in target cell lysis (maximum lysis: 71.65%). In contrast, non-targeting antibodies were unable to induce Ramos cell lysis (maximum detected cell lysis: 5.86%) (Figure 2, Table 16).

[0291] Table 16 shows the induction of maximum cytolysis across a range of antibody doses from Ramos cells and KHYG-1 / ahFc-CD28-CD3 incubated with anti-CD20 antibody (REGN2959) or isotype control (REGN1932). [Table 17]

[0292] Example 2: Biacore binding data for scFv against CD3 antibody idiotypes (09F7 and 7221G) or modified Fc (Fc*) VelocImmune (humanized) mice were immunized with either a CD3 bivalent or CD3 bispecific antibody to generate anti-idiotypic antibodies. Similarly, antibodies recognizing defined features of the modified antibody Fc domain were generated by immunizing VelocImmune mice with Fc-modified antibodies. Antibodies with desired binding properties, as determined by ELISA, were reconstituted into single-chain variable fragments (scFv). Surface plasmon resonance (SPR) technology was used to evaluate the binding of the scFv to the antibody immunogen and to antibodies with similar target specificity but different binding properties.

[0293] Biacore kinetics for binding of scFv supernatants directed against CD3 antibody idiotypes (09F7 and 7221G) or modified Fc (Fc*) to a panel of human antibodies was determined in an scFv capture format at 25°C. The anti-09F7 scFv (also referred to as "PN29950_LCHC") was derived from an anti-idiotypic antibody generated from a mouse immunized with REGN1453 (anti-hCD20 x anti-hCD3-9F07). The anti-7221G scFv (also referred to as "PN77570_HCLC") was derived from an anti-idiotypic antibody generated from a mouse immunized with H4tH7221G (anti-hCD3-7221G). The anti-Fc* scFv (also referred to as "PN78216_HCLC") was derived from an antibody generated from a mouse immunized with an Fc-modified antibody.

[0294] Testing Procedure:

[0295] The equilibrium dissociation constants (KD values) of anti-idiotypic (09F7, 7221G, Fc*) scFvs fused to HA-tagged supernatants that bind to a panel of human antibodies were determined using real-time surface plasmon resonance biosensor technology on a Biacore T-200 or 8k instrument. Briefly, a CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-HA antibody (Abcam, catalog no. ab18181, clone HA.C5). All Biacore binding experiments were performed in a buffer consisting of 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% v / v% surfactant P20 (HBS-EP running buffer). The scFv supernatants (targeting 09F7, 7221G, or Fc* antibodies) were captured on the anti-HA surface by injection for 90 or 120 seconds at a flow rate of 5 or 10 μL / min. A single concentration of antibody (50 or 100 nM) was injected over the captured scFv at a flow rate of 30 μL / min. scFv-antibody association was monitored for 90 or 120 s, and dissociation was monitored for 120 s. At the end of each cycle, the scFv capture surface was regenerated with two 10-s injections of 50 mM NaOH. All binding kinetics experiments were performed at 25°C.

[0296] Data Analysis:

[0297] Specific SPR-Biacore sensorgrams were obtained by a double-referencing procedure. This was performed by first subtracting the signal of each injection at the reference surface (anti-HA) from the signal at the experimental surface (anti-HA captured scFv), thereby eliminating the contribution due to refractive index changes. In addition, a running buffer injection was performed, allowing subtraction of signal changes due to dissociation of the captured scFv from the bound anti-HA surface. The kinetic association (k a ) and dissociation (k d The rate constants for dissociation of the binding (K) were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber v2.0c curve-fitting software or Cytiva Insight v4.0 software. D) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

[0298]

number

[0299] result:

[0300] The kinetic results are presented in Table 17 for the anti-7221G scFv PN29950_LCHC, Table 18 for the anti-09F7 scFv PN77570_HCLC, and Table 19 for the anti-Fc* scFv PN78216_HCLC. All kinetic results were measured at 25°C. [Table 18-1] [Table 18-2] [Table 19-1] [Table 19-2] [Table 20-1] [Table 20-2]

[0301] Example 3: Blocking ELISA data examining human CD3 anti-idiotypic scFv An ELISA-based method was used to assess blocking of anti-hCD3 mAb binding to ELISA plates coated with hCD3ε / δ protein in the presence of dilutions of hCD3 anti-idiotypic scFv.

[0302] reagent [Table 21] [Table 22]

[0303] Testing Procedure:

[0304] Avidin (Thermo Scientific) was coated onto a 96-well microtiter plate at 5.0 μg / mL in PBS and incubated overnight at 4°C. Nonspecific binding sites were then blocked with 0.5% (w / v) BSA in PBS (assay buffer) and incubated at room temperature (RT) for approximately 1 hour. 3.0 μg / mL hCD3ε / δ (Acro Biosystems) was then captured onto the avidin-coated ELISA plate by incubation at room temperature for 1 hour.

[0305] Pre-binding blocking reactions with fixed amounts of anti-human CD3 mAb were set up in a 96-well dilution plate using hCD3 anti-idiotypic scFv and control mAb. For the pre-binding reactions, purified human CD3 anti-idiotypic scFv expressed from Chinese hamster ovary (CHO) cells was serially diluted 3-fold in assay buffer starting from undiluted supernatant; parental control mAb, scFv negative control, and isotype control mAb were serially diluted 3-fold in assay buffer from 5.0 nM to 84.6 fM; and biotin-hCD3ε / δ (used as a positive control) was serially diluted 3-fold in assay buffer from 4.17 μM to 70.5 pM. Serially diluted scFv-PN29950 and control mAb were mixed with 20 pM of each anti-hCD3 mAb, REGN18409 and REGN18411, and serially diluted scFv-PN277570 and control mAb were mixed with 20 pM of REGN2533. Table 23 summarizes the concentrations of each hCD3 anti-idiotypic scFv and control mAb used.

[0306] The prebinding reaction mixture was incubated for 1 hour at room temperature and then transferred to an hCD3 ε / δ-coated ELISA plate and incubated for 1 hour at room temperature. Binding of each anti-hCD3 mAb (REGN18409, REGN18411, and REGN2533) in the presence of the respective scFv and control mAb was detected using an HRP-conjugated anti-human Fc polyclonal antibody (Jackson Immunoresearch) by incubation for 1 hour at room temperature. The assay plate was developed using TMB colorimetric substrate according to the manufacturer's recommended procedure.

[0307] The absorbance at 450 nm for each well was recorded and plotted as a function of dilution of each hCD3 anti-idiotypic scFv tested. Data were analyzed using a four-parameter logistic equation on an 11-point inhibition curve in GraphPad Prism software. Because CHO supernatants did not have measured concentrations, IC values ​​were calculated for the scFv molecules. 50 No value was available (reported as n / a in Table 23). Instead, results showing the hCD3 anti-idiotypic scFv that blocked each anti-hCD3 mAb were reported as percent blocking, as shown in Table 23.

[0308] The percent blocking at the lowest dilution of hCD3 anti-idiotype scFv (i.e., highest concentration) was calculated as a measure of the molecule's ability to block binding of each anti-hCD3 mAb to hCD3ε / δ relative to the assay baseline. The assay baseline signal, defined as 0% binding to hCD3ε / δ, was determined from the OD450 nm readings from anti-hFc detection in wells containing assay buffer alone. The binding signal of 20 pM of each anti-hCD3 mAb (REGN18409, REGN18411, or REGN2533) in the absence of the hCD3 anti-idiotype was defined as 100% binding or 0% blocking.

[0309] Summary of results and conclusions:

[0310] The ability of the hCD3 anti-idiotypic scFv molecule PN29950 to block REGN18409 and REGN18411, as well as the ability of PN77570 to block binding of REGN2533 to plate-coated hCD3ε / δ protein, was assessed using a blocking ELISA. The blocking results are summarized in Table 23 and shown in Figures 4A-4C. Percent blocking calculated at the highest scFv concentration (undiluted CHO supernatant) is reported. All hCD3 anti-idiotypic scFvs tested blocked binding of 20 pM of their respective anti-hCD3 mAbs relative to baseline.

[0311] Parental control mAbs (REGN5766 and REGN2984) blocked 20 pM of anti-hCD3 mAbs (REGN18408 / REGN18411 and REGN2533) with IC50 [M] values ​​of 42 pM / 39 pM and 32 pM, respectively, demonstrating approximately 100% block at the highest mAb concentrations. hCD3ε / δ (ligand control) demonstrated baseline or near-baseline blocking for each anti-hCD3 mAb. The corresponding isotype control mAb (Southern Biotech) and scFv negative control (REGN4393) demonstrated no blocking of anti-hCD3 mAbs under identical assay conditions. [Table 23] 100% unblocked = OD of wells containing HRP-conjugated secondary protein in assay buffer alone 450nm value

number

[0312] Example 4: Evaluation of antibody binding to KHYG1 cell lines engineered to express chimeric antigen receptors with anti-idiotypic scFv Anti-idiotypic scFvs with desired binding strength and specificity were reconfigured into chimeric antigen receptors (CARs) and expressed in KHYG1 cells, a natural killer leukemia cell line. KHYG1 cells engineered to express a CAR targeting an antibody with a modified Fc, KHYG1 / NFAT-Luc / CAR1, or CARs targeting the CD3-binding arm of a CD3 bispecific antibody, KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15, were evaluated for target binding in a flow cytometry assay. KHYG1 / NFAT-Luc / CAR1 cells were evaluated for their ability to bind antibodies containing modified Fc, while the KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines were evaluated for binding to the CD3 bispecific antibody. Detection of antibody binding to KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines was assessed with an Alexa647-conjugated secondary antibody, while antibodies tested for binding to KHYG1 / NFAT-Luc / CAR1 cells were directly conjugated with Alexa647. [Table 24]

[0313] Testing Procedure:

[0314] Description of cell lines expressing chimeric antigen receptor targeting antibody domains:

[0315] Engineering NK reporter cell lines with chimeric antigen receptors directed against antibody domains: The human natural killer cell leukemia-derived KHYG-1 cell line was stably transduced with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct. A puromycin-resistant clone (ACL20834) was isolated and subsequently transduced with a chimeric construct consisting of an mROR signal sequence, an scFv moiety targeting a specific antibody domain, such as a modified human Fc (PN78216) or CD3 anti-idiotype (PN29950 and PN77570), a G4S linker (SEQ ID NO: 89), a CD28 hinge, a transmembrane domain, a cytoplasmic domain, a CD3z cytoplasmic domain, and cytoplasmic eGFP. Blasticidin-resistant cells were maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 10 ng / mL IL-2, 1 μg / mL puromycin, and 5 μg / mL blasticidin. The cell line engineered to recognize the modified Fc domain was designated KHYG1 / NFAT-Luc / PN78216_VH-VL-CD28crosslink-TM-cyto-CD3z-eGFP (ACL22442) and is also referred to as KHYG1 / NFAT-Luc / CAR1. The cell lines engineered to recognize the antigenic determinants of specific CD3 antibodies (also known as CD3 anti-idiotypes) were designated KHYG1 / NFAT-Luc / PN29950_VL-VH-CD28 crosslinked-TM-cyto-CD3z-eGFP highly selected (ACL22550) and KHYG1 / NFAT-Luc / PN77570_VH-VL-CD28 crosslinked-TM-cyto-CD3z-eGFP (ACL22594), and are also referred to as KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15.

[0316] Assay setup:

[0317] For flow binding experiments, KHYG1 / NFAT-Luc / CAR1, KHYG1 / NFAT-Luc / CAR6, and KHYG1 / NFAT-Luc / CAR15 cells were washed and resuspended in staining buffer (PBS containing 2% FBS).5 Cells / well were added to wells of a 96-well V-bottom plate. A 10-point 1:4 titration of antibody ranging from 400 nM to 6.1 pM was added to the cells, with the final point of the antibody-free titration plotted at 1.5 pM. Antibodies tested for binding to KHYG1 / NFAT-Luc / CAR1 cells consisted of antibodies directly conjugated to an Alexa647 fluorophore and bearing a modified Fc, or control antibodies lacking a modified Fc (REGN5949-A647 and REGN7540-A647, respectively). Antibodies tested for binding to KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines consisted of CD3 bispecific antibodies (REGN5949, REGN5950, REGN1979) without a fluorophore conjugated to them, or an isotype-matched control (REGN7540). The cells and antibody were incubated for 30 minutes at 4°C and then washed with staining buffer. For the KHYG1 / NFAT-Luc / CAR6 and CAR15 cell lines, 2 μg / ml of Alexa647-conjugated goat anti-human Fcg fragment-specific secondary antibody diluted in staining buffer was then incubated with the cells for 30 minutes at 4°C. After washing all cells in staining buffer, they were resuspended in a viability dye (reconstituted in DMSO according to the manufacturer's protocol and diluted 1:1000 in PBS). The mixture was incubated for 30 minutes at 4°C and then washed with staining buffer. The cells were resuspended in PFA (2% diluted in staining buffer) for 30 minutes at 4°C. After washing, the cells were resuspended in staining buffer and analyzed by flow cytometry. The EC of the antibody was measured. 50 Values ​​were determined from a four-parameter logistic equation for a 10-point dose-response curve (including only the secondary control) using GraphPad Prism software, where 0 nM was plotted as 1.5 pM.

[0318] Summary of results:

[0319] KHYG1 / NFAT-Luc / CAR1 binding results:

[0320] KHYG-1 cells expressing a CAR directed against an antibody containing a modified Fc (Fc*) bound A647-labeled REGN5949 but failed to bind to a control antibody (REGN7540) with a similar Fc (IgG4) but no modification (Table 25 and Figure 5).

[0321] Binding results of KHYG1 / NFAT-Luc / CAR6 and CAR15:

[0322] KHYG-1 cells expressing CAR (CAR6 or CAR15) directed by anti-idiotypic CD3 antibodies were evaluated for their ability to bind various CD3xCD20 bispecific antibodies (note that the CD3 arms used in antibodies REGN5949, REGN5950, and REGN1979 are not identical). Antibodies REGN5949 and REGN5950 bound to KHYG-1 cells expressing CAR6, whereas binding of REGN1979 was not observed (Table 26 and Figure 6, left panel). Conversely, antibodies REGN5949 and REGN5950 did not bind to KHYG-1 cells expressing CAR15, whereas binding of REGN1979 was observed (Table 26 and Figure 6, right panel). An isotype control antibody (REGN7540) did not bind to either KHYG-1 cells expressing CAR6 or CAR15. [Table 25] [Table 26]

[0323] Example 5: KHYG1 / NFAT-Luc / CAR reporter signaling bioassay ScFvs with the desired binding strength and specificity were reconstituted into chimeric antigen receptors (CARs) and expressed in KHYG1 cells, a natural killer leukemia cell line. To identify ideal candidates, we performed a series of functional assays, including an engineered reporter assay in which activation of the CAR on KHYG1 cells resulted in a luminescent signal. [Table 27]

[0324] Testing Procedure:

[0325] KHYG1 / NFAT-Luc / CAR1(ahFc*-CD28-CD3z) signaling bioassay:

[0326] To assess the targeting and antibody-dependent agonist activity of modified Fc-directed CAR constructs, a cell-based reporter assay was established in which Fc-modified antibodies directed against CD20 (either CD20 bivalent or CD20xCD3 bispecific antibodies, H4H14303N2 and REGN5949, respectively) were co-incubated with Ramos target cells (expressing CD20) and KHYG1 / NFAT-Luc / CAR1 effector cells at a 1:1 (target:effector cell) ratio. Binding of the antibody to CD20 on the target cells and subsequent binding of the modified Fc by the CAR1-expressing KHYG1 cells results in clustering of the CAR construct and subsequent activation of luciferase expression driven by the nuclear factor of activated T cells (NFAT) response element.

[0327] Assay setup:

[0328] Experiments were performed in assay medium containing RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR1 reporter cells were plated at 2.5 × 10 cells / well in a 96-well white flat-bottom plate. 4 Reporter cells were plated at 2.5 × 10 cells / well. 4 Ramos.2G6.4C10(CD20 + ) or HEK293(CD20 -) Target cells / well were added to the plate. CD20 bivalent (H4H14303N2), bispecific (REGN5949), or isotype control (REGN7540) antibodies were serially diluted (1:4) over an 11-point setpoint range (100 nM–95 fM) (Figures and ) and added to the wells to a final volume of 100 μl. The 12th point contained no antibody (expressed as 24 fM). The plate was incubated at 37°C / 5% CO2 for 5 hours, after which 100 μL of detection reagent was added to the well to lyse the cells and detect luciferase activity. Emitted light was measured in RLU using the Envision multilabel plate reader (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation for a 12-point dose-response curve using GraphPad Prism software, where 0 nM was plotted as 24 fM.

[0329] KHYG1 / NFAT-Luc / CAR6 and CAR15 (CD3 anti-idiotype-CD28-CD3z) signaling bioassay:

[0330] To assess the target- and antibody-dependent agonist activity of CD3 anti-idiotype CAR chimeric constructs (CAR6 and CAR15), a cell-based reporter assay was established in which CD3xCD20 bispecific antibodies with different CD3-binding arms (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or the corresponding isotype control (REGN7540) were co-incubated with Ramos target cells (expressing CD20) and KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 effector cells at a 1:1 (target cells:effector cells) ratio. Binding of the antibody to CD20 on the target cells and subsequent engagement of the CD3-binding arms by anti-idiotype CAR1-expressing KHYG1 cells results in clustering of the CAR construct and subsequent activation of luciferase expression driven by the nuclear factor of activated T cells (NFAT) response element.

[0331] Assay setup:

[0332] Experiments were performed in assay medium containing RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 reporter cells were plated at 2.5 × 10 in 96-well white flat-bottom plates. 4 Reporter cells were plated at 2.5 × 10 cells / well. 4 Ramos.2G6.4C10 target cells were added per well to the plate. CD20×CD3 bispecific antibodies (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) containing various CD3-binding arms or an isotype control antibody (REGN7540) were serially diluted (1:5) over a nine-point setpoint range (100 nM–256 fM) (Figures 1 and 2). The tenth point contained no antibody (represented as 51 fM). The final well volume was 100 μl. The plate was incubated at 37°C / 5% CO2 for 4 hours, after which 100 μL of detection reagent was added to the well to lyse the cells and detect luciferase activity. Emitted light was measured in RLU using the Envision multilabel plate reader (PerkinElmer). EC50 values ​​were determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism software, where 0 nM was plotted as 51 fM.

[0333] The induction fold was calculated using the following formula:

[0334]

number

[0335] Summary of results:

[0336] KHYG / NFAT-Luc / CAR1 reporter activation results:

[0337] KHYG-1 cells expressing a CAR directed against an antibody containing a modified Fc (Fc*) were activated in the presence of target cells expressing CD20 and an antibody against CD20 with a modified Fc (Fc*) recognized by the CAR (Table 28 and Figure 7). The control antibody H4sH14303N2, which targets CD20 but does not contain a modified Fc, as well as the additional non-targeting control antibody REGN7540, did not activate KHYG-1 / NFAT-Luc / CAR1 cells (Table 28 and Figure 7). No activation was observed in the presence of target cells that did not express CD20 (data not shown).

[0338] KHYG1 / NFAT-Luc / CAR6 and CAR15 reporter activation results:

[0339] KHYG-1 / NFAT-Luc cells expressing CARs (CAR6 or CAR15) directed against an anti-idiotypic CD3 antibody were activated in the presence of target cells expressing CD20 and a specific CD20xCD3 bispecific antibody. + ) and antibodies REGN5951, REGN5375, REGN5949, REGN5950, and H4sH17400D were activated in a dose-dependent manner in the presence of H4sH17400D (Table 29, Table 30, and Figure 8, left panel). However, antibody REGN1979 did not activate KHYG-1 / NFAT-Luc / CAR6 reporter activity (Table 29, Table 30, and Figure 8, left panel). Conversely, only antibody REGN1979 was able to activate KHYG-1 / NFAT-Luc / CAR15 reporter activity (Table 29, Table 30, and Figure 8, right panel). The isotype control antibody REGN7540 did not activate KHYG-1 / NFAT-Luc / CAR6 or CAR15 cells (Table 29, Table 30, and Figure 8). [Table 28] [Table 29] [Table 30]

[0340] Example 6: KHYG1 / NFAT-Luc / CAR cytotoxic NK bioassay As discussed above in Example 5, scFvs with desired binding strength and specificity were redesigned into chimeric antigen receptors (CARs) and expressed in KHYG1 cells, a natural killer leukemia cell line. A series of functional assays, including cytotoxic NK killing assays with engineered KHYG-1 effector cells and Ramos / GFP-HiBiT target cells, were performed to identify ideal candidates.

[0341] To assess antibody-dependent NK cell activation, we established an NK cell line-based cytolytic assay in which antibodies were co-incubated with target cells (Ramos / HiBit) and KHYG-1 cells expressing the CAR construct. Clustering of the CAR construct leads to cytolysis of the target cells. Lysis of the target cells results in the release of intracellular HiBit into the supernatant. A detection reagent containing the complementary polypeptide LgBiT was added, which spontaneously interacts with the HiBit tag to reconstitute the highly luminescent NanoBiT® enzyme (Promega).

[0342] Engineering B cell reporters for cytolytic activity:

[0343] A clone was isolated from the previously described Ramos / GFP cell line (ACL21777) by single-cell sorting and maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. This cell line was renamed Ramos / GFP-HiBit cl 1B10 (ACL22182).

[0344] The same antibodies were used in this study as shown in Table 27 in Example 5 above.

[0345] Testing Procedure:

[0346] KHYG1 / NFAT-Luc / CAR1(ahFc*-CD28-CD3z) cytotoxicity bioassay:

[0347] To evaluate the targeting and antibody-dependent cellular cytotoxicity activity of the modified Fc-directed CAR constructs, an NK cell-based cytotoxicity assay was established in which Fc-modified antibodies directed against CD20 (either CD20 bivalent or CD20xCD3 bispecific antibodies, H4H14303N2 and REGN5949, respectively) were co-incubated with Ramos / HiBit target cells (expressing CD20) and KHYG1 / NFAT-Luc / CAR1 effector cells at a 1:5 (target:effector cell) ratio. Binding of the antibody to CD20 on the target cells and subsequent binding of the modified Fc by the CAR1-expressing KHYG1 cells resulted in clustering of the CAR construct and subsequent lysis of the target cells, which was measured via the release of HiBit into the supernatant, complemented by LgBiT to form the highly luminescent NanoBiT enzyme.

[0348] Assay setup:

[0349] Experiments were performed in assay medium containing RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR1 cells were plated in 96-well white flat-bottom plates at 2.5 × 10 4 Cells were seeded at 5.0 × 10 cells / well. 3Ramos / HiBit target cells / well were added to the plate. A CD20 bivalent (H4H14303N2), bispecific (REGN5949), or isotype control (REGN7540) antibody was serially diluted (1:4) over a 12-point setpoint range (100 nM–95 fM) (Figures and ) and added to the wells to a final volume of 100 μl, except for the 12th point, which contained no antibody (expressed as 24 fM). The plate was incubated at 37°C / 5% CO2 for 5 hours, after which 100 μL of non-lytic Nano-Glo extracellular detection reagent was added according to the manufacturer's specifications. Emitted light was measured in RLU using the Envision multilabel plate reader (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation for an 11-point dose-response curve using GraphPad Prism software, where 0 nM was plotted as 24 fM.

[0350]

number

[0351] Naturally occurring signal = target cell only (absence of antibody)

[0352] Maximum signal = only target cells lysed at the end of the assay incubation.

[0353] KHYG1 / NFAT-Luc / CAR6 and CAR15 (CD3 anti-idiotype-CD28-CD3z) cytotoxicity bioassay:

[0354] To evaluate the targeting and antibody-dependent cellular cytotoxicity of CD3 anti-idiotype CAR chimeric constructs (CAR6 and CAR15), an assay was established in which CD3xCD20 bispecific antibodies with different CD3-binding arms (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or the corresponding isotype control (REGN7540) were co-incubated with Ramos / HiBit target cells (expressing CD20) and KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 effector cells at a 1:5 (target cells:effector cells) ratio. Binding of the bispecific antibody to CD20 on the target cells and subsequent engagement of the CD3-binding arms by anti-idiotypic CAR-expressing KHYG1 cells results in clustering of the CAR construct and subsequent lysis of the target cells, which is measured via the release of HiBit into the supernatant, complementing LgBiT to form the highly luminescent NanoBiT enzyme.

[0355] Assay setup:

[0356] Experiments were performed in assay medium containing RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 cells were plated in 96-well white flat-bottom plates at 2.5 × 10 4 Cells were plated at 5.0 × 10 cells / well. 3Ramos / HiBit target cells / well were added to the plate. CD20xCD3 bispecific antibodies (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or an isotype control antibody (REGN7540) were serially diluted (1:5) over a nine-point setpoint range (100 nM–256 fM) (Figures and ). The tenth point contained no antibody (represented as 51 fM) and was added to the wells, resulting in a final well volume of 100 μl. The plate was incubated for 4 hours at 37°C / 5% CO2, followed by the addition of 100 μL of non-lytic Nano-Glo extracellular detection reagent according to the manufacturer's specifications. Emitted light was measured in RLU using the Envision multilabel plate reader (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism software, where 0 nM was plotted as 51 fM.

[0357] The cytotoxicity rate was calculated using the following formula:

[0358]

number

[0359] Naturally occurring signal = target cell only (absence of antibody)

[0360] Maximum signal = only target cells lysed at the end of the assay incubation.

[0361] Summary of results:

[0362] Results of KHYG / NFAT-Luc / CAR1 cytotoxic activation:

[0363] KHYG-1 cells expressing a CAR directed against an antibody containing a modified Fc (Fc*) expressed CD20 in the presence of an antibody against CD20 with a modified Fc (Fc*) recognized by the CAR.+ induced killing of Ramos / GFP-HiBit target cells (Table 31 and Figure 9). The control antibody H4sH14303N2, which targets CD20 but does not contain a modified Fc, as well as the non-targeting control antibody REGN7540, did not activate KHYG-1 / NFAT-Luc / CAR1 cells and killed Ramos target cells (Table 31 and Figure 9).

[0364] KHYG1 / NFAT-Luc / CAR6 and CAR15 reporter activation results:

[0365] KHYG-1 / NFAT-Luc cells expressing CARs (CAR6 or CAR15) directed against anti-idiotypic CD3 antibodies were transduced to CD20 in the presence of specific CD20 × CD3 bispecific antibodies. + The antibodies REGN5951, REGN5375, REGN5949, REGN5950, and H4sH17400D killed Ramos / GFP-HiBit target cells. Specifically, the antibodies REGN5951, REGN5375, REGN5949, REGN5950, and H4sH17400D killed CAR6-expressing KHYG-1 / NFAT-Luc / CAR6 cells in a dose-dependent manner (Table 32 and Figure 10, left panel). However, the antibody REGN1979 did not induce KHYG-1 / NFAT-Luc / CAR6 cytotoxicity (Table 32 and Figure 10, left panel). Conversely, only REGN1979 was able to induce the killing of KHYG-1 / NFAT-Luc / CAR15 target cells (Table 32 and Figure 10, right panel). The isotype control antibody REGN7540 did not induce the cytotoxicity of KHYG-1 / NFAT-Luc / CAR6 or CAR15 cells (Table 32 and Figure 10). [Table 31] [Table 32]

[0366] Example 7: CBNK cells engineered with anti-CD3 idiotype chimeric antigen receptor (CAR) mediate target cytotoxicity Similar to the experiments described in Example 6, antibody-dependent NK cell activation was assessed using primary human umbilical cord blood (CBNK)-derived NK cells engineered to express a CAR construct and co-incubated with target cells (Ramos / HiBit).

[0367] Engineering CAR-expressing cord blood (CB)-derived NK cells (CBNK):

[0368] Human CD34 was synthesized using standard synthetic biology and cell engineering (SBCE) protocols. + Cord blood (CB) derived NK cells (CBCE) were generated from HSPCs. Briefly, a two-step serum-free, cytokine-based ex vivo protocol was used to generate NK cells from HSPCs using the StemSpan™ NK Cell Generation Kit (Cat. No. 09960). In the first step, CD34 + HSPCs were cultured for 14 days in medium containing growth complementing agents (mainly SCF, IL7, FLT3, and TPO) to stimulate their proliferation and differentiation into lymphoid progenitor cells. At the end of this initial phase (days 8–10), these cells were engineered with a lentiviral vector (LVV) containing an anti-CD3 idiotypic scFv (CAR6) fused to a 28z-CAR-membrane-bound IL-15 construct. 48–72 hours after transduction, armed CBNK cells were sorted. In a second step, these lymphoid progenitor cells were cultured for an additional 14 days in medium containing differentiation complementing agents (mainly IL15, IL7, SCF, FLT3, and UM729) to stimulate their proliferation and differentiation into lymphoid progenitor cells. 56 These primary aCD3-ID-28z-CAR-mb15-CBNK cells (called CBNK / CAR6) were expanded by culturing them with engineered 41BBL-mbIL21-K562 feeder cells for 1 week, after which they were used in functional assays. [Table 33]

[0369] Testing Procedure:

[0370] CBNK cytotoxicity bioassay:

[0371] To assess the targeting and antibody-dependent cellular cytotoxicity of CBNK expressing a CD3 anti-idiotype CAR chimeric construct (CAR6), an assay was established in which a CD3xCD20 bispecific antibody or an isotype-matched non-targeting xCD3 control was co-incubated with Ramos / HiBit target cells (expressing CD20) and CBNK / CAR6 effector cells at a 1:4 (target cell:effector cell) ratio. Binding of the bispecific antibody to CD20 on the target cells and subsequent engagement of the CD3-binding arm by the anti-idiotype CAR-expressing CBNK cells resulted in clustering of the CAR construct and subsequent lysis of the target cells, as measured via the release of HiBit into the supernatant, complemented by LgBiT to form the highly luminescent NanoBiT enzyme.

[0372] Assay setup:

[0373] The experiment was performed in assay medium containing RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. CBNK / CAR6 cells were plated at 2.0 × 10 in a 96-well white flat-bottom plate. 4 Cells were plated at 5.0 × 10 cells / well. 3Ramos / HiBit target cells were added to the plate. CD20 × CD3 bispecific antibodies (REGN5949, REGN5950, REGN1979) or an isotype-matched non-targeting control × CD3 (REGN4018) were serially diluted (1:5) over a nine-point setpoint range (25 nM–64 fM) (Figures 1 and 2), with the tenth point containing no antibody (represented as 13 fM) added to the wells for a final well volume of 100 μl. Plates were incubated at 37°C / 5% CO2 for 4 hours, after which 25 μl was removed for cytokine assessment. Then, 75 μL of non-lytic Nano-Glo extracellular detection reagent was added according to the manufacturer's specifications. Emitted light was measured in RLU using the Envision multilabel plate reader (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism software, where 0 nM was plotted as 13 fM.

[0374]

number

[0375] Naturally occurring signal = target cell only (absence of antibody)

[0376] Maximum signal = only target cells lysed at the end of the assay incubation.

[0377] To assess the presence of cytokines in the supernatants, we used the iQue Qbeads Assay Builder kit from Sartorius. Cytokine capture beads and standards were prepared according to the manufacturer's instructions. Sample preparation was also performed according to the manufacturer's recommendations. Briefly, 10 μl of supernatant from the 25 μl collected from the assay wells was added to a 96-well v-bottom plate, followed by 10 μl of capture beads. After a brief centrifugation, the plate was incubated in the dark for 60 minutes, followed by the addition of 10 μl of detection cocktail. After a brief centrifugation, the plate was incubated in the dark for 90 minutes, followed by two washes with staining buffer (PBS containing 2% FBS). Samples were resuspended in 25 μl of staining buffer and transferred to a 96-well U-bottom plate. Samples and standards were run on an iQue flow cytometer, and cytokine quantification was performed according to the manufacturer's instructions.

[0378] Summary of results:

[0379] CBNK / CAR6 activation results:

[0380] CBNK cells expressing a CAR (CAR6) directed against an anti-idiotypic CD3 antibody were able to express CD20 in the presence of a specific CD20 × CD3 bispecific antibody. + CBNK / CAR6 resulted in target cell killing in the presence of antibodies REGN5949 and REGN5950, but not REGN1979 (Table 34, Table 35, and Figure 11, upper panel). The non-targeting control x CD3 antibody, REGN4018, also did not result in target cell killing (Table 34, Table 35, and Figure 11, upper panel).

[0381] CBNK cells expressing CARs directed against anti-idiotypic CD3 antibodies express CD20 +In the presence of Ramos / GFP-HiBit target cells and specific CD20xCD3 bispecific antibodies, CBNK / CAR6 induced the release of IFNg, TNFa, granzyme A, granzyme B, CCL5, and FasL in the presence of antibodies REGN5949 and REGN5950, but not REGN1979 (Table 34, Table 35, and Figure 11, middle and bottom panels). The non-targeting control x CD3 antibody, REGN4018, also did not induce cytokine release (Table 34, Table 35, and Figure 11, middle and bottom panels). [Table 34] [Table 35]

[0382] Incorporation by Reference All publications, patents, patent applications, and sequence reference numbers mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions therein, will control.

[0383] equivalent Numerous embodiments disclosed herein have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure herein. Accordingly, other embodiments are within the scope of the following claims. 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 the disclosed invention belongs.

[0384] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed and described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising: (a) (i) the CD3 extracellular domain or a fragment thereof; (ii) an antigen-binding domain specific for the idiotype of an anti-CD3 antibody; or (iii) an extracellular domain comprising an antigen-binding domain specific for the Fc domain; (b) a hinge domain; (c) a transmembrane domain; (d) an intracellular signaling domain.

2. The CAR polypeptide of claim 1, wherein the extracellular domain comprises the extracellular domain of CD3 or a fragment thereof.

3. The CAR polypeptide of claim 2, wherein the extracellular domain of CD3 or a fragment thereof comprises an epitope recognized by an anti-CD3 antibody.

4. 4. The CAR polypeptide of claim 3, wherein the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6.

5. The CAR polypeptide of any one of claims 2 to 4, wherein the extracellular domain of CD3 or a fragment thereof comprises at least 10 consecutive amino acids of SEQ ID NO: 1959.

6. The CAR polypeptide of any one of claims 2 to 5, wherein the extracellular domain of CD3 or a fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1959.

7. The CAR polypeptide of any one of claims 2 to 6, wherein the extracellular domain of CD3 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 1959.

8. The CAR polypeptide of claim 1, wherein the extracellular domain comprises an antigen-binding domain specific for the idiotype of an anti-CD3 antibody.

9. 9. The CAR polypeptide of claim 8, wherein the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6.

10. 10. The CAR polypeptide of claim 8 or 9, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

11. 11. The CAR polypeptide of claim 10, wherein the antigen-binding domain comprises the heavy and light chain CDR sequences of an scFv listed in Table 1.

12. 12. The CAR polypeptide of claim 11 , wherein the antigen-binding domain comprises heavy and light chain variable region sequences of an scFv listed in Table 1.

13. 13. The CAR polypeptide of claim 12, wherein the antigen-binding domain comprises the amino acid sequence of an scFv listed in Table 1.

14. The CAR polypeptide of claim 1, wherein the extracellular domain comprises an antigen-binding domain specific for an Fc domain.

15. 15. The CAR polypeptide of claim 14, wherein the Fc domain is selected from a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain.

16. 16. The CAR polypeptide of claim 15, wherein the Fc domain is an IgG3 Fc domain.

17. 15. The CAR polypeptide of claim 14, wherein the Fc domain comprises the amino acid sequence of Fc shown in Figure 3.

18. The CAR polypeptide of any one of claims 14 to 17, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

19. The CAR polypeptide of any one of claims 1 to 18, wherein the hinge domain is a hinge domain of CD28 or CD8.

20. 20. The CAR polypeptide of claim 19, wherein the hinge domain comprises an amino acid sequence selected from SEQ ID NOs: 1 to 5.

21. The CAR polypeptide of any one of claims 1 to 20, wherein the transmembrane domain is a transmembrane domain of NKG2D, an inverse transmembrane domain of NKG2D, a transmembrane domain of CD28, a transmembrane domain of CD8, a transmembrane domain of CD16, or a transmembrane domain of FcgR1 (CD64).

22. The CAR polypeptide of claim 21, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NOs: 6-13.

23. The CAR polypeptide according to any one of claims 1 to 22, wherein the intracellular signaling domain is the intracellular signaling domain of FcgR1, the intracellular signaling domain of 4-1BB-CD3z, the intracellular signaling domain of 2B4-CD3z, the intracellular signaling domain of CD16, the intracellular signaling domain of CD64, or the intracellular signaling domain of CD28-CD3z.

24. A nucleic acid encoding the CAR polypeptide of any one of claims 1 to 23.

25. A vector comprising the nucleic acid of claim 24.

26. 26. The vector of claim 25, wherein the vector is an expression vector.

27. The vector of claim 25 , wherein the vector is a viral vector.

28. 28. The vector of claim 27, wherein the viral vector is a lentiviral vector.

29. A natural killer (NK) cell comprising the nucleic acid of claim 24.

30. A natural killer (NK) cell expressing the CAR polypeptide according to any one of claims 1 to 23.

31. 31. The NK cell of claim 29 or 30, wherein the cell is a primary NK cell or an induced NK cell differentiated from an induced pluripotent stem cell (iPSC).

32. 1. A method of treating cancer in a subject, said method comprising administering to said subject: (A) a natural killer (NK) cell expressing a CAR polypeptide comprising an extracellular domain; (B) administering together a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to the extracellular domain.

33. The method comprises: (A) a natural killer (NK) cell expressing the CAR polypeptide according to any one of claims 2 to 7; (B) administering to the subject a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to the CD3 extracellular domain or a fragment thereof and a tumor antigen-binding domain that specifically binds to a tumor antigen.

34. 1. A method of treating cancer in a subject, said method comprising administering to said subject: (A) an antigen-binding molecule that binds to a tumor antigen; (B) administering to the subject the antigen-binding molecule, together with natural killer (NK) cells expressing a CAR polypeptide comprising an extracellular domain that binds to the antigen-binding molecule.

35. The method comprises: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen; (B) natural killer (NK) cells expressing the CAR polypeptide of any one of claims 8 to 13, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule.

36. 1. A method of treating cancer in a subject, said method comprising administering to said subject: (a) an antigen-binding molecule that binds to a tumor antigen and includes an Fc domain; (b) administering to the subject a natural killer (NK) cell expressing a CAR polypeptide comprising an extracellular domain that binds to the Fc domain.

37. The method comprises: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; (B) administering to the patient a CAR polypeptide of any one of claims 14 to 18 together with natural killer (NK) cells expressing the CAR polypeptide, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen-binding molecule.

38. 43. The method of any one of claims 40-42, wherein the hinge domain of the CAR polypeptide is a hinge domain of CD28 or CD8.

39. 39. The method of claim 38, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-5.

40. 40. The method of any one of claims 35 to 39, wherein the transmembrane domain of the CAR polypeptide is the transmembrane domain of NKG2D, the reverse transmembrane domain of NKG2D, the transmembrane domain of CD28, the transmembrane domain of CD8, the transmembrane domain of CD16, or the transmembrane domain of FcgR1 (CD64).

41. 41. The method of claim 40, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 6-13.

42. The method of any one of claims 35 to 41, wherein the intracellular signaling domain of the CAR polypeptide is the intracellular signaling domain of FcgR1, the intracellular signaling domain of 4-1BB-CD3z, the intracellular signaling domain of 2B4-CD3z, the intracellular signaling domain of CD16, the intracellular signaling domain of CD64, or the intracellular signaling domain of CD28-CD3z.

43. The method of any one of claims 35 to 42, wherein the antigen-binding molecule and the NK cells are administered simultaneously or sequentially.

44. The method according to any one of claims 35 to 42, wherein the antigen-binding molecule and the NK cells are premixed and administered to the subject simultaneously.

45. The method of any one of claims 35 to 42, wherein the subject is lymphopenic, and the antigen-binding molecule and the NK cells are premixed and administered to the subject simultaneously.

46. The method of any one of claims 35 to 42, wherein the NK cells or the premixed NK cells and antigen-binding molecules are administered after at least one administration of the antigen-binding molecules.

47. The method according to any one of claims 35 to 46, wherein the antigen-binding molecule is a bispecific antigen-binding molecule.

48. 48. The method of any one of claims 35 to 47, wherein the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, BCMA, CA19.9, MSLN, CD22, SLC3A2-APIS, CLDN18.2, and CEACAM5.

49. The method of any one of claims 35 to 48, wherein the antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

50. 50. The method of claim 49, wherein the multispecific antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody.

51. 51. The method of any one of claims 35 to 50, wherein the antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

52. 52. The method of claim 51 , wherein the antigen binding molecule is a multispecific antigen binding molecule listed in Table 6.

53. 1. A pharmaceutical composition comprising: (A) a natural killer (NK) cell expressing the CAR polypeptide according to any one of claims 2 to 7; (B) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to the CD3 extracellular domain or a fragment thereof, and a tumor antigen-binding domain that specifically binds to a tumor antigen.

54. 1. A pharmaceutical composition comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3 and a tumor antigen-binding domain that specifically binds to a tumor antigen; (b) natural killer (NK) cells expressing the CAR polypeptide of any one of claims 8 to 13, wherein the antigen-binding domain of the CAR polypeptide binds to the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule.

55. 1. A pharmaceutical composition comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain that specifically binds to CD3, a tumor antigen-binding domain that specifically binds to a tumor antigen, and an Fc domain; (B) natural killer (NK) cells expressing the CAR polypeptide of any one of claims 14 to 18, wherein the antigen-binding domain of the CAR polypeptide binds to the Fc domain of the multispecific antigen-binding molecule.

56. The pharmaceutical composition of any one of claims 53 to 55, wherein the hinge domain of the CAR polypeptide is the hinge domain of CD28 or CD8.

57. 57. The pharmaceutical composition of claim 56, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-5.

58. 68. The pharmaceutical composition of any one of claims 53 to 67, wherein the transmembrane domain of the CAR polypeptide is the transmembrane domain of NKG2D, the reverse transmembrane domain of NKG2D, the transmembrane domain of CD28, the transmembrane domain of CD8, the transmembrane domain of CD16, or the transmembrane domain of FcgR1 (CD64).

59. 59. The pharmaceutical composition of claim 58, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 6-13.

60. The pharmaceutical composition according to any one of claims 53 to 59, wherein the intracellular signaling domain of the CAR polypeptide is the intracellular signaling domain of FcgR1, the intracellular signaling domain of 4-1BB-CD3z, the intracellular signaling domain of 2B4-CD3z, the intracellular signaling domain of CD16, the intracellular signaling domain of CD64, or the intracellular signaling domain of CD28-CD3z.

61. The pharmaceutical composition of any one of claims 53 to 60, wherein the multispecific antigen-binding molecule is a bispecific antigen-binding molecule.

62. The pharmaceutical composition of any one of claims 53 to 61, wherein the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, BCMA, CA19.9, MSLN, CD22, SLC3A2-APIS, CLDN18.2, and CEACAM5.

63. 63. The pharmaceutical composition of any one of claims 53 to 62, wherein the multispecific antigen-binding molecule comprises a multispecific antibody or antigen-binding fragment thereof.

64. 64. The pharmaceutical composition of claim 63, wherein the multispecific antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody.

65. 65. The pharmaceutical composition of any one of claims 53 to 64, wherein the multispecific antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

66. 66. The pharmaceutical composition of claim 65, wherein the multispecific antigen-binding molecule is a multispecific antigen-binding molecule listed in Table 6.

67. A cell bank comprising NK cells expressing the CAR according to any one of claims 1 to 23.

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