Nucleic acid molecule with natural killer protein 30 transmembrane domain and chimeric antigen receptor comprising same

Abstract

The invention provides a nucleic acid molecule with an NKp30 transmembrane domain and a chimeric antigen receptor containing the nucleic acid molecule. The nucleic acid molecule with the NKp30 transmembrane domain comprises nucleic acid codes of the following sections: (a) an extracellular antigen binding domain comprising a heavy chain variable region; (b) a hinge domain; (c) an NKp30 transmembrane domain; and (d) an NKp30 cytoplasm domain. Nucleic acid sequences of an NKp30 transmembrane structural domain and an NKp30 cytoplasm structural domain are transferred into a T cell, so that the T cell has an NKp30 receptor complex and becomes a multi-chain CAR-T cell, and a stable immune synapse can be formed between the multi-chain CAR-T cell and cancer cells, so that the multi-chain CAR-T cell has excellent cancer cell poisoning ability.

Description

Technical Field

[0001] The present invention relates to a nucleic acid molecule for preparing a chimeric antigen receptor, in particular to a nucleic acid molecule for preparing a chimeric antigen receptor with excellent cancer cell killing ability, and also relates to a chimeric antigen receptor containing the same and its application. Background Art

[0002] Cancer is caused by the transformation of normal cells into tumor cells in a multi-stage process, usually starting from a mild precancerous lesion to a malignant tumor and then metastasizing. The above changes are not caused by a single factor, but the result of the interaction between a person's genetic factors and external factors.

[0003] Cancer is the second leading cause of death in the world. According to statistics from the World Health Organization in 2020, nearly 10 million people died of cancer worldwide, accounting for one-sixth of the global death toll. Among them, the most common cancers in men are lung cancer, prostate cancer, colorectal cancer, stomach cancer and liver cancer, while the most common cancer types in women are breast cancer, colorectal cancer, lung cancer, cervical cancer and thyroid cancer.

[0004] In addition to causing physical and mental pain to patients, cancer also brings serious emotional and financial pressure to patients' families. However, in the high-pressure environment of modern society, it is difficult to completely eliminate adverse external factors, and the number of people suffering from cancer is still increasing year by year.

[0005] Therefore, there is still an urgent need for an effective method for treating cancer. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the advantage of the present invention is to provide a novel CAR-T cell. When the modified T cells are injected back into the human body, they can identify tumor cells expressing specific antigens and then kill them.

[0007] To achieve the aforementioned object, the present invention provides an isolated nucleic acid molecule comprising the nucleic acid sequence of the following segments:

[0008] (a) an extracellular antigen binding domain comprising a heavy chain variable region;

[0009] (b) Hinge domain;

[0010] (c) Natural Killer Protein 30 (NKp30) transmembrane domain; and

[0011] (d) Natural killer protein 30 (NKp30) cytoplasmic domain.

[0012] The present invention transfers the genes of the NKp30 transmembrane domain and the NKp30 cytoplasmic domain into T cells, so that the T cells have a NKp30 receptor complex and become multi-chain CAR-T cells, which can form a stable immune synapse with cancer cells and have excellent cancer cell killing ability.

[0013] Preferably, the aforementioned extracellular antigen binding domain comprises: the heavy chain variable region of an anti-mesothelin antibody, the heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of an anti-CD19 antibody, the heavy chain variable region of an anti-B cell maturation antigen (BCMA) antibody, the heavy chain variable region of an anti-CD123 antibody, the heavy chain variable region of an anti-GPRC5D (Gprotein–coupled receptor, class C, group 5, member D) antibody, or the heavy chain variable region of an anti-TSHR (thyroid stimulating hormone receptor (TSH receptor) antibody.

[0014] In one embodiment, the aforementioned extracellular antigen binding domain may be derived from the heavy chain variable region of a heavy chain antibody (heavy chain variable regions derived from heavy chain antibodies, VHH) or a single-chain variable fragment (scFv), wherein the heavy chain variable region of the heavy chain antibody is a single-chain binding region of a heavy chain antibody from a camelid.

[0015] In one embodiment, the aforementioned extracellular antigen binding domain is the heavy chain variable region of an anti-BCMA heavy chain antibody.

[0016] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of a first anti-BCMA heavy chain antibody and a heavy chain variable region of a second anti-BCMA heavy chain antibody connected in series by G4S, which is shown in the amino acid sequence of SEQ ID NO: 1. The heavy chain variable region of the first anti-BCMA heavy chain antibody is shown in the amino acid sequence of SEQ ID NO: 2, and the heavy chain variable region of the second anti-BCMA heavy chain antibody is shown in the amino acid sequence of SEQ ID NO: 3. The heavy chain variable region of the first anti-BCMA heavy chain antibody comprises complementarity-determining regions (CDRs): CDRH1 is shown in the amino acid sequence of SEQ ID NO: 4; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 5; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 6; and the heavy chain variable region of the second anti-BCMA heavy chain antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 7; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 8; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 9.

[0017] In one embodiment, the aforementioned extracellular antigen binding domain comprises a heavy chain variable region of an anti-CD19 antibody, and the heavy chain variable region of the anti-CD19 antibody comprises a complementary determining region: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 10; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 11; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 12. In one embodiment, the aforementioned extracellular antigen binding domain further comprises a light chain variable region of an anti-CD19 antibody, and the light chain variable region of the anti-CD19 antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 13; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 14; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 15. In one embodiment, the aforementioned extracellular antigen binding domain is a single chain variable region fragment of FMC63, as shown in the amino acid sequence of SEQ ID NO: 16.

[0018] In one embodiment, the aforementioned extracellular antigen binding domain comprises a heavy chain variable region of an anti-mesothelin antibody, and the heavy chain variable region of the anti-mesothelin antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 17; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 18; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 19. In one embodiment, the aforementioned extracellular antigen binding domain further comprises a light chain variable region of an anti-mesothelin antibody, and the light chain variable region of the anti-mesothelin antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 20; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 21; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 22. In one embodiment, the aforementioned extracellular antigen binding domain is a SS1 single chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO: 23.

[0019] In one embodiment, the aforementioned extracellular antigen-binding domain comprises a heavy chain variable region of an anti-BCMA antibody, and the heavy chain variable region of the anti-BCMA antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 24; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 25; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 26. In one embodiment, the aforementioned extracellular antigen-binding domain further comprises a light chain variable region of an anti-BCMA antibody, wherein the light chain variable region of the anti-BCMA antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 27; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 28; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 29. In one embodiment, the aforementioned extracellular antigen-binding domain is a J6M0 single chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO: 30. In another embodiment, the heavy chain variable region of the anti-BCMA antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 31; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 32; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 33. In one embodiment, the aforementioned extracellular antigen-binding domain further comprises a light chain variable region of an anti-BCMA antibody, wherein the light chain variable region of the anti-BCMA antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 34; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 35; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 36. In one embodiment, the aforementioned extracellular antigen-binding domain is a BB2121 single chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO: 37.

[0020] In one embodiment, the aforementioned extracellular antigen binding domain comprises a heavy chain variable region of an anti-CD22 antibody, and the heavy chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 38; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 39; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 40. In one embodiment, the aforementioned extracellular antigen binding domain further comprises a light chain variable region of an anti-CD22 antibody, and the light chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 41; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 42; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 43. In one embodiment, the aforementioned extracellular antigen binding domain is an m971 single chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO: 44. In another embodiment, the aforementioned extracellular antigen binding domain comprises a heavy chain variable region of an anti-CD22 antibody, and the heavy chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO: 45; CDRH2 is shown in the amino acid sequence of SEQ ID NO: 46; CDRH3 is shown in the amino acid sequence of SEQ ID NO: 47. In one embodiment, the aforementioned extracellular antigen binding domain further comprises a light chain variable region of an anti-CD22 antibody, and the light chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO: 48; CDRL2 is shown in the amino acid sequence of SEQ ID NO: 49; CDRL3 is shown in the amino acid sequence of SEQ ID NO: 50. In one embodiment, the aforementioned extracellular antigen binding domain is a LL2 single chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO: 51. In another embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-CD22 antibody, wherein the heavy chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO:52; CDRH2 is shown in the amino acid sequence of SEQ ID NO:53; CDRH3 is shown in the amino acid sequence of SEQ ID NO:54. In one embodiment, the extracellular antigen-binding domain further comprises a light chain variable region of an anti-CD22 antibody, wherein the light chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO:55; CDRL2 is shown in the amino acid sequence of SEQ ID NO:56; CDRL3 is shown in the amino acid sequence of SEQ ID NO:57. In one embodiment, the extracellular antigen-binding domain is a G5 / 44 single-chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO:58.In another embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-CD22 antibody, wherein the heavy chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRH1 is shown in the amino acid sequence of SEQ ID NO:59; CDRH2 is shown in the amino acid sequence of SEQ ID NO:60; CDRH3 is shown in the amino acid sequence of SEQ ID NO:61. In one embodiment, the extracellular antigen-binding domain further comprises a light chain variable region of an anti-CD22 antibody, wherein the light chain variable region of the anti-CD22 antibody comprises the following CDRs: CDRL1 is shown in the amino acid sequence of SEQ ID NO:62; CDRL2 is shown in the amino acid sequence of SEQ ID NO:63; CDRL3 is shown in the amino acid sequence of SEQ ID NO:64. In one embodiment, the extracellular antigen-binding domain is an RFB4 single-chain variable region fragment, as shown in the amino acid sequence of SEQ ID NO:65.

[0021] Preferably, the extracellular antigen binding domain comprises a heavy chain variable region of an anti-CD19 antibody, which may be a single chain variable region fragment of FMC63.

[0022] Preferably, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-mesothelin antibody, which may be a SS1 single chain variable region fragment.

[0023] Preferably, the extracellular antigen binding domain comprises a heavy chain variable region of an anti-BCMA antibody, which may be a J6M0 single chain variable region fragment or a BB2121 single chain variable region fragment.

[0024] Preferably, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-CD22 antibody, which may be an m971 single chain variable region fragment, an LL2 single chain variable region fragment, a G5 / 44 single chain variable region fragment or an RFB4 single chain variable region fragment.

[0025] Preferably, the hinge domain is selected from the group consisting of (G4S)3 (as shown in SEQ ID NO:66), the hinge domain of CD8 (as shown in SEQ ID NO:67), the hinge domain of CD28 (as shown in SEQ ID NO:68), the hinge domain of IgG4 (as shown in SEQ ID NO:69), EAAAKGGGGS (as shown in SEQ ID NO:70), (EAAAK)3 (as shown in SEQ ID NO:71), the hinge domain of GST (as shown in SEQ ID NO:72), the hinge domain of EAAAK-GS (as shown in SEQ ID NO:73), the hinge domain of IgD (as shown in SEQ ID NO:74), the IgG4-CH3 domain (as shown in SEQ ID NO:75) and (AP)6 (as shown in SEQ ID NO:76).

[0026] Preferably, the aforementioned isolated nucleic acid molecule further comprises a nucleic acid sequence of an adapter gene. More preferably, the adapter molecule may be CD3ζ, DAP10, CD79, FcεR1γ, FcRγ or Fcβ / γ. According to the present invention, the full length protein of CD3ζ, DAP10, CD79, FcεR1γ, FcRγ or Fcβ / γ is used as an adapter molecule.

[0027] Preferably, the aforementioned isolated nucleic acid molecule further comprises a nucleic acid sequence of a CD3ζ signaling domain, a nucleic acid sequence of an FcεR1γ adapter molecule, or a nucleic acid sequence of a T2A self-truncating peptide. For example, the nucleic acid sequence of the FcεR1γ adapter molecule may be located at the 5' end of the aforementioned isolated nucleic acid molecule; and the nucleic acid sequence of the T2A self-truncating peptide may be located between the nucleic acid sequence of the FcεR1γ adapter molecule and the nucleic acid sequence of the extracellular antigen binding domain.

[0028] The hinge domain is connected to the NKp30 transmembrane domain via an extracellular stalk region of NKp30.

[0029] Preferably, in the aforementioned isolated nucleic acid molecule, when the aforementioned extracellular antigen binding domain comprises the heavy chain variable region of the anti-CD19 antibody, and the hinge domain is (EAAAK)3. When the combination of the extracellular antigen binding domain and the hinge domain is used, the prepared lentivirus can have an excellent transduction rate, and the prepared CAR-T cells have a better amplification multiple, a higher proportion of stem cell-like memory T cells (stem cell-like memory T-cells, Tscm), and a better cytotoxic ability. More preferably, the heavy chain variable region of the anti-CD19 antibody is an FMC63 single-chain antibody fragment.

[0030] Preferably, in the aforementioned isolated nucleic acid molecule, when the aforementioned extracellular antigen binding domain comprises the heavy chain variable region of the anti-mesothelin antibody, the hinge domain is a hinge domain of (G4S)3. When the combination of the extracellular antigen binding domain and the hinge domain is used, the prepared lentivirus can have an excellent transduction rate, and the prepared CAR-T cells have a better amplification multiple, a higher proportion of Tscm, and a better cytotoxic ability. More preferably, the heavy chain variable region of the anti-mesothelin antibody is an SS1 single-chain antibody fragment.

[0031] Preferably, in the aforementioned isolated nucleic acid molecule, when the aforementioned extracellular antigen binding domain comprises the heavy chain variable region of the anti-BCMA antibody, the hinge domain is the hinge domain of CD28. When the combination of the extracellular antigen binding domain and the hinge domain is used, the prepared lentivirus can have an excellent transduction rate, and the prepared CAR-T cells have a better amplification rate, a higher proportion of Tscm, and a better cytotoxic ability. More preferably, the heavy chain variable region of the anti-BCMA antibody is a J6M0 single-chain antibody fragment.

[0032] To achieve the above-mentioned purpose, the present invention further provides a plasmid, which comprises the above-mentioned isolated nucleic acid molecule, and the plasmid is a DNA plasmid or an RNA plasmid. According to the present invention, the order of the nucleic acid sequence on the plasmid from 5' to 3' is FcεR1γ adapter molecule, T2A self-truncating peptide, extracellular antigen binding domain, hinge domain, NKp30 transmembrane domain and NKp30 cytoplasmic domain. In one embodiment, the order of the nucleic acid sequence on the plasmid from 5' to 3' is 5'LTR, promoter, FcεR1γ adapter molecule, T2A self-truncating peptide, extracellular antigen binding domain, hinge domain, NKp30 transmembrane domain and NKp30 cytoplasmic domain, 3'LTR.

[0033] To achieve the aforementioned object, the present invention further provides a method for preparing a chimeric antigen receptor-cell, which comprises introducing the aforementioned isolated nucleic acid molecule into a nucleated cell.

[0034] Preferably, in the aforementioned method for preparing chimeric antigen receptor-cells, the aforementioned "introduction" is introduction by transduction using lentivirus.

[0035] Preferably, the aforementioned nucleated cells are leukocytes. Preferably, the aforementioned nucleated cells are T cells.

[0036] To achieve the aforementioned object, the present invention further provides an isolated cell containing the isolated nucleic acid molecule as described above.

[0037] Preferably, the isolated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

[0038] To achieve the above-mentioned purpose, the present invention further provides a use of the above-mentioned separated cells, which is used to prepare a drug for treating or alleviating cancer, such as B-cell acute lymphoblastic leukemia, B-cell lymphoma, Leydig cell tumor, ovarian cancer, acute myeloid leukemia, mesothelioma, colorectal cancer, lung cancer, pancreatic cancer, thyroid cancer and / or multiple myeloma.

[0039] To achieve the aforementioned purpose, the present invention further provides a use of the aforementioned isolated cells for preparing a drug for treating or alleviating an autoimmune disease. Wherein, the autoimmune disease is a disease associated with an autoimmune response for which CD19 CAR-T is effective. In one embodiment, the autoimmune disease is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, systemic sclerosis or multiple sclerosis.

[0040] The advantage of the present invention is that by transferring the nucleic acid sequences of the NKp30 transmembrane domain and the NKp30 cytoplasmic domain into T cells, the T cells have NKp30 receptor complexes and become multi-chain CAR-T cells, which can form stable immune synapses with cancer cells and have excellent cancer cell killing ability. The prepared CAR-T cells have excellent cytotoxicity, can kill cancer cells to a large extent, and have persistence; and have a high proportion of Tscm, which can be expected to reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the pLAS5w vector;

[0042] Figure 2 Schematic diagram of plasmids of Examples 1-1A to 1-5A and Comparative Example 1A;

[0043] Figure 3 Schematic diagram of the cell membrane protein structure of CAR-T cells in Examples 1-1C to 1-5C and Control Example 1C;

[0044] Figure 4A , 4B They are the flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of Example 1-4C in Test Example 3;

[0045] Figure 4C , 4D They are the flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of Example 1-5C in Test Example 3;

[0046] Figure 4E , 4F They are the flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of the control example 1C in test example 3;

[0047] FIG. 5A to FIG. 5C They are respectively the flow cytometry analysis results of T cell subset distribution of Example 1-5C, Control Example 1C and the non-transduced group in Test Example 4 on the 7th day;

[0048] Figure 6 The comparison results of luminescent cytotoxicity of the CAR-T cells of Example 1-5C in Test Example 5, the CAR-T cells of Control Example 1C, and the T cells of the untransduced group;

[0049] Fig. 7A The real-time results of the comparison of the cytotoxicity of the CAR-T cells of Example 1-5C in Test Example 6, the CAR-T cells of Control Example 1C, and the T cells of the untransduced group;

[0050] Figure 7B The 72-hour cytotoxicity comparison results of the CAR-T cells of Example 1-5C in Test Example 6, the CAR-T cells of Control Example 1C, and the T cells of the untransduced group;

[0051] Figure 8 Schematic diagram of the plasmids of Example 2A and Comparative Example 2A;

[0052] Fig. 9A , 9B Schematic diagrams of the cell membrane protein structures of CAR-T cells of Example 2C and Control Example 2C, respectively;

[0053] Fig. 10A , 10B They are the flow cytometry results of the CAR-T cells expressing anti-mesothelin receptor and NKp30 in Example 2C of Test Example 8;

[0054] Fig. 10C , 10D They are the flow cytometry results of the CAR-T cells expressing anti-mesothelin receptor and NKp30 of the control example 2C in test example 8;

[0055] Fig.11A , 11B11C are flow cytometric analysis results of T cell subset distribution on day 7 in Example 2C, Control Example 2C and the non-transduced group in Test Example 10, respectively;

[0056] Fig. 12A , Fig. 12B , Fig. 12C The real-time comparison results of the cell cytotoxicity of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C, and the T cells of the untransduced group in Test Example 11 at E:T of 3:1, 1:1, and 0.3:1, respectively;

[0057] Fig.13A , Fig. 13B and Fig. 13C The real-time comparison results of the cytotoxic re-challenge test of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C and the T cells of the untransduced group in Test Example 12 at an E:T of 1:1 on the 1st, 2nd and 3rd days respectively;

[0058] Fig.14 Schematic diagram of the plasmids of Example 3A and Comparative Example 3A;

[0059] Fig.15 Schematic diagram of the cell membrane protein structure of CAR-T cells in Example 3C and Control Example 3C;

[0060] Fig.16A , 16B They are the flow cytometry results of the CAR-T cells expressing anti-BCMA receptor and NKp30 in Example 3C of Test Example 15;

[0061] Fig. 16C The flow cytometry results of the CAR-T cells expressing anti-BCMA receptors in the control example 3C in the test example 15;

[0062] Fig.17A , 17B 17C are flow cytometric analysis results of T cell subset distribution on day 7 in Example 3C, Control Example 3C and the non-transduced group in Test Example 16, respectively;

[0063] Fig.18 The results of the cell toxicity test analyzed by flow cytometry of Example 3C, Control Example 3C and the non-transduced group in Test Example 17;

[0064] Fig.19A , 19B These are the real-time comparison results of the cell cytotoxicity of the CAR-T cells of Example 3C in Test Example 18, the CAR-T cells of Control Example 3C, and the T cells of the untransduced group at E:T of 0.3:1 and 0.1:1. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. These embodiments do not limit the contents disclosed above in the present invention. Those skilled in the art who are familiar with the present invention may make some improvements and modifications without departing from the scope of the present invention.

[0066] 1. Preparation and testing of anti-CD19-NKp30 CAR-T cells

[0067] Examples 1-1 to 1-5: Anti-CD19-NKp30 Nucleic Acid Molecules

[0068] First, a nucleic acid sequence of a full-length FcεR1γ adapter molecule (SEQ ID NO: 77) and a nucleic acid sequence of a single-chain variable region fragment FMC63 of anti-CD19 (SEQ ID NO: 78) are provided, and the 3' ends of the two nucleic acid sequences are connected to the 5' end of a nucleic acid sequence of NKp30 (SEQ ID NO: 79) without an immunoglobulin-like domain, and the nucleic acid sequence of NKp30 itself includes a stem structure region (stalk) and a NKp30 transmembrane domain and a NKp30 cytoplasmic domain. In this way, the anti-CD19-NKp30 nucleic acid molecules of Examples 1-1 to 1-5 are obtained respectively. Among them, the nucleic acid sequences of FcεR1γ and FMC63 are connected by the nucleic acid sequence of T2A self-truncating peptide (SEQ ID NO: 80); and the nucleic acid sequence of NKp30 without immunoglobulin-like domain is connected with the nucleic acid sequence of FMC63 by the nucleic acid sequence of different hinge domains. The hinge domains used in the nucleic acid sequences of Examples 1-1 to 1-5 are: (G4S)3 (SEQ ID NO: 66), hinge domain of CD28 (SEQ ID NO: 68), hinge domain of CD8 (SEQ ID NO: 67), hinge domain of IgG4 (SEQ ID NO: 69), (EAAAK)3 hinge (SEQ ID NO: 71), wherein the hinge domain of CD28, hinge domain of CD8, and hinge domain of IgG4 are all from humans. The nucleic acid sequences of the nucleic acid molecules of Examples 1-1 to 1-5 are shown in SEQ ID NOs: 81-85, respectively.

[0069] The control example 1 which does not have NKp30 without an immunoglobulin-like domain was prepared by the following method: the 3' end of the nucleic acid sequence of FMC63 was connected to the nucleic acid sequence of the CD8 transmembrane domain (SEQ ID NO: 86), and FMC63 and the CD8 transmembrane domain were connected via the CD8 hinge domain, and the nucleic acid sequences of 4-1BB (SEQ ID NO: 87) and CD3ζ signaling domain (SEQ ID NO: 88) were further connected to the 3' end of the nucleic acid sequence of the CD8 transmembrane domain, and the nucleic acid sequence of the nucleic acid molecule of the control example 1 was shown in SEQ ID NO: 89.

[0070] In addition, the hinge domains of Examples 1-1 to 1-5 and Comparative Example 1 are shown in Table 1 below

[0071] Table 1: Hinge domains of Examples 1-1 to 1-5 and Comparative Example 1

[0072]

[0073] Examples 1-1A to 1-5A: Construction of anti-CD19-NKp30 plasmids

[0074] The pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39) with a TAR chimeric 5'LTR nucleic acid sequence and driven by RSV promoter was removed from the pLAS5w vector by removing the hPGK promoter and PAC gene (commissioned by GenScript). Figure 1 Then, the nucleic acid molecules of Examples 1-1 to 1-5 and Control Example 1 were inserted into the pLAS5w vector using HpaI and EcoRI restriction enzymes to obtain the anti-CD19-NKp30 plasmids of Examples 1-1A to 1-5A and the plasmid of Control Example 1A, respectively. Figure 2 shown.

[0075] Examples 1-1B to 1-5B: Preparation of anti-CD19-NKp30 lentivirus

[0076] The target gene was transfected into human embryonic kidney HEK293 cells (ATCC, CRL-3216) using PolyJet transfection reagent (SignaGen Laboratories, SL100688) to prepare the lentiviral particles of Examples 1-1B to 1-5B and Control Example 1B. Specifically, 50 μg of one of the plasmids of Examples 1-1A to 1-5A or the plasmid of Control Example 1A containing the target gene was used as a transfer plasmid; 45 μg of pCMV deltaR8.91 was used as a packaging plasmid vector (purchased from Academia Sinica); and 5 μg of pMD.G was used as an envelope plasmid (purchased from Academia Sinica) and 300 μL of PolyJet transfection reagent were co-cultured in 10 mL of serum-free DMEM culture medium for 15 minutes to obtain a Polyjet / DNA mixed solution. After adding the Polyjet / DNA mixture to 100 mL of DMEM medium containing 10% FBS, add dropwise the 9×10 7 HEK293 cells were cultured in a T875 culture flask. After 12 to 18 hours of culture, the culture medium was replaced with 100 ml of Opti-MEM (Gibco, 31985070) containing 1 mM sodium pyruvate (Sartorius). After 16 hours of transfection, the culture medium was replaced with fresh Opti-MEM. After 48 hours of transfection, the upper layer containing lentiviral particles was collected for the first time, and fresh Opti-MEM culture medium containing 1 mM sodium pyruvate was added for 24 hours of culture, and the upper layer containing lentiviral particles was collected again. The supernatant containing lentiviral particles was centrifuged at 1500 rpm for 10 minutes at 4°C to remove cell debris, and the upper layer after centrifugation was filtered through a 0.45 μm filter membrane (Membrane-solutions). The upper layers collected twice and filtered were mixed together. Lenti-X concentration reagent (Takara Bio) was then used for 3:1 concentration, that is, the volume of the filtered upper layer: Lenti-X was 3:1. Specifically, the mixture of the upper layer and Lenti-X concentrated reagent was mixed overnight at 4°C, then centrifuged at 1500 rpm for 45 minutes at 4°C, the supernatant was removed, and the lower layer pellet was washed with 2 mL of X-VIVO TM 15 culture medium (Lonza) was resuspended to obtain concentrated lentiviruses of Examples 1-1B to 1-5B and lentivirus of Control Example 1B, and stored at -80°C for future use.

[0077] Test Example 1: Test of the Anti-CD19-NKp30 Lentivirus Titer of Examples 1-1B to 1-5B

[0078] The lentiviral titer was determined by transducing 3-fold serial dilutions of the lentiviral sample to 4 × 10 4 The lentivirus samples used in this test example 1 are the concentrated lentiviruses of Examples 1-1B to 1-5B and Control Example 1B. 50 μL of the lentivirus sample was added to 100 μL of X-Vivo TM 15 culture medium, and perform 3-fold serial dilution with the above culture medium, repeat this dilution step until the dilution is 6561 times, and obtain multiple groups of serially diluted lentiviral samples. Then, take 50 μL of each group of serially diluted lentiviral samples and add them to a 96-well plate (U bottom), where each well of the plate has 4×10 4 Jurkat cells / 100 μL culture medium / well were added, and lentivirus transduction was performed for 72 hours. 200 ng of biotinylated human CD19, Fc Tag (ACROBiosystems, CD9-H8259) was used to identify the anti-CD19 receptor, and phycoerythrin (PE)-labeled streptavidin (Invitrogen, 12-4317-87) diluted 500 times was used to detect the expression of the anti-CD19 receptor. The expression of the target protein was analyzed by flow cytometry (Sony, SA3800) to measure the transduction efficiency of the lentivirus. In addition, Jurkat cells that were not transduced with lentivirus were used as a negative control group. Among them, the effective titer of the anti-CD19-NKp30 lentivirus was calculated using the following formula, and the effective titer of the lentivirus was used to determine the amount of lentivirus used in the preparation of CAR-T cells.

[0079] Lentivirus effective titer (TU / mL) = (percentage of cells expressing anti-CD19 receptor) × 4 × 10 4 ×20×dilution factor

[0080] The experimental results are shown in Table 2 below:

[0081] Table 2: Effective titer of lentivirus of Examples 1-1B to 1-5B and Control Example 1B (transducing unit, TU / mL)

[0082]

[0083] If the effective titer of lentivirus is greater than or equal to 1×10 6TU / mL, when preparing CAR-T cells, a smaller volume of lentivirus can be used, which can smoothly transduce T cells into CAR-T cells and enable subsequent T cells to grow well. Therefore, according to the results of Table 2 above, Examples 1-1B, 1-4B, 1-5B and Control Example 1B all meet this standard, which is conducive to the further preparation of CAR-T cells. It can be seen from the results of Table 2 that the effective titer of the lentivirus of Example 1-4B with an IgG4 hinge domain is closest to the effective titer of the lentivirus of Control Example 1B.

[0084] Examples 1-1C, 1-4C and 1-5C: Anti-CD19-NKp30 CAR-T cells

[0085] Fresh blood was collected from healthy donors with their consent. On day 0, human peripheral blood mononuclear cells (PBMCs) were collected using SepMate centrifuge tubes (STEMCELL, 86450). In short, the blood sample was diluted with PBS buffer containing 2% FBS in the same volume as the fresh blood sample, and the diluted sample was dripped into the SepMate centrifuge tube along the tube wall, so that the SepMate tube was kept vertical, and the blood sample was mixed with the density gradient centrifugation solution (ficoll) in the SepMate. Centrifuge at 1200×g for 20 minutes at room temperature. The upper layer (rich in mononuclear cells) was poured into a new tube, washed repeatedly with PBS buffer containing 2% FBS, and then centrifuged at 300×g at room temperature for 8 minutes. The lower layer was taken as PBMC. Next, magnetic beads containing anti-CD3 / CD28 antibodies on the surface (the magnetic beads are CTS (Cell Therapy Systems) Dynabeads CD3 / CD28 (Gibco, Cat. No. 40203D)) were used in combination with DynaMag-Spin (purchased from Thermo) to separate T cells from the collected peripheral blood mononuclear cells (PBMCs). The isolated PBMC cells were tested for the proportion of CD3+T cells in the PBMC using anti-CD3 antibodies (Biolegend, 300441) and flow cytometry, and the number of CD3+T cells was calculated. Magnetic beads containing anti-CD3 / CD28 antibodies on the surface, which are three times the number of CD3+T cells, were added to PBMC cells and incubated at room temperature for 30 to 60 minutes to allow the magnetic beads containing anti-CD3 / CD28 antibodies on the surface to bind to T cells. Then, a magnetic stand (DynaMag-Spin; Thermo) was used to select CD3-positive T cells to achieve the purpose of removing B cells, NK cells and monocytes in PBMCs. Then, a T cell and magnetic bead complex formed by antigen-antibody binding was obtained. The T cell and magnetic bead complex was 2×10 5The cells were dispersed in X-VIVO containing 200 IU / mL of recombinant human IL-2 (R&D Systems) at a density of 100 cells / mL. TM 15 culture medium (Lonza), cultured at 37°C and 5% carbon dioxide for 24 hours to activate T cells. The activated T cells are then transduced for 48 hours by adding one of the lentiviruses of Examples 1-1B, 1-4B, 1-5B, or Control Example 1B with a viral infection dose MOI (multiplicity of infection) of 2 to obtain the CAR-T cells and magnetic bead complexes of Examples 1-1C, 1-4C, 1-5C, and Control Example 1C, and the T cell group without lentivirus treatment is the untransduced group, wherein the MOI here = viral titer (TU / mL) × viral volume (mL) / total number of T cells. On the third day, the suspension of the CAR-T cell and magnetic bead complex was removed from the magnetic beads using a magnetic stand to obtain the anti-CD19-NKp30 CAR-T cells of Examples 1-1C, 1-4C, 1-5C and the CAR-T cells of Control Example 1C. The structural schematics of each group are shown as follows. Figure 3 As shown, the transferred nucleic acid sequences of Examples 1-1C, 1-4C, and 1-5C contain FcεR1γ, so they are multi-chain CAR-T cells; while the transduced nucleic acid sequence of Control Example 1C does not contain FcεR1γ, so it is a single-chain CAR-T cell. The obtained CAR-T cells were transferred to an X-VIVO containing 200 IU / mL of recombinant human IL-2. TM The cells were cultured in a new culture flask with 15% culture medium under the same conditions as the transduction step, and the CAR-T cells were allowed to proliferate until day 7. Finally, the number of CAR-T cells in the non-transduced group and the anti-CD19-NKp30 group was calculated. During the CAR-T culture period, additional culture medium was added every 1 to 3 days to continuously adjust the cell density to 1.0×10 6 cells / mL.

[0086] Test Example 2: Proliferation test of CAR-T cells in Examples 1-1C, 1-4C and 1-5C and Control Example 1C

[0087] The proliferation rate of CAR-T cells in Examples 1-1C, 1-4C and 1-5C was calculated by counting the total number of cells at a specific time point during the preparation of the anti-CD19-NKp30 CAR-T cells in Examples 1-1C, 1-4C and 1-5C. Specifically, on the third day of the preparation of the CAR-T cells, the CAR-T cells expressing anti-CD19-NKp30 were inoculated at a density of 1×10 610 cells / mL were seeded in a 12-well plate. After removing the magnetic beads, the cell survival rate and cell density were measured, including staining the cells with acridine orange / propidium iodide (AO / PI) and then automatically quantifying the cell number using an automatic cell counter (LunaAutomated Cell Counter, Logos Biosystems). The remaining cells were subcultured at a density of 2×10 5 Up to 5×10 5 / mL density was seeded in a culture dish to expand the cells. The number of cells was measured on days 0 and 7 of CAR-T cell preparation to calculate the cell expansion rate. The experimental results are shown in Table 3 below. Among them, the expansion rate of anti-CD19-NKp30 CAR-T cells is the proliferation multiple calculated by standardizing the number of cells on day 0.

[0088] Table 3: Expansion folds of CAR-T cells of Examples 1-1C, 1-4C, 1-5C and Control Example 1C on Day 7

[0089]

[0090] If the expansion multiple can reach 5 times or more, it will be beneficial to the subsequent T cell functional test. Therefore, according to the results in Table 3 above, the CAR-T cells of Example 1-1C, Example 1-4C, and Example 1-5C all have good T cell expansion multiples, among which the CAR-T cells of Example 1-1C with a (G4S)3 hinge domain have the best expansion effect, which is much higher than the CAR-T cells of Control Example 1C.

[0091] Test Example 3: Expression detection of CAR-T cells in Examples 1-1C, 1-4C, 1-5C and Control Example 1C; Lentivirus transduction rate in Examples 1-1B, 1-4B, 1-5B and Control Example 1B

[0092] The CAR-T cells of Examples 1-1C, 1-4C, 1-5C and Control Example 1C were treated with biotinylated human CD19, Fc Tag (ACROBiosystems, CD9-H8259) at a concentration of 100 μg / mL to identify the anti-CD19 receptor and phycoerythrin (PE)-labeled streptavidin (Invitrogen, 12-4317-87) diluted 500 times to detect the expression of the anti-CD19 receptor in iced PBS containing 2% FBS according to the manufacturer's operating manual, while the expression of NKp30 was detected by adding 5 μL of anti-NKp30 antibody (BioLegend) labeled with BV421 fluorescent dye to each sample. Among them, Figure 4A , Figure 4B These are the flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of Examples 1-4C, respectively; Figure 4C , Figure 4D These are the flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of Examples 1-5C, respectively; Figure 4E , Figure 4F The flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 of the control example 1C are shown respectively. The percentage of cells expressing anti-CD19 receptor compared to the total number of cells is used as the transduction rate of the lentivirus. The experimental results are shown in Table 4 below.

[0093] Table 4: Transduction rate of lentivirus of Examples 1-1B, 1-4B, 1-5B and Control Example 1B

[0094]

[0095] If the transduction rate of preparing CAR-T cells is greater than 25%, it will be beneficial to the functional test of CAR-T cells after subsequent amplification. Therefore, according to the results of Table 4 above, it can be concluded that the lentivirus of Example 1-4B having an IgG4 hinge domain and the lentivirus of Example 1-5B having an (EAAAK)3 hinge domain both have excellent transduction rates.

[0096] Test Example 4: T cell phenotype analysis of CAR-T cells of Examples 1-1C, 1-4C, 1-5C and Control Example 1C

[0097] T cell typing was performed by antibody fluorescence labeling and flow cytometry (Sony Biotechnology, SA3800) with SA3800 software to detect T cell surface markers, the antibodies used were: CD3-FITC (BioLegend, 300406), CD4-PE-Cy7 (BioLegend, 300512), CD8-AF700 (Beckman, B76279), CD95-APC (BioLegend, 305612), CD45RA-PE (BioLegend, 304108), CCR7-BV421 (BioLegend, 353208), and a fluorescence-minus-one (FMO) control group containing all other fluorescent pigments except CCR7-BV421 and CD45RA-PE was set up. In this test example, non-transduced activated T cells were used as the control group (non-transduced group). The staining results obtained by the above method were used to determine the distribution (%) of each T cell subset according to Annals of Oncology, Vol. 32, No. 11, pp. 1366-1380: Tscm, central memory T cells (T CM ), effector memory T cells (effector memory T cells, T EM ), terminally differentiated effector memory T cells (T EMRA ) (or called effector T cell (effector T cell, T EFF ), and the ratio of Tscm in the CAR-T cells of Examples 1-1C, 1-4C, 1-5C and Control Example 1C is shown in Table 5 below. The results of Example 1-5C and the flow cytometry results of the T cell subset distribution of Control Example 1C and the non-transduced group are shown in Table 5. Figure 5A , 5B Among them, T cells expressing CD45RA and CCR7 are Tscm, and T cells expressing CD45RA but not CCR7 are T EMRA (or T EFF ), those that do not express CCR7 and CD45RA are T EM , those expressing CCR7 but not CD45RA are T CM .

[0098] Table 5: Percentage of Tscm in CAR-T cells of Examples 1-1C, 1-4C, 1-5C, Control Example 1C, and T cells in the untransduced group

[0099]

[0100] The optimal range of the percentage of stem cell-like memory T cells (Tscm) of CAR-T cells is above 40%, because a high proportion of Tscm can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Figures 5A to 5C It can be seen that Example 1-1C having a (G4S)3 hinge domain, Example 1-4C having an IgG4 hinge domain, and Example 1-5C having a (EAAAK)3 hinge domain all have a high proportion of Tscm, and are comparable to the CAR-T cells of the traditional single-chain control Example 1C. Therefore, it can be expected that the CAR-T cells of Examples 1-1C, 1-4C, and 1-5C can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0101] Test Example 5: Luciferase killing assay

[0102] First, the PLL-CMV-rFluc-T2A-GFP-mPGK Lenti-Labeler lentiviral vector (SystemBiosciences) was transduced into the Raji human lymphoma cell line (EP-CL-0189, Elabscience) (hereinafter referred to as Raji cells), and then a single stable expression cell line was screened to obtain GFP / Luc+Raji cells that simultaneously express green fluorescent protein (GFP) and luciferase. Since Raji cells themselves can express CD19 in large quantities, these GFP / Luc+Raji cells were cultured in RPMI-1640 medium (Gibco) containing 10% FBS as the target cells for the luciferase cytotoxicity test.

[0103] In order to test the anti-tumor activity of the CAR-T cells prepared in Examples 1-4C and 1-5C against CD19-NKp30 on the 7th day of preparation, the aforementioned GFP / Luc+Raji cancer cell line expressing CD19 was used as the target cell for cytotoxicity test. The effector cells used in this test example were the CAR-T cells of Examples 1-4C, 1-5C and Control Example 1C, respectively, and untransduced T cells were used as a control group. Specifically, the effector cells and target cells were co-cultured in a 96-well plate at an effector-to-target ratio (E:T ratio) of 3:1, cultured at 37°C and 5% carbon dioxide for 24 hours, and three replicates were performed.

[0104] The fluorescence produced by the luciferase expressed by the surviving cancer cells was measured using the luciferase assay system reagent kit (Promega, E1501) to estimate the relative number of surviving cancer cells that were not lysed by CAR-T cells. The cell-specific lysis ratio was calculated using the following formula:

[0105] Cell-specific lysis % = 100% × (number of target cells lysed experimentally – number of target cells lysed spontaneously) ÷ (number of target cells lysed maximally – number of target cells lysed spontaneously)

[0106] in,

[0107] The number of target cells lysed in the experiment = the number of target cells lysed in the group co-cultured with effector cells

[0108] The number of spontaneously lysed target cells = the number of lysed target cells in the group without effector cells, that is, the number of lysed target cells after culturing GFP / Luc+Raji cells in RPMI-1640 medium containing 10% FBS at 37°C and 5% carbon dioxide for 24 hours.

[0109] Maximum number of target cells lysed = the number of target cells lysed after adding 100 μl (microliters) of 1% Triton X-100 to the group without effector cells

[0110] The experimental results are shown in Table 6 below; among them, the cytotoxicity comparison results of the CAR-T cells of Example 1-5C, the CAR-T cells of Control Example 1C, and the T cells of the untransduced group are shown in Figure 6 shown.

[0111] Table 6: Luciferase toxicity test results of CAR-T cells of Examples 1-4C, 1-5C, Control Example 1C and T cells of the untransduced group (specific lysis %)

[0112]

[0113] According to Table 6 and Figure 6 The results show that the cytotoxicity of CAR-T cells in Example 1-4C and Example 1-5C is better than that of the non-transduced group, and the cytotoxicity of CAR-T cells in Example 1-5C is better than that of the control group 1C. In addition, if the cytotoxicity of CAR-T cells is >50%, it will have sufficient cytotoxicity when CAR-T is prepared with patient cells in the future. According to the results of Table 6 above, the CAR-T cells in Example 1-4C and Example 1-5C have good cytotoxic effects. And Example 1-5C with the (EAAAK)3 hinge domain has an excellent cytotoxic effect that is better than the control example 1C of the traditional single-chain CAR-T cell.

[0114] Test Example 6: Real-time cytotoxicity assay (RTCA) using xCELLigence

[0115] First, a human CD19 cDNA fragment was selected from Raji cells (purchased from Elabscience, CL-0189). A cDNA gene fragment with a truncated CD19 (SEQ ID NO: 90) was amplified using PCR. The aforementioned truncated CD19 cDNA fragment (1-342 amino acids) refers to a fragment that does not have an intracellular domain, but has a transmembrane domain and does not contain a cytoplasmic signaling domain. The aforementioned truncated CD19 cDNA fragment was inserted into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39) with a puromycin resistance gene using the HpaI and NheI restriction enzyme sites to obtain a vector containing a truncated CD19. The aforementioned vector containing a truncated CD19 was transfected into the SKOV3 human ovarian cancer cell line using Polyjet (SignaGen Laboratories, SL100688). The transfected SKOV3 cells were cultured in McCoy 5A Medium (Gibco, 16600082) containing 2 μg / mL puromycin and 10% FBS to screen out drug-resistant SKOV3 cell lines that express CD19. Finally, flow cytometry was used to confirm the expression of CD19 in the cells. The SKOV3 cell lines expressing CD19 were used as target cells in the real-time cytotoxicity test recorded by the xCELLigence system.

[0116] The xCELLigence RTCA system (Agilent Technologies, RTCA SP) was placed in a 37°C, 5% carbon dioxide cell culture incubator and connected to the interface of the external analysis and control unit via a cable. The RTCA SoftwarePro software was used to control and monitor the instrument in real time, including real-time data display and analysis functions. Specifically, the xCELLigence RTCA system was used to continuously monitor the cytotoxic effect of tumor cells for 90 hours: First, McCoy 5A culture medium containing 10% FBS was added to the holes of the E-plate 96-well plate (Agilent Technologies, 300600910), and the background resistance of the culture medium was briefly measured. Then, the aforementioned SKOV3 cells expressing CD19 were used as target cells, and 2×10 4 The density of cells / mL was 1:1, and the cells were seeded in a culture dish with background correction and cultured overnight. After the target cells were attached and impedance was measured for 29 hours, the effector cells (E) were added to the culture dish at a ratio of 0.1:1 effector cells: target cells (E:T ratio) to make the final volume of each well 200 μL. The effector cells used in this test example were the CAR-T cells of Example 1-5C and Control Example 1C. Untransduced T cells were used as a control group (untransduced group). 100 μl (microliters) of 1% Triton X-100 was added to the culture dish with 2×10 4 cells / mL in the cell culture medium of the target cells, which is the full lysis group. The cell index (CI) is detected every 15 minutes for at least 72 hours. The original RTCA impedance background value is standardized with the cell index data before the effector cells are added. The aforementioned cell index is a dimensionless parameter, which is obtained by measuring the relative change of electrical impedance to represent the cell state. For example: when the cell does not exist or is not attached to the electrode, the CI is zero; and under the same physiological conditions, the more cells attached to the electrode, the greater the CI value. In addition, changes in cell status, such as changes in cell morphology or cell adhesion to the resistor, will lead to changes in CI.

[0117] The experimental results are as follows Fig. 7A and 7B As shown, it can be seen that the cytotoxic effect of the CAR-T cells of Example 1-5C having the (EAAAK)3 hinge domain is much better than that of the CAR-T cells of the control group 1C and the untransduced T cells, and is more than 3 times the cytotoxic ability of the CAR-T cells of the control group 1C, and has an excellent cytotoxic effect.

[0118] In summary, the lentivirus of Example 1-5B having the (EAAAK)3 hinge domain has an excellent transduction rate, and the CAR-T cells of Example 1-5C have an excellent CAR-T cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm cell populations, which can be expected to reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Therefore, the (EAAAK)3 hinge domain is the most suitable hinge domain for matching with the anti-CD19 receptor.

[0119] 2. Preparation and testing of anti-mesothelin-NKp30 CAR-T cells

[0120] Example 2: Anti-mesothelin-NKp30 nucleic acid molecules

[0121] The preparation method of Example 2 is similar to the preparation method of Example 1-1, except that the single-chain variable region fragment used in Example 2 is not the anti-CD19 FMC63 used in the preparation method of Example 1-1, but the single-chain variable region fragment SS1 nucleic acid sequence of anti-mesothelin (as shown in SEQ ID NO: 91), and the hinge domain selected is (G4S) 3. The nucleic acid sequence of the nucleic acid molecule of Example 2 is shown in SEQ ID NO: 92.

[0122] The preparation method of Control Example 2 without NKp30 is similar to that of Control Example 1, except that the single-chain variable region fragment used in Control Example 2 is not the single-chain variable region fragment of the anti-CD19 antibody used in the preparation method of Control Example 1: FMC63, but the nucleic acid sequence of the single-chain variable region fragment SS1 of anti-mesothelin. The nucleic acid sequence of the nucleic acid molecule of Control Example 2 is shown in SEQ ID NO:93.

[0123] Example 2A: Construction of anti-mesothelin-NKp30 plasmid

[0124] The preparation method of Example 2A is similar to the preparation method of the plasmid of Example 1-1A, except that Example 2A uses BstBI and EcoRI restriction enzymes to insert the anti-mesothelin-NKp30 nucleic acid molecule of Example 2 into the aforementioned pLAS5w vector. While Control Example 2A commissioned GenScript to insert the nucleic acid molecule of Control Example 2 into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39) using HpaI and EcoRI restriction enzymes. The obtained anti-mesothelin-NKp30 plasmid of Example 2A and the plasmid of Control Example 2A are as follows: Figure 8 shown.

[0125] Example 2B: Preparation of anti-mesothelin-NKp30 lentivirus

[0126] The preparation method of Example 2B is similar to the preparation method of Example 1-1B, except that Example 2B uses the plasmid of Example 2A as the transfer plasmid, while the lentivirus of Control Example 2B uses the plasmid of Control Example 2A as the transfer plasmid.

[0127] Test Example 7: Effective titer of the anti-mesothelin-NKp30 lentivirus of Example 2B

[0128] The experimental method of Test Example 7 is similar to that of Test Example 1, except that biotinylated human mesothelin protein, His, Avitag (, ACROBiosystems, MSN-H82E9) is used to identify anti-mesothelin receptors, and phycoerythrin (PE)-labeled streptavidin (Streptavidin conjugated PE) (Invitrogen, 12-4317-87) diluted 500 times is used to detect the expression of anti-mesothelin receptors.

[0129] The test results are shown in Table 7 below:

[0130] Table 7: Effective titer of lentivirus in Example 2B and Control Example 2B

[0131] Group Example 2B Comparative Example 2B Lentivirus effective titer (TU / mL) <![CDATA[3.9×10 7 ]]> <![CDATA[3×10 7 Up to 7×10 7 ]]>

[0132] If the effective titer of lentivirus is greater than or equal to 1×10 6 TU / mL, a smaller volume of lentivirus can be used to prepare CAR-T cells, that is, T cells can be smoothly transduced into CAR-T cells, and subsequent T cells can grow well. Therefore, according to the results of Table 7 above, Example 2B with a (G4S)3 hinge domain meets this standard, which is conducive to further preparation of CAR-T cells.

[0133] Example 2C: Anti-mesothelin-NKp30 CAR-T cells

[0134] The method for preparing the anti-mesothelin-NKp30 CAR-T cells of Example 2C is similar to the method for preparing Example 1-1C, except that the T cells are transduced with the lentivirus of Example 2B, and the MOI during the lentivirus transduction is 3. The CAR-T cells of Control Example 2C are transduced with the lentivirus of Control Example 2B, and the MOI during the lentivirus transduction is 3. The schematic diagrams of the structures of the anti-mesothelin-NKp30 CAR-T cells of Example 2C and the CAR-T cells of Control Example 2C are shown in FIG. Fig. 9A and 9B shown.

[0135] Test Example 8: Proliferation test of anti-mesothelin-NKp30 CAR-T cells

[0136] The test method of Test Example 8 is similar to that of Test Example 2, except that this test example is to detect the expansion rate of the anti-mesothelin-NKp30 CAR-T cells of Example 2C. The results are shown in Table 8 below.

[0137] Table 8: Expansion folds of CAR-T cells of Example 2C and Control Example 2C on day 7

[0138] Group Example 2C Comparative Example 2C The fold increase on the 7th day of expansion compared to the 0th day 29.4X 18.9X

[0139] Since if the expansion multiple can reach 5 times or more, it will be beneficial to the subsequent T cell functional test, therefore, according to the results of Table 8 above, it can be seen that the CAR-T cells of Example 2C have a good T cell expansion multiple and are better than the CAR-T cells of Control Example 2C.

[0140] Test Example 9: Expression detection of CAR-T cells in Example 2C and Control Example 2C; Lentivirus transduction rate in Example 2B and Control Example 2B

[0141] The test method of Test Example 9 is similar to that of Test Example 3, except that this test example uses biotinylated human mesothelin protein, His, Avitag (ACROBiosystems, MSN-H82E9) to identify anti-mesothelin receptors, and uses phycoerythrin (PE)-labeled streptavidin (Streptavidin conjugated PE) (Invitrogen, 12-4317-87) diluted 500 times to detect the expression of anti-mesothelin receptors and NKp30 in the CAR-T cells of Example 2C and Control Example 2C. The test results of Example 2C and Control Example 2C are as follows: Fig. 10A , Fig. 10B and Fig. 10C , Fig. 10D The flow cytometry results are shown. The percentage of the number of cells expressing anti-mesothelin receptor compared to the total number of cells is used as the transduction rate of the lentivirus of Example 2B or Control Example 2B. The experimental results are shown in Table 9 below.

[0142] Table 9: Transduction rate of lentivirus of Example 2B and Control Example 2B

[0143] Group Example 2B Comparative Example 2B Transduction rate 50% to 85% 60% to 79%

[0144] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial to the functional test of the subsequent amplified CAR-T cells. Fig. 10A The results showed that the lentivirus of Example 2B had a good transduction rate.

[0145] Test Example 10: T cell phenotype analysis of CAR-T cells of Example 2C

[0146] The test method of Test Example 10 is similar to that of Test Example 4, except that this test example is to prepare the anti-mesothelin-NKp30 CAR-T cells of Example 2C, and the cells on the 7th day of preparation are tested. The ratio of Tscm in the CAR-T cells of Example 2C and Control Example 2C and the T cells of the non-transduced group is shown in Table 10 below; the results of Example 2C and the flow cytometer results of the distribution of T cell subsets of Control Example 2C and the non-transduced group are shown in Table 10 below. Fig.11A , 11B As shown in Figure 11C, T cells expressing CD45RA and CCR7 are Tscm.

[0147] Table 10: Percentage of Tscm in activated T cells in Example 2C, Control Example 2C and non-transduced groups

[0148] Group Example 2C Comparative Example 2C Untransduced group %Tscm 76％ 74％ 79％

[0149] If the percentage of stem cell-like memory T cells in CAR-T cells is above 40%, the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy can be further reduced. Fig.11A , 11B From Figure 11C, it can be seen that Example 2C with a (G4S)3 hinge domain has a high proportion of Tscm, which is better than the traditional single-chain CAR-T cells of Control Example 2C, and is similar to the activated T cells of the untransduced group. Therefore, it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0150] Test Example 11: Real-time Cytotoxicity Assay Using xCELLigence

[0151] HeLa cervical cancer cells (hereinafter referred to as HeLa cells) are mesothelin-positive cells. HeLa cells (purchased from Elabscience, CL-0101) were cultured in DMEM medium containing 10% FBS and cultured in an environment of 5% CO2 and 37°C, and used as target cells in the subsequent real-time cytotoxicity test of xCELLigence.

[0152] The experimental method of Test Example 11 is similar to that of Test Example 6, except that the CAR-T cells of Example 2C are used as effector cells and the aforementioned Hela cells are used as target cells. After 18 hours of background impedance measurement, the target cells are incubated at 1.2×10 4 The density of cells / mL was 1:1, 1:1 and 0.3:1, and the effector cells were added to the culture dish at an E:T ratio of 3:1, 1:1 and 0.3:1. In this test case, the whole lysis group was not used as a control.

[0153] The experimental results of E:T ratios of 3:1, 1:1 and 0.3:1 are as follows Figures 12A to 12C As shown, it can be seen from the results in the figure that the toxicity results of the three E:T ratios all show that 58 hours after the start of the experiment, that is, 40 hours after the target cells and effector cells were co-cultured, the CAR-T cells of Example 2C can kill 100% of the HeLa cells, which is equivalent to the CAR-T cell toxicity of the single-chain control example 2C.

[0154] Test Example 12: Re-challenge test of real-time cell killing using xCELLigence

[0155] The purpose of this experiment is to test whether the effector cells still have a toxic effect after being stimulated by the target cells again. First, DMEM medium containing 10% FBS was added to the holes of the E-plate 96-well plate (Agilent Technologies, 300600910), and the background resistance of the culture medium was briefly measured. Then Hela cells (purchased from Elabscience, CL-0101) were used as target cells and seeded in the 96-well plate containing DMEM medium. The target cells were cultured at 1.2×10 4 The cells were seeded at a density of 10 cells / mL in a background-corrected culture dish and cultured overnight to allow the target cells to attach. After 18 hours of background impedance measurement, effector cells were added to co-culture at an E:T ratio of 1:1, and HeLa cells were added for re-challenge one and two days later (i.e., 2.4×10 cells were seeded on the second and third days, respectively). 4 Cells / mL), the effector cells used in this test example are the CAR-T cells of Example 2C and Control Example 2C, and untransduced T cells are used as a control group. The cell index is detected every 15 minutes for at least 72 hours. The original RTCA impedance data is normalized by the cell index data before adding effector cells.

[0156] The results of the re-challenge experiments on the first day of co-culture and on the second and third days are as follows: Fig.13A , 13B As shown in Figure 13C, the killing effect of Example 2C is better than that of Control Example 2C, and the re-challenge effect is also better than that of Control Example 2C. Therefore, in addition to the excellent killing effect of cancer cells in Example 2C, it also has excellent killing persistence. Therefore, this experiment can confirm that the CAR-T cells of Example 2C are superior to the CAR-T cells of Control Example 2C in terms of killing intensity and persistence of cancer cells.

[0157] In summary, the lentivirus of Example 2B using (G4S)3 as the hinge domain has an excellent transduction rate, and the CAR-T cells of Example 2C have an excellent CAR-T cell proliferation rate and excellent cytotoxicity and persistence, and have a high proportion of Tscm, which can be expected to reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Therefore, the (G4S)3 hinge domain is a hinge domain suitable for use with anti-mesothelin receptors.

[0158] 3. Preparation and testing of anti-BCMA-NKp30 CAR-T cells

[0159] Example 3: Anti-BCMA-NKp30 Nucleic Acid Molecules

[0160] The preparation method of Example 3 is similar to the preparation method of Example 1-1, except that the single-chain variable region fragment used in Example 3 is not FMC63, which is the single-chain variable region fragment of the anti-CD19 antibody prepared in the preparation method of Example 1-1, but a nucleic acid sequence of the single-chain variable region fragment J6M0 with anti-BCMA (as shown in SEQ ID NO: 94), and the hinge domain selected is the hinge domain of CD28. The nucleic acid sequence of the nucleic acid molecule of Example 3 is shown in SEQ ID NO: 95.

[0161] The preparation method of Control Example 3 without NKp30 is similar to that of Control Example 1, except that the single-chain variable region fragment used in Control Example 3 is not the single-chain variable region fragment of the anti-CD19 antibody: FMC63 used in the preparation method of Control Example 1, but the nucleic acid sequence of the single-chain variable region fragment J6M0 of anti-BCMA is used. The nucleic acid sequence of the nucleic acid molecule of Control Example 3 is shown in SEQ ID NO:96.

[0162] Example 3A: Construction of anti-BCMA-NKp30 plasmid

[0163] The preparation method of Example 3A is similar to the preparation method of the plasmid of Example 1-1A, except that Example 3A uses NheI and EcoRI restriction enzymes to insert the anti-BCMA-NKp30 nucleic acid molecule of Example 3 into the aforementioned pLAS5w vector. In contrast, in Control Example 3A, GenScript was commissioned to insert the nucleic acid molecule of Control Example 3 into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39) using HpaI and EcoRI restriction enzymes. The anti-BCMA-NKp30 plasmid of Example 3A and the plasmid of Control Example 3A are obtained as follows. Fig.14 shown.

[0164] Example 3B: Preparation of anti-BCMA-NKp30 lentivirus

[0165] The preparation method of Example 3B is similar to the preparation method of Examples 1-2B, except that Example 3B uses the plasmid of Example 3A as the transfer plasmid, while the lentivirus of Control Example 3B uses the plasmid of Control Example 3A as the transfer plasmid.

[0166] Test Example 13: Effective titer of anti-BCMA-NKp30 lentivirus of Example 3B

[0167] The experimental method of Test Example 13 is similar to that of Test Example 1, except that biotinylated BCMA protein, His, Avitag (His & AVI Tag, Sino Biological. Cat: 10620-H40H-B) is used to identify anti-BCMA receptors, and phycoerythrin (PE)-labeled streptavidin (Streptavidin conjugated PE) (Invitrogen, 12-4317-87) diluted 500 times is used to detect the expression of anti-BCMA receptors.

[0168] The test results are shown in Table 11 below:

[0169] Table 11: Effective titer of lentivirus in Example 3B and Control Example 3B

[0170] Group Example 3B Comparative Example 3B Lentivirus effective titer (TU / mL) <![CDATA[1.44×10 7 ]]> <![CDATA[1.5×10 7 ]]>

[0171] If the effective titer of lentivirus is greater than or equal to 1×10 6 , a smaller volume of lentivirus can be used to prepare CAR-T cells, and T cells can be smoothly transduced into CAR-T cells, and subsequent T cells can grow well. Therefore, according to the results of Table 11 above, Example 3B meets this standard, is conducive to the preparation of CAR-T cells, and the effective titer is similar to that of Control Example 3B, which is conducive to the further preparation of CAR-T cells.

[0172] Example 3C: Anti-BCMA-NKp30 CAR-T cells

[0173] The method for preparing anti-BCMA-NKp30 CAR-T cells in Example 3C is similar to the method for preparing in Example 1-2C, except that the T cells are transduced with the lentivirus of Example 3B, and the MOI during the lentivirus transduction is 3. The CAR-T cells in Control Example 3C are transduced with the lentivirus of Control Example 3B, and the MOI during the lentivirus transduction is 3. The schematic diagram of the structure of the anti-BCMA-NKp30 CAR-T cells in Example 3C and the CAR-T cells in Control Example 3C is as follows Fig.15 shown.

[0174] Test Example 14: Proliferation test of anti-BCMA-NKp30 CAR-T cells

[0175] The test method of Test Example 14 is similar to that of Test Example 2, except that this test example is to detect the proliferation rate of the anti-BCMA-NKp30 CAR-T cells of Example 3C. The results are shown in Table 12 below.

[0176] Table 12: Expansion folds of CAR-T cells of Example 3C and Control Example 3C on day 7

[0177] Group Example 3C Comparative Example 3C The fold increase on the 7th day of expansion compared to the 0th day 20.2X 12.5X

[0178] Since if the expansion multiple can reach 5 times or more, it will be beneficial to the subsequent T cell functional test, therefore according to the results of Table 12 above, it can be seen that the CAR-T cells of Example 3C have a good T cell expansion multiple, which is much higher than the CAR-T cells of Control Example 3C.

[0179] Test Example 15: Expression detection of CAR-T cells in Example 3C and Control Example 3C; Lentivirus transduction rate in Example 3B and Control Example 3B

[0180] The test method of test example 15 is similar to that of test example 3, except that this test example uses biotinylated BCMA protein, His, Avitag (His & AVI Tag, Sino Biological. Cat: 10620-H40H-B) to identify anti-BCMA receptors, and uses phycoerythrin (PE) labeled streptavidin conjugated PE (Invitrogen, 12-4317-87) diluted 500 times to detect the expression of anti-BCMA receptors. The test results of Example 3C and Control Example 3C are shown as follows: Fig.16A , Fig. 16B and Fig. 16C The flow cytometry results are shown. The percentage of cells expressing anti-BCMA receptors compared to the total number of cells is used as the transduction rate of the lentivirus of Example 3B and Control Example 3B, and the experimental results are shown in Table 13 below.

[0181] Table 13: Transduction rate of lentivirus of Example 3B and Control Example 3B

[0182] Group Example 3B Comparative Example 3B Transduction rate 25% to 80% 65% to 80%

[0183] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial to the functional test of the subsequent amplified CAR-T cells. Fig.16A The results showed that the lentivirus of Example 3B had a good transduction rate.

[0184] Test Example 16: T cell phenotype analysis of CAR-T cells of Example 3C

[0185] The test method of Test Example 16 is similar to that of Test Example 4, except that the test example is to detect the CAR-T cells obtained on the 7th day of preparation of the anti-BCMA-NKp30 CAR-T cells of Example 3C. The ratio of Tscm in Example 3C, the CAR-T cells of Example 3C and the untransduced T cells is shown in Table 14 below; the results of Example 3C and the flow cytometer results of the distribution of T cell subsets of the control example 3C and the untransduced T cells are shown in Table 14. Fig.17A , 17B As shown in Figure 17C, T cells expressing CD45RA and CCR7 are Tscm.

[0186] Table 14: Percentage of Tscm in CAR-T cells of Example 3C, Control Example 3C, and T cells in the untransduced group

[0187] Group Example 3C Comparative Example 3C Untransduced group %Tscm 68.8％ 45.3％ 70.5％

[0188] The optimal range of stem cell-like memory T cell percentage of CAR-T cells is above 40%, because a high proportion of Tscm can reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Fig.17A , 17B It can be seen from Figure 17C that Example 3C with the hinge domain of CD28 has a high proportion of Tscm, and is significantly better than the traditional single-chain CAR-T cells of Control Example 3C, which is similar to the activated T cells of the untransduced group. Therefore, it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0189] Test Example 17: Cytotoxicity test analyzed by flow cytometry

[0190] In order to test the anti-BCMA-NKp30 CAR-T's anti-tumor activity, this test example used H929 myeloma cells (hereinafter referred to as H929 cells) (purchased from BCRC, 60386) calibrated with CellTraceTM Violet and cultured in RPMI-1640 medium (Gibco) for subsequent flow cytometer analysis of biological toxicity tests. The effector cells used in this test example are Example 3C and Control Example 3C, and untransduced T cells are used as a control group. Specifically, the target cells were cultured at 1×10 4Cells were seeded in a 96-well plate at a density of 10 cells / well, and effector cells were added to make the ratio of effector cells to target cells (E:T) 0.1:1, 0.3:1, and 1:1. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours, and the experiment was repeated three times. Next, the 96-well plate was centrifuged at 300g for 5 minutes, the supernatant was removed, and each well was washed with 200μL of PBS containing 2% fetal calf serum (FCS). After removing the PBS used for washing, the cells were collected with 300μL of flow buffer (PBS containing 2% FCS), and 15μL of counting beads (CountBright TM Plus Absolute Counting Beads, C36995 Invitrogen), collect 3000 micro beads to convert the number of cells. Then calculate the cell killing rate using the following formula:

[0191] Cytotoxicity rate % = (the number of target cells in the group without effector cells - the number of target cells after co-culture with effector cells) ÷ the number of target cells in the group without effector cells × 100%

[0192] The experimental results are as follows Fig.18 As shown, when E:T is 0.1:1, the cytotoxic effect of Example 3C is 73.54%, which is similar to 74.78% of Control Example 3C; when E:T is 0.3:1 and 1:1, the cytotoxic effect of Example 3C is about 80% to 85%, which is better than the cytotoxic effect of Control Example 3C.

[0193] Test Example 18: Real-time Cytotoxicity Assay Using xCELLigence

[0194] A human BCMA fragment was cloned from the H929 cell line (purchased from BCRC, 60386). A truncated BCMA cDNA fragment (SEQ ID NO: 97) was amplified using PCR. The truncated BCMA cDNA fragment (1-98 amino acids) refers to a fragment having an extracellular domain and a transmembrane domain but lacking an intracellular domain and a cytoplasmic signaling domain. The truncated BCMA cDNA fragment was inserted into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39) using the HpaI and NheI restriction enzyme sites to obtain a vector containing truncated BCMA. The truncated BCMA vector was transfected into the SKOV3 human ovarian cancer cell line (purchased from ATCC, HTB-77) using Polyjet (SignaGen Laboratories, SL100688). The transfected SKOV3 cells were cultured in McCoy 5A Medium (Gibco) containing 2 μg / mL puromycin and 10% FBS to screen out drug-resistant SKOV3 cell lines that express BCMA. Finally, flow cytometry was used to confirm the expression of BCMA in the cells. The SKOV3 cell lines expressing BCMA were used as target cells for subsequent cytotoxicity tests with the xCELLigence system.

[0195] The test method of Test Example 18 is similar to that of Test Example 6, except that the CAR-T cells of Example 3C are used as effector cells, the aforementioned SKOV3 cell line expressing BCMA is used as target cells, and the target cells are cultured at 1.2×10 4 The cells were seeded at a density of 100 cells / mL in a background-corrected culture plate (E-plate 96-well plate, Agilent Technologies, 300600910) and cultured overnight to allow the target cells to attach. After 22 hours of background impedance measurement, the effector cells were added to the culture plate at an E:T ratio of 0.3:1 and 0.1:1 for testing.

[0196] The experimental results of E:T of 0.3:1 and 0.1:1 are as follows Fig.19A and 19BAs shown, it can be seen from the results in the figure that the cytotoxicity results of the two E:T ratios both show that after the effector cells and the target cells are co-cultured for 36 hours, that is, the total test time is about 58 hours, the CAR-T cells of Example 3C can kill 100% of the SKOV3 cells expressing BCMA, and the normalized cell index of Example 3C overlaps with the curve of the full lysis group, and is slightly better than the CAR-T cell cytotoxicity of the single-chain control example 3C.

[0197] In summary, the lentivirus of Example 3B with the hinge domain of CD28 has an excellent transduction rate, and the CAR-T cells of Example 3C have an excellent CAR-T cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm, which can be expected to reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Therefore, the hinge domain of CD28 is a hinge domain suitable for use with anti-BCMA receptors.

[0198] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific 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. An isolated nucleic acid molecule comprising the nucleic acid sequence of the following segments: (a) an extracellular antigen binding domain comprising a heavy chain variable region; (b) Hinge domain; (c) Natural Killer Protein 30 (NKp30) transmembrane domain; and (d) Natural killer protein 30 (NKp30) cytoplasmic domain.

2. The isolated nucleic acid molecule of claim 1, wherein The extracellular antigen binding domain comprises: the heavy chain variable region of an anti-mesothelin antibody, the heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of an anti-CD19 antibody, the heavy chain variable region of an anti-B cell maturation antigen (BCMA) antibody, the heavy chain variable region of an anti-CD123 antibody, the heavy chain variable region of an anti-GPRC5D (G protein–coupled receptor, class C, group 5, member D) antibody, or the heavy chain variable region of an anti-TSHR (thyroid stimulating hormone receptor (TSH receptor) antibody.

3. The isolated nucleic acid molecule of claim 1, wherein The hinge domain is selected from the group consisting of (G4S)3, the hinge domain of CD8, the hinge domain of CD28, the hinge domain of IgG4, EAAAKGGGGS, (EAAAK)3, the hinge domain of GST, the hinge domain of EAAAK-GS, the hinge domain of IgD, the IgG4-CH3 domain and (AP)6.

4. The isolated nucleic acid molecule of claim 1 further comprises a nucleic acid sequence of an adapter gene.

5. The isolated nucleic acid molecule of claim 1, wherein The hinge domain is connected to the NKp30 transmembrane domain via an extracellular stalk region of NKp30.

6. The isolated nucleic acid molecule of claim 1, wherein The extracellular antigen binding domain comprises the heavy chain variable region of an anti-mesothelin antibody and the hinge domain is (G4S)3.

7. A plasmid comprising the isolated nucleic acid molecule according to any one of claims 1 to 6, wherein the plasmid is a DNA plasmid or an RNA plasmid.

8. A method for preparing a chimeric antigen receptor-cell, comprising introducing the isolated nucleic acid molecule according to any one of claims 1 to 6 into a nucleated cell.

9. The method for preparing a chimeric antigen receptor-cell according to claim 8, wherein the introduction is by transduction using a lentivirus.

10. An isolated cell comprising the isolated nucleic acid molecule according to any one of claims 1 to 6.

11. The isolated cell of claim 10, wherein The separated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

12. Use of the isolated cells according to claim 10 or 11 for the preparation of a medicament for treating or slowing down cancer.

13. Use of the isolated cells according to claim 10 or 11, for preparing a medicament for treating or alleviating an autoimmune disease, wherein the autoimmune disease is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, systemic sclerosis or multiple sclerosis.

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