Inducible NK cell responsive to CD3 / TAA bispecific antibody

Abstract

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

Description

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

[0002] Sequence Listing This application includes a sequence list electronically submitted in XML format, which is incorporated herein by reference in its entirety. This XML copy was created on February 16, 2024, 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 therapies for many cancers are either ineffective for certain patient populations or produce toxic side effects that severely impact patients' quality of life. Adoptive immunotherapy, involving the transfer of antigen-specific immune cells (such as T cells or NK cells) generated in vitro, is a promising cancer treatment strategy. Immune cells used in adoptive immunotherapy can be generated, for example, by genetically engineering and retargeting immune cells (e.g., by engineering them to express chimeric antigen receptors, or "CARs"). Compared to CAR-T cells, CAR-NK cells have better safety profiles, minimal cytokine release, and fewer graft-versus-host diseases. However, designing unique CAR-NK cells targeting each potential cancer antigen is a cumbersome and time-consuming process. Therefore, there is an urgent need in the art to improve adoptive immunotherapy for cancer treatment. Summary of the Invention

[0004] This disclosure is partly based on the finding that co-administration of a cancer antigen-binding molecule (e.g., a cancer antigen-specific antibody) and NK cells expressing a CAR containing a specific binding domain against the cancer antigen-binding molecule induces cytotoxicity in tumor cells expressing that cancer antigen. Therefore, in some embodiments, this document provides CAR-NK cells that can be used as readily available therapeutic agents and can target a variety of different cancers by co-administering a cancer antigen-specific antibody.

[0005] Therefore, in some respects, this article provides a chimeric antigen receptor (CAR) polypeptide comprising: (a) an extracellular domain comprising: (i) a CD3 extracellular domain or a fragment thereof; (ii) a unique antigen-binding domain specific to an anti-CD3 antibody; or (iii) an antigen-binding domain specific to an Fc domain; (b) a hinge domain; (c) a transmembrane domain; and (d) an intracellular signal transduction domain.

[0006] In some embodiments, the extracellular domain includes a CD3 extracellular domain or a fragment thereof. In some embodiments, the CD3 extracellular domain or a fragment thereof contains 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 CD3 extracellular domain or a fragment thereof contains at least 10 consecutive amino acids of SEQ ID NO: 1959. In some embodiments, the CD3 extracellular domain or a fragment thereof contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 1959. In some embodiments, the CD3 extracellular domain or a fragment thereof contains the amino acid sequence of SEQ ID NO: 1959.

[0007] In some embodiments, the extracellular domain comprises a uniquely type-specific antigen-binding domain against the 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 heavy and light chain CDR sequences of the 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 the scFv listed in Table 1.

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

[0009] In some implementations, the antigen-binding domain is a single-stranded 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 NO: 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 NO: 6-13. In some embodiments, intracellular signal transduction includes any combination of an FcgR1 intracellular signal transduction domain, a CD3z intracellular signal transduction domain, a 4-1BB intracellular signal transduction domain, a 2B4 intracellular signal transduction domain, a CD16 intracellular signal transduction domain, a CD64 intracellular signal transduction domain, or a CD28 intracellular signal transduction domain. In some implementations, the intracellular signal transduction domain is the FcgR1 intracellular signal transduction domain, the 4-1BB-CD3z intracellular signal transduction domain, the 2B4-CD3z intracellular signal transduction domain, the CD16 intracellular signal transduction domain, the CD64 intracellular signal transduction domain, or the CD28-CD3z intracellular signal transduction domain.

[0011] In some embodiments, this document provides vectors comprising the nucleic acids described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0012] In some aspects, this document provides natural killer (NK) cells comprising the nucleic acids described herein. In some aspects, this document provides natural killer (NK) cells expressing the CAR peptides described herein. In some embodiments, the cells are primary NK cells or inducible NK cells differentiated from induced pluripotent stem cells (iPSCs). In some aspects, this document provides immune cells (e.g., phagocytes) comprising the nucleic acids described herein or expressing the CAR peptides described herein.

[0013] In some embodiments, this article provides a method for treating cancer in a subject, the method comprising administering to the subject in combination: (A) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain; and (B) a multispecific antigen-binding molecule comprising a first antigen-binding domain binding to a tumor antigen and a second antigen-binding domain binding to an extracellular domain. In some embodiments, the method comprises administering to the subject in combination: (A) natural killer (NK) cells expressing a CAR peptide 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 specifically binding to a CD3 extracellular domain or a fragment thereof and a tumor antigen-binding domain specifically binding to a tumor antigen.

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

[0015] In some aspects, this article provides a method for treating cancer in a subject, the method comprising co-administering to the subject: (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 peptide, said CAR peptide including an extracellular domain that binds to the Fc domain. In some embodiments, the method comprises co-administering to the subject: (A) a multispecific antigen-binding molecule including 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 peptide including an extracellular domain that includes an Fc domain-specific antigen-binding domain, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule.

[0016] In some embodiments, antigen-binding molecules (e.g., multispecific antigen-binding molecules) and NK cells are administered simultaneously or sequentially. In some embodiments, antigen-binding molecules (e.g., multispecific antigen-binding molecules) and NK cells are pre-mixed and administered simultaneously to the subject. In some embodiments, the subject is a patient with lymphopenia, and antigen-binding molecules (e.g., multispecific antigen-binding molecules) and NK cells are pre-mixed and administered simultaneously to the subject. In some embodiments, NK cells are administered after at least one dose of antigen-binding molecules (e.g., multispecific antigen-binding molecules) or after pre-mixing NK cells and antigen-binding molecules (e.g., pre-mixing NK cells and multispecific antigen-binding molecules).

[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) comprises a multispecific antibody or an antigen-binding fragment thereof. In some embodiments, the multispecific antibody or its antigen-binding fragment is chimeric, humanized, or human.

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

[0019] In some respects, this article provides pharmaceutical compositions comprising: (A) natural killer (NK) cells expressing a CAR polypeptide, said 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 specifically binding to a CD3 extracellular domain or a fragment thereof and a tumor antigen-binding domain specifically binding to a tumor antigen.

[0020] In some respects, this document provides pharmaceutical compositions comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3 and a tumor antigen-binding domain specifically binding to a tumor antigen; and (B) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising a idiotype-specific antigen-binding domain of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR peptide binds the idiotype of the CD3-binding domain of the multispecific antigen-binding molecule.

[0021] In some respects, this article provides pharmaceutical compositions comprising: (A) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding CD3, a tumor antigen-binding domain specifically binding a tumor antigen, and an Fc domain; and (B) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising an Fc domain and a specific antigen-binding domain, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule.

[0022] In some respects, this article provides a cell bank containing NK cells expressing the CAR described herein. Attached Figure Description

[0023] Figure 1A-1B Jurkat / NFAT-Luc cl. 3C7 cells were shown. Figure 1A ) or Jurkat / NFAT-Luc / ahFc-CD28-CD3 cells ( Figure 1B NFAT activity was assessed. Two cell lines were incubated together with titrated isotype controls (REGN1932, gray squares) or single-arm anti-CD20 antibody (REGN2959, black) and target cells lacking CD20 expression (Jurkat, hollow symbol / dashed line) or positive for CD20 expression (Ramos.2G6.4C10, solid symbol / solid line). NFAT activity was assessed by luminescent readings after 5 hours.

[0024] Figure 2The cytotoxicity of KHYG / ahFc-CD28-CD3 cells was demonstrated. KHYG / ahFc-CD28-CD3 cells were incubated with either a titrated isotype control (REGN1932, gray square, gray dashed line) or a single-arm anti-CD20 antibody (REGN2959, black circle, black solid line) in the presence of a fixed amount of Ramos / GFP target cells. Tag release was detected using an extracellular detection system after 4 hours.

[0025] Figure 3 The sequence alignment between the stealth* constant regions of hIgG3 and IgG4 is shown. hIgG3, REGN2280, and REGN7075 have 100% sequence identity in the CH3 region containing the asterisk mutation: FSCSVMHEALHN RF TQKSLSLSPGK (SEQ ID NO: 14). SEQ ID NOs 91-94 are disclosed in the figure in the order of appearance.

[0026] Figure 4 shows a single-chain variable fragment (scFv) derived from an anti-human CD3 idiotype monoclonal antibody (mAb) that blocks the binding of anti-hCD3 mAb to immobilized hCD3 ε / δ (ε / δ heterodimer). Figure 4A and 4B show 1 PN29950_ 2 HCLC (hollow circle) and PN29950 2 LCHC (hollow cube) blocked 20.0 pM REGN18409 ( Figure 4A Anti-hCD3, 7221G) or REGN18411 ( Figure 4B The binding of anti-hCD3 (7221G20) to immobilized hCD3 ε / δ. Figure 4C The diagram shows the binding of PN77570_HCLC (hollow circle) and PN77570_LCHC (hollow square) to immobilized hCD3 ε / δ at 20.0 pM. The inset diagram shows REGN18409 (…). Figure 4A REGN18411 ( Figure 4B ) and REGN2533 ( Figure 4C (Solid inverted triangle) and immobilized hCD3 ε / δ dose-dependent binding, EC 50 The corresponding values ​​were 7.5 pM, 11.3 pM, and 8.1 pM. Parental bivalent mAb REGN5766 (solid circle) blocked 20.0 pM. REGN18409 (…). Figure 4A ) or REGN18411 ( Figure 4B The binding of ) with hCD3 ε / δ, IC 50 The values ​​were 42.0 pM and 40.0 pM, respectively. Figure 4C The results showed that the parental divalent mAb REGN2984 (solid circle) blocked the binding of REGN2533 to hCD3 ε / δ at 20.0 pM, IC50. 50 The value was 32.0 pM. Isotype control mIgG1 ( Figure 4A and Figure 4B ) and mIgG2a ( Figure 4C (Southern Biotech #0102-01 and 0103-01, respectively; solid triangle) and negative scFv control ( Figure 4A , Figure 4B and Figure 4C (REGN4393; solid cube) showed no inhibition under the same measurement conditions. The X-axis of the inset plot represents Log. 10 Molar concentration; the X-axis of the external plot represents the dilution factor (derived from Log). 10 (Dilution factor). The Y-axis of all graphs shows the absorbance at 450 nm. 1 PN indicates the root protein number. 2 HCLC or 2 LCHC corresponds to the heavy chain, joint, and light chain orientation of scFv.

[0027] Figure 5 The antibody was shown to bind to KHYG-1 / NFAT-Luc / CAR1 cells.

[0028] Figure 6 The antibody was shown to bind to KHYG-1 / NFAT-Luc / CAR6 and CAR15 cells.

[0029] Figure 7 The activation of reporter genes in KHYG-1 / NFAT-Luc / CAR1 cells was demonstrated.

[0030] Figure 8 The activation of reporter genes in KHYG-1 / NFAT-Luc / CAR6 and CAR15 cells was shown.

[0031] Figure 9 The study demonstrated cytotoxic activation of KHYG-1 / NFAT-Luc / CAR1 cells.

[0032] Figure 10 The study demonstrated cytotoxic activation in KHYG-1 / NFAT-Luc / CAR6 and CAR15 cells.

[0033] Figure 11The cytotoxicity and cytokine release of CBNK / CAR6 cells were demonstrated. Detailed Implementation

[0034] Overview This article provides methods and compositions for treating cancer. As disclosed herein, administration of NK cells expressing a CAR in combination with a bispecific antibody that binds CD3 and a tumor antigen (TAA) induces cytotoxicity in tumor cells expressing a specific tumor antigen, wherein the CAR comprises a unique type-specific antigen-binding domain against a CD3 antibody, or an antigen-binding domain specific to an Fc domain.

[0035] Therefore, in some respects, this paper provides a chimeric immune receptor (CAR) comprising an extracellular domain including a CD3 extracellular domain, a unique antigen-binding domain specific to anti-CD3 antibodies, or an antigen-binding domain specific to an Fc domain.

[0036] In some respects, this article provides a method for treating cancer using NK cells expressing the CAR described herein (e.g., inducible NK cells) in combination with antigen-binding molecules that bind to tumor antigens, wherein the CAR-NK cells bind to the antigen-binding molecules, and then the antigen-binding molecules target cancer cells expressing tumor antigens to induce antitumor activity (e.g., cytotoxicity).

[0037] In some implementations, the method may include administering to a subject in combination: (A) natural killer (NK) cells expressing a CAR polypeptide containing an extracellular domain (e.g., the CD3 extracellular domain or a fragment thereof); and (B) a multispecific antigen-binding molecule containing a first antigen-binding domain that binds to a tumor antigen and a second antigen-binding domain that binds to an extracellular domain (e.g., the CD3 extracellular domain or a fragment thereof).

[0038] In some implementations, the method may include administering to a subject in combination: (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 peptide comprising an extracellular domain that binds to the antigen-binding molecule (e.g., an extracellular domain comprising an antigen-binding domain that is specific to the idiotype of the CD3 multispecific antigen-binding molecule).

[0039] In some implementations, the method may include administering to a subject in combination: (a) an antigen-binding molecule that binds to a tumor antigen and contains an Fc domain (e.g., a multispecific antigen-binding molecule containing 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 that express a CAR peptide containing an extracellular domain that binds to an Fc domain.

[0040] In some aspects, this document provides pharmaceutical compositions comprising the CAR-NK cells and the antigen-binding molecules described herein, wherein the CAR-NK cells bind to the antigen-binding molecules. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] Compared to CAR-T cells, CAR-NK cells offer better safety, minimal cytokine release, and fewer graft-versus-host diseases. Instead of modifying NK cells each time to express a CAR specific to a particular tumor antigen, the CAR-NK cells disclosed herein can be used as off-the-shelf “universal” CAR-NK cells, which can be combined with a variety of tumor antigen-binding molecules to target different types of tumors. For example, CAR-expressing NK cells can be used in combination with a variety of CD3 bispecific antibodies known in the art, said CAR containing a CD3 extracellular domain or a unique antigen-binding domain specific to anti-CD3 antibodies. CAR-expressing NK cells can be used in combination with a variety of CD3 bispecific antibodies with an Fc domain or any other antibody that binds to tumor antigens and contains an Fc domain, said CAR containing an antigen-binding domain specific to the Fc domain.

[0042] definition For convenience, certain terms used in the specification, embodiments and appended claims are collected herein.

[0043] As used herein, the term “about” when used in conjunction with a particular referenced numerical value means that the value may differ from the referenced value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0044] The article “a / an” is used in this text to refer to one or more (i.e., at least one) grammatical object of the article. For example, “a / an element” means one or more elements.

[0045] As used herein, “administering / administration” means providing a drug or composition to a subject, including but not limited to administration by a medical professional or self-administration. Such agents may contain, for example, CAR T cells as described herein.

[0046] As used herein, the term "antibody" can refer to both the complete antibody and its antigen-binding fragment. A complete antibody is a glycoprotein consisting of at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as V in this document). H The light chain contains a constant region (V) and a variable region (V) for the heavy chain. L ) and the constant region of the light chain. V H and V L The region can be further subdivided into highly variable regions known as complementary determinant regions (CDRs), interspersed with more conservative regions known as framing regions (FRs). Each V H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "antibody" includes, 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.

[0047] As used herein, the terms “antigen-binding fragment” and “antigen-binding moiety” of an antibody refer to one or more fragments of an antibody that retain the ability to bind antigens. 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 region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the term “antigen-binding fragment” as used herein.

[0048] In a broad sense, "cancer" refers to the uncontrolled, abnormal growth of the host's own cells, leading to their invasion of surrounding tissues and potentially tissues distal to the initial site of abnormal cell growth within the host. Major categories 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); leukemias, which are cancers of hematopoietic tissues (e.g., bone marrow); lymphomas and myelomas, which are cancers of immune cells; and cancers of the central nervous system, including brain and spinal cord tissues. The terms "cancer" and "vesicle" are used interchangeably in this article. As used herein, "cancer" refers to all types of cancer or vesicles or malignant tumors, including new or recurrent leukemia, carcinomas, and sarcomas. Specific examples of cancer include carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Non-limiting examples of cancer include newly diagnosed or recurrent cancers of the brain, melanoma, bladder, breast, cervical, colon, head and neck, kidney, lung, non-small cell lung cancer, mesothelioma, ovarian, prostate, sarcoma, stomach, uterine, and medulloblastoma. In some implementations, cancer includes solid tumors. In some implementations, cancer includes metastases.

[0049] 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., antibody-based specificity for a desired antigen, such as a tumor antigen) with an intracellular domain that activates a T-cell receptor to produce a chimeric protein exhibiting specific anti-target cell immune activity. Generally, a CAR is formed by fusing an extracellular single-chain antigen-binding domain (scFv) with an intracellular signaling domain of the ζ chain of the T-cell antigen receptor complex, and when expressed in T cells, it can redirect antigen recognition based on the specificity of a monoclonal antibody.

[0050] As used herein, the phrase "combined administration" or "combined administration" refers to the administration of two or more different therapeutic agents in any form, such that the second agent is administered while the previously administered agent remains effective in the body (e.g., both agents are effective simultaneously in the subject, which may include a synergistic effect between the two agents). For example, the different therapeutic agents may be administered simultaneously or sequentially, either in the same formulation or as separate formulations. In some embodiments, the different therapeutic agents may be administered within approximately one hour, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 72 hours, or approximately one week of each other. Therefore, a subject receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0051] A “co-stimulatory domain” or “co-stimulatory molecule” refers to a homologous binding partner on immune cells (such as B cells, T cells, NK cells, or bone marrow cells) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in the cell, such as, but not limited to, proliferation. The co-stimulatory domain can be a human co-stimulatory domain. Exemplary co-stimulatory molecules include CD28, CD27, 4-1BB (CD137), OX40, CD30, CD40, ICOS, CD2, LIGHT, CD244 (2B4), and NKG2C.

[0052] "Costimulatory ligands" refer to molecules on antigen-presenting cells that specifically bind to relevant costimulatory molecules on immune cells (e.g., B cells, T cells, NK cells, or bone marrow cells), thereby providing signals that mediate immune cell responses, including but not limited to proliferation activation and differentiation. Costimulatory ligands may 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.

[0053] "Co-stimulatory signals" refer to signals that, in combination with the initial signal, lead to the proliferation of immune cells (e.g., B cells, T cells, NK cells, or bone marrow cells) and / or the upregulation or downregulation of key molecules.

[0054] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions of the antigen and may have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposing amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated from adjacent amino acid residues in a polypeptide chain. In some cases, epitopes may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0055] A "gene construct" refers to a nucleic acid containing a polypeptide "coding sequence" or a nucleic acid that can be transcribed into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), such as a vector, plasmid, or viral genome, which can be transfected into cells (e.g., mammalian cells) and induce the expression of the coding sequence in cells transfected with the construct. A gene construct may include one or more regulatory elements operatively linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0056] The terms “ligand-binding domain” and “antigen-binding domain” are used interchangeably in this document and refer to the portion of a chimeric antigen receptor that specifically binds to a predetermined antigen.

[0057] The term "connector" is recognized in the art and refers to a molecule or group of molecules that connects two compounds (e.g., two polypeptides). A connector may consist of a single linker molecule or may include linker molecules and spacer molecules designed to separate the linker molecule and the compound at a specific distance.

[0058] The term "operably linked" refers to the functional relationship between one nucleic acid sequence and another. Promoters, enhancers, transcription and translation termination sites, and other signaling sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, the operably linked DNA to a transcriptional control element refers to the physical and functional relationship between DNA and a promoter, such that transcription of this DNA is initiated by the promoter through an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0059] As used herein, the phrase “pharmaceutically acceptable” means those agents, compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the limits of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0060] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting an agent from one organ or part of the body to another. Each carrier must be “acceptable” in the sense of compatibility with other components of the formulation and harmless to the patient. Some examples of materials that can serve 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; and (9) oils, such as peanut oil and cottonseed oil. (10) Safflower oil, sesame oil, olive oil, corn oil and soybean oil; (11) Diols, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Atherless water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) pH buffer solution; (22) Polyesters, polycarbonates and / or polyanhydrides; and (23) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0061] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to natural or synthetic molecules, or combinations thereof, containing a single nucleotide or two or more nucleotides linked to the 5' end of another nucleotide by a phosphate group at the 3' position of one nucleotide. The polymerized form of nucleotides is not limited by length and can include deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, loci (loci / locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with any sequence, isolated RNA with any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. Polynucleotides can be further modified, such as by conjugation with labeled components. In all the nucleic acid sequences provided in this article, U nucleotides are interchangeable with T nucleotides. Polynucleotides are not necessarily associated with the cells in which the nucleic acid is found in nature, and / or operatively linked to polynucleotides in nature to which they are linked.

[0062] As used in this article, a "preventive" treatment agent for a disease refers to a compound that, when administered to a statistical sample prior to the onset of a disease or illness, reduces the incidence of the disease or illness in a treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disease or illness or reduces its severity relative to an untreated control sample.

[0063] The “signal transduction domain” or “signaling domain” of the CAR used in this article is responsible for intracellular signal transduction following the binding of the extracellular ligand-binding domain to the target, leading to the activation of immune cells and the immune response. In other words, the signal transduction domain is responsible for activating at least one normal effector function of immune cells expressing the CAR. For example, the effector function of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term “signal transduction domain” refers to the part of a protein that transduces effector function signals and directs the cell to perform a specific function. Examples of signal transduction domains used for CARs can be cytoplasmic sequences of T cell receptors and co-receptors that work together to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. In some cases, the signal transduction domain contains two distinct classes of cytoplasmic signaling sequences, one initiating antigen-dependent primary activation and the other providing secondary or co-stimulatory signals in an antigen-independent manner. The primary cytoplasmic signaling sequence may contain a signaling motif, referred to as the tyrosine-based activation motif of the immune receptor for ITAM. ITAMs are well-defined signal transduction motifs located in the cytoplasmic tails of various receptors and can serve as binding sites for syk / zap70-type tyrosine kinases. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD28, 4-1BB, CD79a, CD79b, and CD66d.

[0064] As used in this article, "spacer" refers to a peptide that connects proteins (such as proteins in fusion proteins). Generally, spacers have no specific biological activity other than connecting proteins or maintaining a minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer can be chosen to affect certain properties of the molecule, such as folding, net charge, or hydrophobicity.

[0065] The term "substantially identical" or "substantially identical" when referring to a nucleic acid or fragment thereof means that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as measured by any known sequence identity algorithm such as FASTA, BLAST, or Gap, at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases have nucleotide sequence identity, as discussed below. In some cases, a nucleic acid molecule substantially identical to a reference nucleic acid molecule may encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0066] When applied to peptides, the terms "substantially similar" or "substantially alike" mean that, when best aligned using default gap weights, such as through procedures like GAP or BESTFIT, two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity. Preferably, the different residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue whose side chain (R group) has similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity or similarity can be increased to correct for the conservation of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with similar chemical properties in their side chains 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: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0067] As used herein, the term "specific binding" or "specific binding" when referring to a polypeptide refers to a binding reaction that identifies the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological agents. Therefore, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its specific "target" (e.g., antibody specific binding to endothelial antigens) when it does not bind extensively to other proteins present in the sample or to other proteins that the ligand or antibody might encounter in the organism. Typically, the affinity constant (Ka) between the first and second molecules for "specific binding" to a second molecule is greater than approximately 10. 5 M –1 (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 (or greater). For example, in terms of the ability of a PIG-specific CAR to bind peptides presented on an MHC (e.g., class I or class II MHC); typically, the CAR specifically binds its peptide / MHC with an affinity of at least about 10⁻⁴ M or less, and binds the intended antigen / binding partner with an affinity (in KD) of at least 1 / 10, at least 1 / 100, or at least 1 / 1000 that binds to non-specific and unrelated peptide / MHC complexes (e.g., complexes containing BSA peptides or casein peptides).

[0068] As used in this article, "subject" means a human or non-human animal selected for treatment or therapy.

[0069] As used herein, the term "treatment" refers to a clinical intervention in clinicopathology aimed at altering the natural course of a disease in an individual being treated. The desired effects of treatment include slowing the rate of progression, improving or alleviating the pathological condition, and mitigating or improving the prognosis of a particular disease, symptom, or disorder. For example, if one or more symptoms associated with a particular disease, symptom, or disorder are reduced or eliminated, the individual is considered to have been successfully "treated."

[0070] The term "variant" refers to an amino acid or peptide sequence that has conserved amino acid substitutions, non-conserved amino acid substitutions (e.g., degenerate variants), substitutions within the wobble position of each codon encoding an amino acid (e.g., DNA and RNA), amino acids added to the C-terminus of a peptide, or a peptide that has 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence.

[0071] The term "vector" refers to a means by which nucleic acids can be replicated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phage particles, transposons, and artificial chromosomes, to which nucleic acids are attached and which may or may not autonomously replicate or integrate into the host cell's chromosome. Such vectors can include any vector (e.g., plasmids, kinasemids, or phage chromosomes) containing gene constructs (e.g., those linked to transcriptional control elements) in a form suitable for cellular expression.

[0072] Chimeric antigen receptor (CAR) A chimeric antigen receptor (CAR) is a receptor comprising a targeting moiety associated with one or more signaling domains and / or co-stimulatory domains in a single fusion molecule. In some aspects, the binding moiety of the CAR comprises an extracellular domain including: (i) a CD3 extracellular domain or a fragment thereof; (ii) a idiotype-specific antigen-binding domain against an anti-CD3 antibody; or (iii) an antigen-binding domain specific to an Fc domain. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising a light and heavy chain variable fragment of a monoclonal antibody linked by a flexible linker. In some embodiments, the CAR also comprises a transmembrane domain, a transmembrane domain, and an intracellular signaling domain.

[0073] CD3 extracellular domain In some implementations, this document provides a CAR that includes an extracellular domain, said extracellular domain comprising the CD3 extracellular domain or a fragment thereof.

[0074] As used in this article, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR), which consists of homodimers or heterodimers formed by the binding of two of the four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-ε comprises the amino acid sequence shown in SEQ ID NO:116 of U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-δ comprises the amino acid sequence shown in SEQ ID NO:117 of U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-ζ comprises the amino acid sequence shown in SEQ ID NO:118 of U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; and CD3-γ comprises the amino acid sequence shown in SEQ ID NO:119 of U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. Unless expressly specified as originating from a non-human species, all references to proteins, peptides, and protein fragments herein are intended to refer to the respective human-type proteins, peptides, or protein fragments. Therefore, unless specified as originating from a non-human species, such as "mouse CD3" or "monkey CD3", the expression "CD3" refers to human CD3.

[0075] As used herein, the term "cell surface expressed CD3" refers to one or more CD3 proteins that are expressed on the cell surface in vitro or in vivo, such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. Cell surface expressed CD3 includes CD3 proteins contained within the cell membrane in the case of functional T cell receptors. Cell surface expressed CD3 includes CD3 proteins expressed as part of homodimers or heterodimers (e.g., γ / ε, δ / ε, and ξ / ξ CD3 dimers) on the cell surface. Cell surface expressed CD3 also includes CD3 chains (e.g., CD3-ε, CD3-δ, or CD3-γ) that are spontaneously expressed on the cell surface (without other CD3 chain types). Cell surface expressed CD3 may include or consist of CD3 proteins expressed on the surface of cells that normally express CD3 proteins. Alternatively, CD3 expressed on the cell surface may comprise or consist of CD3 protein expressed on the cell surface, or the cell may not normally express human CD3 on its surface, but may be engineered to express CD3 on its surface.

[0076] In some embodiments, the CD3 extracellular domain or a fragment thereof is the human CD3 extracellular domain or a fragment thereof, such as the human CD3ε extracellular domain or a fragment thereof, the human CD3δ extracellular domain or a fragment thereof, the human CD3γ extracellular domain or a fragment thereof, or the human CD3ζ extracellular domain or a fragment thereof. In some embodiments, the extracellular domain of human CD3ε is shown as SEQ ID NO: 33, the extracellular domain of human CD3δ is shown as SEQ ID NO: 34, and the extracellular domain of human CD3γ is shown as SEQ ID NO: 35 of WO 2016 / 085889, which is incorporated herein by reference in its entirety.

[0077] In some embodiments, the CD3 extracellular domain 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 WO 2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below). In some embodiments, the CD3 extracellular domain or a fragment thereof comprises an amino acid sequence having 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% identity with SEQ ID NO: 33 of WO 2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below). In some embodiments, the CD3 extracellular domain or a fragment thereof contains the amino acid sequence of SEQ ID NO: 33 of WO 2016 / 085889 (i.e., SEQ ID NO: 1959 shown in Table 22 below).

[0078] Table 22: Examples of CD3 extracellular domains GVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSV (SEQ ID NO: 1959) In some embodiments, the CD3 extracellular domain 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 consecutive amino acids of SEQ ID NO: 34 of WO 2016 / 085889. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises an amino acid sequence having 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% identity with SEQ ID NO: 34 of WO 2016 / 085889. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 34 of WO 2016 / 085889.

[0079] In some embodiments, the CD3 extracellular domain 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 consecutive amino acids of SEQ ID NO: 35 of WO 2016 / 085889. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises an amino acid sequence having 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% identity with SEQ ID NO: 35 of WO 2016 / 085889. In some embodiments, the CD3 extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 35 of WO 2016 / 085889.

[0080] In some embodiments, the extracellular domain of CD3 or a fragment thereof contains an epitope recognized by the anti-CD3 antibody. As used herein, "antibody that binds CD3" or "anti-CD3 antibody" includes antibodies that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ) and their antigen-binding fragments, as well as antibodies that specifically recognize dimer complexes of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) and their antigen-binding fragments. The antibodies and antigen-binding fragments disclosed herein can bind soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants, such as monomeric and dimer CD3 constructs, which lack a transmembrane domain or do not bind to the cell membrane. In some embodiments, the anti-CD3 antibody is selected from the anti-CD3 antibodies listed in Table 6.

[0081] Unique types of anti-CD3 antibodies have specific antigen-binding domains. In some embodiments, the binding domains and / or extracellular domains of the CARs provided herein provide the CAR with the ability to bind to the target antigen of interest. The binding domain (e.g., a ligand-binding domain or an antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide with the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or tumor protein, or a component thereof). The binding domain includes binding partners of any naturally occurring, synthetic, semi-synthetic, or recombinantly generated biomolecule of interest. For example, as further described herein, the binding domain can be a variable region of the antibody light and heavy chains, or the light and heavy chain variable regions can be in single-chain form in either orientation (e.g., V...). L -V H or V H -V LThe components are linked together. Various assays are known for identifying the binding domains of this disclosure that specifically bind to a particular target, including Western blotting, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). Exemplary methods for generating anti-idiotype antibodies are described in Example 1 of U.S. Patent Nos. 10,150,817 B2 and WO 2017 / 162587 A1, each of which is incorporated herein by reference in its entirety.

[0082] In some embodiments, the binding domain and / or extracellular domain of the CAR provided herein comprise a unique antigen-binding domain specific to the 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 anti-CD3 antibody is the anti-CD3 antibody designated as CH2527 in WO 2017 / 162587 A1. For example, in some embodiments, the anti-CD3 antibody comprises the CDR H1, CDR H2, and CDR H3 sequences disclosed in WO 2017 / 162587, SEQ ID NOs:11, 12, and 13, respectively, which are incorporated herein by reference in their entirety. In some embodiments, the anti-CD3 antibody comprises the CDRH1, CDR H2, and CDR H3 sequences disclosed in WO 2017 / 162587, SEQ ID NOs:44, 45, and 46, which are incorporated herein by reference in their entirety. In some implementations, the anti-CD3 antibody comprises the variable heavy chain (VH) sequence of SEQ ID NO: 43 disclosed in WO 2017 / 162587, which is incorporated herein by reference in its entirety.

[0083] In some embodiments, the antigen-binding domain described herein is a idiotype-specific single-chain variable fragment (scFv) against CD3 antibodies, and may be mouse, human, or humanized scFv. In some embodiments, the idiotype-specific single-chain variable fragment (scFv) against CD3 antibodies is the scFv specified as 4.15.64 or 4.32.63 disclosed in WO 2017 / 162587, which is incorporated herein by reference in its entirety.

[0084] In some embodiments, the antigen-binding domain comprises the heavy chain and / or light chain CDR sequences of the scFv listed in Table 1. In some embodiments, the antigen-binding domain comprises the heavy chain and / or light chain variable region sequences of the scFv listed in Table 1. In some embodiments, the heavy chain variable region disclosed herein may contain 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 region sequences of the scFv listed in Table 1. In some embodiments, the light chain variable region disclosed herein may contain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 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 region sequences of the scFv listed in Table 1. In some embodiments, the antigen-binding domain comprises the amino acid sequence of the scFv listed in Table 1. In some embodiments, the scFv described herein may contain 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 scFv sequences listed in Table 1.

[0085] Table 1: Examples of unique antigen-binding domains specific to anti-CD3 antibodies In some embodiments, this document provides a chimeric antigen receptor (CAR) peptide comprising (a) an extracellular domain containing a uniquely type-specific antigen-binding domain of an anti-CD28 antibody; (b) a hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. This CAR can be used in combination with anti-CD28 x TAA to target tumor cells expressing TAA.

[0086] Antigen-binding domains specific to the Fc domain In some embodiments, the extracellular domain includes an antigen-binding domain specific to the Fc domain. In some embodiments, the Fc domain is selected from the human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, and human IgG4 Fc domain. In some embodiments, the Fc domain is the IgG3 Fc domain. In some embodiments, the Fc domain includes... Figure 3 The amino acid sequence of Fc is shown.

[0087] In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the single-chain variable fragment (scFv) specific to the idiotype or Fc domain of an anti-CD3 antibody can be a mouse, human, or humanized scFv. Single-chain antibodies can be cloned from the V region gene of a hybridoma targeting a specific target. Techniques that can be used to clone the variable region heavy chain (VH) and variable region light chain (VL) have been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. Therefore, in some embodiments, the binding domain comprises an antibody-derived binding domain, but can 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 that participates in binding to the antigen.

[0088] In some embodiments, the CAR of this disclosure may include a linker between the various domains, the linker being added to obtain appropriate molecular spacing and conformation. For example, in one embodiment, the linker length between the binding domains VH or VL may be 1-10 amino acids. In other embodiments, the linker length between any domains of the chimeric antigen receptor may be 1-20 or 20 amino acids. In this regard, the linker length 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 a further embodiment, the linker length may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The range including the numbers described herein is also included herein, for example, a linker with a length of 10-30 amino acids.

[0089] In some embodiments, the connector suitable for the CAR described herein is a flexible connector. Suitable connectors can be readily selected and can have any suitable different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0090] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively structureless and therefore can serve as neutral linkers between domains of fusion proteins (such as the CARs described herein). Glycine has more access to the phi-psi space than alanine and is much less restricted than residues with longer side chains. Those skilled in the art will recognize that CAR designs can include wholly or partially flexible linkers, such that the linker can include a flexible linker and one or more portions imparting a lower flexibility structure to provide the desired CAR structure.

[0091] Hinge domain, transmembrane domain and intracellular signal transduction domain The binding domain of a CAR may be followed by a "spacer region" or "hinge," which refers to the region that removes the extracellular domain or binding domain (e.g., the CD3 extracellular domain, or a unique type of anti-CD3 antibody or an antigen-binding domain specific to the Fc domain described herein) from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in a CAR is typically located between the transmembrane (TM) region and the extracellular domain or binding domain (e.g., the CD3 extracellular domain, or a unique type of anti-CD3 antibody or an antigen-binding domain specific to the Fc domain described herein). In some embodiments, the hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CAR 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 derived from these molecules or may be altered.

[0092] In some embodiments, the CAR described herein further comprises a hinge domain. The hinge domain may be selected from the hinge domains listed in Table 2 below. In some embodiments, the hinge domain is a CD28 or CD8 hinge domain. In some embodiments, the CAR further comprises an amino acid sequence having 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% identity with the amino acid sequences listed in Table 2. In a specific embodiment, the hinge domain comprises an amino acid sequence selected from SEQ ID NO: 1-5.

[0093] Table 2: Examples of hinge structural domains In some embodiments, the CAR described herein also includes a transmembrane domain. The “transmembrane” region or domain is part of the CAR that anchors the extracellular binding portion to the plasma membrane of immune effector cells and facilitates the binding of the extracellular domain or binding domain (e.g., the CD3 extracellular domain or an antigen-binding domain targeting an anti-CD3 antibody idiotype or the Fc domain described herein) to its binding partner. In some embodiments, the transmembrane domain may be the CD28 transmembrane domain. Other transmembrane domains that may be used in some embodiments include transmembrane domains obtained from CD8, CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is synthetic, in which case it primarily contains hydrophobic residues such as leucine and valine.

[0094] 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 listed in Table 3 below. In some embodiments, the transmembrane domain is the NKG2D transmembrane domain, the NKG2D reverse transmembrane domain, the CD28 transmembrane domain, the CD8 transmembrane domain, the CD16 transmembrane domain, or the FcgR1 (CD64) transmembrane domain. In some embodiments, the CAR further comprises a transmembrane domain containing an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences listed in Table 3. In a specific embodiment, the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 6-13.

[0095] Table 3: Examples of transmembrane domains In some embodiments, the CARs provided herein also include 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 participates in transducing information about effective CAR binding to target antigens into immune effector cells to trigger effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses triggered by antigen binding to the extracellular CAR domain.

[0096] In some embodiments, the CARs provided herein comprise one or more immune receptor tyrosine-based activation motifs or ITAMs. Examples of available ITAMs containing major cytoplasmic signaling sequences include sequences derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD28, 4-1BB, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain of the CAR described herein is derived from CD3ζ.

[0097] In some embodiments, the CARs provided herein also include a co-stimulatory domain. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that, upon binding to an antigen, provides a second signal required for the effective activation and function of immune cells (e.g., T lymphocytes or NK cells). Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), CD2, LIGHT, and NKD2C. Thus, while this disclosure provides an exemplary co-stimulatory domain derived from CD28, including one or more co-stimulatory signaling domains can enhance the efficacy and expansion of T cells expressing CAR receptors. CAR peptides are also disclosed herein, wherein the intracytoplasmic / co-stimulatory region of the CAR peptide also includes a 4-1BB domain (e.g., in addition to the CD28 / ζ domain). The co-stimulatory region of such CAR peptides may include the complete 4-1BB domain or a fragment thereof, and / or the complete CD28 / ζ domain or a fragment thereof. Intracellular signal transduction and co-stimulatory signal transduction domains can be tandemly connected to the carboxyl terminus of transmembrane domains in any order.

[0098] In some implementations, the intracellular signal transduction domain is the FcgR1 intracellular signal transduction domain, the 4-1BB-CD3z intracellular signal transduction domain, the 2B4-CD3z intracellular signal transduction domain, the CD16 intracellular signal transduction domain, the CD64 intracellular signal transduction domain, or the CD28-CD3z intracellular signal transduction domain.

[0099] In some respects, the CAR peptides provided herein comprise at least one intracytoplasmic / co-stimulatory region containing a differentiation cluster 28ζ (CD28 / ζ) domain. Additionally, the hinge / spacer region and / or transmembrane region of the CAR, or the transmembrane region of the CAR, may contain a CD28 / ζ domain. The CAR may comprise at least one differentiation cluster 28ζ (CD28 / ζ) amino acid sequence selected from the amino acid sequences shown in SEQ ID NO. 5, 6, and 22. The CAR peptides disclosed herein may comprise at least one differentiation cluster 28ζ (CD28 / ζ) amino acid sequence having 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 amino acid sequences shown in SEQ ID NO. 5, 6, and 22. The CAR peptide may comprise all three sequences shown in SEQ ID NO. 5, 6, and 22. For example, the CAR hinge domain may include SEQ ID NO: 5, the transmembrane domain may include SEQ ID NO: 6, and the co-stimulatory domain may include SEQ ID NO: 22.

[0100] Table 4: Exemplary CD28 / ζ sequences. The CAR peptide sequences disclosed herein may comprise any one of the amino acid sequences shown in SEQ ID NO: 26 to 28. The CAR peptides disclosed herein may comprise amino acid sequences having 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 amino acid sequences shown in SEQ ID NO: 26 to 28.

[0101] Table 5: Additional exemplary CAR sequences: Nucleic acid and vector In some respects, nucleic acid and polynucleotide vectors encoding the CAR peptides disclosed herein are also disclosed.

[0102] The nucleic acid sequences encoding the disclosed CAR and its regions can be obtained using recombination methods known in the art, such as by screening libraries from cells expressing the gene, obtaining the gene from vectors known to contain the gene, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be generated synthetically rather than by cloning.

[0103] Expression of CAR-encoding nucleic acids is typically achieved by operatively linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter to regulate the expression of the desired nucleic acid sequence.

[0104] In some embodiments, a polynucleotide encoding the CAR described herein is inserted into a vector. A vector is a medium into which a polynucleotide encoding a protein is covalently inserted to achieve expression of the protein and / or cloning of the polynucleotide. Such a vector may also be referred to as an "expression vector". The isolated polynucleotide can be inserted into the vector using any suitable method known in the art, for example, but not limited to, digestion of the vector with a suitable restriction enzyme, followed by ligation to the isolated polynucleotide having a matching restriction end. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences that encode at least a portion of a gene product capable of being transcribed in a cell. In most cases, the RNA molecule is subsequently translated into a protein. Expression vectors may contain a variety of control sequences, which are nucleic acid sequences necessary for transcription and possibly translation of the operablely linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, as discussed below. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host.

[0105] Expression vectors may contain necessary 5′ upstream and 3′ downstream regulatory elements, such as promoter sequences (e.g., CMV, PGK, and EF1alpha), promoters, ribosome recognition and binding TATA boxes, and 3′UTR AAUAAA transcription termination sequences, to enable efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters of the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoters, MoMuLV promoters, avian leukosis virus promoters, EBV immediate early promoters, and Rous sarcoma virus promoters. Human gene promoters may also be used, including but not limited to actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. In some embodiments, inducible promoters are also considered as part of the vector expressing the chimeric antigen receptor. This provides a molecular switch capable of turning the expression of the polynucleotide sequence of interest on or off. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters.

[0106] Expression vectors may have additional sequences, such as 6×-histidine (SEQ ID NO: 88), c-Myc, and FLAG tags, incorporated into the expressed CAR. Thus, expression vectors can be engineered to contain 5′ and 3′ untranslated regulatory sequences, which can sometimes act as enhancer sequences, promoter regions, and / or terminator sequences, thereby promoting or enhancing the efficient transcription of the nucleic acid of interest carried on the expression vector. Expression vectors can also be engineered to replicate and / or express functionally (e.g., transcription and translation) in specific cell types, cell locations, or tissue types. Expression vectors may include optional markers for maintaining the vector in host or recipient cells.

[0107] In various implementations, the vector is a plasmid, an autonomously replicating sequence, and a transposon element. Other exemplary vectors include, but are not limited to, plasmids, phagemids, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Examples of animal virus classes that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary multivoid viruses (e.g., SV40). Examples of expression vectors include the Lenti-X™ bicistronic expression system (Neo) vector (Clontech) and the pCl-neo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The CAR coding sequence disclosed in this paper can be ligated into such expression vectors to express chimeric proteins in mammalian cells.

[0108] In some embodiments, the nucleic acid encoding the CAR is provided in a viral vector. The viral vector can be derived from, for example, a retrovirus (e.g., foamy virus) or a lentivirus. As used herein, the term "viral vector" refers to a nucleic acid vector construct containing at least one virally derived element and having the ability to be packaged into viral vector particles. The viral vector may contain coding sequences for various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particles can be used for the purpose of transferring DNA, RNA, or other nucleic acids into cells, either in vitro or in vivo. Various forms of viral vectors are known in the art.

[0109] In some implementations, 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 containing structural and functional genetic elements primarily derived from retroviruses. The term "lentiviral vector" refers to a vector containing structural and functional genetic elements primarily derived from lentiviruses other than their LTRs.

[0110] The retroviral vectors used in this article can be derived from any known retrovirus (e.g., type c retroviruses such as Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retroviruses" also include human T-cell leukemia virus, HTLV-1, and HTLV-2, as well as retroviruses from the lentivirus family, such as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0111] The lentiviral vectors used in this article refer to vectors derived from lentiviruses, which are a class (or genus) of retroviruses that cause slowly progressing diseases. This group includes viruses such as HIV (human immunodeficiency virus; including HIV types 1 and 2), visna-maedi virus, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Recombinant lentiviruses can be prepared according to the methods of 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).

[0112] The desired retroviral vector (i.e., lentivirus and non-lentivirus) can be formed by combining the required DNA sequences in the order and orientation described herein using standard cloning techniques (Current Protocols in Molecular Biology, Ausubel, FM 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. (1990) Proc. Natl. Acad. Sci. USA 87:6141–6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et 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. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; US Patent Nos. 4,868,116; 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0113] Suitable sources for obtaining retroviral (i.e., lentivirus and non-lentivirus) sequences used to form vectors include, for example, genomic RNA and cDNA available from commercial sources, including the American Type Culture Collection (ATCC) in Rockville, Maryland, USA. These sequences can also be chemically synthesized.

[0114] To express a CAR, a vector can be introduced into a host cell to allow the peptide to be expressed within the host cell. The expression vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection markers, and signal sequences. As described above, those skilled in the art can appropriately select these elements. For example, a promoter sequence can be selected to initiate the transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence can be selected to enhance the transcription of the polynucleotide. A selection marker can be selected to allow the selection of host cells containing the vector from host cells that have never had the vector inserted; for example, the selection marker could be a gene conferring antibiotic resistance. A signal sequence can be selected to allow the expressed peptide to be transported outside the host cell.

[0115] To clone polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to allow the vector itself to replicate, thereby amplifying copies of the polynucleotides it contains. Cloning vectors may contain sequence components, typically including, but not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selection markers. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication can be selected to facilitate autonomous replication of the vector within the host cell.

[0116] In some embodiments, this disclosure provides isolated host cells containing the vectors provided herein. Host cells containing the vectors can be used to express or clone the polynucleotides contained in the vectors. Suitable host cells may 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, such as Enterobacteriaceae such as Escherichia coli. Escherichia ), such as Escherichia coli ( E. coli ), Enterobacteriaceae ( Enterobacter Erwinia ( ) Erwinia Klebsiella spp. Klebsiella ), Proteus spp. Proteus Salmonella ( Salmonella ), such as Salmonella typhimurium ( Salmonella typhimurium ), Serratia ( Serratia ), such as Serratia marcescens ( Serratia marcescans ), and Shigella spp. Shigella ), and Bacilli species such as Bacillus subtilis ( B. subtilis ) and Bacillus licheniformis ( B. licheniformis ), Pseudomonas species, such as Pseudomonas aeruginosa ( P. aeruginosa ), and Streptomyces ( Streptomyces ).

[0117] CARs are introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms “transfection” and “transduction” refer to the process of introducing a foreign nucleic acid sequence into a host cell. The nucleic acid may integrate into the host cell DNA or may remain extrachromosomally. The nucleic acid may be temporarily maintained or stably introduced. Transfection can be performed by a variety of methods known in the art, including but not limited to calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and gene gun methods. Transduction refers to the delivery of a gene via viral infection rather than transfection using a virus or retroviral vector. In some embodiments, the retroviral vector is transduced by packaging the vector into a virion prior to contact with the cell. For example, a nucleic acid encoding a CAR carried by a retroviral vector can be transduced into the cell via infection and provirus integration.

[0118] To assess the expression of CAR peptides or portions thereof, the expression vector to be introduced into cells may also contain an optional marker gene or reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells attempting to be transfected or infected via a viral vector. In other respects, the optional marker may be carried on a separate DNA fragment and used in co-transfection procedures. Both the optional marker and the reporter gene can be linked to appropriate regulatory sequences to enable their expression in host cells. Useful selection markers include, for example, antibiotic resistance genes.

[0119] Reporter genes are used to identify potentially transfected cells and assess the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and the expression of its encoded polypeptide is indicated by easily detectable properties such as enzyme activity. Reporter gene expression is detected at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well-known and can be prepared using known techniques or obtained commercially. Generally, constructs with minimal 5′ flanking regions that exhibit the highest reporter gene expression levels are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of drugs to regulate promoter-driven transcription.

[0120] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0121] In the case of using a non-viral delivery system, an exemplary delivery medium is liposomes. Alternatively, nucleic acids can bind to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within a lipid bilayer of liposomes, attached to liposomes by linkers that bind to both liposomes and oligonucleotides, encapsulated within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as bilayer structures, micelles, or “collapsed” structures. They may also simply be dispersed in solution, potentially forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic. For example, lipids include naturally occurring fat droplets in the cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids are available from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma (St. Louis, MO); dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; and dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc (Birmingham, AL).

[0122] Induced natural killer (NK) cells In some respects, this paper also discloses natural killer (NK) cells engineered to express the disclosed CAR peptide. Natural killer (NK) cells are CD56 cells. + CD3 – Large granular lymphocytes can kill virus-infected and transformed cells and constitute a key cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666–1676). They are associated with cytotoxic CD8+.+ Unlike T lymphocytes, NK cells can initiate cytotoxicity against tumor cells without prior sensitization and can eliminate MHC-I negative cells (Narni-Mancinelli E et al., Int Immunol 2011 23:427–431). NK cells are safer effector cells because they can avoid cytokine storms (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 potentially fatal complications of targeted, off-tumor effects.

[0123] In some implementations, NK cells are obtained from the subject to be treated (i.e., autologous). However, in other implementations, NK cell lines or donor effector cells (allogeneic) are used. In some implementations, NK cells are inducible NK cells differentiated from induced pluripotent stem cells (iPSCs).

[0124] NK cells can be obtained from many sources using methods well-known in the art, including peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte isolates (PBSCs), bone marrow, umbilical cord blood, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs). NK cells can be detected by specific surface markers, such as expressing CD16, CD56, and CD8 in humans, but not CD3.

[0125] In some implementations, NK cells can be obtained from blood collected from a subject using any of the many techniques known to those skilled in the art. For example, the initial population of NK cells can be obtained by separating monocytes using Ficoll density gradient centrifugation. Specific subpopulations of NK cells can be further separated using positive or negative selection techniques. For example, NK cells can be separated using a combination of antibodies targeting surface markers specific to positively selected cells, for example, by incubating with antibody-conjugated beads for a sufficient time to positively select NK cells. Alternatively, the NK cell population can be enriched by negative selection using a combination of antibodies targeting surface markers specific to negatively selected cells. For example, any cells expressing CD3, CD14, and / or CD19 in a cell culture can be depleted and characterized to determine CD56. + / CD3 − Percentage of cells or NK cells.

[0126] In some embodiments, cord blood (CB) is used to obtain NK cells. In some embodiments, NK cells are isolated and expanded using previously described methods for in vitro expansion of NK cells. For example, CB mononuclear cells can be isolated by ficoll density gradient centrifugation and cultured in a bioreactor containing IL-2 and artificial antigen-presenting cells (aAPCs). After several days, cells expressing CD3 in the cell culture are depleted, and then cultured for several more days. These cells are again depleted by CD3 and characterized to determine CD56. + / CD3 − Percentage of cells or NK cells. In other embodiments, cord blood is used to isolate CD34. + NK cells were derived from cells and CD34 cells were isolated by culturing in a medium containing SCF, IL-7, IL-15, and IL-2. + Cells differentiate it into CD56 + / CD3 − cell.

[0127] In some implementations, NK cells are derived from pluripotent stem cells. Pluripotent stem cells, whether human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), can grow indefinitely in an undifferentiated state through self-renewal. Therefore, the ability to routinely derive NK cells from human embryonic stem cells and pluripotent stem cells allows for the generation of an unlimited number of homogeneous NK cells from a starting pluripotent stem cell population, providing a standardized, off-the-shelf approach. iPSC-derived NK (iNK) cells have been reported to produce inflammatory cytokines and exhibit potent cytotoxicity against a variety of hematologic malignancies and solid tumors. Genetic engineering methods such as transposon and lentiviral delivery can be used to engineer hESCs and iPSCs to express the CARs described herein, thereby ensuring efficient transgene insertion and stable expression in iPSCs. TALENS and CRISPR / Cas9 can also be used for more precise knock-in or deletion of specific genes. Engineered and undifferentiated hESCs or iPSCs can then be differentiated into NK cells expressing the CARs described herein. The methods for differentiating hESCs or iPSCs into NK cells are well known in the art, including those described in Cichocki et al., SciTransl 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, all of which are incorporated herein by reference in their entirety.

[0128] This disclosure provides a method for manufacturing NK cells expressing the CARs described herein. In one embodiment, the method includes transfecting or transducing NK cells isolated from a subject such that the NK cells express one or more CARs as described herein. In some embodiments, NK cells are isolated from an individual and genetically modified without further in vitro manipulation. These cells can then be directly re-administered to the individual. In a further embodiment, NK cells are expanded in vitro and then genetically modified to express CARs. In this respect, NK cells can be cultured before or after genetic modification (i.e., transduction or transfection to express CARs as described herein). NK cells may be expanded in the presence of artificial antigen-presenting cells (aAPCs). The expanded culture may also contain cytokines that promote expansion, such as IL-2, IL-21, and / or IL-18. Cytokines may be supplemented during the expanded culture, for example, every 2-3 days. APCs may be added to the culture at least a second time, for example, after CAR transduction.

[0129] In some respects, this paper also discloses other immune cells (e.g., phagocytes) engineered to express the disclosed CAR peptides.

[0130] Binding properties of chimeric antigen receptors As used herein, the term "binding" is used in the context of the binding of a chimeric antigen receptor containing the extracellular domain described herein to, for example, an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and tumor antigens). Binding generally refers to an interaction or association between at least two entities or molecular structures, such as antigen-binding domain:antigen interaction. For example, when using an antigen as a ligand and a chimeric antigen receptor as an analyte (or antiligand) in a BIAcore 3000 instrument, measured by, for example, surface plasmon resonance (SPR) technology, binding affinity typically corresponds to K. D The value is approximately 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller. 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).

[0131] Therefore, in some embodiments, the chimeric antigen receptor of this disclosure binds to antigen-binding molecules (e.g., multispecific antigen-binding molecules that bind to CD3 and tumor antigens), the affinity corresponding to K D The value is at least one-tenth of its affinity for binding to non-specific antigens (e.g., BSA, casein). According to this disclosure, in some embodiments, K... D Affinity of chimeric antigen receptors with a value equal to or less than one-tenth of that of the nonspecific binding partner can be considered undetectable binding.

[0132] Term "K" D "(M) refers to the dissociation equilibrium constant of a specific antigen-binding domain: antigen-antigen interaction. K" D There is an inverse relationship between K and binding affinity, therefore K D The smaller the value, the higher the affinity, i.e., the stronger. Therefore, the terms "higher affinity" or "stronger affinity" are associated with a higher ability to form interactions and thus with a smaller K value. D The value is related to the strength of the interaction, while conversely, the terms "lower affinity" or "weaker affinity" are associated with a lower ability to form interactions and therefore with a larger K. D The value is related. In some cases, the binding affinity (or K-value) of a specific molecule (e.g., a chimeric antigen receptor) to its interacting partner molecule (e.g., antigen X) is related to the binding affinity. D A higher binding affinity of a molecule (e.g., a chimeric antigen receptor) to another interacting partner molecule (e.g., antigen Y) can be expressed as the binding affinity of a larger Kx is used. D The value (lower or weaker affinity) divided by the smaller K D The binding ratio determined by (higher or stronger affinity), for example, expressed as a binding affinity of 5 times or 10 times, depending on the case.

[0133] The term "k" d "(sec⁻¹ or 1 / s)" refers to the dissociation rate constant of a specific antigen-binding domain: antigen-antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. This value is also called k. off value.

[0134] The term "k" a "(M-1 x sec-1 or 1 / M) refers to the binding rate constant of a specific antigen-binding domain: antigen-antigen interaction, or the association rate constant of a chimeric antigen receptor."

[0135] Term "K" A "(M-1 or 1 / M) refers to the association equilibrium constant of a specific antigen-binding domain: the association equilibrium constant of antigen-antigen interactions, or chimeric antigen receptors. This is determined by using k..." a Divide by k d To obtain the association equilibrium constant.

[0136] The term "EC50" or "EC" 50 "" refers to the half-maximal effective concentration, which includes the concentration of chimeric antigen receptors that induces half of the responses between baseline and maximum after a specified exposure time. 50 This generally represents the chimeric antigen receptor concentration at 50% of the observed maximum effect. In some implementations, EC... 50 The value is equal to the concentration of the chimeric antigen receptor of this disclosure, which gives the half-maximal binding to cells expressing antigens (e.g., tumor-associated antigens), as determined by, for example, a FACS binding assay. Therefore, in increased EC... 50 A reduced or weaker binding was observed at half the maximum effective concentration value.

[0137] In one embodiment, a decrease in binding can be defined as the ability of ECs to bind to half of the maximum number of target cells. 50 Increased concentration of chimeric antigen receptors.

[0138] This disclosure provides a chimeric antigen receptor having an antibody-derived antigen-binding domain, said antibody having high affinity (e.g., nanomolar or sub-nanomolar K). D (Value) combines with human antigen.

[0139] According to certain embodiments, this disclosure 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 tumor antigens) (e.g., at 25°C), with K measured by surface plasmon resonance. D Less than approximately 5 nM. In some embodiments, the K of the corresponding antibody-binding antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and tumor antigens) D 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 1 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.

[0140] This disclosure also provides chimeric antigen receptors having an antigen-binding domain derived from a corresponding antibody that binds an antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds CD3 and tumor antigens) with a dissociation half-life (t½) greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25°C. In some embodiments, the t½ of the corresponding antibody binding to the antigen-binding molecule (e.g., a multispecific antigen-binding molecule that binds to CD3 and tumor antigens) is 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.

[0141] Therapeutic antibodies Overview In some respects, the methods and compositions provided herein relate to the use of therapeutic antibodies, such as bispecific CD3-binding antibodies.

[0142] As described above, and as used herein, the term "antibody" encompasses both the complete antibody molecule and the antigen-binding fragment of the complete antibody molecule. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) heavy chain (Fd) fragments of Fab; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) domain-specific antibody (dAb) fragments; and (vii) minimal recognition units consisting of amino acid residues of a hypervariable region mimicking an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a restrictive FR3-CDR3-FR4 peptide. The term "antigen-binding fragment" as used herein also includes other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0143] Antigen-binding fragments of antibodies will typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will typically contain at least one core-delimited domain (CDR), which is adjacent to or co-frames with one or more frame sequences. In the presence of V... L V associated with the structural domain H In the antigen-binding fragment of the domain, V H and V LDomains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimer and contain V. H -V H 、V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V. H or V L Structural domain.

[0144] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found in the antigen-binding fragments of the antibodies disclosed herein include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (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 the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected via fully or partially hinged or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which creates flexible or semi-flexible bonding between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies disclosed herein may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are connected to each other and / or to one or more monomers V. H or V L Non-covalent association of structural domains (e.g., via disulfide bonds).

[0145] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. Any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed herein, can be applied to the antigen-binding fragments of the antibodies disclosed herein using conventional techniques available in the art. In some embodiments provided herein, at least one variable domain of the multispecific antibody is capable of specifically binding to CD3.

[0146] 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 cells expressing an antigen by the antibody 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), such as natural killer (NK) cells, neutrophils, and macrophages, recognize an antibody bound to a target cell and thereby induce lysis of the target cell. CDC and ADCC can be measured using assays well-known and available in the art. (See, for example, 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 in its ability to immobilize complement and mediate cell-dependent cytotoxicity. Therefore, antibody isotypes can be selected based on whether antibody-mediated cytotoxicity is required.

[0147] In some 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. Human antibodies disclosed herein may include, for example, amino acid residues in the CDR, particularly in CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0148] In some embodiments, the antibodies provided herein may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, formed, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (described further below); antibodies isolated from recombinant, combined human antibody libraries (described further below); antibodies isolated from animals that are transgenic for human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies prepared, expressed, formed, or isolated by any other means involving splicing 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 some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody is... H and V L The amino acid sequence of the region, although derived from human lineage V, is H and V L The sequence and related sequences, but which may not naturally exist in the human antibody pedigree in vivo.

[0149] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150–160 kDa, where dimers are linked together by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, but instead form a molecule of approximately 75–80 kDa composed of covalently coupled light and heavy chains (half-antibodies). These forms are extremely difficult to separate, even after affinity purification.

[0150] The frequent occurrence of the second form in various intact IgG isotypes is attributed, but not limited to, structural differences associated with hinge region isotypes of antibodies. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993)). Molecular Immunology 30:105), reaching levels typically observed with human IgG1 hinges. In some embodiments, this disclosure covers the hinge, C H 2 or C H An antibody with one or more mutations in region 3, said mutations being, for example, necessary to improve the yield of the desired antibody form during preparation.

[0151] The antibodies disclosed herein may be isolated antibodies. As used herein, "isolated antibody" refers to an antibody that has been identified, isolated, and / or recovered from at least one component of its natural environment. For example, an antibody that has been isolated or removed from at least one component of an organism or from tissues where antibodies are naturally present or produced is an "isolated antibody" for the purposes of this disclosure. Isolated antibodies also include in situ antibodies within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0152] In some embodiments, the methods and compositions provided herein include a single-arm antibody that binds to a tumor antigen (TAA). As used herein, "single-arm antibody" means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain.

[0153] Sequence variants In some embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame regions and / or CDR regions of the heavy chain variable domain and light chain variable domain, compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. In some embodiments, this disclosure includes antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more frame regions and / or CDR regions are mutated to corresponding residues of the germline sequence from which the antibody is derived, or corresponding residues of another human germline sequence, or conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In some embodiments, V H and / or VL All frame regions and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as mutated residues found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence different 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 frame and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. After obtaining an antibody and antigen-binding fragment containing one or more germline mutations, 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., K0), can be readily tested. D It can improve or enhance antagonistic or agonistic biological properties (depending on the case), or reduce immunogenicity, etc.

[0154] In some embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein comprise variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. For example, in some embodiments, the CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein have HCVR, LCVR, and / or CDR amino acid sequences having conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, such as 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.

[0155] Fc variant According to certain embodiments provided herein, antibodies and multispecific antigen-binding molecules are provided comprising one or more mutated Fc domains, wherein the mutation, for example, enhances or weakens the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. In some embodiments, this disclosure includes a C0 in the Fc domain. H 2 or C HThe antibody contains a mutation in region 3, wherein the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome with a pH range of about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to animals. 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 250 and / or 428; 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).

[0156] In some embodiments, this disclosure includes a CD3 multispecific antigen-binding molecule (e.g., an anti-CD3 / anti-MUC16 bispecific, anti-BCMA x anti-CD3, or anti-CD3 / anti-CD20 bispecific antibody) comprising an Fc domain containing one or more pairs of mutations or mutation sets selected from 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 aforementioned Fc domain mutations, as well as other mutations within the variable domains of the disclosed antibodies, are contemplated.

[0157] In some embodiments, the CD3xTAA bispecific antibody of this disclosure comprises an IgG Fc sequence with amino acid substitutions, such as using two residues derived from IgG3. For example, the CD3xTAA bispecific antibody of this disclosure may comprise an IgG1 Fc sequence with amino acid substitutions H365R and Y366F in the CH3 region. Exemplary Fc sequences are also shown. Figure 3 middle.

[0158] Bioequivalents This document provides antigen-binding molecules having amino acid sequences that differ from the exemplary molecules disclosed herein but retain the ability to bind the same one or more antigens. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions when compared to the parental sequence, but exhibit biological activity substantially equivalent to that of the described bispecific antigen-binding molecules.

[0159] In some embodiments, this disclosure includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they do not show significant differences in absorption rate and extent when administered at the same molar dose (single or multiple doses) under similar experimental conditions. Some antigen-binding proteins are equivalent in their extent of absorption but not in their rate of absorption, yet may still be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling, are not necessary to achieve effective in vivo drug concentrations after, for example, prolonged use, and are considered medically insignificant for the particular pharmaceutical product under investigation.

[0160] In one implementation, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0161] In one implementation, if a patient can switch between the reference product and the biological product once or multiple times without an expected increase in risk of adverse effects compared to continuous therapy without such switching, including clinically significant changes in immunogenicity or reduced efficacy, then the two antigen-binding proteins are bioequivalent.

[0162] In one implementation, two antigen-binding proteins are bioequivalent if they function through one or more common mechanisms of action (where such mechanisms are known) for one or more conditions of use.

[0163] Bioequivalence can be demonstrated through in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolites over time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and appropriately predict bioavailability in humans; (c) in vivo tests in humans or other mammals that measure the appropriate acute pharmacological effects of an antibody (or its target) over time; and (d) fully controlled clinical trials that determine the safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0164] Bioequivalent variants of the exemplary bispecific antigen-binding molecules illustrated herein can be constructed, for example, by various substitutions or deletions of terminal or internal residues or sequences that are not required for biological activity. For instance, cysteine ​​residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inappropriate intramolecular disulfide bridges after renaturation. In other cases, bioequivalent antigen-binding proteins may comprise variants of the exemplary bispecific antigen-binding molecules illustrated herein, which include amino acid changes that modify the glycosylation characteristics of the molecule, such as mutations that eliminate or remove glycosylation.

[0165] antibody binding As used herein, in cases where an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein binds to, for example, a predetermined antigen such as a cell surface protein or a fragment thereof, the term “binding” generally refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0166] For example, when using an antigen as a ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as an analyte (or antiligand) in a BIAcore 3000 instrument using, for example, surface plasmon resonance (SPR) technology, binding affinity typically corresponds to K. D The value is approximately 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller. 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).

[0167] Therefore, the antibodies or antigen-binding proteins described herein bind to predetermined antigens or cell surface molecules (receptors) with an affinity corresponding to at least one-tenth of their affinity for nonspecific antigens (e.g., BSA, casein). D Value. In some implementations, the antibody affinity corresponds to a K value equal to or one-tenth the value of the nonspecific antigen. D The value can be considered an undetectable binding; however, such antibodies can pair with a second antigen binding arm to produce the bispecific antibodies disclosed herein.

[0168] Term "K" D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment bound to an antigen. K" D There is an inverse relationship between K and binding affinity, therefore K D The smaller the value, the higher the affinity, i.e., the stronger. Therefore, the terms "higher affinity" or "stronger affinity" are associated with a higher ability to form interactions and thus with a smaller K value. D The value is related to the strength of the interaction, while conversely, the terms "lower affinity" or "weaker affinity" are associated with a lower ability to form interactions and therefore with a larger K. D The value is related. In some cases, the binding affinity (or K-value) of a specific molecule (e.g., an antibody) to its interacting partner molecule (e.g., antigen X) is related to the binding affinity. D A higher binding affinity between a molecule (e.g., an antibody) and another interacting partner molecule (e.g., antigen Y) can be expressed as the binding affinity of a larger Kx is used. D The value (lower or weaker affinity) divided by the smaller K D The binding ratio determined by (higher or stronger affinity), for example, expressed as 5 or 10 times greater binding affinity, depending on the case.

[0169] The term "k" d "(sec⁻¹ or 1 / s)" refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is also known as k. off value.

[0170] The term "k" a "(M-1 x sec-1 or 1 / M)" refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0171] Term "K" A "(M-1 or 1 / M)" refers to the association equilibrium constant of a specific antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. This is determined by using k... a Divide by k d To obtain the association equilibrium constant.

[0172] The term "EC50" or "EC" 50 "" refers to the half-maximal effective concentration (MCI), which includes the antibody concentration that induces half the response between baseline and maximum after a specified exposure time. 50 Roughly representing the antibody concentration at which 50% of the maximum observed effect was achieved. In some implementations, EC... 50The value is equal to the concentration of the antibody disclosed herein, which produces a half-maximal binding to cells expressing CD3 or tumor-associated antigens (e.g., CD123, STEAP2, CD20, PSMA, SSTR2, CD38, STEAP1, 5T4, ENPP3, MUC16, or BCMA), as determined by, for example, a FACS binding assay. Therefore, in increased EC... 50 A reduced or weaker binding was observed at half the maximum effective concentration value.

[0173] In one implementation, reduced binding of an antibody (e.g., a CD3 multispecific antibody) can be defined as achieving EC50 binding with half the maximum number of target cells. 50 Antibody concentration increased.

[0174] In another implementation scheme, EC 50 The value represents the concentration of an antibody (e.g., the CD3 multispecific antibody disclosed herein) that induces half-maximal depletion of target cells through cytotoxic activity of effector cells (e.g., T cells or NK cells). Therefore, an increase in cytotoxic activity (e.g., T cell or NK cell-mediated tumor cell killing) was observed, while EC50... 50 Or half of the maximum effective concentration values ​​decrease.

[0175] In yet another implementation plan, EC 50 The value represents the concentration of an antibody (e.g., a CD3 multispecific antibody disclosed herein) that induces half-maximal activation of target cells through effector cell (e.g., T cells or NK cells) activation. For example, T cell activation can be measured using a Jurkat NFAT reporter bioassay (e.g., the Jurkat / NFAT-Luc bioassay described in Example 1). Therefore, an increase in effector cell activation (e.g., T cell or NK cell activation) is observed, while EC... 50 Or half of the maximum effective concentration values ​​decrease.

[0176] pH-dependent binding In some embodiments, this disclosure includes antibodies and multispecific antigen-binding molecules having pH-dependent binding properties. For example, the CD3 multispecific antibody disclosed herein may bind to CD3 with less force at acidic pH than it does at neutral pH. Alternatively, the CD3 multispecific antibody disclosed herein may bind to CD3 with greater force at acidic pH than it does at neutral pH. “Acidic pH” includes pH values ​​below 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, or lower. As used herein, the expression “neutral pH” means a pH from about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0177] In some cases, "reduced binding at acidic pH compared to neutral pH" is based on the Kc of antibody binding to its antigen at acidic pH. D The value of Kc at neutral pH is related to the binding of antibodies to their antigens. D The ratio of values ​​is expressed (or vice versa). For example, for the purposes of this disclosure, if a CD3 multispecific antibody or its antigen-binding fragment exhibits an acidic / neutral K ratio of approximately 3.0 or greater... D The ratio, then, indicates that the CD3 multispecific antibody or its antigen-binding fragment can be considered to exhibit "reduced binding to CD3 at acidic pH compared to neutral pH". In some exemplary embodiments, the acidic / neutral KA of the antibody or antigen-binding fragment disclosed herein is... D The ratio can be approximately 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 greater.

[0178] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for binding to a specific antigen that is reduced (or enhanced) at acidic pH compared to neutral pH. Additionally, modifications to the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For instance, by replacing one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies exhibiting reduced antigen binding at acidic pH relative to neutral pH can be obtained.

[0179] Preparation of antigen-binding domains and construction of multispecific molecules Antigen-binding domains specific to a particular antigen can be prepared using any antibody production technique known in the art. After obtaining two different antigen-binding domains specific to two different antigens (e.g., CD3 and human tumor antigens (e.g., MUC16, BCMA, CD20, etc.)), they can be appropriately arranged relative to each other to produce the bispecific antigen-binding molecule disclosed herein using conventional methods. In some embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecule disclosed herein are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing 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 molecule disclosed herein can be prepared using the VELOCIMMUNE™ technology. Using the VELOCIMMUNE™ technology (or any other human antibody production technique), a high-affinity chimeric antibody having a human variable region and a mouse constant region against a specific antigen (e.g., CD3 or human tumor antigens (e.g., MUC16, BCMA, CD20, etc.)) is initially isolated. Antibodies are characterized and selected for desired features, including affinity, selectivity, and epitopes. Mouse constant regions are replaced with desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules disclosed herein.

[0180] Human bispecific antigen-binding molecules can be prepared using genetically engineered animals. For example, genetically modified mice can be used that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences, wherein the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operatively linked to a mouse κ constant gene at an endogenous mouse κ locus. Such genetically modified mice can be used to generate fully human bispecific antigen-binding molecules comprising two different heavy chains associated with the same light chain, the same light chain comprising a variable domain derived from one of two different human light chain variable region gene segments (see, for example, US 2011 / 0195454). Fully human refers to an antibody or its antigen-binding fragment or immunoglobulin domain comprising, along its entire length, an amino acid sequence encoded by DNA derived from a human sequence. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, the fully human protein or protein sequence comprises a chimeric sequence where each component sequence is derived from a human sequence. While not bound by any particular theory, chimeric proteins or sequences are typically designed to minimize the formation of immunogenic epitopes at component sequence junctions, for example, compared to any wild-type human immunoglobulin region or domain.

[0181] CD3 multispecific antigen-binding molecule In some 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 some embodiments, the CD3 multispecific antigen-binding molecules provided herein further comprise an antigen-binding domain that binds to cancer antigens (i.e., antigens expressed on cancer cells). In some embodiments, the CD3 multispecific antigen-binding molecules provided herein further comprise an antigen-binding domain that binds to a co-stimulatory receptor (e.g., CD28).

[0182] In some implementations, the CD3 antibody is any of the CD3 antibodies listed in Table 6.

[0183] Table 6: CD3 Antibodies As used herein, the term "multispecific antigen-binding molecule" refers to a protein, peptide, or molecular complex that comprises 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 individual CDR, or a combination of at least one CDR with one or more additional CDRs and / or FRs, specifically binding to a particular antigen. In the context of this disclosure, 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).

[0184] In some embodiments, the CD3 multispecific antigen-binding molecule is a CD3 multispecific antibody. The CD3 multispecific antibodies provided herein can be, for example, bispecific or trispecific. Multispecific antibodies may be specific to different epitopes of a target polypeptide or may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The CD3 bispecific antibodies provided herein can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0185] In some embodiments, this disclosure includes a bispecific antibody, wherein one arm of an immunoglobulin binds CD3, and the other arm of the immunoglobulin is specific for a cancer antigen (also referred to herein as a tumor antigen or “TAA”). In some embodiments, this disclosure includes a trispecific antibody, wherein the first arm of an immunoglobulin binds CD3, the second arm of the immunoglobulin is specific for a tumor antigen, and the third arm of the immunoglobulin binds to an additional T-cell antigen (e.g., CD28) or another tumor antigen.

[0186] In some embodiments, the CD3 binding arm may comprise any HCVR / LCVR or CDR amino acid sequence disclosed in WO 2014 / 047231 or WO 2017 / 053856. In some embodiments, the CD3 binding arm binds to human CD3 and induces human T cell activation. In some embodiments, the CD3 binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, in the case of bispecific or multispecific antibodies, the CD3 binding arm binds weakly to human CD3 and induces the killing of cells expressing tumor-associated antigens. In other embodiments, the CD3 binding arm binds or weakly associates with human and cynomolgus monkey CD3, but the binding interaction cannot be detected by in vitro assays known in the art.

[0187] In some implementations, the multispecific antibody or antigen-binding fragment includes an antigen-binding arm that binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2 to induce T cell activation.

[0188] In some implementations, the CD3 multispecific antigen-binding molecule includes an antigen-binding domain that is specific to cancer antigens. In some implementations, the cancer antigen is selected from AIM-2, ALDH1A1, α-actin-4, alpha-fetoprotein (“AFP”), ARTC1, β-RAF, BAGE-1, BCLX(L), BCMA, BCR-ABL fusion protein b3a2, β-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-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, Phosphatidylinositol Polysaccharide-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, KM-HN-1, KMHN1 (Also known as CCDC110), LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic acid oxidase, lactoglobulin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, metaphase factor, MMP-2, MMP-7, MSLN, MUC1, MUC5AC, MUC16, mucin, MUM-1, MUM-2, MUM-3, myosin, type I myosin, N-raw, NA88-A neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P-peptide, p53, PAP, PAX5, PBF, pml-RARα fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, seternin 1. 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-βRII, TPBG, TRAG-3, triose phosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, 5T4, and XAGE-1b / GAGED2a.

[0189] In some implementations, cancer antigens include ADAM 17, 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, FORLR1, GD-2, phosphatidylinositol polysaccharide-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 implementations, 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.

[0190] In some implementations, 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.

[0191] In some implementations, the cancer antigen is CD20, MUC16, BCMA, PSMA, or STEAP2.

[0192] 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 in over 95% of B-cell non-Hodgkin lymphomas (NHL) and other B-cell malignancies, but it is not present in precursor B cells, dendritic cells, or plasma cells. The human CD20 protein has the amino acid sequence shown in SEQ ID NO: 5 of U.S. Patent Application Publication No. US 2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0193] MUC16 refers to mucin 16. MUC16 is a fully glycosylated membrane glycoprotein with a single transmembrane domain, which is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is listed in SEQ ID NO: 1899 of U.S. Patent Application Publication No. US 2018 / 0118848A1, the entire contents of which are incorporated herein by reference.

[0194] BCMA stands for 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 the human BCMA protein is shown in SEQ ID NO:115 of U.S. Patent Application Publication No. US 2020 / 0024356, the entire contents of which are incorporated herein by reference. It can also be found in GenBank accession number NP_001183.2.

[0195] PSMA refers to prostate-specific membrane antigen, also known as folic acid hydrolase 1 (FOLH1). PSMA is an intact, non-shedding membrane 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 Publication No. US 2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0196] STEAP2 refers to prostatic six-span membrane epithelial antigen 2. STEAP2 is a complete six-span membrane protein highly expressed in prostatic 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 region 7q21 of human chromosome 2. The amino acid sequence of human STEAP2 is shown in SEQ ID NO: 9 of U.S. Patent Application Publication No. US 2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0197] In some implementations, 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.

[0198] In some embodiments, this disclosure includes antibodies having the HCVR, LCVR, and / or CDR amino acid sequences of the antibodies described herein, anti-CD3 antibodies disclosed in WO 2014 / 047231 or WO 2017 / 053856, bispecific anti-CD20 x anti-CD3 antibodies disclosed in WO 2014 / 047231, bispecific anti-PSMA x anti-CD3 antibodies disclosed in WO 2017 / 023761, bispecific anti-MUC16 x anti-CD3 antibodies disclosed in WO 2018 / 067331, bispecific anti-STEAP2 x anti-CD3 antibodies disclosed in WO 2018 / 058001, or bispecific anti-BCMA x anti-CD3 antibodies disclosed in WO 2020 / 018820, each of which is incorporated herein by reference.

[0199] In some embodiments, the multispecific antigen-binding molecule is a multispecific antibody or an antigen-binding fragment thereof. Each antigen-binding domain of the multispecific antibody includes a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the case of a bispecific antigen-binding molecule comprising first and second antigen-binding domains (e.g., a bispecific antibody), the prefix "A1" may be used to designate the CDR of the first antigen-binding domain and the prefix "A2" may be used to designate the CDR of the second antigen-binding domain. Therefore, the CDR of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDR of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3. In the case of a trispecific antigen-binding molecule comprising a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain (e.g., a trispecific antibody), the prefix "A1" can be used to specify the CDR of the first antigen-binding domain, the prefix "A2" can be used to specify the CDR of the second antigen-binding domain, and the prefix "A3" can be used to specify the CDR of the third antigen-binding domain. Therefore, 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.

[0200] The bispecific antigen-binding molecules discussed above or herein can be bispecific antibodies. In some cases, bispecific antibodies contain a constant region of the human IgG heavy chain. In some cases, the constant region of the human IgG heavy chain is isotype IgG1. In some cases, the constant region of the human IgG heavy chain is isotype IgG4. In various embodiments, the bispecific antibody contains a chimeric hinge that reduces Fcɣ receptor binding relative to a wild-type hinge of the same isotype.

[0201] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule disclosed herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerizing domain. Association of one multimerizing domain with the other multimerizing domain facilitates association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerizing domain of the same or similar structure or construction. For example, a multimerizing domain may be a domain containing immunoglobulin C. H3-domain polypeptides. A non-limiting example of a polymerized component is the Fc region of an immunoglobulin (containing C... H 2-C H 3 domains), such as the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype within each isotype group.

[0202] The bispecific antigen-binding molecules disclosed herein will typically comprise two multimerizing domains, such as two Fc domains, each individually part of a separate antibody heavy chain. The first and second multimerizing domains may belong to the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may belong to different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0203] In some embodiments, the polymerizing domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids in length and containing at least one cysteine ​​residue. In other embodiments, the polymerizing domain is a cysteine ​​residue, or a short cysteine-containing peptide. Other polymerizing domains include peptides or polypeptides comprising leucine zippers, helical-cyclic motifs, or coiled-helical motifs, or composed thereof.

[0204] The bispecific antigen-binding molecules disclosed herein can be prepared using any form of bispecific antibody or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity may be functionally linked (e.g., by chemical conjugation, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific forms that may be used in the context of this disclosure include, but are not limited to, for example, scFv-based or biantibody bispecific forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetrads, club-and-mortar structures, common light chains (e.g., common light chains with club-and-mortar structures, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab. 2 Bispecific form (for a review of the aforementioned forms, see, for example, Klein et al. 2012, mAbs4:6, 1-11, and the references cited therein).

[0205] In the case of the bispecific antigen-binding molecules provided herein, the polymerized domain (e.g., the Fc domain) may contain one or more amino acid variations (e.g., insertions, deletions, or substitutions) compared to wild-type, naturally occurring Fc domains. In some embodiments, this disclosure includes bispecific antigen-binding molecules in which the Fc domain contains one or more modifications such that the modified Fc domain has a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is located at C H 2 or C H Region 3 includes modifications that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with pH values ​​ranging from about 5.5 to about 6.0). 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., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; 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).

[0206] In some implementations, this document provides a method that includes a first C H 3 structural domains and second Ig C H A bispecific antigen-binding molecule with three domains, wherein the first and second IgC H The three domains differ from each other by at least one amino acid, and this difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking said amino acid difference. In one embodiment, the first IgC H 3. The domain binds to protein A and the second IgC. H Domain 3 contains mutations that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; according to EU numbering, it is H435R). Second CH 3 may also include a Y96F modification (according to IMGT; Y436F according to the EU). See, for example, U.S. Patent No. 8,586,713. Second C H Other modifications that may be found within 3 include: D16E, L18M, N44S, K52N, V57M, and V82I in the case of IgG1 antibody (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); N44S, K52N, and V82I in the case of IgG2 antibody (IMGT; N384S, K392N, and V422I according to EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I in the case of IgG4 antibody (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU).

[0207] In some implementations, the Fc domain may be a chimeric combination of Fc sequences derived from more than one immunoglobulin isotype. For example, the chimeric Fc domain may contain sequences derived from human IgG1, human IgG2, or human IgG4 C. H Part or all of Zone 2, C H 2 sequence, and partial or complete C derived from human IgG1, human IgG2 or human IgG4 H 3. Sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise a combination of an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 and a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that may be included in any of the antigen-binding molecules described herein contain, from the N-terminus to the C-terminus: [IgG4 C H 1] - [IgG4 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from the N-terminus to the C-terminus: [IgG1 C H [1] - [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. Publication 2014 / 0243504, published August 28, 2014, which is incorporated herein by reference in its entirety. Chimeric Fc domains having these general structural arrangements, and their variants, may have altered Fc receptor binding, thereby affecting Fc effector function.

[0208] Compared to the corresponding germline sequence from which the antibody is derived, the CD3 multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame regions and / or CDR regions of the heavy chain variable domain and light chain variable domain. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. In some embodiments, this disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frame regions and / or CDR regions are mutated to corresponding residues of the germline sequence from which the antibody is derived, or corresponding residues of another human germline sequence, or conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In some embodiments, V H and / or V L All frame regions and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as mutated residues found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence different 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 frame and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. After obtaining an antibody and antigen-binding fragment containing one or more germline mutations, 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., K0), can be readily tested. D Antibody-antigen binding fragments obtained in this general manner may exhibit improved or enhanced antagonistic or agonistic biological properties (as the case may be), or reduced immunogenicity, etc. This disclosure covers antibody-antigen binding fragments obtained in this general manner.

[0209] This document also provides CD3 multispecific (e.g., bispecific or trispecific) antibodies comprising variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. In some embodiments, this disclosure includes CD3 multispecific (e.g., bispecific or trispecific) antibodies having HCVR, LCVR, and / or CDR amino acid sequences having conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, such as 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.

[0210] Example CD3xMUC16 antibody In some embodiments, the methods and compositions provided herein include a bispecific antibody, wherein one arm of an immunoglobulin binds to human CD3, and the other arm of the immunoglobulin is specific for human MUC16. Unless otherwise specified as originating from a non-human species (e.g., “mouse MUC16,” “monkey MUC16,” etc.), the term “MUC16” as used herein refers to the human MUC16 protein. The human MUC16 protein has the amino acid sequence shown in SEQ ID NO: 1899 of U.S. Patent Application Publication No. US 2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein as, for example, “anti-CD3 / anti-MUC16,” “anti-CD3xMUC16,” or “CD3xMUC16” bispecific molecules or other similar terms (e.g., anti-MUC16 / anti-CD3). Such a bispecific antigen-binding molecule consists of a first antigen-binding arm that binds to MUC16 and a second antigen-binding arm that binds to CD3. The MUC16 binding arm may contain any HCVR / LCVR or CDR amino acid sequence listed in Table 7 herein. The CD3 binding arm may contain any HCVR / LCVR or CDR amino acid sequence listed in Tables 8-12 herein. The sequences in Tables 7-12 are disclosed in U.S. Patent Application Publication No. US 2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety.

[0211] Table 7 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-MUC16 antibodies disclosed herein.

[0212] Table 7: Amino Acid Sequence Identifiers Table 8 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-CD3 antibodies disclosed herein. The method for preparing anti-CD3 antibodies disclosed herein can also be found in U.S. Publication 2014 / 0088295.

[0213] Table 8: Amino Acid Sequence Identifiers Tables 9 and 10 list the amino acid sequence identifiers and their corresponding CDRs for the heavy chain variable regions (Table 9) and light chain variable regions (Table 10) of other useful anti-CD3 HCVR and LCVR antibodies disclosed herein.

[0214] Table 9 (Amino acid sequence of the heavy chain variable region) Table 10 (Amino acid sequence of the light chain variable region) Table 11 lists the amino acid sequence identifiers of the heavy chain variable region and CDR of the engineered anti-CD3 antibody disclosed herein. The amino acid sequence identifiers of the light chain variable region and CDR are also determined in Table 12 below.

[0215] Table 11: Heavy chain amino acid sequence identifiers Table 12: Light chain amino acid sequence identifiers In some exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 includes heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) from the heavy chain variable region (HCVR) selected from SEQ ID NO: 1730, 1762, 1778, 1786, and 1866, and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) from the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 117.

[0216] In some 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 SEQ ID NO: 1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 1734, 1766, 1782, 1790, and 1870; A1-HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 1736, 1768, 1784, 1792, and 1872; A1-LCDR1 comprises the amino acid sequence SEQ ID NO: 119; A1-LCDR2 comprises the amino acid sequence TAS; and A1-LCDR3 comprises the amino acid sequence SEQ ID NO: 119. The amino acid sequence of NO:312.

[0217] In some exemplary embodiments, the first antigen-binding domain that specifically binds to human CD3 comprises heavy and light chain CDRs selected from the following HCVR / LCVR amino acid sequence pairs: SEQ ID NO: 1730 / 117, 1762 / 117, 1778 / 117, 1786 / 117 and 1866 / 117.

[0218] In some exemplary embodiments, the first antigen-binding domain specifically binding 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), and the second antigen-binding domain specifically binding 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 amino acid sequences selected from SEQ ID NO: 1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises amino acid sequences selected from SEQ ID NO: 1734, 1766, 1782, 1790, and 1870; and A1-HCDR3 comprises amino acid sequences selected from SEQ ID NO: 1734, 1766, 1782, 1790, and 1870. The amino acid sequences 1736, 1768, 1784, 1792, and 1872; A1-LCDR1 contains the amino acid sequence SEQ ID NO: 119; A1-LCDR2 contains the amino acid sequence TAS; and A1-LCDR3 contains the amino acid sequence SEQ ID NO: 312; wherein A2-HCDR1 contains the amino acid sequence SEQ ID NO: 111; A2-HCDR2 contains the amino acid sequence SEQ ID NO: 113; A2-HCDR3 contains the amino acid sequence SEQ ID NO: 115; A2-LCDR1 contains the amino acid sequence SEQ ID NO: 119; A2-LCDR2 contains the amino acid sequence TAS; and A2-LCDR3 contains the amino acid sequence SEQ ID NO: 312.

[0219] Other bispecific anti-MUC16 x anti-CD3 antibodies are disclosed, for example, in WO 2018 / 067331, which is incorporated herein by reference.

[0220] Example CD3xBCMA antibody In some embodiments, the methods and compositions provided herein include a bispecific antibody, wherein one arm of an immunoglobulin binds to human CD3, and the other arm of the immunoglobulin is specific for human BCMA. Unless specifically indicated as originating from a non-human species (e.g., “mouse BCMA,” “monkey BCMA,” etc.), the term “BCMA” as used herein refers to the human BCMA protein. The human BCMA protein has the amino acid sequence shown in SEQ ID NO: 115 of U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein as, for example, “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 HCVR / LCVR or CDR amino acid sequence as shown in Table 13 herein. The CD3 binding arm may contain any HCVR / LCVR or CDR amino acid sequence listed in Table 14 herein, or an anti-CD3 antibody disclosed in WO 2014 / 047231 or WO 2017 / 053856. The sequences in Tables 13 and 14 are disclosed in U.S. Patent Application Publication No. US 2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0221] Table 13 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-BCMA antibodies disclosed herein.

[0222] Table 13: Amino Acid Sequence Identifiers Table 14 lists the heavy and light chain variable regions and the amino acid sequence identifiers of the CDRs of the selected anti-CD3 antibodies. Other anti-CD3 antibodies used to prepare bispecific antibodies according to this disclosure can be found, for example, in WO 2014 / 047231.

[0223] Table 14: Amino Acid Sequence Identifiers In some exemplary embodiments, the 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) containing the amino acid sequence SEQ ID NO: 217; and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence SEQ ID NO: 394. In some cases, the isolated bispecific antigen-binding molecule comprises HCDR1 containing the amino acid sequence SEQ ID NO: 219, HCDR2 containing the amino acid sequence SEQ ID NO: 221, and HCDR3 containing the amino acid sequence SEQ ID NO: 223. In some cases, the isolated bispecific antigen-binding molecule comprises LCDR1 containing the amino acid sequence SEQ ID NO: 396, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence SEQ ID NO: 312. In some cases, the first antigen-binding domain comprises an HCVR containing the amino acid sequence of SEQ ID NO: 217 and an LCVR containing the amino acid sequence of SEQ ID NO: 394.

[0224] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule comprises a first antigen-binding domain, the second antigen-binding domain comprising: (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) containing the amino acid sequence 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) containing the amino acid sequence SEQ ID NO: 394. In some cases, the second antigen-binding domain comprises: (a) HCDR1 containing the amino acid sequence SEQ ID NO: 740; (b) HCDR2 containing the amino acid sequence SEQ ID NO: 438 or SEQ ID NO: 406; and (c) HCDR3 containing the amino acid sequence SEQ ID NO: 1512 or SEQ ID NO: 1848. In some cases, the second antigen-binding domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 396, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 312. In some cases, the second antigen-binding domain comprises: (a) HCDR1, HCDR2, and HCDR3 domains containing the amino acid sequences of SEQ ID NO: 740, 438, and 1512, respectively; and LCDR1, LCDR2, and LCDR3 domains containing the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively; or (b) HCDR1, HCDR2, and HCDR3 domains containing the amino acid sequences of SEQ ID NO: 740, 406, and 1848, respectively; and LCDR1, LCDR2, and LCDR3 domains containing the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively. In some cases, the second antigen-binding domain comprises: (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 1610 and an LCVR comprising the amino acid sequence of SEQ ID NO: 394; or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 1866 and an LCVR comprising the amino acid sequence of SEQ ID NO: 394.

[0225] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 219, 221, and 223, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 740, 438, and 1512, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCVR containing the amino acid sequence of SEQ ID NO: 217 and an LCVR containing the amino acid sequence of SEQ ID NO: 394; and (b) a second antigen-binding domain comprising an HCVR containing the amino acid sequence of SEQ ID NO: 1610 and an LCVR containing the amino acid sequence of SEQ ID NO: 394.

[0226] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 219, 221, and 223, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 740, 406, and 1848, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 396, AAS, and SEQ ID NO: 312, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCVR containing the amino acid sequence of SEQ ID NO: 217 and an LCVR containing the amino acid sequence of SEQ ID NO: 394; and (b) a second antigen-binding domain comprising an HCVR containing the amino acid sequence of SEQ ID NO: 1866 and an LCVR containing the amino acid sequence of SEQ ID NO: 394.

[0227] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain that specifically binds to human BCMA and includes a CDR of an HCVR comprising an amino acid sequence selected from SEQ ID NO: 165, 179, 191, 203, 217, 227 and 231, and a CDR of an LCVR comprising an amino acid sequence selected from SEQ ID NO: 173, 187, 197, 211, 225, 394, 229 and 125; and (b) a second antigen-binding domain that specifically binds to human CD3. In some cases, the first antigen-binding domain comprises a CDR from the HCVR / LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 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 comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from the group consisting of: SEQ ID NO: 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 ,167-169-171-396-AAS-312, 181-183-185-396-AAS-312, 1740-193-195 -396-AAS-312, 205-207-209-396-AAS-312 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 NO: 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 a CDR of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 1610 / 394 and 1866 / 394.

[0228] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule competitively binds to BCMA or to the same epitope on BCMA with a reference antibody, wherein the reference antibody comprises a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain comprising an HCVR / LCVR pair containing the amino acid sequence SEQ ID NO:217 / 394, and the second antigen-binding domain comprising an HCVR / LCVR pair containing the amino acid sequences SEQ ID NO:1610 / 394 or SEQ ID NO:1866 / 394.

[0229] In some exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen-binding molecule competitively binds to human CD3 or to the same epitope on human CD3 with a reference antibody, wherein the reference antibody comprises a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain comprising an HCVR / LCVR pair containing the amino acid sequence SEQ ID NO: 217 / 394, and the second antigen-binding domain comprising an HCVR / LCVR pair containing the amino acid sequences SEQ ID NO: 1610 / 394 or SEQ ID NO: 1866 / 394.

[0230] Other bispecific anti-BCMA x anti-CD3 antibodies are disclosed, for example, in WO 2020 / 018820.

[0231] CD3xCD20 antibody In some embodiments, this document provides bispecific antibodies in which one arm of an immunoglobulin binds to human CD3 and the other arm of the immunoglobulin is specific for human CD20. Unless specified as originating from a non-human species (e.g., “mouse CD20,” “monkey CD20,” etc.), the term “CD20” as used herein refers to the human CD20 protein. The human CD20 protein has the amino acid sequence shown in SEQ ID NO:1369 of U.S. Patent No. 9,657,102B2, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein as, for example, “anti-CD3 / anti-CD20,” “anti-CD3×CD20,” or “CD3×CD20” bispecific molecules, or other similar terms.

[0232] In some embodiments, the first antigen-binding domain that specifically binds to CD3 includes a heavy chain variable region (HCVR) having an amino acid sequence selected from SEQ ID NO: 1250, 1266, 1282, 1298, 1314, and 1329, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. All sequences of antigen-binding domains that specifically bind to CD3 or CD20 disclosed in this section (i.e., the “CD3xCD20 antibody” section) and their corresponding SEQ ID NOs are derived from U.S. Patent No. US 9,657,102B2, the contents of which are incorporated herein by reference in their entirety.

[0233] In some embodiments, the first antigen-binding domain that specifically binds to CD3 includes a light chain variable region (LCVR) having an amino acid sequence selected from SEQ ID NO: 1258, 1274, 1290, 1306, 1322 and 1333, or a sequence substantially similar to it having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0234] In some embodiments, the first antigen-binding domain that specifically binds to CD3 comprises HCVR and LCVR (HCVR / LCVR) amino acid sequence pairs selected from SEQ ID NO:1250 / 1258, 1266 / 1274, 1282 / 1290, 1298 / 1306, 1314 / 1322 and 1329 / 1333.

[0235] In some embodiments, the first antigen-binding domain that specifically binds to CD3 includes a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from SEQ ID NO: 1252, 1268, 1284, 1300, 1316, and 1330, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from SEQ ID NO: 1254, 1270, 1286, 1302, 1318, and 1331, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from SEQ ID NO: The amino acid sequences 1256, 1272, 1288, 1304, 1320, and 1332, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from SEQ ID NO: 1260, 1276, 1292, 1308, 1324, and 1334, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them; a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from SEQ ID NO: 1262, 1278, 1294, 1310, 1326, and 1335, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from SEQ ID NO: The amino acid sequences of 1264, 1280, 1296, 1312, 1328 and 1336, or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with them.

[0236] In some embodiments, the first antigen-binding domain that specifically binds to CD3 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, each having an amino acid sequence selected from the group consisting of: SEQ ID NO: 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.

[0237] In some 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0238] In some embodiments, the second antigen-binding domain that specifically binds to CD20 includes a light chain variable region (LCVR) having an amino acid sequence selected from SEQ ID NO: 1258, 1274, 1290, 1306, 1322 and 1333, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0239] In some embodiments, the second antigen-binding domain that specifically binds to CD20 comprises HCVR and LCVR (HCVR / LCVR) amino acid sequence pairs selected from the group consisting of SEQ ID NO: 1242 / 1258, 1242 / 1274, 1242 / 1290, 1242 / 1306, 1242 / 1322 and 1242 / 1333.

[0240] In some embodiments, the second antigen-binding domain that specifically binds to CD20 includes a heavy chain CDR1 (HCDR1) domain having the amino acid sequence of SEQ ID NO: 1244 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith; a heavy chain CDR2 (HCDR2) domain having the amino acid sequence of SEQ ID NO: 1246 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith; a heavy chain CDR3 (HCDR3) domain having the amino acid sequence of SEQ ID NO: 1248 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith; and a light chain CDR1 domain having the amino acid sequence selected from SEQ ID NO: 1260, 1276, 1292, 1308, 1324, and 1334. The light chain CDR2 (LCDR2) domain has an amino acid sequence selected from SEQ ID NO: 1262, 1278, 1294, 1310, 1326 and 1335, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the light chain CDR3 (LCDR3) domain has an amino acid sequence selected from SEQ ID NO: 1264, 1280, 1296, 1312, 1328 and 1336, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0241] In some embodiments, the second antigen-binding domain that specifically binds to CD20 comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, each having an amino acid sequence selected from the following: SEQ ID NO: 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.

[0242] Other bispecific anti-CD20 / anti-CD3 antibodies are disclosed, for example, in U.S. Patent No. 9,657,102, which is incorporated herein by reference in its entirety.

[0243] Other exemplary CD3 multispecific antibodies Other exemplary CD3 multispecific antibodies that can be used in the compositions and methods disclosed herein include, but are not limited to, bispecific CD3xCD123 antibodies disclosed in, for example, U.S. Patent No. 10,787,521B2, U.S. Patent Application Publication Nos. 2018 / 0222987A1 and US 2019 / 0241657A1, and international application publications WO 2016 / 036937A1, WO 2017 / 210443A1, WO 2019 / 050521A1, WO 2019 / 210147A1, WO 2019 / 232528A1 and WO 2020 / 092404A1; bispecific CD3xSTEAP2 antibodies disclosed in international application publication No. WO 2018 / 058001A1; WO 2014 / 047231A1, WO 2015 / 143079A1, WO Bispecific CD3xCD20 antibodies disclosed in WO 2016 / 081490A1, WO 2017 / 112775A1, WO 2017 / 210485A1, WO 2018 / 114748A1, WO 2018 / 093821A8, WO 2018 / 223004A1, WO 2018 / 188612A1, WO 2019 / 155008A1, WO 2019 / 228406A1, WO 2020 / 088608A1, WO 2020 / 156405A1 and US Patent Application Publication Nos. US2020 / 0199231A1 and US 2020 / 0172627A1; International Application Publication No. WO Bispecific CD3xSSTR 2 antibody disclosed in 2018 / 005706A1; bispecific CD3xCD38 antibody disclosed in international applications WO 2015 / 149077A1 and WO2020 / 018556A1 and US patent application publications US 2018 / 0305465A1 and US 2020 / 0102403A1; bispecific CD3xSTEAP1 antibody disclosed by Olivier Nolan-Stevaux (2020) in the abstract of the proceedings of the 2020 American Association for Cancer Research Annual Meeting; bispecific CD3x5T4 antibody disclosed in international application publication WO 2013 / 041687A1, US patent application publications US 2017 / 0342160A1 and US 20200277397A1; international application publication WO The bispecific CD3xENPP3 antibody described in 2020 / 180726A1; the bispecific CD3xMUC16 antibody disclosed in international application publication numbers WO 2018 / 067331A9 and WO 2019 / 246356A1;International application publication numbers WO 2013 / 072406A1, WO 2014 / 140248A1, WO 2016 / 166629A1, WO 2017 / 031104A1, WO2017 / 134134A1, WO 2017 / 095267A1, WO 2019 / 220369A3, WO 2019 / 075359A1, WO 2019 / 226761A1, WO 2020 / 025596A1, WO 2020 / 191346A1, WO 2020018820A1; US ​​patent application publication number US 2013 / 0273055A1, US Bispecific CD3xBCMA antibody disclosed in 2019 / 0263920A1; International Application Publications WO 2012 / 055961A1, WO 2016 / 048938A1, WO 2017 / 087603A1, WO 2017 / 096368A1, WO2018 / 188612A1, WO 2019 / 237081A1, WO 2020 / 048525A1, WO 2020 / 135335A1; US ​​Patent Application Publications US 2016 / 0326249A1, US 2020 / 0283523A1, US 2019 / 0284279A1; US ​​Patent Publications US 9,315,567B2, US 7,575,923B2, US... The bispecific CD3xCD19 antibody disclosed in 7,635,472B2; the bispecific CD3xGPRC5D antibody disclosed in International Application Publication Nos. WO 2018 / 017786A3 and WO 2019 / 220369A3; the bispecific CD3xPSMA antibody disclosed in US Patent Application Publication No. US 2017 / 0320947A1; the trispecific CD3xCD28xCD38 antibody disclosed in US Patent Application Publication No. US 2020 / 0140552A1; or other CD3 multispecific antibodies disclosed in International Application Publication Nos. WO 2016 / 086189A2, WO 2020 / 088608A1, WO2019191120A1, and WO 2016 / 105450A3, the contents of which are incorporated herein by reference in their entirety.

[0244] In some embodiments, the aforementioned multispecific (e.g., bispecific or trispecific) antigen-binding molecules that specifically bind to CD3 and tumor antigens may contain K+ as measured by an in vitro affinity binding assay, exhibiting a K+ concentration greater than about 40 nM. DThe aforementioned bispecific antigen-binding molecules may include anti-CD3 antigen-binding molecules that bind to CD3 with weak binding affinity. These molecules may also include anti-CD3 antigen-binding molecules that bind to CD3 and exhibit an EC50 greater than about 100 nM as measured by FACS titration. Furthermore, these bispecific antigen-binding molecules may include anti-CD3 antigen-binding molecules that do not exhibit measurable or observable binding to CD3 as measured by in vitro affinity binding assays or FACS titrations, but still retain the ability to activate human PBMCs and / or induce cytotoxic activity against tumor antigen-expressing cell lines.

[0245] Therapeutic preparations and administration In some aspects, this document provides pharmaceutical compositions comprising NK cells expressing the CAR described herein. In some aspects, this document provides pharmaceutical compositions comprising the CD3 multispecific antigen-binding molecule described herein. In some aspects, this document provides pharmaceutical compositions wherein the CD3 multispecific antigen-binding molecule described herein is co-formulated with NK cells expressing the CAR described elsewhere herein.

[0246] The pharmaceutical compositions described herein can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to medicinal chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm Sci Technol 52:238-311.

[0247] The dosage of antigen-binding molecules administered to patients can be varied depending on the patient's age and body size, the target disease, condition, and route of administration.

[0248] Various delivery systems are known and can be used to administer the pharmaceutical compositions provided herein, such as encapsulations in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., (1987) J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or rapid injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered with other bioactive agents. Systemic or local administration is possible.

[0249] In some embodiments, the pharmaceutical compositions disclosed herein can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, compared to subcutaneous delivery, pen delivery devices have been readily applied to deliver pharmaceutical compositions as disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. After all 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 are no replaceable cartridges. Instead, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. After the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0250] Many reusable pen and auto-injector delivery devices have been used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (NovoNordisk), KWIKPEN™ (Eli Lilly), 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.

[0251] In some cases, drug compositions can be delivered using a controlled-release system. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115–138). Other controlled-release systems are discussed in a review in Langer, 1990, Science 249:1527–1533.

[0252] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable formulations can be prepared using methods known to the public. For example, injectable formulations can be prepared, for instance, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Therefore, the prepared injectable is preferably filled in a suitable ampoule.

[0253] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared into dosage forms in unit doses suitable for the dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0254] In some respects, this article provides pharmaceutical compositions comprising NK cells (e.g., inducible NK cells) expressing the CAR described herein.

[0255] In some embodiments, CAR-NK cell populations can be administered alone or as a pharmaceutical composition in combination with pharmaceutically or physiologically acceptable carriers, diluents, excipients, and / or other components or cell populations. Such compositions may contain buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions disclosed herein are formulated for intravenous administration.

[0256] CAR-NK cell administration can be performed in any convenient manner, including injection, transfusion, or implantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (iv), or intraperitoneally. In some embodiments, the disclosed compositions are administered to patients via intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered via intravenous injection. The compositions can also be injected directly into tumors or lymph nodes.

[0257] Methods of treating cancer In some embodiments, this disclosure includes methods for treating a subject. In some embodiments, the method may include administering to a subject in need in combination (e.g., simultaneously or sequentially): (1) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds a tumor antigen and a second antigen-binding domain that binds an extracellular domain. In some embodiments, the method may include administering to a subject in need in combination (e.g., simultaneously or sequentially): (1) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising a CD3 extracellular domain or a fragment thereof; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds a tumor antigen and a second antigen-binding domain that binds a CD3 extracellular domain or a fragment thereof. In some embodiments, the CD3 extracellular domain 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 CD3 extracellular domain or a fragment thereof comprises at least 10 consecutive 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 that of 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.

[0258] In some embodiments, the method may include administering a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising (1) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain including a CD3 extracellular domain or a fragment thereof; and (2) a multispecific antigen-binding molecule comprising a first antigen-binding domain binding to a tumor antigen and a second antigen-binding domain binding to the CD3 extracellular domain or a fragment thereof. The therapeutic composition may also comprise a pharmaceutically acceptable carrier or diluent.

[0259] In some respects, the method may include administering, in combination (e.g., simultaneously or sequentially) to a subject in need: (1) an antigen-binding molecule that binds to a tumor antigen; and (2) natural killer (NK) cells expressing a CAR peptide that contains an extracellular domain that binds to the antigen-binding molecule.

[0260] In some embodiments, the method may include administering, in combination (e.g., simultaneously or sequentially) to a subject in need: (1) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3 and a tumor antigen-binding domain specifically binding to a tumor antigen; and (2) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising a idiotype-specific antigen-binding domain of an anti-CD3 antibody, wherein the antigen-binding domain of the CAR peptide binds idiotype-specifically to 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 the 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 the scFv listed in Table 1.

[0261] In some embodiments, the method may include administering a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising (1) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3 and a tumor antigen-binding domain specifically binding to a tumor antigen; and (2) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising a idiotype-specific antigen-binding domain against an anti-CD3 antibody, wherein the antigen-binding domain of the CAR peptide binds idiotype to the CD3-binding domain of the multispecific antigen-binding molecule. The therapeutic composition may also comprise a pharmaceutically acceptable carrier or diluent.

[0262] In some aspects, the method may include administering, in combination (e.g., simultaneously or sequentially) to a subject in need: (a) an antigen-binding molecule that binds to a tumor antigen and contains an Fc domain; and (b) natural killer (NK) cells that express a CAR polypeptide containing an extracellular domain that binds an Fc domain.

[0263] In some embodiments, the method may include administering, in combination (e.g., simultaneously or sequentially) to a subject in need of: (1) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3, a tumor antigen-binding domain specifically binding to a tumor antigen, and an Fc domain; and (2) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising an antigen-binding domain specific to the Fc domain, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule.

[0264] 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 the 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 the scFv listed in Table 1.

[0265] In some embodiments, the method may include administering a pharmaceutical composition to a subject in need, the pharmaceutical composition comprising (1) a multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3, a tumor antigen-binding domain specifically binding to a tumor antigen, and an Fc domain; and (2) natural killer (NK) cells expressing a CAR peptide comprising an extracellular domain comprising an antigen-binding domain specific to the Fc domain, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule. The therapeutic composition may also comprise a pharmaceutically acceptable carrier or diluent.

[0266] The term “treatment” or similar terms used in this article refers to the temporary or permanent relief or elimination of symptoms or their cause. For example, “treating cancer” can refer to slowing or inhibiting tumor growth, reducing tumor cell burden or tumor load, promoting tumor regression, causing tumor shrinkage, necrosis and / or disappearance, preventing tumor recurrence, and / or prolonging the survival time of the subject.

[0267] As used herein, the term "subject in need" refers to a human or non-human mammal who exhibits one or more symptoms or indications of cancer and / or has been diagnosed with cancer and requires treatment. In many implementations, the term "subject" may be used interchangeably with the term "patient."

[0268] In some implementations, cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, tongue base cancer, laryngeal cancer, tonsil cancer, bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gingival cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, non-melanoma skin cancer (NMSC), squamous cell carcinoma of the skin (SCC), gastric cancer, testicular cancer, tongue cancer, or uterine cancer. In addition, cancer can be specifically classified into the following histological types, but is not limited to: malignant growths; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial colonic polyps. Sarcoma adenocarcinoma; Solid carcinoma; Malignant carcinoid tumor; Bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma; Chromophobe carcinoma; Eosinophilic cell carcinoma; Eosinophilic adenocarcinoma; Basophilic granulocyte carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-capsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin appendage carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cyst Adenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant blastoma; Setori cell carcinoma; Malignant stromal cell tumor; Malignant lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Globosa Sarcoma (glomangiosarcoma); Malignant melanoma; Amelanotic melanoma; Superficial disseminated melanoma; Malignant melanoma in giant nevi; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian duct mixed tumor Mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner's tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma; Malignant ovarian thyroid tumor; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Subcortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma;Malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma Tumors; granulomatoid tumors; small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specific non-Hodgkin lymphomas; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel diseases; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0269] In some embodiments, cancers treated with the methods and compositions provided herein express tumor antigens targeted by antigen-binding molecules or multispecific antigen-binding molecules (e.g., CD3 multispecific antigen-binding molecules). In some embodiments, the cancers treated with the methods and compositions provided herein may be tumors in which ≥20% of tumor cells express tumor antigens as determined by flow cytometry. Specifically, the compositions and methods disclosed herein can be used to treat, prevent, and / or improve cancers expressing or active, or containing, for example, CD20, PSMA, MUC16, STEAP2, or BCMA. + PSMA + MUC16 + STEAP2 + or BCMA + Any disease or condition related to or mediated by cell proliferation. The mechanisms of action of the treatments disclosed herein include killing cells expressing this antigen in the presence of effector cells, for example, through CDC, apoptosis, ADCC, phage activity, or a combination of two or more of these mechanisms.

[0270] 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 can be used to treat PSMA-expressing cancers, including prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0271] 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-MUC16 antibody. The compositions or methods can be used to treat cancers expressing MUC16, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma-mass-forming type, cervical adenocarcinoma, and gastrointestinal adenocarcinoma. In some embodiments, the cancer is ovarian cancer.

[0272] 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-STEAP2 antibody. The compositions or methods can be used to treat cancers expressing 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).

[0273] 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-BCMA antibody. The compositions or methods can be used to treat cancers expressing BCMA, including multiple myeloma or other B-cell or plasma cell carcinomas, such as Waldenström macroglobulinemia, Burkitt lymphoma and diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma. In some embodiments, the cancer is multiple myeloma.

[0274] 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-CD20 antibody. The compositions or methods can be used to treat CD20-expressing cancers, including non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenström macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Burkitt lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0275] In some implementations, the methods disclosed herein are used on subjects who have been treated with certain cancer drugs (e.g., cancer immunotherapy, CAR-T cell therapy, or CD3 multispecific antigen-binding molecules, such as those described herein).

[0276] In some implementations, for any of the methods disclosed herein, the subject receiving treatment or the subject being evaluated is a subject to be treated or a subject already treated with cancer immunotherapy (such as the CD3 multispecific antigen-binding molecule described herein).

[0277] In some embodiments, the methods provided herein treat, delay, or inhibit tumor growth, or induce tumor cell death. In some embodiments, the methods provided herein promote tumor regression. In some embodiments, the methods provided herein reduce tumor cell burden or alleviate tumor burden. In some embodiments, the methods provided herein prevent tumor recurrence.

[0278] In some embodiments, the disclosed natural killer (NK) cells expressing the CAR peptide and / or antigen-binding molecules (e.g., CD3 multispecific antigen-binding molecules) are administered to the patient in combination with any number of relevant therapeutic modalities (including, but not limited to, additional cancer treatments) (e.g., before, during, or after).

[0279] Additional therapeutically active ingredients may be administered before, simultaneously with, or shortly after administration of natural killer (NK) cells expressing the disclosed CAR peptide and / or antigen-binding molecule (e.g., CD3 multispecific antigen-binding molecule); (for the purposes of this disclosure, such administration regimen is considered as the combined administration of the disclosed natural killer (NK) cells expressing the disclosed CAR peptide and / or antigen-binding molecule (e.g., CD3 multispecific antigen-binding molecule) with “an additional therapeutically active ingredient”).

[0280] As mentioned above, combined administration can be simultaneous, single, or sequential. For simultaneous administration, the drugs can be administered as a single composition or as separate compositions, depending on the method of administration.

[0281] Application plan In some implementations, this document provides a method comprising administering to a subject NK cells expressing the CAR described herein at a dosing frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, provided that a therapeutic response is achieved.

[0282] In some implementations, this document provides a method comprising administering a CD3 multispecific antigen-binding molecule to a subject at a dosing frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is achieved.

[0283] In some implementations, the method includes administering a combination of NK cells expressing the CAR described herein with a CD3 multispecific antigen-binding molecule at a dosing frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is achieved.

[0284] According to certain embodiments, multiple doses of a combination of NK cells expressing the CAR described herein and a CD3 multispecific antigen-binding molecule can be administered to a subject over a predetermined time period. Methods according to this aspect of the disclosure may include sequentially administering multiple doses of NK cells expressing the CAR described herein and a CD3 multispecific antigen-binding molecule to a subject. As used herein, “sequential administration” means administering each dose of CAR-NK cells or antigen-binding molecule to a subject at different time points, such as on different dates at predetermined intervals (e.g., hours, days, weeks, or months). In some embodiments, the invention includes a method comprising sequentially administering to a patient a single initial dose of NK cells expressing the CAR described herein, followed by one or more secondary doses of NK cells expressing the CAR described herein, and optionally one or more tertiary doses of NK cells expressing the CAR described herein. In some embodiments, this disclosure also includes sequentially administering to a patient a single initial dose of a CD3 multispecific antigen-binding molecule, followed by one or more secondary doses of a CD3 multispecific antigen-binding molecule, and optionally followed by one or more tertiary doses of a CD3 multispecific antigen-binding molecule.

[0285] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the temporal sequence of administration of the antigen-binding molecules disclosed herein. Thus, “initial dose” is the dose administered at the start of a treatment regimen (also known as the “baseline dose”); “secondary dose” is the dose administered after the initial dose; and “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of the therapeutic agent described herein, but may generally differ from each other in terms of frequency of administration. However, in some embodiments, the amounts of antigen-binding molecules contained in the initial, secondary, and / or tertiary doses differ from each other during the treatment process (e.g., up- or down-adjusted as appropriate). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the start of a treatment regimen as a “loading dose,” followed by subsequent doses administered less frequently (e.g., a “maintenance dose”).

[0286] In one exemplary embodiment disclosed herein, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1½, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26) or more after the immediately preceding dose. As used herein, the phrase “immediately preceding dose” means, in a sequence of multiple administrations, the dose of the therapeutic agent described herein administered to the patient immediately before the next dose is administered, provided there is no intermediate dose in the sequence.

[0287] The methods disclosed herein may include administering any number of secondary and / or tertiary doses of the therapeutic agent described herein to a patient. For example, in some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in some embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0288] In implementations involving multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in implementations involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency of administration of secondary and / or tertiary doses to the patient may be varied during the treatment regimen. The administration frequency may also be adjusted by the physician according to the individual patient's needs after a clinical examination during the treatment process.

[0289] example Example 1: Evaluation of target dependence induced by anti-CD20 single-arm antibody using Jurkat / NFAT-Luc / ahFc-CD28-CD3z The ability of ahFc-CAR to induce alpha-CAR-dependent signaling, and the evaluation of ahFc-CAR induction using KHYG1 / ahFc-CD28-CD3z. The ability of ahFc-CAR to lyse Ramos cells.

[0290] Experimental procedure: Cell line engineering Engineering of reporter cell lines based on luciferase:The Jurkat E6 cell line, derived from human acute T-cell leukemia, was transduced using an NFAT-driven luciferase reporter construct. Puromycin-resistant cells were maintained in RPMI1640 cells supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, and 1 mg / mL puromycin. Single-cell sorting identified a single clone, which was renamed Jurkat / NFAT-Luc cl. 3C7 (ACL8722). Jurkat / NFAT-Luc cl. 3C7 was transduced using a vector encoding a chimeric construct consisting of an mROR signaling sequence, an anti-human FcscFv moiety, a G4S linker (SEQ ID NO: 89), a CD28 hinge, transmembrane and cytoplasmic domains, and a CD3z cytoplasmic domain. The blast fungicide-resistant cells were maintained in RPMI1640 cells supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 1 mg / mL puromycin, and 10 mg / mL blast fungicide. This cell line was renamed Jurkat / NFAT-Luc / ahFc-CD28-CD3z (ACL21770).

[0291] Engineering of B cell line reporter cells for evaluating cell lysis activity: The human B lymphocyte line Ramos.2G6.4C10 was transduced using a vector encoding a chimeric construct consisting of enhanced GFP (eGFP) (WP_031943942.1M1-K239), a GSGGSG adapter (SEQ ID NO: 90), and a HiBiT tag (VSGWRLFKKIS) (SEQ ID NO: 1960). The eGFP was then... + Cells were sorted and maintained in RPMI 1640 medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. The cell line was renamed Ramos / GFP (ACL21777).

[0292] Engineering of NK cell lines with cell lysis activity: The human natural killer cell leukemia-derived KHYG-1 cell line was transduced using a vector encoding a chimeric construct consisting of an mROR signaling sequence, a scFv portion targeting human Fc, a G4S linker (SEQ ID NO: 89), a CD28 hinge, transmembrane and cytoplasmic domains, and a CD3z cytoplasmic domain. Phenythiocarbamate-resistant cells were maintained in RPMI1640 cells supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 10 ng / mL IL2, and 5 mg / mL pyrocarbamate. This cell line was renamed KHYG / ahFc-CD28-CD3z (ACL21772).

[0293] Jurkat / NFAT-Luc / ahFc-CD28-CD3z signal transduction bioassay: To evaluate the target and antibody-dependent agonistic 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. Upon CAR construct aggregation, this led to activation of T-cell nuclear factor-dependent luciferase expression driven by the nuclear factor-dependent agonist (NFAT). The effects of the anti-CD20 antibody (REGN2959: anti-CD20 (10F2) single-arm antibody IgG1) and the non-target isotype control (REGN1932: isotype control IgG1) were assessed in the presence of CD20-positive (Ramos.2G6.4C10) or CD20-negative (Jurkat) cells.

[0294] RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin was used as the assay medium to prepare cell suspensions and antibody diluents. The day before screening, all reporter cells and target cells were cultured at 3 x 10⁻⁶ ppm. 5 Resuspend cells / mL. On the day of assay, resuspend Jurkat / NFAT-Luc cl.3C7 or Jurkat / NFAT-Luc / ahFc-CD28-CD3z reporter cells at 2.5 x 10⁻⁶ cells / mL. 4 Reporter cells were seeded per well in a 96-well white plate. Anti-CD20 single-arm antibody [REGN2959] or isotype control [REGN1932] was serially diluted (1:4) within a 9-point titration range (25 nM to 0.38 pM). Figure 1A and 1B Point 10 contains no antibody (represented as 0.10 pM), and 2.5 x 10⁻⁶ ppm is added. 4 Ramos.2G6.4C10 or Jurkat target cells / well were added to the cells beforehand. The plate was incubated at 37°C / 5% CO2 for 5 hours, and then 100 µL of the assay reagent was added to the wells to lyse the cells and detect luciferase activity. Emission was measured in RLUs on an Envision (PerkinElmer) multilabel plate reader.

[0295] Calculate multiple induction using the following formula: KHYG / ahFc-CD28-CD3z target cell lysis assay: To evaluate antibody-dependent NK cell activation via an anti-Fc chimeric construct, a cell lysis assay based on an NK cell line was established, in which antibodies were co-incubated with target cells and KHYG cells expressing an anti-hFc CAR construct. Aggregation of the CAR construct led to cell lysis of the target cells. Detection of the released tag in the supernatant served as a proxy for target lysis.

[0296] RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin was used as the assay medium to prepare cell suspensions and antibody diluents. The day before screening, transduced NK cell lines and target cells were cultured at 3 x 10⁻⁶ ppm. 5 KHYG / ahFc-CD28-CD3z cells were resuspended at a concentration of 10 cells / mL. On the day of testing, the cells were resuspended at 2.5 x 10⁻⁶ cells / mL. 4 Report cells / well were seeded in a 96-well white flat plate. Add 5 x 10 3 Ramos / GFP. The anti-CD20 single-arm antibody [REGN2959] or the isotype control [REGN1932] was serially diluted (1:4) within a 9-point titration range (25 nM to 0.38 pM). Figure 1A and 1B Point 10, which contains no antibody (expressed as 0.10 pM), was added to the cells, and the plate was incubated at 37°C / 5% CO2 for 5 hours before adding 100 μL of detection reagent to detect the extracellular tag. Emission was measured in RLUs on an Envision (PerkinElmer) multi-label plate reader.

[0297] Calculate the percentage of cytotoxicity using the following formula: Maximum percentage cytotoxicity is the highest repeatable average cytotoxicity within the antibody dose range.

[0298] result: Jurkat / NFAT-Luc / ahFc-CD28-CD3z signal transduction bioassay: like Figure 1A and 1B As shown, in the presence of CAR-expressing Jurkat reporter cells, target-expressing cells (Ramos), and anti-CD20 (REGN2959), a dose-dependent increase in NFAT-driven luciferase reporter gene expression was detected (maximum signal: 4.54-fold). Conversely, the absence of CAR expression on Jurkat reporter cells, target expression (using Jurkat as the target cell), or antibody targeting (using the isotype control REGN1932) did not lead to an increase in luciferase reporter gene expression (maximum signal: 1.33-fold). Figure 1A and 1B (Table 15).

[0299] Table 15 shows the maximum fold induction of signal within the antibody dose range when Jurkat / NFAT-Luc cl.3C7 or Jurkat / NFAT-Luc / ahFc-CD28-CD3 is incubated with Jurkat or Ramos and anti-CD20 (REGN2959) or isotype control (REGN1932).

[0300] Table 15. Maximum fold induction of Jurkat reporter cells in the presence or absence of ahFc-CD28-CD3 receptor, Ramos target cells, and CD20 antibody. *Maximum fold induction is defined as the highest average RLU within the ab dose range divided by the average RLU in the absence of protein.

[0301] KHYG / ahFc-CD28-CD3z target cell lysis assay: like Figure 2 As shown, in the presence of a CAR-expressing KHYG-1 NK cell line and target cells (Ramos / GFP), the addition of anti-CD20 (REGN2959) resulted in a dose-dependent increase in target cell lysis (maximum detected cell lysis: 71.65%). Conversely, the non-targeting antibody failed to induce Ramos cell lysis (maximum detected cell lysis: 5.86%). Figure 2 (Table 16).

[0302] Table 16 shows the maximum percentage of cell lysis induced within the antibody dose range when KHYG-1 / ahFc-CD28-CD3 was incubated with Ramos and anti-CD20 (REGN2959) or isotype control (REGN1932).

[0303] Table 16. Maximum percentage of cell lysis induced when KHYG-1 cells expressing ahFc-CD28-CD3 were incubated with Ramos / GFP cells and CD20 antibody. *Maximum cell lysis percentage is defined as the highest percentage of cell lysis within the ab dose range.

[0304] Example 2: Biacore of scFv targeting CD3 antibody idiotypes (09F7 and 7221G) or modified Fc (Fc*). Combining data VelocImmune (humanized) mice were immunized with either a CD3 bivalent or CD3 bispecific antibody to generate anti-idiotype antibodies. Similarly, immunization of VelocImmune mice with the aforementioned Fc-modified antibody generated antibodies that recognized a well-defined characteristic of the modified antibody's Fc domain. Antidrugs with the desired binding properties, as determined by ELISA, were reconstructed into single-chain variable fragments (ScFvs). Surface plasmon resonance (SPR) technology was used to evaluate the binding of ScFvs to antibody immunogens and antibodies with similar target specificity but different binding properties.

[0305] Biacore kinetics of binding of scFv supernatants against CD3 antibody idiotypes (09F7 and 7221G) or modified Fc (Fc*) to a panel of human antibodies were determined at 25 °C in scFv capture mode. Anti-09F7 scFv (also known as “PN29950_LCHC”) was derived from anti-idiotype antibodies produced in mice immunized with REGN1453 (anti-hCD20 x anti-hCD3-9F07). Anti-7221G scFv (also known as “PN77570_HCLC”) was derived from anti-idiotype antibodies produced in mice immunized with H4tH7221G (anti-hCD3-7221G). Anti-Fc* scFv (also known as “PN78216_HCLC”) was derived from antibodies produced in mice immunized with Fc-modified antibodies.

[0306] Experimental procedure: The equilibrium dissociation constant (KD value) of anti-idiotypic (09F7, 7221G, Fc*) scFv fused with HA-tagged supernatants bound to a set of human antibodies was determined using real-time surface plasmon resonance biosensor technology on a Biacore T-200 or 8k instrument. In brief, the CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-HA antibody (Abcam, Cat #ab18181, Clone HA.C5). All Biacore binding studies 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). scFv supernatants (targeting 09F7, 7221G, or Fc* antibodies) were captured onto the anti-HA modified surface by injection at a flow rate of 5 or 10 µL / min for 90 or 120 seconds. A single concentration (50 or 100 nM) of antibody was injected onto the captured scFv at a flow rate of 30 µL / min. scFv-antibody binding was monitored for 90 or 120 seconds, and dissociation for 120 seconds. At the end of each cycle, the scFv capturing surface was regenerated using two 10-second injections of 50 mM NaOH. All binding kinetics experiments were performed at 25 °C.

[0307] Data Analysis: Specific SPR-Biacore sensor maps were obtained using a dual-reference procedure. First, the signal from each injection on the reference surface (anti-HA) was subtracted from the signal on the experimental surface (anti-HA trapped scFv) to eliminate the effect of refractive index variations. Additionally, run buffer injections were performed to subtract signal variations caused by the dissociation of trapped scFv from the coupled anti-HA surface. The real-time sensor maps were fitted to a 1:1 binding model using Scrubber v2.0c curve fitting software or Cytiva Insight v4.0 software to determine the kinetic binding (…). k a ) and dissociation k d The rate constant is calculated from the kinetic rate constant and then combined with the dissociation equilibrium constant. K D ) and dissociation half-life (t½): K D (M) = And t½ (min) = result: Table 17 presents the kinetic results for PN29950_LCHC (resistance to 7221G scFv); Table 18 presents the kinetic results for PN77570_HCLC (resistance to 09F7 scFv); and Table 19 presents the kinetic results for PN78216_HCLC (resistance to Fc* scFv). All kinetic results were determined at 25 °C.

[0308] Table 17: Summary of kinetic and equilibrium binding parameters of various forms of anti-CD3, Fc* and control antibodies with surface-captured anti-7221G scFv (PN29950_LCHC). *NB = Not bound Table 18: Summary of kinetic and equilibrium binding parameters of various forms of anti-CD3, Fc* and control antibodies with surface-captured anti-09F7 scFv (PN77570_HCLC). *NB = Not bound **IC = Uncertain** Table 19: Summary of kinetic and equilibrium binding parameters of various forms of anti-CD3, Fc* and control antibodies with surface-captured anti-Fc* scFv (PN78216_HCLC). *NB = Not bound Example 3: Blocking ELISA data testing human CD3 anti-idiotype scFv An ELISA-based approach was used to assess the blocking effect of anti-hCD3 mAb on ELISA plates coated with hCD3 ε / δ protein in the presence of an anti-hCD3 idiotype scFv dilution.

[0309] reagents Table 20: IDs of scFvs derived from human CD3 anti-idiotype mAbs: VH – Variable heavy chain; VL – Variable light chain; CAR – Chimeric antigen receptor Table 21: Parental mAbs, ligands, and controls: mAb-monoclonal antibody Experimental procedure: Avidin (Thermo Scientific) in 5.0 µg / mL PBS was coated onto a 96-well microtiter plate and incubated overnight at 4°C. Non-specific binding sites were then blocked with 0.5% (w / v) BSA in PBS (assay buffer), and the plate was incubated at room temperature (RT) for approximately 1 hour. Then, 3.0 µg / mL hCD3 ε / δ (Acro Biosystems) was captured onto the avidin-coated ELISA plate by incubation at room temperature for 1 hour.

[0310] In 96-well dilution plates, a pre-binding blocking reaction was established using hCD3 anti-idiotype scFv and a control mAb with a constant amount of anti-human CD3 mAb. For the pre-binding reaction, human CD3 anti-idiotype scFv expressed and purified from Chinese hamster ovary (CHO) cells was serially diluted three-fold in assay buffer starting from pure supernatant; parental control mAb, scFv negative control, and isotype control mAb were serially diluted three-fold in assay buffer from 5.0 nM to 84.6 fM; biotin-hCD3 ε / δ (used as a positive control) was serially diluted three-fold in assay buffer from 4.17 µM to 70.5 pM. Serially diluted scFv-PN29950 and control mAb were mixed with 20 pM each of anti-hCD3 mAb, REGN18409, and REGN18411, and serially diluted scFv-PN277570 and control mAb were mixed with 20 pM REGN2533. Table 23 summarizes the concentrations of each hCD3 anti-idiotype scFv and control mAb used.

[0311] The pre-bound reaction mixture was incubated at room temperature for 1 hour, then transferred to an hCD3 ε / δ-coated ELISA plate and incubated at room temperature for 1 hour. Binding of each anti-hCD3 mAb (REGN18409, REGN18411, and REGN2533) was detected in the presence of their respective scFv and control mAb for 1 hour at room temperature. The test plate was developed using a TMB colorimetric substrate according to the manufacturer's recommended procedure.

[0312] The absorbance at 450 nM was recorded for each well and plotted as a function of the dilution of each hCD3 anti-idiotype scFv tested. The 11-point inhibition curves were analyzed using a four-parameter logistic equation with GraphPad Prism software. Since the concentration of the CHO supernatant was not measured, the IC50 of the scFv molecule was not calculated. 50Values ​​(reported as n / a in Table 23). Conversely, as shown in Table 23, the results of each anti-hCD3 mAb blocking each unique scFv against hCD3 are reported as a percentage of blocking.

[0313] The percentage of blockade at the lowest hCD3 anti-idiotype scFv dilution (i.e., the highest concentration) was calculated as an indicator of the molecular blocking ability of each anti-hCD3 mAb to bind to hCD3 ε / δ relative to the assay baseline. The assay baseline signal was defined as 0% binding to hCD3 ε / δ, determined based on the OD450 nM reading of the anti-hFc assay in the wells using a separate assay buffer. In the absence of hCD3 anti-idiotypes, the binding signal at 20 pM for each anti-hCD3 mAb (REGN18409, REGN18411, or REGN2533) was defined as 100% binding or 0% blockade.

[0314] Summary of results and conclusions: The ability of hCD3 anti-idiotype scFv molecules PN29950 to block REGN18409 and REGN18411, and the ability of PN77570 to block REGN2533 from binding to hCD3 ε / δ proteins on coated plates were evaluated using a blocking ELISA method. The blocking results are summarized in Table 23, and are as follows: Figures 4A-4C As shown. The percentage of blockade calculated at the highest scFv concentration (undiluted CHO supernatant) is reported. All tested hCD3 anti-idiotype scFv blocked binding to baseline at 20 pM of their respective anti-hCD3 mAb.

[0315] Parental control mAbs (REGN5766 and REGN2984) blocked 20 pM anti-hCD3 mAbs (REGN18408 / REGN18411 and REGN2533), IC 50 [M] values ​​were 42 pM / 39 pM and 32 pM, respectively, showing approximately 100% blocking activity at the highest mAb concentration. hCD3 ε / δ (ligand control) showed blocking activity at or near baseline for each anti-hCD3 mAb. Under the same assay conditions, the corresponding isotype control mAb (Southern Biotech) and scFv negative control (REGN4393) showed no blocking activity against any anti-hCD3 mAb.

[0316] Table 23: Percentage of hCD3 anti-idiotype scFv and control blocking 20pM anti-hCD3 mAb binding to immobilized hCD3 ε / δ, blocking percentage (%) and IC50. 50 Summary of [M] values. 100% non-blocking = OD of pores containing HRP-conjugated secondary proteins in a separate assay buffer. 450 nM value Experimental signal = OD of anti-hCD3 mAb binding observed in the presence of hCD3 anti-idiotype scFv or control mAb. 450 Maximum signal = OD of 20 pM anti-hCD3 mAb binding in the absence of hCD3 anti-idiotype scFv or control mAb. 450 Background signal = OD of anti-human Fc HRP binding measured only in the buffer control. 450 n / a: The IC50 value of ScFv was not calculated because the concentration was unknown.

[0317] Example 4: Evaluation of antibody and KHYG1 engineered to express chimeric antigen receptor with anti-idiotype scFv cell line combination Anti-idiotype scFvs with the desired binding strength and specificity were reconstructed into chimeric antigen receptors (CARs) and expressed in the natural killer leukemia cell line KHYG1. Target binding in KHYG1 cells engineered to express CARs targeting antibodies with modified Fc, KHYG1 / NFAT-Luc / CAR1, or CARs targeting the CD3-binding arm of CD3 bispecific antibodies, KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15, was assessed by flow cytometry. The ability of KHYG1 / NFAT-Luc / CAR1 cells to bind antibodies containing modified Fc was evaluated, as were the binding abilities of KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines to CD3 bispecific antibodies. Alexa 647-conjugated secondary antibodies were used to assess antibody binding to KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines, while antibodies testing binding to KHYG1 / NFAT-Luc / CAR1 cells were directly conjugated to Alexa 647.

[0318] Table 24: Reagent / Antibody Information / Materials Experimental procedure: Description of cell lines expressing chimeric antigen receptors targeting antibody domains: Engineering of NK reporter cell lines targeting chimeric antigen receptors with antibody domains:The human natural killer cell leukemia-derived KHYG-1 cell line was stably transduced using an activated T cell nuclear factor (NFAT)-luciferase reporter construct. A puromycin-resistant clone (ACL20834) was isolated and transduced using a chimeric construct containing an mROR signaling sequence, an scFv portion targeting a specific antibody domain such as modified human Fc (PN78216) or CD3 anti-idiotype (PN29950 and PN77570), a G4S linker (SEQ ID NO: 89), a CD28 hinge, transmembrane and cytoplasmic domains, a CD3z cytoplasmic domain, and cytoplasmic eGFP. The blast fungicide-resistant cells were maintained in RPMI1640 supplemented with 10% FBS, L-glutamine, penicillin and streptomycin, 10 ng / mL IL2, 1 g / mL puromycin, and 5 µg / mL blast fungicide. Cell lines engineered to recognize modified Fc domains were named KHYG1 / NFAT-Luc / PN78216_VH-VL-CD28bridge-TM-cyto-CD3z-eGFP (ACL22442), also known as KHYG1 / NFAT-Luc / CAR1. Cell lines engineered to recognize antigenic determinants of specific CD3 antibodies (also known as CD3 anti-idiotype) were named KHYG1 / NFAT-Luc / PN29950_VL-VH-CD28 bridge-TM-cyto-CD3z-eGFP High Sort (ACL22550) and KHYG1 / NFAT-Luc / PN77570_VH-VL-CD28 bridge-TM-cyto-CD3z-eGFP (ACL22594), also known as KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15.

[0319] Measurement setup: For the flow binding assay, KHYG1 / NFAT-Luc / CAR1, KHYG1 / NFAT-Luc / CAR6, and KHYG1 / NFAT-Luc / CAR15 cells were washed and resuspended in staining buffer (PBS with 2% FBS). 3 x 10⁻⁶ cells were then placed in the solution. 5Cells / well were added to the wells of a 96-well V-plate. 10-point 1:4 titration of antibody (range from 400 nM to 6.1 pM) was added to the cells, with the final titration point unconjugated at 1.5 pM. Antibodies used to test binding to KHYG1 / NFAT-Luc / CAR1 cells were directly conjugated to the Alexa 647 fluorophore and consisted of either an antibody with a modified Fc or a control antibody without a modified Fc (REGN5949-A647 and REGN7540-A647, respectively). Antibodies used to test binding to KHYG1 / NFAT-Luc / CAR6 and KHYG1 / NFAT-Luc / CAR15 cell lines consisted of non-fluorophore-conjugated CD3 bispecific antibodies (REGN5949, REGN5950, REGN1979) or a matched allotype control (REGN7540). Cells and antibodies were incubated at 4°C for 30 min and then washed in staining buffer. Subsequently, for the KHYG1 / NFAT-Luc / CAR6 and CAR15 cell lines, 2 µg / ml of Alexa 647-conjugated goat anti-human Fcg fragment-specific secondary antibody was diluted in staining buffer and incubated with the cells at 4°C for 30 min. After washing all cells in staining buffer, they were resuspended in viability dye (reconstituted in DMSO according to the manufacturer's protocol and diluted 1:1000 in PBS). The mixture was incubated at 4°C for 30 min, followed by washing in staining buffer. Cells were then resuspended in PFA (2% diluted in staining buffer) at 4°C for 30 min. After washing, cells were resuspended in staining buffer and analyzed by flow cytometry. The EC50 of the antibody was determined by a 4-parameter logistic equation on a 10-point dose-response curve (including secondary controls only) using GraphPad Prism software. 50 Value. In Prism, a concentration of 0 nM is plotted as 1.5 pM.

[0320] Summary of results: KHYG1 / NFAT-Luc / CAR1 binding results: KHYG-1 cells expressing an antibody against a modified Fc (Fc*) bound A647-labeled REGN5949 but could not bind to a control antibody (REGN7540) with a similar Fc (IgG4) but without modification (Table 25 and 2010). Figure 5 ).

[0321] Results of KHYG1 / NFAT-Luc / CAR6 and CAR15 combination: The ability of KHYG-1 cells expressing CARs (CAR6 or CAR15) against anti-idiotype CD3 antibodies to bind to multiple CD3xCD20 bispecific antibodies was evaluated (note that the CD3 arm used in antibodies REGN5949, REGN5950, and REGN1979 is different). Antibodies REGN5949 and REGN5950 bound to CAR6-expressing KHYG-1 cells, while binding to REGN1979 was not observed (Table 26 and...). Figure 6 (Left figure). Conversely, antibodies REGN5949 and REGN5950 did not bind to KHYG-1 cells expressing CAR15, while binding to REGN1979 was observed (Table 26 and...). Figure 6 (Right figure). The isotype control antibody (REGN7540) does not bind to KHYG-1 cells expressing CAR6 or CAR15.

[0322] Table 25: Potential value and EC50 of binding to KHYG-1 / NFAT / CAR1 cells 50 [M] and maximum multiple: Abbreviation: ND: Undetermined because no dose-dependent response was observed. Max (gMFI) is the highest gMFI value within the tested dose range.

[0323] Table 26: Potential values ​​and EC50 values ​​for binding to KHYG-1 / NFAT / CAR6 and CAR15 cells 50 [M] and maximum multiple: Abbreviation: ND: Undetermined because no dose-dependent response was observed. Max (gMFI) is the highest gMFI value within the tested dose range.

[0324] Example 5: KHYG1 / NFAT-Luc / CAR reporter signal transduction bioassay scFv with the desired binding strength and specificity was reconstructed into a chimeric antigen receptor (CAR) and expressed in the natural killer leukemia cell line KHYG1. A series of functional assays were performed to identify the ideal candidate, including an engineering reporter assay in which activation of the CAR on KHYG1 cells resulted in a luminescent signal.

[0325] Table 27: Reagent / Antibody Information / Materials: Experimental procedure: KHYG1 / NFAT-Luc / CAR1 (ahFc*-CD28-CD3z) signal transduction bioassay: To evaluate the target and antibody-dependent agonistic activity of CAR constructs targeting modified Fc, a cell-based reporter assay was established in which antibodies against CD20-modified Fc (CD20 bivalent antibodies 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 target cells, and subsequent binding of CAR1-expressing KHYG1 cells to the modified Fc, led to the aggregation of the CAR construct and subsequent activation of luciferase expression driven by the nuclear factor (NFAT) response element in activated T cells.

[0326] Measurement setup: The experiment was conducted in RPMI 1640 assay medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR1 reporter cells were cultured at 2.5 x 10⁻⁶ cells / mL. 4 Report cells / wells were seeded in 96-well white flat-bottomed plates. Subsequently, 2.5 x 10⁶ cells were seeded. 4 Ramos.2G6.4C10 (CD20) + ) or HEK293 (CD20) - Target cells / wells were added to the plate. CD20 bivalent (H4H14303N2), bispecific (REGN5949), or isotype control (REGN7540) antibodies were serially diluted (1:4) in 11 titration ranges (100 nM to 95 fM) (Figures 1 and 2), with the 12th well containing no antibody (denoted as 24 fM), and added to bring the final volume in the well to 100 µl. The plate was incubated at 37°C / 5% CO2 for 5 hours, and then 100 µL of assay reagent was added to the well to lyse the cells and detect luciferase activity. Emission was measured in RLUs on an Envision (PerkinElmer) multilabel plate reader. EC5 was determined using a 4-parameter logistic equation on the 12-point dose-response curve using GraphPad Prism software. 50 Value. In Prism, a concentration of 0 nM is plotted as 24 fM.

[0327] KHYG1 / NFAT-Luc / CAR6 and CAR15 (CD3 anti-idiotype-CD28-CD3z) signal transduction bioassay: To evaluate the target- and antibody-dependent agonistic activity of CD3 anti-idiotype CAR chimeric constructs (CAR6 and CAR15), a cell-based reporter assay was established in which CD3 x CD20 bispecific antibodies (containing different CD3-binding arms: REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or matched allotype controls (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:effector cell) ratio. Binding of the bispecific antibodies to CD20 on target cells, followed by binding of the CD3-binding arms to KHYG1 cells expressing anti-idiotype CARs, led to the aggregation of the CAR constructs and subsequent activation of luciferase expression driven by nuclear factor (NFAT) response elements in activated T cells.

[0328] Measurement setup: Experiments were performed in RPMI 1640 assay medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 reporter cells were cultured at 2.5 x 10⁻⁶ cells / mL. 4 One cell / well was seeded into a 96-well white flat-bottom plate. Then, 2.5 x 10⁶ cells were placed in each well. 4 Ramos.2G6.4C10 target cells / well were added to the plate. CD20xCD3 bispecific antibodies (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or allotype control antibodies (REGN7540) containing various CD3 binding arms were serially diluted (1:5) in a 9-point titration range (100 nM to 256 fM) (Figures 1 and 2), with the 10th point free of antibody (indicated as 51 fM), and added to the wells, for a final volume of 100 µl. The plate was incubated at 37°C / 5% CO2 for 4 hours, and then 100 µL of assay reagent was added to the wells to lyse the cells and detect luciferase activity. Emission was measured in RLUs on an Envision (PerkinElmer) multilabel plate reader. EC50 values ​​were determined using a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software. In Prism, a 0 nM concentration was plotted as 51 fM.

[0329] Calculate multiple induction using the following formula: Summary of results: KHYG / NFAT-Luc / CAR1 reporter gene activation results: In the presence of target cells expressing CD20 and an anti-CD20 antibody with a modified Fc (Fc*) recognized by the CAR, KHYG-1 cells expressing CARs containing antibodies against the modified Fc (Fc*) are activated (Table 28 and 2019). Figure 7 The control antibody H4sH14303N2, which targets CD20 but does not contain modified Fc, and the additional non-targeting control antibody REGN7540, did not activate KHYG-1 / NFAT-Luc / CAR1 cells (Table 28 and 2019). Figure 7 No activation was observed in the presence of target cells that do not express CD20 (data not shown).

[0330] KHYG1 / NFAT-Luc / CAR6 and CAR15 reporter gene activation results: KHYG-1 / NFAT-Luc cells expressing CARs (CAR6 or CAR15) targeting anti-idiotype CD3 antibodies are activated in the presence of target cells expressing CD20 and specific CD20xCD3 bispecific antibodies. Specifically, KHYG-1 / NFAT-Luc cells expressing CAR6 are activated in Ramos cells (CD20... + It was activated in a dose-dependent manner in the presence of antibodies REGN5951, REGN5375, REGN5949, REGN5950 and H4sH17400D (Tables 29, 30 and 4sH17400D). Figure 8 (Left figure). However, the antibody REGN1979 does not activate KHYG-1 / NFAT-Luc / CAR6 reporter gene activity (Tables 29, 30 and 4). Figure 8 (Left figure). Conversely, only REGN1979 was able to activate the KHYG-1 / NFAT-Luc / CAR15 reporter gene activity (Tables 29, 30, and...). Figure 8 (Right figure). The isotype control antibody REGN7540 does not activate KHYG-1 / NFAT-Luc / CAR6 or CAR15 cells (Tables 29, 30 and 40). Figure 8 ).

[0331] Table 28: Potency value and EC50 of KHYG-1 / NFAT / CAR1 cells 50 [M] and maximum activation factor: Abbreviation: ND: Undetermined because no dose-dependent response was observed. The maximum activation factor is the highest value above the antibody-free control value within the test dose range.

[0332] Table 29: Potency values ​​(EC) of KHYG-1 / NFAT / CAR6 and CAR15 cells 50 : Abbreviation: ND: Undetermined because no dose-dependent response was observed. Table 30: Maximum activation fold of KHYG-1 / NFAT / CAR6 and CAR15 cells: The maximum activation factor is the highest value above the antibody-free control value within the test dose range.

[0333] Example 6: KHYG1 / NFAT-Luc / CAR cytotoxic NK bioassay As discussed in Example 5 above, the anti-idiotype scFv with the desired binding strength and specificity was reconstructed into a chimeric antigen receptor (CAR) and expressed in the natural killer leukemia cell line KHYG1. A series of functional assays were performed to identify the ideal candidate, including cytotoxic NK killing assays using engineered KHYG-1 effector cells and Ramos / GFP-HiBiT target cells.

[0334] To assess antibody-dependent NK cell activation, a cell lysis assay based on the NK cell line was established, in which antibodies were co-incubated with target cells (Ramos / HiBit) and KHYG-1 cells expressing a CAR construct. Aggregation of the CAR construct led to cell lysis of the target cells. Target cell lysis resulted in the release of intracellular HiBit into the supernatant. An assay reagent containing the complementary peptide LgBiT was added, which spontaneously interacts with the HiBiT tag to reconstitute the bright, luminescent NanoBiT® enzyme (Promega).

[0335] Engineering of B cell reporter genes for cell lysis activity: Clones were isolated from the previously described Ramos / GFP cell line (ACL21777) by single-cell sorting and maintained in RPMI1640 supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. This cell line was renamed Ramos / GFP-HiBiT cl 1B10 (ACL22182).

[0336] The same antibody shown in Table 27 of Example 5 above was also used in this study.

[0337] Experimental procedure: KHYG1 / NFAT-Luc / CAR1(ahFc*-CD28-CD3z) Cytotoxicity Bioassay: To evaluate the target- and antibody-dependent cytotoxic activity of CAR constructs against modified Fc, an NK cell-based cytotoxicity assay was established. In this assay, Fc-modified antibodies against CD20 (CD20 bivalent antibodies 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 target cells, and subsequent binding of CAR1-expressing KHYG1 cells to the modified Fc, led to the aggregation of the CAR construct and subsequent lysis of the target cells. This was measured by the release of HiBiT into the supernatant, where it complemented lgBiT to form a highly luminescent nanobiT enzyme.

[0338] Measurement setup: The experiment was conducted in RPMI 1640 assay medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR1 cells were cultured at 2.5 x 10⁻⁶ cells / mL. 4 One cell / well was seeded into a 96-well white flat-bottom plate. Then, 5.0 x 10⁻⁶ cells were placed in the plate. 3 Ramos / HiBit target cells / wells were added to the plate. CD20 bivalent (H4H14303N2), bispecific (REGN5949), or isotype control (REGN7540) antibodies were serially diluted (1:4) in 12 titration ranges (100 nM to 95 fM) (Figures 1 and 2), with the 12th spot free of antibody (indicated as 24 fM), and added to the wells to bring the final volume to 100 µl. The plate was incubated at 37°C / 5% CO2 for 5 hours, and then 100 µL of non-lytic Nano-Glo extracellular assay reagent was added according to the manufacturer's instructions. Emission was measured in RLUs on an Envision (PerkinElmer) multilabeled plate reader. EC50 was determined using a 4-parameter logistic equation on an 11-point dose-response curve using GraphPad Prism software. 50 Value. In Prism, a concentration of 0 nM is plotted as 24 fM.

[0339] Cytotoxicity percentage x 100 Spontaneous signal = isolated target cell (without antibody) Maximum signal = determination of individual target cells lysed at the end of incubation.

[0340] KHYG1 / NFAT-Luc / CAR6 and CAR15 (CD3 anti-idiotype-CD28-CD3z) cytotoxicity bioassay: To evaluate the target and antibody-dependent cytotoxic activity of CD3 anti-idiotype CAR chimeric constructs (CAR6 and CAR15), an assay was established in which CD3 x CD20 bispecific antibodies (with a series of different CD3-binding arms, REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or matched allotype controls (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:effector cell) ratio. The binding of bispecific antibodies to CD20 on target cells and subsequent binding of the CD3 binding arm to KHYG1 cells expressing anti-idiotype CARs led to the aggregation of CAR constructs and subsequent lysis of target cells. This was measured by the release of HiBiT into the supernatant, in which HiBiT complemented LgBiT to form highly luminescent nanobiT enzymes.

[0341] Measurement setup: The experiment was conducted in RPMI 1640 assay medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. KHYG1 / NFAT-Luc / CAR6 or KHYG1 / NFAT-Luc / CAR15 cells were cultured at 2.5 x 10⁻⁶ cells / year. 4 One cell / well was seeded into a 96-well white flat-bottom plate. Then, 5 x 10 cells / well were seeded into the plate. 3 Ramos / HiBiT target cells / wells were added to the plate. CD20xCD3 bispecific antibodies (REGN5949, REGN5950, H4sH17400D, REGN1979, REGN5951, REGN5375) or isotype control antibody (REGN7540) were serially diluted (1:5) in a 9-point titration range (100 nM to 256 fM) (Figures 1 and 2), with the 10th point free of antibody (indicated as 51 μm), and added to the wells to bring the final volume to 100 µl. The plate was incubated at 37°C / 5% CO2 for 4 hours, and then 100 µL of non-lytic Nano-Glo extracellular assay reagent was added according to the manufacturer's instructions. Emission was measured in RLUs on an Envision (PerkinElmer) multilabel plate reader. EC5 was determined using a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software. 50 Value. In Prism, a concentration of 0 nM is plotted as 51 fM.

[0342] Calculate the percentage of cytotoxicity using the following formula: Cytotoxicity% x 100 Spontaneous signal = isolated target cell (without antibody) Maximum signal = determination of individual target cells lysed at the end of incubation.

[0343] Summary of results: KHYG / NFAT-Luc / CAR1 cytotoxic activation results: KHYG-1 cells expressing a CAR containing an antibody against a modified Fc (Fc*) induce CD20 in the presence of a CD20 antibody against a modified Fc (Fc*) recognized by the CAR. + Killing of target cells by Ramos / GFP-HiBiT (Table 31 and...) Figure 9 The control antibody H4sH14303N, which targets CD20 but does not contain modified Fc, and the non-targeting control antibody REGN7540 do not activate KHYG-1 / NFAT-Luc / CAR1 cells to kill Ramos target cells (Table 31 and 32). Figure 9 ).

[0344] KHYG1 / NFAT-Luc / CAR6 and CAR15 reporter gene activation results: KHYG-1 / NFAT-Luc cells expressing CARs (CAR6 or CAR15) against anti-idiotype CD3 antibodies showed a decrease in CD20 in the presence of specific CD20xCD3 bispecific antibodies. + Ramos / GFP-HiBiT kills target cells. In other words, CAR6-expressing KHYG-1 / NFAT-Luc cells induce target cell killing in a dose-dependent manner in the presence of antibodies REGN5951, REGN5375, REGN5949, REGN5950, and H4sH17400D (Table 32 and...). Figure 10 (Left figure). However, the antibody REGN1979 does not induce KHYG-1 / NFAT-Luc / CAR6 cytotoxic activity (Table 32 and...). Figure 10 (Left figure). Conversely, only REGN1979 was able to induce the killing of KHYG-1 / NFAT-Luc / CAR15 target cells (Table 32 and...). Figure 10 (Right figure). The isotype control antibody REGN7540 does not induce cytotoxic activity in KHYG-1 / NFAT-Luc / CAR6 or CAR15 cells (Table 32 and...). Figure 10 ).

[0345] Table 31: Potency values ​​and EC50 values ​​using KHYG-1 / NFAT / CAR1 cells 50 [M] and maximum lysis rate of target cells: Abbreviation: ND: Undetermined because no dose-dependent response was observed. Maximum lysis rate is the highest percentage of cytotoxicity calculated within the test dose range.

[0346] Table 32: Potency values ​​and EC50 of KHYG-1 / NFAT / CAR6 and CAR15 cells 50 [M] and maximum cytotoxicity: Abbreviation: ND: Undetermined because no dose-dependent response was observed. Maximum lysis rate is the highest percentage of cytotoxicity calculated within the test dose range.

[0347] Example 7: Target cell degeneration mediated by CBNK cells engineered with anti-CD3 idiotype chimeric antigen receptor (CAR). Cytotoxicity.

[0348] Similar to the experiment described in Example 6, antibody-dependent NK cell activation was assessed using NK cells derived from primary human cord blood (CBNK), which were engineered to express CAR constructs and co-incubated with target cells (Ramos / HiBit).

[0349] Engineering of CAR-expressing cord blood (CB) derived NK cells (CBNK): Human CD34 + -HSPC cord blood (CB) derived NK cells (CBNK) are generated using a standard synthetic biology and cell engineering (SBCE) protocol. In short, HSPC NK cell generation is facilitated using the StemSpan™ NK cell generation kit (catalog number 09960) via a two-step, serum-free, cytokine-based in vitro protocol. The first step involves introducing CD34... + HSPCs were cultured for 14 days in a medium containing amplification supplements (mainly SCF, IL7, FLT3, and TPO) to stimulate their proliferation and differentiation into lymphoprogenitor cells. At the end of this initial phase (days 8-10), the cells were engineered with a lentiviral vector (LVV) containing anti-CD3 idiotype ScFv (CAR6) and fused with a 28z-CAR membrane-bound IL-15 construct. Armored CBNK cells were sorted 48-72 hours post-transduction. In the second step, these lymphoprogenitor cells were cultured again for 14 days in a medium containing differentiation supplements (mainly IL15, IL7, SCF, FLT3, and UM729) to promote their expansion and differentiation into CD56 cells. +NK cells. These primary aCD3-ID-28z-CAR-mb15-CBNK cells (referred to as CBNK / CAR6) were ultimately expanded by culturing them for one week with engineered 41BBL-mbIL21-K562 feeder cells and then used for functional assays.

[0350] Table 33: Reagent / Antibody Information / Materials: Experimental procedure: CBNK cytotoxicity bioassay: To evaluate the target-cell and antibody-dependent cytotoxic activity of CBNK cells expressing a CD3-anti-idiotype CAR chimeric construct (CAR6), an assay was established in which a CD3 x CD20 bispecific antibody or an isotype-matched non-target x CD3 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, followed by binding of the CD3 binding arm to the anti-idiotype CAR-expressing CBNK cells, led to CAR construct aggregation and subsequent target cell lysis, which was measured by the release of HiBiT into the supernatant, where HiBiT complemented LgBiT to form a highly luminescent nanoplatelet.

[0351] Measurement setup: The experiment was conducted in RPMI 1640 assay medium supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. CBNK / CAR6 cells were cultured at 2.0 x 10⁻⁶ cells / mL. 4 One cell / well was seeded into a 96-well white flat-bottom plate. Then, 5 x 10 cells / well were seeded into the plate. 3Ramos / HiBiT target cells / wells were added to the plate. CD20xCD3 bispecific antibodies (REGN5949, REGN5950, REGN1979) or allotype-matched non-targeted control xCD3 (REGN4018) were serially diluted (1:5) in a 9-point titration range (25 nM to 64 fM) (Figures 1 and 2), with the 10th point free of antibody (indicated as 13 fM), and added to the wells to bring the final volume to 100 µL. The plate was incubated at 37°C / 5% CO2 for 4 hours, and then 25 µL was removed for cytokine assessment. Subsequently, 75 μL of non-lytic Nano-Glo extracellular assay reagent was added to the wells according to the manufacturer's instructions. Emission was measured in RLUs on a multi-label plate reader Envision (PerkinElmer). EC50 was determined using a 4-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software. 50 Value. In Prism, a concentration of 0 nM is plotted as 13 fM.

[0352] Cytotoxicity% x 100 Spontaneous signal = isolated target cell (without antibody) Maximum signal = determination of individual target cells lysed at the end of incubation.

[0353] To assess the presence of cytokines in the supernatant, the Sartorius iQue Qbeads AssayBuilder kit was used. Cytokine trap 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 the 25 µl supernatant collected from the assay wells was added to a 96-well V-plate, followed by 10 µl of trap beads. After brief centrifugation, the plate was incubated in the dark for 60 min, followed by the addition of 10 µl of assay mixture. After brief centrifugation, the plate was incubated in the dark for 90 min, followed by two washes with staining buffer (2% FBS in PBS). The sample was resuspended in 25 µl of staining buffer and transferred to a 96-well U-plate. Samples and standards were run on an iQue flow cytometer, and cytokine quantification was performed according to the manufacturer's instructions.

[0354] Summary of results: CBNK / CAR6 activation results: CBNK cells expressing a CAR against an anti-idiotype CD3 antibody (CAR6) exhibited CD20 seroconversion in the presence of a specific CD20xCD3 bispecific antibody. +Ramos / GFP-HiBiT kills target cells. That is, CBNK / CAR6 causes target cell killing in the presence of antibodies REGN5949 and REGN5950, but not in REGN1979 (Tables 34, 35, and 36). Figure 11 (See above figure). The non-targeted control x CD3 antibody REGN4018 also did not induce target cell killing (Tables 34, 35 and...). Figure 11 (See above image).

[0355] CBNK cells expressing a CAR against an anti-idiotype CD3 antibody (CAR6) in the presence of CD20 + In the presence of Ramos / GFP-HiBiT target cells and a specific CD20xCD3 bispecific antibody, cytokine release is induced. Specifically, in the presence of antibodies REGN5949 and REGN5950, CBNK / CAR6 leads to the release of IFNγ, TNFα, granzyme A, granzyme B, CCL5, and FasL, but not the release of REGN1979 (Tables 34, 35, and 36). Figure 11 (Middle and bottom figures). The non-targeted control x CD3 antibody REGN4018 also did not induce cytokine release (Tables 34, 35 and). Figure 11 (Middle and bottom images).

[0356] Table 34: Potency values ​​of CBNK / CAR6 cells, EC50 50 [M] Abbreviation: ND: Undetermined because no dose-dependent response was observed. NC: Not calculated because although a dose-dependent response was observed, the curve could not be fitted. Table 35: Maximum viability of CBNK / CAR6 cells Maximum activity is the highest percentage of cytotoxicity or cytokine release (pg / ml) within the tested dose range.

[0357] Incorporate by reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application had been specifically and individually cited and incorporated by reference. In case of any conflict, this application, including any definitions herein, shall prevail.

[0358] Equivalent solution Numerous embodiments disclosed herein have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of this disclosure. Therefore, other embodiments are also within the scope of the appended 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 pertains.

[0359] Those skilled in the art will recognize or be able to identify numerous equivalents of the specific embodiments disclosed herein using only conventional experiments. These equivalents are intended to be covered by the appended claims.

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising: (a) An extracellular domain comprising: (i) CD3 extracellular domain or a fragment thereof; (ii) Unique types of anti-CD3 antibodies possess a specific antigen-binding domain; or (iii) An antigen-binding domain that is specific to the Fc domain; (b) Hinge structural domain; (c) Transmembrane domains; and (d) Intracellular signal transduction domains.

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

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

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

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

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

8. The CAR peptide of claim 1, wherein the extracellular domain comprises a unique antigen-binding domain specific to the anti-CD3 antibody.

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

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

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

12. The CAR peptide of claim 11, wherein the antigen-binding domain comprises a heavy chain and light chain variable region sequence of one of the scFvs listed in Table 1.

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

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

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

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

17. The CAR polypeptide of claim 14, wherein the Fc domain comprises the amino acid sequence of the Fc shown in FIG3.

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

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

20. The CAR polypeptide of claim 19, wherein the hinge domain comprises an amino acid sequence selected from SEQ ID NO: 1-5.

21. The CAR peptide according to any one of claims 1-20, wherein 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.

22. The CAR polypeptide of claim 21, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 6-13.

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

24. A nucleic acid encoding a CAR polypeptide according to any one of claims 1-23.

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

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

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

28. The vector of any one of claims 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. The NK cell of claim 29 or 30, wherein the cell is a primary NK cell or an inducible NK cell differentiated from an induced pluripotent stem cell (iPSC).

32. A method of treating a subject's cancer, the method comprising administering, in combination with, the following to the subject: (A) Natural killer (NK) cells expressing a CAR polypeptide containing 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.

33. The method of claim 32, wherein the method comprises jointly administering to the subject: (A) Natural killer (NK) cells expressing the CAR polypeptide of any one of claims 2-7; 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 tumor antigens.

34. A method of treating a subject's cancer, the method comprising administering, in combination with, the following to the subject: (A) Antigen-binding molecules that bind to tumor antigens; and (B) Natural killer (NK) cells expressing a CAR peptide, wherein the CAR peptide contains an extracellular domain that binds to the antigen-binding molecule.

35. The method of claim 34, wherein the method comprises jointly 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 tumor antigens; and (B) Natural killer (NK) cells expressing the CAR polypeptide of any one of claims 8-13, wherein the antigen-binding domain of the CAR polypeptide binds idiotype to the CD3-binding domain of the multispecific antigen-binding molecule.

36. A method of treating a subject's cancer, the method comprising administering, in combination with, the following to the subject: (a) An antigen-binding molecule that binds to a tumor antigen and contains an Fc domain; and (b) Natural killer (NK) cells expressing a CAR peptide, wherein the CAR peptide comprises an extracellular domain that binds to the Fc domain.

37. The method of claim 36, wherein the method comprises jointly administering to the subject: (A) A multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3, a tumor antigen-binding domain specifically binding to tumor antigens, and an Fc domain; and (B) Natural killer (NK) cells expressing any one of the CAR peptides of claims 14-18, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule.

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

39. The method of claim 38, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NO:1-5.

40. The method of any one of claims 35-39, wherein the transmembrane domain of the CAR peptide 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.

41. The method of claim 40, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NO:6-13.

42. The method according to any one of claims 35-41, wherein the intracellular signal transduction domain of the CAR peptide is an FcgR1 intracellular signal transduction domain, a 4-1BB-CD3z intracellular signal transduction domain, a 2B4-CD3z intracellular signal transduction domain, a CD16 intracellular signal transduction domain, a CD64 intracellular signal transduction domain, or a CD28-CD3z intracellular signal transduction domain.

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

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

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

46. ​​The method of any one of claims 35-42, wherein NK cells or a premixed NK cell and antigen-binding molecule are administered after at least one dose of the antigen-binding molecule.

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

48. The method of any one of claims 35-47, 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 35-48, wherein the antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

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

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

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

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

54. 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 tumor antigens; and (B) Natural killer (NK) cells expressing the CAR polypeptide of any one of claims 8-13, wherein the antigen-binding domain of the CAR polypeptide binds idiotype to the CD3-binding domain of the multispecific antigen-binding molecule.

55. A pharmaceutical composition comprising: (A) A multispecific antigen-binding molecule comprising a CD3-binding domain specifically binding to CD3, a tumor antigen-binding domain specifically binding to tumor antigens, and an Fc domain; and (B) Natural killer (NK) cells expressing any one of the CAR peptides of claims 14-28, wherein the antigen-binding domain of the CAR peptide binds to the Fc domain of the multispecific antigen-binding molecule.

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

57. The pharmaceutical composition of claim 56, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1-5.

58. The pharmaceutical composition of any one of claims 53-67, wherein the transmembrane domain of the CAR peptide 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.

59. The pharmaceutical composition of claim 58, wherein the hinge domain of the CAR polypeptide comprises an amino acid sequence selected from SEQ ID NO: 6-13.

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

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

62. The pharmaceutical composition according to any one of claims 53-61, 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. The pharmaceutical composition of any one of claims 53-62, wherein the multispecific antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

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

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

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

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

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