Chimeric antigen receptor and its uses

Abstract

The present invention discloses a chimeric antigen receptor (CAR), which relates to the field of biopharmaceuticals. The CAR comprises a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof. The enhancer is selected from IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, their receptors, functional variants thereof, or combinations thereof. The present application also relates to CAR-NK cells comprising the CAR. The CAR targeted to tumors effectively kills tumor cells and can be used as a CAR-NK cell drug for tumor treatment.

Description

[Technical Field]

[0001] The present invention claims priority to a Chinese patent application filed with the China Patent Office on June 2, 2022, bearing application number 202210620008.2 and entitled "Chimeric Antigen Receptor and Use Thereof," and to a Chinese patent application filed with the China Patent Office on May 31, 2023, bearing application number 202310639033.X and entitled "Chimeric Antigen Receptor and Use Thereof," each of which is incorporated by reference in its entirety. The present invention relates to the field of biomedicine technology, and more particularly to a chimeric antigen receptor and its use. [Background technology]

[0002] The two main approaches to immunotherapy are immune checkpoint inhibitors, such as anti-PD1 / PDL1 antibodies and anti-CTLA4 antibodies, and chimeric antigen receptor T cells (CAR-T). The CAR-T treatment process involves isolating T cells from human peripheral blood, modifying them with CAR genes in vitro, expanding and culturing them to a certain number, and then infusing them into the patient. The core technology of CAR-T is the design and development of CAR genes. After introducing the engineered CAR gene into the T cell genome, the T cell can express the corresponding CAR molecule. The CAR molecule allows T cells to directly recognize tumor cells via the single-chain variable fragment (scFv) without requiring MHC restriction, simultaneously receiving primary and costimulatory signals for "one-touch activation." CAR-T cells can replicate in the body, forming a "living drug" with sustained killing capacity. CAR-T can also release cytokines in the body, recruiting and activating more immune cells to coordinately kill tumor cells. Although existing CAR-T drug technology is mature and has significant therapeutic effects, many issues remain. For example, patients must wait for manufacturing time (non-available), during which time tumor progression can increase treatment risk or result in missed treatment opportunities. There is also the risk of manufacturing failure and a lack of available drugs. Other issues, such as safety issues and high retail prices, remain. To address the current shortages (non-available, high prices) of autologous CAR-T cell drugs, generic, non-available cell drugs, such as generic CAR-T and generic CAR-NK, can be developed. These use allogeneic T cells or NK cells, or iPS cell-induced differentiation to obtain T cells or NK cells, to produce genuine CAR-T and CAR-NK. This allows for the production of tens of minutes of cell drug per lot. Compared to autologous CAR-T cell drugs, which require one minute of drug production per lot, generic CAR-T and CAR-NK drugs can reduce costs and retail prices.CAR-NK technology is an immune cell therapy strategy that emerged alongside autologous CAR-T technology. Initially, to achieve the goal of in vivo cell therapy, CAR-modified NK cells primarily used the NK cell line NK-92 (which can expand indefinitely). However, due to the risk of tumorigenesis of NK-92, irradiation was required before use, and CAR-NK-92 could not expand in vivo, resulting in less than ideal reported clinical efficacy. The CAR structure used in NK cells requires special design. Currently, new CAR-NK therapies are still needed to meet patient needs. Summary of the Invention [Problem to be solved by the invention]

[0003] The objective of the present invention is to provide a chimeric antigen receptor (CAR-NK) that targets BCMA, which has good tumor cell killing effect. [Means for solving the problem]

[0004] In order to achieve the above object of the invention, the technical solution of the present invention is as follows:

[0005] In one aspect, the present invention provides a fusion polypeptide, comprising a chimeric antigen receptor (CAR) according to the present invention and an enhancer capable of enhancing one or more activities of an immune effector cell, wherein said chimeric antigen receptor comprises a BCMA-binding domain and a non-antigen-binding domain, wherein said non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof, and said enhancer is one or more selected from IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, receptors thereof, and functional variants thereof.

[0006] In some embodiments, the BCMA binding domain comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4.

[0007] In some embodiments, the HCDR1 comprises an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:2, the HCDR2 comprises an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:3, and the HCDR3 comprises an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4.

[0008] In some embodiments, wherein said BCMA binding domain comprises a single domain antibody.

[0009] In some embodiments, wherein the BCMA binding domain comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:8.

[0010] In some embodiments, the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0011] In some embodiments, wherein the signaling domain of CD3ζ comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:5.

[0012] In some embodiments, wherein the non-antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:10.

[0013] In some embodiments, wherein the antigen binding domain specifically binds to a tumor antigen.

[0014] In some embodiments, wherein said tumor antigen is associated with a non-solid tumor.

[0015] In some embodiments, wherein the non-solid tumor is selected from the group consisting of lymphocytoma, Hodgkin's lymphoma, chronic and acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, and multiple myeloma.

[0016] In some embodiments, wherein said tumor antigen is associated with a solid tumor.

[0017] In some embodiments, wherein the solid tumor is selected from the group consisting of liver cancer, gastric cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer and ovarian cancer.

[0018] In some embodiments, the tumor antigen comprises a tumor antigen selected from the group consisting of CD5, CD7, CD38, CS1, BAFFR, TACI, CD19, CD20, CD22, BCMA, GPRC5D, Mesothelin, DLL1, GPC3, EGFR, PSMA, PSCA, Claudin18.2, HER2, and CD70.

[0019] In some embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment being linked to the N-terminus of the BCMA binding domain, and the signal peptide fragment comprising the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0020] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:11 or the amino acid sequence set forth in SEQ ID NO:12.

[0021] In some embodiments, wherein said enhancer comprises a cytokine and / or a cytokine receptor.

[0022] In some embodiments, wherein the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising the IL-15 receptor (IL15R).

[0023] In some embodiments, wherein the enhancer comprises the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:7.

[0024] In some embodiments, the chimeric antigen receptor and the enhancer are linked via a linker.

[0025] In some embodiments, wherein said linker comprises a cleavable linker.

[0026] In some embodiments, wherein said linker comprises a self-cleaving peptide.

[0027] In some embodiments, wherein the self-cleaving peptide comprises P2A, F2A, E2A, or T2A.

[0028] In some embodiments, the self-cleaving peptide is T2A, and the N-terminus of the chimeric antigen receptor and the C-terminus of the enhancer can be linked via T2A, resulting in a structure of IL-15-T2A-CD8SP-anti-BCMA sdAb-CD28(hinge / TM / Intracellular)-CD3ζ; or the C-terminus of the chimeric antigen receptor and the N-terminus of the enhancer can be linked via T2A, resulting in a structure of CD8SP-anti-BCMA sdAb-CD28(hinge / TM / Intracellular)-CD3ζ-T2A~IL-15.

[0029] In some embodiments, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:6.

[0030] In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:13, or an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:13.

[0031] In another aspect, the present invention provides an isolated nucleic acid molecule, which encodes a fusion polypeptide according to the present invention.

[0032] In another aspect, the present invention provides one or more isolated nucleic acid molecules comprising a nucleotide sequence encoding a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of an immune effector cell.

[0033] In some embodiments, wherein the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence set forth in SEQ ID NO:9.

[0034] In some embodiments, the nucleic acid molecule herein may comprise the nucleotide sequence set forth in SEQ ID NO:17.

[0035] In some embodiments, wherein the signaling domain of CD3ζ comprises the amino acid sequence set forth in SEQ ID NO:5.

[0036] In some embodiments, the nucleic acid molecule herein may comprise the nucleotide sequence set forth in SEQ ID NO:18.

[0037] In some embodiments, wherein the non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:10.

[0038] In some embodiments, wherein the antigen binding domain specifically binds to a tumor antigen.

[0039] In some embodiments, wherein said tumor antigen is associated with a non-solid tumor.

[0040] In some embodiments, wherein the non-solid tumor is selected from the group consisting of B lymphoma, Hodgkin's lymphoma, chronic myeloid leukemia, and acute myeloid leukemia.

[0041] In some embodiments, wherein said tumor antigen is associated with a solid tumor.

[0042] In some embodiments, wherein the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, and non-small cell lung cancer.

[0043] In some embodiments, wherein the tumor antigen comprises a tumor antigen selected from the group consisting of CD5, CD19, CD20 and BCMA.

[0044] In some embodiments, the BCMA binding domain comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4.

[0045] In some embodiments, wherein said BCMA binding domain comprises a single domain antibody.

[0046] In some embodiments, wherein the BCMA binding domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0047] In some embodiments, the nucleic acid molecule herein may comprise the nucleotide sequence set forth in SEQ ID NO:16.

[0048] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:11.

[0049] In some embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, wherein the C-terminus of the signal peptide fragment is linked to the N-terminus of the BCMA binding domain.

[0050] In some embodiments, the signal peptide fragment comprises the amino acid sequence set forth in SEQ ID NO:1.

[0051] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:12.

[0052] In some embodiments, the nucleic acid molecule herein may comprise the nucleotide sequence set forth in SEQ ID NO:21, or a nucleotide molecule having 50% identity to the nucleotide sequence set forth in SEQ ID NO:21.

[0053] In some embodiments, wherein the nucleotide sequence encoding the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NO:22.

[0054] In some embodiments, wherein the nucleotide sequence encoding the chimeric antigen receptor comprises a nucleotide sequence having 50% identity to the nucleotide sequence set forth in SEQ ID NO:22.

[0055] In some embodiments, wherein said enhancer comprises a cytokine and / or a cytokine receptor.

[0056] In some embodiments, wherein the enhancer is selected from IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, receptors thereof, functional variants thereof, and combinations thereof.

[0057] In some embodiments, wherein the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising the IL-15 receptor (IL15R).

[0058] In some embodiments, wherein the enhancer comprises the amino acid sequence shown in SEQ ID NO:7.

[0059] In some embodiments, wherein the nucleotide sequence encoding the enhancer is set forth in SEQ ID NO:20.

[0060] In some embodiments, the chimeric antigen receptor and the enhancer are linked via a linker.

[0061] In some embodiments, wherein said linker comprises a cleavable linker.

[0062] In some embodiments, wherein said linker comprises a self-cleaving peptide.

[0063] In some embodiments, wherein the self-cleaving peptide comprises P2A, F2A, E2A, or T2A.

[0064] In some embodiments, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:6.

[0065] In some embodiments, wherein the nucleic acid encoding the linker comprises the nucleotide sequence set forth in SEQ ID NO:19.

[0066] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:23.

[0067] In another aspect, the present invention provides an isolated construct or constructs comprising a nucleic acid molecule or isolated nucleic acid molecule or molecules according to the present invention.

[0068] In some embodiments, it comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a nucleotide sequence encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0069] In some embodiments, wherein the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence set forth in SEQ ID NO:9.

[0070] In some embodiments, wherein the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:17.

[0071] In some embodiments, wherein the signaling domain of CD3ζ comprises the amino acid sequence set forth in SEQ ID NO:5.

[0072] In some embodiments, wherein the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:18.

[0073] In some embodiments, wherein the non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:10.

[0074] In some embodiments, the BCMA binding domain comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4, and the second nucleic acid molecule comprises a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of an immune effector cell.

[0075] In some embodiments, wherein said BCMA binding domain comprises a single domain antibody.

[0076] In some embodiments, wherein the BCMA binding domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0077] In some embodiments, wherein the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:16.

[0078] In some embodiments, wherein the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:21.

[0079] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:11.

[0080] In some embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, wherein the C-terminus of the signal peptide fragment is linked to the N-terminus of the BCMA binding domain.

[0081] In some embodiments, the signal peptide fragment comprises the amino acid sequence set forth in SEQ ID NO:1.

[0082] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:12.

[0083] In some embodiments, wherein the first nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:22.

[0084] In some embodiments, wherein said enhancer comprises a cytokine and / or a cytokine receptor.

[0085] In some embodiments, wherein the enhancer is selected from IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, a receptor thereof, a functional variant thereof, or a combination thereof.

[0086] In some embodiments, wherein the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising the IL-15 receptor (IL15R).

[0087] In some embodiments, wherein the enhancer comprises the amino acid sequence shown in SEQ ID NO:7.

[0088] In some embodiments, wherein the second nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:20.

[0089] In some embodiments, wherein the first nucleic acid molecule and the second nucleic acid molecule are located on the same construct.

[0090] In some embodiments, the chimeric antigen receptor and the enhancer are linked via a linker.

[0091] In some embodiments, wherein said linker comprises a cleavable linker.

[0092] In some embodiments, wherein said linker comprises a self-cleaving peptide.

[0093] In some embodiments, wherein the self-cleaving peptide comprises P2A, F2A, E2A, or T2A.

[0094] In some embodiments, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:6.

[0095] In some embodiments, it comprises the nucleotide sequence shown in SEQ ID NO:23.

[0096] In some embodiments, wherein the first nucleic acid molecule and the second nucleic acid molecule are located on separate constructs.

[0097] The constructs according to the invention may be present in a vector or may be integrated into the genome.

[0098] In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0099] In another aspect, the present invention provides a cell, which comprises a nucleic acid molecule according to the invention or one or more of the isolated nucleic acid molecules described above or a construct according to the invention, and / or a chimeric antigen receptor according to the invention or a fusion polypeptide expressing the invention.

[0100] In some embodiments, wherein said cells comprise immune effector cells.

[0101] In some embodiments, the immune effector cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0102] In some embodiments, it comprises engineered immune effector cells.

[0103] In some embodiments, wherein said engineered immune effector cells comprise CAR-T cells or CAR-NK cells.

[0104] In some embodiments, the cells The present invention expresses (i) an enhancer capable of enhancing one or more activities of immune effector cells, and (ii) a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0105] The cell may be a CAR-NK cell, which expresses (i) an enhancer capable of enhancing one or more activities of the CAR-NK cell, and (ii) a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0106] In some embodiments, the cells express (i) IL-15 or a functional variant thereof, and (ii) a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0107] The cells may be CAR-NK cells that express (i) IL-15 or a functional variant thereof, and (ii) a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0108] The present invention provides an IL-15-expressing immune effector cell, wherein the cell expresses IL-15 and a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0109] In some embodiments, the IL-15 or functional variant thereof is expressed as a membrane-bound and / or secreted polypeptide. In some embodiments, the IL-15 or functional variant thereof may be expressed as a secreted polypeptide. In other embodiments, the IL-15 or functional variant thereof is fused to a transmembrane protein. A "functional variant" of IL-15 includes a portion of IL-15 that retains one or more functions of full-length or mature IL-15. IL-15 or a functional variant thereof may be expressed in multiple ways by one or more NK cells. IL-15 may be expressed in NK cells, secreted by NK cells, and / or linked directly or indirectly using any one of several linkers known in the art. In some specific aspects, IL-15 may be linked to all or a portion of a transmembrane protein. In one aspect, NK cells express a fusion protein comprising IL-15 fused to a transmembrane protein or its transmembrane domain. In some embodiments, the cell comprises (i) a first nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and (ii) a second nucleic acid molecule comprising a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of the engineered immune effector cell. In some embodiments, the cells comprise: (i) a first nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and (ii) a second nucleic acid molecule comprising a nucleotide sequence encoding the exogenous cytokine IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor.

[0110] In another aspect, the present invention provides a method of engineering a cell, said method comprising introducing a first nucleic acid molecule and a second nucleic acid molecule into said cell, wherein: (i) the first nucleic acid molecule encodes a chimeric antigen receptor, the chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and (ii) the second nucleic acid molecule encodes a nucleotide sequence of an enhancer capable of enhancing one or more activities of the engineered immune effector cell.

[0111] In another aspect, the present invention provides a pharmaceutical composition, which comprises a fusion polypeptide according to the invention, a nucleic acid molecule according to the invention, an isolated construct or constructs according to the invention or a cell according to the invention, and optionally a pharmaceutically acceptable carrier.

[0112] In another aspect, the present invention provides the use of a fusion polypeptide according to the invention, a nucleic acid molecule according to the invention, one or more isolated nucleic acid molecules according to the invention, one or more isolated constructs according to the invention, or a cell according to the invention for the manufacture of a medicament, wherein said medicament is used to treat a tumor.

[0113] The beneficial effects of the present invention are as follows:

[0114] The present invention provides a chimeric antigen receptor (CAR) molecule that uses a single-domain antibody to target BCMA. The extracellular hinge region, transmembrane region, and cytoplasmic region of the CD28 molecule are linked to the intracellular domain of CD3ζ, and the wild-type IL-15 cytokine is linked via T2A. Cell and animal experiments have shown that BCMA-targeting CAR-NKs have good tumor cell killing effects, and further development could lead to their use as CAR-NK cell drugs for the treatment of multiple myeloma. Both BCMA-targeting CAR-NKs, whether produced by cytokine amplification or by incubating and activating K562 feeder cells engineered to express mbIL-21, CD137L, and CD86 genes, have good tumor killing effects. Animal experiments have shown that CAR-NKs produced by feeder cell activation can effectively inhibit the proliferation of the bone marrow tumor cell line MM.1S. [Brief explanation of the drawings]

[0115] Specific inventive features relating to the present invention are set forth in the appended claims. A better understanding of the inventive features and advantages relating to the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, a brief description of which follows:

[0116] [Figure 1] 1 shows a schematic diagram of the structure of the anti-BCMA CAR-NK of the present invention, in which CD8SP represents the signal peptide of the CD8 molecule, BCMA sdAb represents the single domain antibody sequence targeting BCMA, CD28Hinge+TM+Intracellular represents the extracellular region, transmembrane region and cytoplasmic region sequences of the CD28 molecule, CD3ζ represents the cytoplasmic region sequence of the CD3ζ molecule, T2A represents the self-cleavage sequence, IL-15 represents the IL-15 sequence, IgG1hinge represents the IgG1 hinge region, and 2B4 Intracellular represents the 2B4 cytoplasmic region sequence. [Figure 2A]1 shows the results of detecting the positive rate of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 2 of the present invention. [Figure 2B] 1 shows the results of detecting the positive rate of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 2 of the present invention. [Figure 3] 1 shows the killing rate of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 2 of the present invention against the multiple myeloma cell line MM.1S in vitro. [Figure 4A] 1 shows the results of detecting the positive rate of anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention. [Figure 4B] 1 shows the results of detecting the positive rate of anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention. [Figure 5] 1 shows the killing rate of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention against the multiple myeloma cell line MM.1S in vitro. [Figure 6] 1 shows the comparative results of the killing rates of the multiple myeloma cell line MM.1S in vitro by anti-BCMA CAR-NK (CD28 hinge) cells and anti-BCMA CAR-NK (IgG1 hinge) cells prepared in Example 3 of the present invention. [Figure 7] 1 shows the comparative results of the killing rates of the multiple myeloma cell line MM.1S in vitro by the anti-BCMA CAR-NK (CD28 hinge) cells and anti-BCMA CAR-NK (2B4 Intracellular) cells prepared in Example 3 of the present invention. [Figure 8] 1 shows the results of comparing the killing rates of the multiple myeloma cell line MM.1S in vitro by anti-BCMA CAR-NK (CD28 hinge) cells and anti-BCMA CAR-NK (CD28 hinge + no IL-15) cells prepared in Example 3 of the present invention. [Figure 9]1 shows the results of the expression levels of the cytokine IFN-γ in anti-BCMA CAR-NK (CD28 hinge) cells and anti-BCMA CAR-NK (IgG1 hinge) cells produced in Example 3 of the present invention. [Figure 10] 1 shows the results of in vitro killing of tumor cells expressing different levels of BCMA by anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention. [Figure 11] 1 shows the effects of MM.1S anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention in an animal experiment. DETAILED DESCRIPTION OF THE INVENTION

[0117] In order to facilitate understanding of the technical means, creative features, objectives and effects achieved by the present invention, the present invention will be further described below in conjunction with specific examples. However, the following examples are only preferred examples of the present invention and are not all inclusive. Any other examples obtained by those skilled in the art without exerting creative efforts based on the examples in the embodiments are also within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment materials used in each example are the same.

[0118] Definition of Terms In the present invention, the term "BCMA" generally refers to a cell maturation antigen, which belongs to the tumor death factor receptor superfamily.

[0119] In the present invention, the term "BCMA binding domain" generally includes a humanized anti-BCMA antibody or antigen-binding fragment thereof that is capable of specifically binding to a BCMA polypeptide expressed on a B cell.

[0120] In the present invention, the term "antigen-binding fragment" (also referred to herein as "targeting moiety" or "antigen-binding moiety") generally refers to a portion of an antibody molecule, which comprises the amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope," as described above. The antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), although it does not necessarily have to comprise both.

[0121] In the present invention, the term "complementarity determining region" (CDR) generally refers to a complementarity determining region in an antigen-binding fragment variable region. In the present invention, the heavy chain variable region has three CDRs, which are designated HCDR1, HCDR2, and HCDR3 for each variable region.

[0122] In the present invention, the term "FR" generally refers to the more highly conserved parts of antibody variable domains, which are called framework regions.

[0123] In the present invention, the term "single domain antibody (sdAb)" or "VHH" refers to a type of antibody that generally lacks the antibody light chain and only has a heavy chain variable region.

[0124] As used herein, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, of which there are generally two types in the simplest embodiments, that, when present in an immune effector cell, provide the cell with specificity for a target cell (typically a cancer cell) and generate an intracellular signal. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain (e.g., a VHH, scFv, or portion thereof), a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), which includes functional signaling domains derived from stimulatory molecules and / or costimulatory molecules, as defined below.

[0125] In the present invention, the term "functional variant" generally includes an amino acid sequence that has substantially the same function and has at least 85% sequence identity therewith.

[0126] In the present invention, the term "isolated nucleic acid molecule" refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, generally isolated from their natural environment or artificially synthesized.

[0127] In the present invention, the term "construct" generally refers to a nucleic acid molecule capable of autonomous replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells.

[0128] In the present invention, the term "immune effector cell" is an immune cell that generally participates in an immune response and exerts effector functions.

[0129] In the present invention, the term "natural killer cells" ("NK cells") generally refers to a type of cytotoxic lymphocyte of the immune system.

[0130] In the present invention, the term "interleukin-15" refers to a cytokine that generally regulates the activation and proliferation of T and NK cells. In one embodiment, the IL-15 is wild-type IL-15.

[0131] In the present invention, the term "expression" generally refers to the transcription and / or translation of a particular nucleotide sequence.

[0132] In the present invention, the terms "tumor" and "cancer" are used interchangeably and generally refer to diseases characterized by the rapid and uncontrolled growth of abnormal cells. Examples of cancer herein include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. It includes pre-cancerous and malignant cancers and tumors, and covers solid tumors and non-solid tumors.

[0133] In the present invention, the term "administration" generally refers to the delivery of a protein to a human or animal in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art.

[0134] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonicity and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, and the like, that are compatible with drug administration.

[0135] As used herein, the term "effective amount" or "effective dose" is generally an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of drug that promotes disease regression when used alone or in combination with another therapeutic agent.

[0136] In the present invention, the term "comprise" generally means to include, generalize, contain or encompass.

[0137] In the present invention, the term "subject" generally refers to a human or non-human animal.

[0138] Example 1 1.1 Construction of chimeric antigen receptors As shown in Figure 1, the anti-BCMA single-domain antibody sequence (SEQ ID NO: 8) was used, and the extracellular hinge region, transmembrane region, and cytoplasmic region of the CD28 molecule (SEQ ID NO: 9) was combined with the intracellular domain of CD3ζ (SEQ ID NO: 5). The wild-type IL-15 cytokine (SEQ ID NO: 7) was linked via T2A (SEQ ID NO: 6) to construct a BCMA-targeting CAR construct suitable for NK cells. The lentiviral Pre-Lenti-EF1-CAR V2 vector and the BCMA CAR gene (SEQ ID NO: 13) were enzymatically cleaved, ligated, transformed, cloned, plasmid extracted, and sequenced to obtain the correct lentiviral vector Pre-Lenti-EF1-CAR V2 NCI-NK.

[0139] 1.2 Production of BCMA chimeric antigen receptor lentivirus 1) 293TS cells were cultured in a 37°C, 5% CO2 incubator at 150 rpm. The cells to be transfected were taken and counted, and the cell density was 6x 10 6 cells / ml-7× 10 6 The cell viability is greater than 90% and available for transfection.

[0140] 2) The Pre-Lenti-EF1-CAR V2 NCI-NK plasmid, packaging plasmids ZL004:ZL006, and envelope plasmid ZL003 were thoroughly mixed in a ratio of 9:7:7:7, and the mixture of these four plasmids was added to a tube containing 293TS base medium. PEIpro was added to another tube at a ratio of 1:2 plasmid:PEIpro. After incubating the two tubes at room temperature for 5 min, the PEIpro-containing mixture was slowly added to the DNA-containing mixture. The centrifuge tube was gently shaken to mix uniformly and incubated at room temperature for approximately 15 min. The DNA-PEIpro mixture was added to the cells to be transfected, mixed thoroughly, and cultured with shaking at 37°C and 5% CO2. After 6 h, the cells were replenished. After 48 h, the lentivirus-containing culture supernatant was collected. The virus-containing culture supernatant was transferred to a 50 ml centrifuge tube and centrifuged to remove 293TS cells. The virus-containing supernatant was filtered, concentrated, aliquoted, and stored at -80°C for storage.

[0141] Example 2: Production of CAR-NK by cytokine-amplifying activation 1) Isolation of PBMCs In a sterile environment, 60 ml of venous blood was drawn from a volunteer. The blood was transferred to two 50 ml centrifuge tubes, mixed thoroughly, and centrifuged at 800 g and 18–22°C for 15 min. Plasma was aspirated into the 50 ml centrifuge tube. After removing the plasma, an equal volume of 0.9% NaCl was added to the whole blood for dilution and mixed thoroughly. 15 ml of human lymphocyte isolation solution was added to each new 50 ml centrifuge tube. 20 ml of diluted blood was slowly added to the isolation solution along the side of the tube and centrifuged at 400 g and 20°C for 30 min in a horizontal rotor (ascent rate: 3, descent rate: 0). The white film layer was carefully aspirated into a new 50 ml centrifuge tube, diluted to 40 ml with 0.9% NaCl injection, and the tube was mixed thoroughly by pipetting. The tube was centrifuged at 450 g and 20°C for 10 min in a horizontal rotor. The supernatant was discarded and the washing procedure was repeated once. After centrifugation, the supernatant was discarded, and the cells were resuspended in 20 ml of saline and counted.

[0142] 2) Depletion of CD3 T cells Miltenyi CD3 Reagent was used to deplete CD3 T cells. 6× 10 7 PBMC cells were collected and centrifuged at 300 g at 20°C for 10 minutes. The supernatant was discarded, and magnetic bead separation solution (80 μl / 1× 10 7 Resuspend the cells by adding CD3 Reagent (5 μl / 1× 10 7 The cells were added, mixed well, and placed in a refrigerator at 2-8°C for 15 minutes. Five ml of magnetic bead separation solution was added to the cells, mixed well by pipetting, and centrifuged at 300 g and 20°C for 10 minutes. The LS Columns separation column was attached to a magnet and rinsed with 3 ml of magnetic bead separation solution. The supernatant was discarded, and the cells were resuspended in magnetic bead separation solution (100 μl / 1×). 10 7 The cells were mixed well by pipetting and passed through the column. The column was washed three times with 3 ml of magnetic bead separation solution. The flow-through was collected.

[0143] 3) NK cell purification The effluent after T cell removal was sampled and counted. The cells were centrifuged at 300 g for 10 min. Magnetic bead separation solution (80 μl / 1×) was added. 10 7 Resuspend the cells by adding CD56 MicroBeads (20 μl / 1× 10 7 The cells were added to the magnetic bead separation solution, mixed thoroughly, and left in a refrigerator at 2-8°C for 15 minutes. The cells were resuspended by adding magnetic bead separation solution, mixed thoroughly, and centrifuged at 300 g for 10 minutes. The LS Columns separation column was attached to a magnet and rinsed with 3 ml of magnetic bead separation solution. The supernatant was discarded, and the cells were resuspended in magnetic bead separation solution (500 μl / 1×). 10 8The cells were mixed well by pipetting and passed through a column. The column was washed three times with 3 ml of magnetic bead separation solution. The column was removed, 5 ml of magnetic bead separation solution was added, and the mixture was pushed into a 15 ml centrifuge tube. The mixture was centrifuged at 300 g and 20°C for 10 min. The supernatant was discarded, and the cells were resuspended in 10 ml of 0.9% sodium chloride injection, mixed well, counted, and centrifuged at 300 g and 20°C for 10 min. The supernatant was discarded, and the cells were resuspended in NK medium (1% of NK MACS Supplement + NK MACS Basal Medium + IL-2 (500 IU / ml) + IL-15 (140 IU / ml) + 3% autologous plasma). The cell density was adjusted to 1x 10 6 / ml and inoculated into a 12-well plate at 1 ml / well.

[0144] 4) Infection of NK cells with lentivirus On day 3, NK cells were observed and most of them were found to have aggregated into clumps. They were mixed thoroughly by pipetting. Vectofusin-1 was added to a final concentration of 10 μg / ml, and virus was added at an MOI of 15. On day 4, the medium was replenished. On day 5, the cells were transferred to a 6-well plate and the medium was replenished. On day 7, the medium was replenished. On day 8, the CAR-positive rate of BCMA CAR NK cells was detected using target protein binding flow cytometry.

[0145] The results are shown in Figures 2A and 2B. Figure 2A is a negative control, and Figure 2B shows the positive rate (71%) of CAR molecule expression that recognizes BCMA using a BCMA target protein.

[0146] Example 3 Production of anti-BCMA CAR-NK by feeder cell expansion activation 1) Isolation of PBMCs Blood samples were collected from healthy volunteers. They were transferred to 50ml centrifuge tubes and diluted with 1:1 volume of 0.9% NaCl injection. New 50ml centrifuge tubes were taken and 15ml of room-temperature human lymphocyte isolation solution was added to each tube. 20ml of diluted blood was slowly added to the isolation solution along the side of the tube. The tubes were placed in a centrifuge at 400g and 20°C for 30 minutes (ascending speed: 3, descending speed: 3). The white film layer was carefully aspirated into the other two 50ml centrifuge tubes. 0.9% sodium chloride injection was added to the tubes to make up to 40ml, and the tubes were mixed well by pipetting using a pipette gun. The tubes were centrifuged at 400g and 20°C for 10 minutes. The supernatant was discarded, and 0.9% sodium chloride injection was added to resuspend the lymphocytes. The injection solution was then added to make up to 40ml. The tubes were centrifuged at 400g and 20°C for 10 minutes. After centrifugation, the supernatant was discarded and 20 ml of 0.9% sodium chloride injection was added. After thorough mixing, the cells were sampled and counted.

[0147] 2) Depletion of CD3 T cells Miltenyi CD3 Reagent was used to deplete CD3 T cells. 1× 10 8 PBMC cells were collected and centrifuged at 300 g at 20°C for 10 minutes. The supernatant was discarded, and magnetic bead separation solution (80 μl / 1× 10 7 Resuspend the cells by adding CD3 Reagent (5 μl / 1× 10 7 The cells were added, mixed well, and placed in a refrigerator at 2-8°C for 15 minutes. Five ml of magnetic bead separation solution was added to the cells, mixed well by pipetting, and centrifuged at 300 g and 20°C for 10 minutes. The LS Columns separation column was attached to a magnet and rinsed with 3 ml of magnetic bead separation solution. The supernatant was discarded, and the cells were resuspended in magnetic bead separation solution (100 μl / 1×). 10 7 The cells were mixed well by pipetting and passed through the column. The column was washed three times with 3 ml of magnetic bead separation solution. The flow-through was collected.

[0148] 3) Purification of NK cells. The effluent after T cell removal was sampled and counted. The cells were centrifuged at 300 g for 10 min. Magnetic bead separation solution (80 μl / 1×) was added. 10 7 Resuspend the cells by adding CD56 MicroBeads (10 μl / 1× 10 7 The cells were added to the magnetic bead separation solution, mixed thoroughly, and left in a refrigerator at 2-8°C for 15 minutes. The cells were resuspended by adding magnetic bead separation solution, mixed thoroughly, and centrifuged at 300 g for 10 minutes. The LS Columns separation column was attached to a magnet and rinsed with 3 ml of magnetic bead separation solution. The supernatant was discarded, and the cells were resuspended in magnetic bead separation solution (500 μl / 1×). 10 8 The cells were mixed thoroughly by pipetting and passed through a column. The column was washed three times with 3 ml of magnetic bead separation solution. The column was removed, 5 ml of magnetic bead separation solution was added, and the mixture was placed in a 15 ml centrifuge tube. The mixture was centrifuged at 300 g and 20°C for 10 minutes. The supernatant was discarded, and the cells were resuspended in 10 ml of 0.9% sodium chloride injection, mixed thoroughly, counted, and centrifuged at 300 g and 20°C for 10 minutes. The supernatant was discarded, and the cells were resuspended in NK medium.

[0149] 4) NK cell activation 30 ml NK cell medium, 7.5 × 10 6 NK cells (2.5×10 5 / ml) and 7.5× 10 6 K562-mbIL-21 cells and (irradiation, 100 Gy) (2.5 × 10 5 The cells were placed in a T-75 vial and mixed thoroughly at a 1:1 ratio. The cells were then placed in a 37°C, 5% CO2 incubator and cultured. The cells were observed on days 2, 3, and 4. On day 5, the liquid was exchanged by centrifugation, the cells were transferred to a 50 ml centrifuge tube, mixed thoroughly, counted, and centrifuged at 300 g, 20°C, for 10 min. The supernatant was discarded, and the cells were resuspended in NK cell medium (1% of NKMACS Supplement + NKMACS Basal Medium + IL-2 (200 IU / ml) + 5% autologous plasma). The cell density was adjusted to 1× 106 The concentration was adjusted to / ml.

[0150] 5) Infection of NK cells with lentivirus On day 6, NK cells were observed and found to be fully activated and actively proliferating. Plates were coated with Retronectin, cells were inoculated, Vectofusin-1 was added, and virus was added at a final concentration of 10 μg / ml and an MOI of 10.

[0151] On day 7, the medium was replenished.

[0152] On day 8, secondary activation of CAR NK cells was performed. CAR NK cells and K562-mbIL-21 cells were mixed thoroughly at a 1:1 ratio. The mixture was placed in a 37°C, 5% CO2 incubator and cultured.

[0153] The cells were observed on the 9th and 10th days.

[0154] On day 11, the liquid was exchanged by centrifugation, and the cells were transferred to a 50 ml centrifuge tube, mixed thoroughly, counted, and centrifuged at 300 g, 20°C, for 10 min. The supernatant was discarded, and the cells were resuspended in NK cell medium (1% of NK MACS Supplement + NK MACS Basal Medium + IL-2 (200 IU / ml) + 3% autologous plasma). The cell density was adjusted to 1× 10 6 The concentration was adjusted to / ml.

[0155] The cells were observed on day 12.

[0156] On day 13, the cells were replenished and the cell density was increased to 1× 10 6 / ml.

[0157] On day 14, target protein-binding flow cytometry was used to detect the CAR positivity rate of BCMA CAR NK cells.

[0158] The results are shown in Figures 4A and 4B. Figure 4A is a negative control, and Figure 4B shows the positive rate (62%) of CAR molecule expression that recognizes BCMA using a BCMA target protein.

[0159] Example 4 Evaluation of anti-BCMA CAR-NK cell function In vitro cell killing experiments were performed using an LDH detection kit (Promega). Four gradients were set up with the anti-BCMA CAR-NK cells prepared in Examples 2 or 3 and target cells (MM.1S cells, which highly express BCMA molecules, purchased from ATCC) at ratios of 1:2, 1:4, 1:8, and 1:16 (i.e., effector cell-target cell ratio). 3 x 10 target cells were used. 4 Each well contained 100 cells / well, and 200 μL of XVIVO-containing medium / 1640 medium was added to all remaining wells. The 96-well plate was then placed in a 37°C, 5% CO2 incubator for incubation. After 17 hours, 20 μL of lysis solution was added to the well with the highest release, mixed thoroughly to completely disrupt the cells, and the 96-well plate was then placed in a CO2 incubator for 2 hours. After 2 hours, the wells with the highest release were observed. After all target cells were lysed, 50 μL of supernatant was aspirated from each well and placed in a flat-bottom 96-well plate. 50 μL of substrate solution was added to each well and allowed to develop for 30 minutes, protected from light. After 30 minutes, the color change was observed, with the highest release MM.1S well and the well containing anti-BCMA CAR-NK cells showing a relatively darker color. Measurements were performed using a microplate reader at 490 nm. Killing activity was detected using an LDH detection kit.

[0160] The killing activity of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 2 against MM.1S cells is shown in Figure 3. The results demonstrated that the anti-BCMA CAR-NK cells prepared in Example 2 were able to specifically kill BCMA-positive cells, with high killing activity of over 20% in all cases.

[0161] The killing activity of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 against MM.1S cells is shown in Figure 5. The results demonstrated that the anti-BCMA CAR-NK cells prepared in Example 3 were able to specifically kill BCMA-positive cells, with high killing activity of over 40% in all cases.

[0162] The killing activity of the anti-BCMA CAR-NK (CD28 hinge; CAR sequence is shown in SEQ ID NO: 13) cells, anti-BCMA CAR-NK (IgG1 hinge; CAR sequence is shown in SEQ ID NO: 14) cells, anti-BCMA CAR-NK (2B4 Intracellular; CAR sequence is shown in SEQ ID NO: 15) cells, and anti-BCMA CAR-NK (CD28 hinge + no IL-15; CAR sequence is shown in SEQ ID NO: 12) cells prepared in Example 3 on MM.1S cells was shown in Figures 6 to 8. The results showed that the in vitro killing effect of anti-BCMA CAR-NK cells whose hinge region was the extracellular region of CD28 was superior to that of anti-BCMA CAR-NK cells whose hinge region was the hinge region of an IgG1 molecule (Figure 6), the in vitro killing effect of anti-BCMA CAR-NK cells whose cytoplasmic region was CD28 was superior to that of anti-BCMA CAR-NK cells whose cytoplasmic region was 2B4 (Figure 7), and the in vitro killing effect of anti-BCMA CAR-NK cells capable of secreting IL-15 was superior to that of anti-BCMA CAR-NK cells that expressed CAR molecules but did not secrete IL-15 (Figure 8).

[0163] After killing, the supernatant was collected and the level of the cytokine IFN-γ was measured. The results, as shown in Figure 9, showed that compared to anti-BCMA CAR-NK cells whose hinge region was the IgG1 molecule hinge region, anti-BCMA CAR-NK cells whose hinge region was the CD28 extracellular region significantly upregulated the expression level of the cytokine IFN-γ, which has tumor-killing activity in vitro, verifying the specific killing activity of anti-BCMA CAR-NK (CD28 hinge) cells.

[0164] Example 5 In vitro cell killing experiments were performed using an LDH detection kit (Promega). The anti-BCMA CAR-NK cells prepared in Example 3 and target cells (RS4 and 11 tumor cells that do not express BCMA, and Daudi and NCI-1299 tumor cells that express BCMA) were used in four gradients (effector cell-target cell ratios) of 1:2, 1:4, 1:8, and 1:16. Here, 3 x 10 target cells were used. 4 Cells were released per well, and 200 μL of X-VIVO-containing medium / 1640 medium was added to all remaining wells. The 96-well plate was then placed in a 37°C, 5% CO2 incubator for incubation. After 17 hours, 20 μL of lysis solution was added to the well with the highest release, mixed thoroughly, and the cells were completely disrupted. The 96-well plate was then placed in a CO2 incubator and incubated for 2 hours. After 2 hours, the wells with the highest release were observed. After all target cells were lysed, 50 μL of supernatant was aspirated from each well and placed in a flat-bottom 96-well plate. 50 μL of substrate solution was added to each well and allowed to develop for 30 minutes in the dark. After 30 minutes, the color change was observed, with the highest release MM.1S wells and wells containing anti-BCMA CAR-NK cells showing a relatively darker color. Measurements were performed using a microplate reader at 490 nm. Killing activity was detected using an LDH detection kit.

[0165] Figure 10 shows that anti-BCMA CAR-NK (CD28 hinge) cells kill tumor cells expressing different levels of BCMA in vitro. The results show that 11 did not kill RS4, which does not express BCMA, but did kill both Daudi and NCI-1299 tumor cells, which express BCMA. This demonstrates that the anti-BCMA CAR-NK (CD28 hinge) cells of the present application are BCMA specific.

[0166] Example 6 The human multiple myeloma MM.1S cell line was transduced to express the luciferase reporter gene (luciferase) to obtain MM.1S-luc. The cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) in a 5% carbon dioxide, 37°C incubator. The MM.1S-luc cell line was cultured at 1.5× 10 6 NSG mice were injected with 200 μL of cells / 200 μL of PBS (50 mice, half male and half female) via the tail vein. 17 days later, all animals were imaged. Mice with uniform tumor burden were included in the experimental group and randomly divided into five groups (half male and half female, 8 mice per group): PBS, NK cell, anti-BCMA CAR-NK (CD28 hinge) cell, and BCMA CAR-T (CD28 hinge) cell groups. PBS was injected into the extracellular solution group, NK cells were injected into the NK cell group, anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 were injected into the anti-BCMA CAR-NK (CD28 hinge) cell group, and BCMA CAR-T (CD28 hinge) cell group were injected into the tail vein. Three days later, the mice were anesthetized and intraperitoneally injected with luciferase substrate. The tumor burden in the control and treatment groups was observed using a small animal in vivo imaging system. The results on days 3, 6, 12, and 30 after treatment are shown in Figure 11.

[0167] As can be seen from the results, there was almost no tumor cell imaging in the anti-BCMA CAR-NK cell treatment group, while the control group mice had severe tumor burden, indicating that anti-BCMA CAR-NK cells can effectively eliminate BCMA-positive tumor cells. After intravenous administration of anti-BCMA CAR-NK cells, on day 3, the fluorescent signal intensity in the mice began to decrease compared to the PBS and NK cell groups, indicating a decrease in tumor burden. As can be seen from Figure 11, on day 6, the fluorescent signal intensity in the mice was essentially undetectable, demonstrating that anti-BCMA CAR-NK demonstrated potent therapeutic effects against BCMA-associated diseases in in vivo animal experiments.

[0168] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent changes, improvements, etc. made without departing from the spirit and principle of the present invention are all included within the protection scope of the present invention.

Claims

1. 1. A fusion polypeptide comprising: a chimeric antigen receptor; and an enhancer capable of enhancing one or more activities of an immune effector cell, wherein the chimeric antigen receptor comprises a BCMA-binding domain and a non-antigen-binding domain, the non-antigen-binding domain comprising the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof, and the enhancer is one or more selected from IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, their receptors, and functional variants thereof.

2. 2. The fusion polypeptide of claim 1, wherein the BCMA-binding domain comprises a heavy chain complementarity determining region 1, a heavy chain complementarity determining region 2, and a heavy chain complementarity determining region 3, wherein the heavy chain complementarity determining region 1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the heavy chain complementarity determining region 2 comprises the amino acid sequence set forth in SEQ ID NO: 3, and the heavy chain complementarity determining region 3 comprises the amino acid sequence set forth in SEQ ID NO:

4.

3. The fusion polypeptide of claim 2 , wherein the BCMA binding domain comprises a single domain antibody.

4. The fusion polypeptide of claim 3, wherein the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 8 or a functional variant thereof.

5. The fusion polypeptide of claim 1, wherein the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 comprise the amino acid sequence shown in SEQ ID NO:

9.

6. The fusion polypeptide of claim 1, wherein the signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

5.

7. The fusion polypeptide of claim 1, wherein the non-antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The fusion polypeptide of claim 1, wherein the chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment being linked to the N-terminus of the antigen-binding domain, and the signal peptide fragment comprises the amino acid sequence set forth in SEQ ID NO:

1.

9. The fusion polypeptide of claim 8, wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 11 or the amino acid sequence shown in SEQ ID NO:

12.

10. The fusion polypeptide of claim 1, wherein the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor.

11. The fusion polypeptide of claim 10, wherein the enhancer comprises the amino acid sequence shown in SEQ ID NO:

7.

12. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO:

13.

13. An isolated nucleic acid molecule, characterized in that it encodes a fusion polypeptide according to any one of claims 1 to 12.

14. 1. The isolated nucleic acid molecule or molecules, wherein the isolated nucleic acid molecule or molecules comprise a nucleotide sequence encoding a chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of an immune effector cell.

15. 15. The isolated nucleic acid molecule or molecules of claim 14, wherein the nucleotide sequence encoding the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NO:

22.

16. 15. The isolated nucleic acid molecule or molecules according to claim 14, wherein the nucleotide sequence encoding the enhancer is set forth in SEQ ID NO:

20.

17. 15. The isolated nucleic acid molecule or molecules according to claim 14, comprising the nucleotide sequence set forth in SEQ ID NO:

23.

18. 18. An isolated construct or constructs, characterized in that they comprise a nucleic acid molecule according to claim 13 or an isolated nucleic acid molecule or molecules according to any one of claims 14 to 17.

19. 19. The isolated construct or constructs of claim 18, comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a nucleotide sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof, and the second nucleic acid molecule comprises a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of an immune effector cell.

20. A cell, characterized in that it contains a nucleic acid molecule according to claim 13 or one or more isolated nucleic acid molecules according to any one of claims 14 to 17 or one or more isolated constructs according to claim 18 or 19, and / or it expresses a fusion polypeptide according to any one of claims 1 to 12.

21. 21. The cell of claim 20, wherein the cell comprises an immune effector cell.

22. 22. The cell of claim 21, wherein the immune effector cells comprise T cells, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

23. 23. The cell of claim 22, comprising an engineered immune effector cell.

24. The cell of claim 23, wherein the engineered immune effector cell comprises a CAR-T cell or a CAR-NK cell.

25. 1. A method of engineering a cell, comprising introducing into the cell a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a chimeric antigen receptor, the chimeric antigen receptor comprising a BCMA-binding domain and a non-antigen-binding domain, wherein the non-antigen-binding domain comprises the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; and the second nucleic acid molecule encodes a nucleotide sequence of an enhancer capable of enhancing one or more activities of the engineered immune effector cell.

26. A pharmaceutical composition comprising a nucleic acid molecule according to claim 13, or one or more isolated nucleic acid molecules according to any one of claims 14 to 17, or one or more isolated constructs according to claims 18 or 19, or a cell according to any one of claims 20 to 25, and optionally a pharmaceutically acceptable carrier.

27. 26. Use of a fusion polypeptide according to any one of claims 1 to 12, a nucleic acid molecule according to claim 13, or one or more isolated nucleic acid molecules according to any one of claims 14 to 17, or one or more isolated constructs according to claims 18 or 19, or a cell according to any one of claims 20 to 25, for the manufacture of a drug, wherein the drug is used to treat a tumor.

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