Chimeric antigen receptor and use thereof

Abstract

A chimeric antigen receptor, which relates to the technical field of biomedicine. The chimeric antigen receptor comprises a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain comprises an extracellular domain, a transmembrane domain and an intracellular co-stimulatory domain of CD28 or functional variants thereof, and a 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, a receptor thereof, a functional variant thereof, or a combination thereof. The present application also relates to a CAR-NK cell comprising the chimeric antigen receptor. The tumor-targeting CAR-NK cell has a good killing effect on tumor cells, and can be used as a CAR-NK cell drug for treating tumors.

Description

Chimeric antigen receptors and their uses

[0001] This application claims priority to Chinese patent application No. 202210620008.2, filed with the Patent Office of China on June 2, 2022, entitled “Chimeric Antigen Receptor and Uses Thereof,” and to Chinese patent application No. 202310639033X, filed with the Patent Office of China on May 31, 2023, entitled “Chimeric Antigen Receptor and Uses Thereof.” The entire contents of each of these applications are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor and uses thereof. Background Art

[0003] 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 the CAR gene in vitro, amplifying and culturing them to a certain number, and then returning them to the patient. The design and development of the CAR gene is the core technology of CAR-T. By transferring the designed CAR gene into the T cell genome, the T cell can express the corresponding CAR molecule. The CAR molecule enables T cells to directly recognize tumor cells through the single-chain variable region fragment scFv without MHC restriction, simultaneously obtaining the primary stimulation signal and the co-stimulation signal, and being "activated with one click." CAR-T can proliferate in vivo, acting as a "living drug" with sustained lethality. CAR-T can also release cytokines in the body to recruit and activate more immune cells to collaboratively kill tumor cells. Although existing CAR-T drug technology is mature and has significant efficacy, many problems still exist. For example, patients need to wait for preparation time (not available off the shelf), during which time the tumor may progress, increasing the risk of treatment or losing the opportunity for treatment; preparation failure may also lead to the drug being unavailable; there are also safety issues and high prices. To address the main shortcomings of current autologous CAR-T cell drugs (not available off the shelf and high prices), it is necessary to develop universal off-the-shelf cell drugs, such as universal CAR-T and universal CAR-NK. These use allogeneic T cells or NK cells, or T cells or NK cells induced and differentiated from iPS cells, to prepare off-the-shelf CAR-T and CAR-NK. Dozens of cell drugs can be prepared in a single batch. Compared to autologous CAR-T cell drugs, which only prepare one drug in a batch, universal CAR-T or CAR-NK reduces costs and can therefore lower the price. CAR-NK technology is an immune cell therapy strategy that emerged alongside autologous CAR-T technology. Initially, to achieve the goal of off-the-shelf cell therapy, CAR-modified NK cells primarily used the NK cell line NK-92 (which can expand indefinitely). However, due to the tumorigenicity risk of NK-92, irradiation is required before use. This CAR-NK-92 cannot expand in vivo, and reported clinical efficacy has been suboptimal. The CAR structure used in NK cells requires specialized design. New CAR-NK therapies are still needed to meet patient needs.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a chimeric antigen receptor, which targets BCMA and has a better effect in killing tumor cells.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] On the one hand, 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 immune effector cells, wherein the chimeric antigen receptor comprises a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof; the enhancer is selected from one or more of 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 their functional variants.

[0008] In certain embodiments, the BCMA-binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), and a heavy chain complementary 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.

[0009] In certain embodiments, the HCDR1 comprises an amino acid sequence that is at least about 50% identical to the amino acid sequence of SEQ ID NO:2, the HCDR2 comprises an amino acid sequence that is at least about 50% identical to the amino acid sequence of SEQ ID NO:3, and the HCDR3 comprises an amino acid sequence that is at least about 50% identical to the amino acid sequence of SEQ ID NO:4.

[0010] In certain embodiments, the BCMA-binding domain comprises a single domain antibody.

[0011] In certain embodiments, the BCMA-binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least about 50% identical to the amino acid sequence of SEQ ID NO: 8.

[0012] In certain embodiments, the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence shown in SEQ ID NO: 9;

[0013] Or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO:9.

[0014] In certain embodiments, the signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:5; or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO:5.

[0015] In certain embodiments, the non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 10; or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 10.

[0016] In certain embodiments, the antigen binding domain specifically binds to a tumor antigen.

[0017] In certain embodiments, the tumor antigen is associated with a non-solid tumor.

[0018] In certain embodiments, the non-solid tumor is selected from the group consisting of lymphocytic neoplasms, Hodgkin's lymphoma, chronic and acute lymphocytic leukemias, chronic myeloid leukemia, acute myeloid leukemia, and multiple myeloma.

[0019] In certain embodiments, the tumor antigen is associated with a solid tumor.

[0020] In certain embodiments, 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.

[0021] In certain 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.

[0022] In certain embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment being connected to the N-terminus of the BCMA-binding domain; the signal peptide fragment comprises the amino acid sequence set forth in SEQ ID NO: 1; or an amino acid sequence having at least about 50% identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0023] In certain embodiments, 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.

[0024] In certain embodiments, the enhancer comprises a cytokine and / or a cytokine receptor.

[0025] In certain embodiments, the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor (IL15R).

[0026] In certain embodiments, the enhancer comprises the amino acid sequence shown in SEQ ID NO: 7; or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7.

[0027] In certain embodiments, the chimeric antigen receptor and the enhancer are connected by a linker.

[0028] In certain embodiments, the linker comprises a cleavable linker.

[0029] In certain embodiments, the linker comprises a self-cleaving peptide.

[0030] In certain embodiments, the self-cleaving peptide comprises P2A, F2A, E2A or T2A.

[0031] In certain embodiments, wherein the self-cleaving peptide is T2A, the N-terminus of the chimeric antigen receptor and the C-terminus of the enhancer can be connected by T2A, and have the following structure: 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 connected by T2A, and have the following structure: CD8SP-anti-BCMA sdAb-CD28 (hinge / TM / Intracellular)-CD3ζ-T2A-IL-15.

[0032] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 6; or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6.

[0033] In certain embodiments, the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13; or an amino acid sequence that is at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 13.

[0034] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide of the present invention.

[0035] In another aspect, the present invention provides one or more isolated nucleic acid molecules comprising: 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 includes the extracellular domain, transmembrane domain and intracellular co-stimulatory 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.

[0036] In certain embodiments, the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence shown in SEQ ID NO:9.

[0037] In certain embodiments, the nucleic acid molecule may comprise the nucleotide sequence described in SEQ ID NO:17.

[0038] In certain embodiments, the signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:5.

[0039] In certain embodiments, the nucleic acid molecule may comprise the nucleotide sequence described in SEQ ID NO:18.

[0040] In certain embodiments, the non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:10.

[0041] In certain embodiments, the antigen binding domain specifically binds to a tumor antigen.

[0042] In certain embodiments, the tumor antigen is associated with a non-solid tumor.

[0043] In certain embodiments, the non-solid tumor is selected from the group consisting of B-lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia, and acute myeloid leukemia.

[0044] In certain embodiments, the tumor antigen is associated with a solid tumor.

[0045] In certain embodiments, the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, and non-small cell lung cancer.

[0046] In certain embodiments, the tumor antigen comprises a tumor antigen selected from the group consisting of CD5, CD19, CD20, and BCMA.

[0047] In certain embodiments, the BCMA-binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), and a heavy chain complementary 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.

[0048] In certain embodiments, the BCMA-binding domain comprises a single domain antibody.

[0049] In certain embodiments, the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0050] In certain embodiments, the nucleic acid molecule may comprise the nucleotide sequence described in SEQ ID NO:16.

[0051] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:11.

[0052] In certain embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, wherein the C-terminus of the signal peptide fragment is connected to the N-terminus of the BCMA-binding domain.

[0053] In certain embodiments, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:1.

[0054] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:12.

[0055] In certain embodiments, the nucleic acid molecule may comprise the nucleotide sequence of SEQ ID NO: 21; or a nucleotide molecule having 50% identity with the nucleotide sequence of SEQ ID NO: 21.

[0056] In certain embodiments, the nucleotide sequence encoding the chimeric antigen receptor comprises the nucleotide sequence shown in SEQ ID NO:22.

[0057] In certain embodiments, the nucleotide sequence encoding the chimeric antigen receptor comprises a nucleotide sequence having 50% identity with the nucleotide sequence shown in SEQ ID NO:22.

[0058] In certain embodiments, the enhancer comprises a cytokine and / or a cytokine receptor.

[0059] In certain embodiments, 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, their functional variants and combinations thereof.

[0060] In certain embodiments, the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor (IL15R).

[0061] In certain embodiments, the enhancer comprises the amino acid sequence shown in SEQ ID NO:7.

[0062] In certain embodiments, the nucleotide sequence encoding the enhancer is shown in SEQ ID NO: 20.

[0063] In certain embodiments, the chimeric antigen receptor and the enhancer are connected by a linker.

[0064] In certain embodiments, the linker comprises a cleavable linker.

[0065] In certain embodiments, the linker comprises a self-cleaving peptide.

[0066] In certain embodiments, the self-cleaving peptide comprises P2A, F2A, E2A or T2A.

[0067] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:6.

[0068] In certain embodiments, the nucleic acid encoding the linker comprises the nucleotide sequence shown in SEQ ID NO:19.

[0069] In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:23.

[0070] 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.

[0071] In certain 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.

[0072] In certain embodiments, the extracellular domain, transmembrane domain, and intracellular costimulatory domain of CD28 comprise the amino acid sequence shown in SEQ ID NO:9.

[0073] In certain embodiments, the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:17.

[0074] In certain embodiments, the signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:5.

[0075] In certain embodiments, the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:18.

[0076] In certain embodiments, the non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:10.

[0077] In certain embodiments, the BCMA-binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), and a heavy chain complementary 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 immune effector cells.

[0078] In certain embodiments, the BCMA-binding domain comprises a single domain antibody.

[0079] In certain embodiments, the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0080] In certain embodiments, the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:16.

[0081] In certain embodiments, the first nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:21.

[0082] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:11.

[0083] In certain embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, wherein the C-terminus of the signal peptide fragment is connected to the N-terminus of the BCMA-binding domain.

[0084] In certain embodiments, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:1.

[0085] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:12.

[0086] In certain embodiments, the first nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:22.

[0087] In certain embodiments, the enhancer comprises a cytokine and / or a cytokine receptor.

[0088] In certain embodiments, 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, their functional variants, or a combination thereof.

[0089] In certain embodiments, the enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor (IL15R).

[0090] In certain embodiments, the enhancer comprises the amino acid sequence shown in SEQ ID NO:7.

[0091] In certain embodiments, the second nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:20.

[0092] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule are in the same construct.

[0093] In certain embodiments, the chimeric antigen receptor and the enhancer are connected by a linker.

[0094] In certain embodiments, the linker comprises a cleavable linker.

[0095] In certain embodiments, the linker comprises a self-cleaving peptide.

[0096] In certain embodiments, the self-cleaving peptide comprises P2A, F2A, E2A or T2A.

[0097] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:6.

[0098] In certain embodiments, it comprises the nucleotide sequence shown in SEQ ID NO:23.

[0099] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule are located in separate constructs.

[0100] The constructs of the present invention may be present in a vector, or may be integrated into the genome.

[0101] In certain embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0102] In another aspect, the present invention provides a cell comprising the nucleic acid molecule of the present invention or one or more isolated nucleic acid molecules described above or the construct of the present invention, and / or expressing the chimeric antigen receptor of the present invention or the fusion polypeptide of the present invention.

[0103] In certain embodiments, the cells comprise immune effector cells.

[0104] In certain embodiments, the immune effector cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0105] In certain embodiments, they include engineered immune effector cells.

[0106] In certain embodiments, the engineered immune effector cells comprise CAR-T cells or CAR-NK cells.

[0107] In certain embodiments, the cells express:

[0108] (i) an enhancer capable of enhancing one or more activities of immune effector cells; (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 co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0109] The cell can be a CAR-NK cell, which expresses: (i) an enhancer capable of enhancing one or more activities of the CAR-NK cell; (ii) a chimeric antigen receptor, which comprises a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0110] In certain embodiments, the cells express: (i) IL-15 or a functional variant thereof; (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 co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0111] The cell can be a CAR-NK cell, which expresses: (i) IL-15 or a functional variant thereof; (ii) a chimeric antigen receptor comprising a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0112] The present invention provides an immune effector cell expressing IL-15, wherein the cell expresses IL-15 and 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 co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0113] In certain embodiments, wherein the IL-15 or its functional variant is expressed as a membrane-bound polypeptide and / or a secreted polypeptide. In some embodiments, the IL-15 or its functional variant can be expressed as a secreted polypeptide. In other embodiments, the IL-15 or its functional variant is fused to a transmembrane protein. The "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 its functional variant can be expressed by one or more NK cells in a variety of ways. IL-15 can be expressed in NK cells and secreted by NK cells and / or can be directly or indirectly connected using any of a variety of linkers known in the art. In some specific aspects, IL-15 can be connected to all or part 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 certain 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 certain 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 exogenous cytokine IL-15 or a functional variant thereof or a nucleotide sequence encoding a chimeric cytokine receptor comprising an IL-15 receptor.

[0114] In another aspect, the present invention provides a method for modifying a cell, the method comprising: introducing a first nucleic acid molecule and a second nucleic acid molecule into the cell, wherein:

[0115] (i) the first nucleic acid molecule encodes a chimeric antigen receptor comprising a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory 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.

[0116] In another aspect, the present invention provides a pharmaceutical composition comprising the fusion polypeptide of the present invention, the nucleic acid molecule of the present invention, the isolated one or more constructs of the present invention or the cell of the present invention, and optionally a pharmaceutically acceptable carrier.

[0117] In another aspect, the present invention provides a use of the fusion polypeptide of the present invention, the nucleic acid molecule of the present invention, the isolated one or more nucleic acid molecules of the present invention, the isolated one or more constructs of the present invention, or the cell of the present invention for preparing a medicament, wherein the medicament is for treating a tumor.

[0118] The beneficial effects of the present invention are:

[0119] 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 use the extracellular region, transmembrane region, and cytoplasmic region of the CD28 molecule, connected to the intracellular domain of CD3ζ, and connected to the wild-type IL-15 cytokine via T2A. Cell experiments and animal experiments have shown that the CAR-NK targeting BCMA has a good effect in killing tumor cells. Further development can be used as a CAR-NK cell drug for the treatment of multiple myeloma. Whether it is CAR-NK prepared by cytokine amplification or CAR-NK prepared by incubation and activation of K562 feeder cells genetically modified to express mbIL-21, CD137L, and CD86, the CAR-NK targeting BCMA has a good killing effect. Animal experiments have shown that the CAR-NK prepared by feeder cell activation can effectively inhibit the proliferation of the myeloma cell line MM.1S. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The specific features of the invention are as shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0121] Figure 1 shows a schematic diagram of the anti-BCMA CAR-NK cell structure of the present invention; wherein 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, transmembrane, and cytoplasmic regions of the CD28 molecule; CD3ζ represents the cytoplasmic region 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.

[0122] FIG2A-FIG2B show the positive rate detection results of the anti-BCMA CAR-NK (CD28hinge) cells prepared in Example 2 of the present invention.

[0123] FIG3 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.

[0124] FIG4A-FIG4B show the positive rate detection results of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention.

[0125] FIG5 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.

[0126] FIG6 shows the comparative results of the killing rates of the anti-BCMA CAR-NK (CD28 hinge) cells and the anti-BCMA CAR-NK (IgG1 hinge) cells prepared in Example 3 of the present invention against the multiple myeloma cell line MM.1S in vitro.

[0127] FIG7 shows the comparative results of the killing rates of the anti-BCMA CAR-NK (CD28 hinge) cells and the anti-BCMA CAR-NK (2B4 Intracelluar) cells prepared in Example 3 of the present invention against the multiple myeloma cell line MM.1S in vitro.

[0128] FIG8 shows a comparison of the killing rates of the anti-BCMA CAR-NK (CD28 hinge) cells and the anti-BCMA CAR-NK (CD28 hinge + no IL-15) cells prepared in Example 3 of the present invention against the multiple myeloma cell line MM.1S in vitro.

[0129] FIG9 shows the expression results of the cytokine IFN-γ in the anti-BCMA CAR-NK (CD28 hinge) cells and the anti-BCMA CAR-NK (IgG1 hinge) cells prepared in Example 3 of the present invention.

[0130] FIG10 shows the results of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention killing tumor cells with different BCMA expression levels in vitro.

[0131] FIG11 shows the animal experimental results of MM.1S of anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0132] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0133] Definition of terms

[0134] In the present invention, the term "BCMA" generally refers to a cell maturation antigen, which is a member of the tumor necrosis factor receptor superfamily.

[0135] In the present invention, the term "BCMA-binding domain" generally refers to a humanized anti-BCMA antibody or an antigen-binding fragment thereof that can specifically bind to a BCMA polypeptide expressed on B cells.

[0136] In the present invention, the term "antigen-binding fragment" (also referred to herein as "targeting portion" or "antigen-binding portion") generally refers to a portion of an antibody molecule that 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 referred to as an "epitope" as described above. An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both.

[0137] In the present invention, the term "complementarity determining region" (CDR) generally refers to the complementarity determining region within the variable region of an antigen-binding fragment. In the present invention, the heavy chain variable region has three CDRs, which are named HCDR1, HCDR2 and HCDR3 for each variable region.

[0138] In the present invention, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, which are called the framework regions.

[0139] In the present invention, the term "single domain antibody (sdAb)" or "VHH" generally refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain.

[0140] In the present invention, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, which are generally two kinds in the simplest embodiment, which, when in immune effector cells, provide cells with specificity for target cells (usually cancer cells) and produce intracellular signals. In some embodiments, CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or part thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below.

[0141] In the present invention, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function as the functional variant and has at least 85% sequence identity therewith.

[0142] In the present invention, the term "isolated nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length or their analogs that are separated from their natural environment or artificially synthesized.

[0143] In the present invention, the term "construct" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into the host cell and / or between host cells.

[0144] In the present invention, the term "immune effector cells" generally refers to immune cells that participate in immune responses and perform effector functions.

[0145] In the present invention, the term "natural killer cell" ("NK cell") generally refers to a type of cytotoxic lymphocyte of the immune system.

[0146] In the present invention, the term "interleukin-15" generally refers to a cytokine that regulates T and NK cell activation and proliferation. In one aspect, IL-15 is wild-type IL-15.

[0147] In the present invention, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0148] 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. These include pre-malignant and malignant cancers and tumors, and encompass both solid and non-solid tumors.

[0149] In the present invention, the term "administering" generally refers to delivering 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.

[0150] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration.

[0151] As used herein, the term "effective amount" or "effective dose" generally refers to 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 the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease.

[0152] In the present invention, the term "include" generally means to include, encompass, contain or encompass.

[0153] In the present invention, the term "subject" generally refers to a human or a non-human animal.

[0154] Example 1

[0155] 1.1 Chimeric Antigen Receptor Construction

[0156] As shown in Figure 1, the anti-BCMA single-domain antibody sequence (SEQ ID NO: 8) was used, combined with the extracellular hinge region, transmembrane region and cytoplasmic region of the CD28 molecule (SEQ ID NO: 9), connected to the intracellular domain of CD3ζ (SEQ ID NO: 5), and connected to the wild-type IL-15 cytokine (SEQ ID NO: 7) through T2A (SEQ ID NO: 6) to construct a BCMA-targeting CAR structure that can be used for NK cells; the lentiviral Pre-Lenti-EF1-CAR V2 vector and BCMA CAR gene (SEQ ID NO: 13) were enzymatically digested, connected, transformed, cloned, plasmid extracted, and sequenced to obtain the sequence-corrected lentiviral vector Pre-Lenti-EF1-CAR V2 NCI-NK.

[0157] 1.2 Preparation of BCMA chimeric antigen receptor lentivirus

[0158] 1) Culture 293TS cells in a 37°C, 5% CO2 incubator at 150 rpm. Count the cells to be transfected. Cell density should be approximately 6×106 cells / ml to 7×106 cells / ml, and cell viability should be greater than 90%. Cells can be used for transfection.

[0159] 2) Mix the Pre-Lenti-EF1-CAR V2 NCI-NK plasmid with the packaging plasmids ZL004:ZL006:envelope plasmid ZL003 in a ratio of 9:7:7:7. Add this mixture of four plasmids to a tube containing 293TS basal medium. Add PEIpro to another tube at a ratio of plasmid:PEIpro = 1:2. Incubate each tube at room temperature for 5 minutes. Slowly add the PEIpro mixture to the DNA mixture, gently shake the tube to mix thoroughly, and incubate at room temperature for approximately 15 minutes. Add the DNA-PEIpro mixture to the cells to be transfected, mix thoroughly, and incubate with shaking at 37°C, 5% CO2. After 6 hours, feed the cells. After 48 hours, collect the supernatant containing the lentivirus. Transfer the virus-containing supernatant to a 50ml centrifuge tube, centrifuge to remove the 293TS cells, filter the virus-containing supernatant, concentrate, aliquot, and store at -80°C until needed.

[0160] Example 2 Preparation of CAR-NK Cells by Cytokine Amplification and Activation

[0161] 1) PBMC isolation

[0162] In a sterile environment, draw 60ml of venous blood from volunteers. Transfer to two 50ml centrifuge tubes, mix well, and centrifuge at 800g, 18-22℃, for 15min. Aspirate plasma into a 50ml centrifuge tube. After removing the plasma, add an equal volume of 0.9% NaCl to dilute the whole blood and mix well. Take a new 50ml centrifuge tube, add 15ml of human lymphocyte separation solution respectively, take 20ml of diluted blood and slowly add it to the separation solution along the side wall of the tube; centrifuge at 400g in a horizontal rotor at 20℃ for 30min (speed increase 3, speed decrease 0). Carefully aspirate the buffy coat layer into a new 50ml centrifuge tube, dilute to 40ml with 0.9% NaCl injection solution, and mix well by pipetting the centrifuge tube. Centrifuge at 450g in a horizontal rotor at 20℃ for 10min, discard the supernatant, and repeat the wash once. After the centrifugation is completed, pour out the supernatant, add 20ml of normal saline to resuspend and count.

[0163] 2) CD3 T cell depletion

[0164] Deplete CD3 T cells using Miltenyi CD3 Regent. Take 6 × 107 PBMCs and centrifuge at 300g for 10 minutes at 20°C. Discard the supernatant and resuspend the cells in magnetic bead separation buffer (80μl / 1 × 107 cells). Add CD3 Reagent (5μl / 1 × 107 cells), mix well, and refrigerate at 2-8°C for 15 minutes. Add 5ml of magnetic bead separation buffer to the cells, pipette to mix, and centrifuge at 300g for 10 minutes at 20°C. Mount the LS Columns on the magnet and rinse the column with 3ml of magnetic bead separation buffer. Discard the supernatant and resuspend the cells in magnetic bead separation buffer (100μl / 1 × 107 cells), pipette to mix, and pass through the column. Rinse three times with 3ml of magnetic bead separation buffer. Collect the flow-through.

[0165] 3) NK cell purification

[0166] After T cell removal, sample the effluent and count it. Centrifuge at 300g for 10 minutes. Add magnetic bead separation solution (80μl / 1×107 cells) to resuspend the cells. Add CD56 MicroBeads (20μl / 1×107 cells), mix well, and place in a refrigerator at 2-8°C for 15 minutes. Add magnetic bead separation solution to resuspend the cells, and centrifuge at 300g for 10 minutes. Mount the LS Columns on the magnet and rinse the column with 3ml of magnetic bead separation solution. Discard the supernatant, resuspend the cells in magnetic bead separation solution (500μl / 1×108 cells), pipette to mix well, and pass through the column. Rinse three times with 3ml of magnetic bead separation solution. Remove the column, add 5ml of magnetic bead separation solution, and transfer to a 15ml centrifuge tube. Centrifuge at 300g at 20°C for 10 minutes. Discard the supernatant and resuspend the cells in 10 ml of 0.9% sodium chloride injection. Mix well, count, and centrifuge at 300 g at 20°C for 10 min. Discard the supernatant and resuspend the cells in NK culture medium (1% of NK MACS Supplement + NK MACS Basal Medium + IL-2 (500 IU / ml) + IL-15 (140 IU / ml) + 3% autologous plasma). Adjust the cell density to 1 × 106 / ml and seed into a 12-well plate, 1 ml per well.

[0167] 4) Lentiviral infection of NK cells

[0168] On day 3, NK cells were observed, and most were aggregated. Mix 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, culture medium was added. On day 5, the cells were transferred to a 6-well plate and culture medium was added. On day 7, culture medium was added again. On day 8, the CAR-positive rate of BCMACAR NK cells was determined using target protein-binding flow cytometry.

[0169] The results are shown in Figures 2A-2B, where Figure 2A is a negative control and Figure 2B is the positive expression rate (71%) of CAR molecules that recognize BCMA using BCMA target protein detection.

[0170] Example 3: Preparation of anti-BCMA CAR-NK cells by expansion and activation of feeder cells

[0171] 1) PBMC isolation

[0172] Collect single blood samples from healthy volunteers. Transfer to a 50ml centrifuge tube and dilute the blood sample with an equal amount of 0.9% NaCl injection. Take a new 50ml centrifuge tube and add 15ml of room temperature human lymphocyte separation solution to each tube. Take 20ml of the diluted blood and slowly add it to the separation solution along the side of the tube. Place in a centrifuge, set the centrifugal force to 400g, the temperature to 20℃, and centrifuge for 30 minutes (speed increase 3, speed decrease 3). Carefully aspirate the buffy coat layer into two other 50ml centrifuge tubes. Add 0.9% sodium chloride injection to 40ml and use a pipette to mix. Centrifuge at 400g, temperature 20℃, for 10 minutes. Discard the supernatant, add 0.9% sodium chloride injection to resuspend the lymphocytes, and add more injection solution to 40ml. Centrifuge at 400g, temperature 20℃, for 10 minutes. After centrifugation, discard the supernatant and add 20ml of 0.9% sodium chloride injection. Mix well and take samples for counting.

[0173] 2) CD3 T cell depletion

[0174] Deplete CD3 T cells using Miltenyi CD3 Regent. Take 1×108 PBMCs and centrifuge at 300g for 10 minutes at 20°C. Discard the supernatant and resuspend the cells in magnetic bead separation buffer (80μl / 1×107 cells). Add CD3 Reagent (5μl / 1×107 cells), mix well, and refrigerate at 2-8°C for 15 minutes. Add 5ml of magnetic bead separation buffer to the cells, pipette to mix, and centrifuge at 300g for 10 minutes at 20°C. Mount the LS Columns on the magnet and rinse the column with 3ml of magnetic bead separation buffer. Discard the supernatant and resuspend the cells in magnetic bead separation buffer (100μl / 1×107 cells), pipette to mix, and pass through the column. Rinse three times with 3ml of magnetic bead separation buffer. Collect the flow-through.

[0175] 3) NK Cell Purification: After T cell removal, sample and count the effluent. Centrifuge at 300g for 10 minutes. Add magnetic bead separation buffer (80μl / 1×107 cells) to resuspend and mix the cells. Add CD56 MicroBeads (10μl / 1×107 cells), mix, and place in a refrigerator at 2-8°C for 15 minutes. Resuspend and mix the cells in magnetic bead separation buffer and centrifuge at 300g for 10 minutes. Mount the LS Columns on the magnet and rinse the column with 3ml of magnetic bead separation buffer. Discard the supernatant and resuspend the cells in magnetic bead separation buffer (500μl / 1×108 cells), pipette and mix, and pass through the column. Rinse three times with 3ml of magnetic bead separation buffer. Remove the column, add 5ml of magnetic bead separation buffer, and transfer to a 15ml centrifuge tube. Centrifuge at 300g at 20°C for 10 minutes. Discard the supernatant and resuspend the cells in 10 ml of 0.9% sodium chloride injection. Mix well, count, and centrifuge at 300 g for 10 min at 20°C. Discard the supernatant and resuspend in NK culture medium.

[0176] 4) NK cell activation

[0177] 30ml NK cell culture medium was added with 7.5×106NK cells (2.5×105 / ml) and 7.5×106

[0178] K562-mbIL-21 cells (irradiated, 100 Gy) (2.5 × 10⁵ / ml) were mixed 1:1 in a T-75 flask. Cultured in a 37°C, 5% CO₂ incubator. Observed cells on days 2, 3, and 4. On day 5, centrifuged and replaced the medium. Transferred cells to a 50ml centrifuge tube, mixed, counted, and centrifuged at 300g at 20°C for 10 minutes. Discard the supernatant and resuspend in NK cell culture medium (1% of NKMACS Supplement + NK MACS Basal Medium + IL-2 (200 IU / ml) + 5% autologous plasma). Adjust the cell density to 1 × 10⁶ / ml.

[0179] 5) Lentiviral infection of NK cells

[0180] On day 6, NK cells were observed and found to be fully activated and proliferating vigorously. Retronectin-coated plates were inoculated with cells, and Vectofusin-1 was added at a final concentration of 10 μg / ml. Virus was added at an MOI of 10.

[0181] On day 7, the culture medium was replenished.

[0182] On day 8, CAR NK cells were secondary activated and mixed with K562-mbIL-21 cells at a ratio of 1:1. The cells were cultured in a 37°C, 5% CO2 incubator.

[0183] The cells were observed on the 9th and 10th days.

[0184] On day 11, centrifuge and replace the medium. Transfer the cells to a 50 ml centrifuge tube, mix well, count, and centrifuge at 300 g at 20°C for 10 min. Discard the supernatant and resuspend in NK cell culture medium (1% of NK MACS Supplement + NK MACS Basal Medium + IL-2 (200 IU / ml) + 3% autologous plasma). Adjust the cell density to 1 × 106 / ml.

[0185] The cells were observed on day 12.

[0186] On the 13th day, the cells were replenished and the cell density was adjusted to 1×106 / ml.

[0187] On day 14, the CAR positivity rate of BCMA CAR NK cells was detected using target protein-binding flow cytometry.

[0188] The results are shown in Figures 4A-4B, where Figure 4A is a negative control and Figure 4B is the positive expression rate (62%) of CAR molecules that recognize BCMA using BCMA target protein detection.

[0189] Example 4 Evaluation of Anti-BCMA CAR-NK Cell Function

[0190] The in vitro cell killing experiment was detected using an LDH detection kit (Promega). The anti-BCMA CAR-NK cells prepared in Example 2 or 3 and the target cells (MM.1S cells that highly express BCMA molecules, purchased from ATCC) were set up with four gradients according to the number ratio of the two (ie, the effective target ratio) of 1:2, 1:4, 1:8, and 1:16. Among them, 3×104 target cells / well were added to 200 μL of the system containing XVIVO medium / 1640 medium in all other wells. The 96-well plate was placed in a 37 ° C, 5% CO2 incubator for culture. After 17 hours, 20 μL of lysate was added to the well with maximum release, mixed to completely rupture the cells, and the 96-well plate was placed in a CO2 incubator and incubated for 2 hours. After 2 hours, observe the wells with the highest release. After complete lysis of the target cells, aspirate 50 μL of supernatant from each well into a flat-bottom 96-well plate. Add 50 μL of substrate solution to each well and develop in the dark for 30 minutes. After 30 minutes, observe the color change. The wells with the highest release of MM.1S and those containing anti-BCMA CAR-NK cells will be darker. Measure the cytotoxicity using a microplate reader at a wavelength of 490 nm. Use an LDH assay kit to determine the cytotoxicity.

[0191] The killing effect of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 2 on MM.1S cells is shown in Figure 3. The results show that the anti-BCMA CAR-NK cells prepared in Example 2 can specifically kill BCMA-positive cells, and the killing activity is very high, all above 20%.

[0192] The killing effect of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3 on MM.1S cells is shown in Figure 5. The results show that the anti-BCMA CAR-NK cells prepared in Example 3 can specifically kill BCMA-positive cells, and the killing activity is very high, all above 40%.

[0193] The killing effects of anti-BCMA CAR-NK (CD28 hinge, CAR sequence as shown in SEQ ID NO: 13) cells, anti-BCMA CAR-NK (IgG1 hinge, CAR sequence as shown in SEQ ID NO: 14) cells, anti-BCMA CAR-NK (2B4 Intracelluar, CAR sequence as shown in SEQ ID NO: 15) cells, and anti-BCMA CAR-NK (CD28 hinge + no IL-15, CAR sequence as shown in SEQ ID NO: 12) on MM.1S cells prepared in Example 3 are shown in Figures 6-8. The results showed that the anti-BCMA CAR-NK cells with the CD28 extracellular region as the hinge region had a better killing effect in vitro than the anti-BCMA CAR-NK cells with the IgG1 molecule hinge region as the hinge region (Figure 6); the anti-BCMA CAR-NK cells with the CD28 cytoplasmic region had a better killing effect in vitro than the anti-BCMA CAR-NK cells with the 2B4 cytoplasmic region (Figure 7); the anti-BCMA CAR-NK cells that could secrete IL-15 had a better killing effect in vitro than the anti-BCMA CAR-NK cells that expressed CAR molecules but did not secrete IL-15 (Figure 8).

[0194] After killing, the supernatant was taken and the level of cytokine IFN-γ was measured. The results are shown in Figure 9. Compared with the anti-BCMA CAR-NK cells whose hinge region is the hinge region of the IgG1 molecule, the anti-BCMA CAR-NK cells whose hinge region is the extracellular region of CD28 can significantly upregulate the expression of cytokine IFN-γ with tumor killing effect in vitro, verifying the specific killing effect of anti-BCMA CAR-NK (CD28 hinge) cells.

[0195] Example 5

[0196] The in vitro cell killing experiment was detected using an LDH detection kit (Promega). The anti-BCMA CAR-NK cells and target cells (RS4; 11 that do not express BCMA, Daudi and NCI-1299 tumor cells that express BCMA) prepared in Example 3 were set up in four gradients according to the number ratio of the two (i.e., the effective target ratio) of 1: 2, 1: 4, 1: 8, and 1: 16. Among them, 3×104 target cells / well were added to 200 μL of the system containing X-VIVO medium / 1640 medium in all other wells. The 96-well plate was placed in a 37°C, 5% CO2 incubator for culture. After 17 hours, 20 μL of lysate was added to the well with maximum release, mixed to completely rupture the cells, and the 96-well plate was placed in a CO2 incubator and incubated for 2 hours. After 2 hours, observe the wells with the highest release. After complete lysis of the target cells, aspirate 50 μL of supernatant from each well into a flat-bottom 96-well plate. Add 50 μL of substrate solution to each well and develop in the dark for 30 minutes. After 30 minutes, observe the color change. The wells with the highest release of MM.1S and those containing anti-BCMA CAR-NK cells will be darker. Measure the cytotoxicity using a microplate reader at a wavelength of 490 nm. Use an LDH assay kit to determine the cytotoxicity.

[0197] Figure 10 shows the in vitro killing of tumor cells with varying levels of BCMA expression by anti-BCMA CAR-NK (CD28 hinge) cells. The results showed no killing of RS4;11, which does not express BCMA, but killing of both Daudi and NCI-1299 tumor cells, which express BCMA. The anti-BCMA CAR-NK (CD28 hinge) cells of the present application are BCMA-specific.

[0198] Example 6

[0199] The human multiple myeloma MM.1S cell line was transduced to express the luciferase reporter gene (luciferase) to generate MM.1S-luc. The cells were routinely cultured in 1640 medium containing 10% fetal bovine serum (FBS) in a 5% CO2, 37°C incubator. MM.1S-luc cells were injected into the tail vein of NSG mice (50 mice, half male and half female) at a density of 1.5×106 cells / 200μl PBS. Seventeen days later, all animals were imaged. Tumor-bearing mice were randomly divided into five groups (half male and half female, 8 mice per group): PBS group, NK cell group, anti-BCMA CAR-NK (CD28 hinge) cell group, and BCMA CAR-T (CD28 hinge) cell group. The extracellular fluid group received a tail vein injection of PBS, the NK cell group received a tail vein injection of NK cells, the anti-BCMA CAR-NK (CD28 hinge) cell group received a tail vein injection of the anti-BCMA CAR-NK (CD28 hinge) cells prepared in Example 3, and the BCMA CAR-T (CD28 hinge) cell group received a tail vein injection of BCMA CAR-T (CD28 hinge) cells. Three days later, the mice were anesthetized and intraperitoneally injected with luciferase substrate. 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, respectively.

[0200] The results showed that almost no tumor cells were imaged in the anti-BCMA CAR-NK cell treatment group, while the control group mice had a severe tumor burden, indicating that anti-BCMA CAR-NK cells can effectively eliminate BCMA-positive tumor cells. After intravenous infusion of anti-BCMA CAR-NK cells, the fluorescence signal intensity in the mice began to decrease compared with the PBS group and NK cell group on day 3, indicating that the tumor burden was decreasing. As shown in Figure 11, the fluorescence signal intensity in the mice was basically undetectable on day 6, indicating that anti-BCMA CAR-NK also demonstrated a strong therapeutic effect against BCMA expression-related diseases in animal experiments.

[0201] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Fusion peptide, It is characterized in that The invention relates to a chimeric antigen receptor and an enhancer capable of enhancing one or more activities of immune effector cells, wherein the chimeric antigen receptor comprises a BCMA binding domain and a non-antigen binding domain, wherein the non-antigen binding domain comprises an extracellular domain, a transmembrane domain and an intracellular co-stimulatory domain of CD28 or a functional variant thereof, and a signaling domain of CD3ζ or a functional variant thereof; and the enhancer is selected from one or more of 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 their functional variants.

2. The fusion polypeptide according to claim 1, It is characterized in that The BCMA-binding domain comprises a heavy chain complementary determining region 1, a heavy chain complementary determining region 2 and a heavy chain complementary determining region 3, wherein the heavy chain complementary determining region 1 comprises the amino acid sequence shown in SEQ ID NO: 2, the heavy chain complementary determining region 2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain complementary determining region 3 comprises the amino acid sequence shown in SEQ ID NO:

4.

3. The fusion polypeptide according to claim 2, It is characterized in that The BCMA binding domain comprises a single domain antibody.

4. The fusion polypeptide according to claim 3, It is characterized in that The BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 8 or a functional variant thereof.

5. The fusion polypeptide according to claim 1, It is characterized in that The extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 comprise the amino acid sequence shown in SEQ ID NO:

9.

6. The fusion polypeptide according to claim 1, It is characterized in that The signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

5.

7. The fusion polypeptide according to claim 1, It is characterized in that The non-antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The fusion polypeptide according to claim 1, It is characterized in that The chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment is connected to the N-terminus of the antigen binding domain; the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:

1.

9. The fusion polypeptide according to claim 8, It is characterized in that 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 according to claim 1, It is characterized in that The enhancer comprises IL-15 or a functional variant thereof, or a chimeric cytokine receptor comprising an IL-15 receptor.

11. The fusion polypeptide according to claim 10, It is characterized in that The enhancer comprises the amino acid sequence shown in SEQ ID NO:

7.

12. The fusion polypeptide according to claim 1, It is characterized in that The fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO:

13.

13. Isolated nucleic acid molecules, It is characterized in that Encodes the fusion polypeptide according to any one of claims 1 to 12.

14. Isolated one or more nucleic acid molecules, It is characterized in that The invention 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 an extracellular domain, a transmembrane domain and an intracellular co-stimulatory domain of CD28 or a functional variant thereof, and a signaling domain of CD3ζ or a functional variant thereof; and a nucleotide sequence encoding an enhancer capable of enhancing one or more activities of immune effector cells.

15. The isolated one or more nucleic acid molecules according to claim 14, It is characterized in that The nucleotide sequence encoding the chimeric antigen receptor comprises the nucleotide sequence shown in SEQ ID NO:

22.

16. The isolated one or more nucleic acid molecules according to claim 14, It is characterized in that The nucleotide sequence encoding the enhancer is shown in SEQ ID NO:

20.

17. The isolated one or more nucleic acid molecules according to claim 14, It is characterized in that It comprises the nucleotide sequence shown in SEQ ID NO:

23.

18. The isolated construct(s), It is characterized in that Comprising the nucleic acid molecule of claim 13 or the isolated one or more nucleic acid molecules of any one of claims 14-17.

19. The isolated one or more constructs according to claim 18, It is characterized in that The invention 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 an extracellular domain, a transmembrane domain and an intracellular co-stimulatory domain of CD28 or a functional variant thereof, and a 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 immune effector cells.

20. Cells, It is characterized in that Comprising the nucleic acid molecule of claim 13 or the isolated one or more nucleic acid molecules of any one of claims 14-17 or the isolated one or more constructs of any one of claims 18-19, and / or expressing the fusion polypeptide of any one of claims 1-12.

21. The cell according to claim 20, It is characterized in that The cells include immune effector cells.

22. The cell according to claim 21, It is characterized in that The immune effector cells include T cells, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

23. The cell according to claim 22, It is characterized in that Including engineered immune effector cells.

24. The cell according to claim 23, It is characterized in that The engineered immune effector cells include CAR-T cells or CAR-NK cells.

25. A method for modifying cells, It is characterized in that The method comprises: introducing a first nucleic acid molecule and a second nucleic acid molecule into the cell, 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 an extracellular domain, a transmembrane domain and an intracellular co-stimulatory domain of CD28 or a functional variant thereof, and a 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, It is characterized in that A method comprising: comprising: comprising: providing a nucleic acid molecule according to claim 13, or one or more nucleic acid molecules separated according to any one of claims 14-17, or one or more constructs separated according to any one of claims 18-19, or a cell according to any one of claims 20-25, and optionally a pharmaceutically acceptable carrier.

27. Use of the fusion polypeptide of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the isolated one or more nucleic acid molecules of any one of claims 14 to 17, the isolated one or more constructs of any one of claims 18 to 19, or the cell of any one of claims 20 to 25 for the preparation of a medicament, It is characterized in that The medicine is used for treating tumors.

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