Nucleic acid molecule having intracellular signaling domain and natural killer protein 30 transmembrane domain, and chimeric antigen receptor comprising same

Abstract

The present invention provides a nucleic acid molecule having an intracellular signaling domain and an NKp30 transmembrane domain, and a chimeric antigen receptor comprising the same. The nucleic acid molecule comprises the following nucleic acid sequences: (a) an extracellular antigen binding domain; (b) a hinge domain; (c) an NKp30 transmembrane domain; (d) an NKp30 cytoplasm domain; and (e) an intracellular signaling domain. Nucleic acid sequences of an intracellular signal transduction structural domain, 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 cell is injected back into the human body, it 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) Extracellular antigen binding domain;

[0009] (b) Hinge domain;

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

[0011] (d) natural killer protein 30 (NKp30) cytoplasmic domain; and

[0012] (e) Intracellular signaling domain.

[0013] The present invention transfers the genes of NKp30 transmembrane domain, NKp30 cytoplasmic domain and intracellular signal transduction domain into T cells, so that 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.

[0014] Preferably, the aforementioned extracellular antigen binding domain is the extracellular domain of NKp30.

[0015] Preferably, the aforementioned extracellular antigen binding domain comprises a heavy chain variable region.

[0016] Preferably, 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 (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.

[0017] In one embodiment, the heavy chain variable region of the aforementioned extracellular antigen binding domain may be derived from the heavy chain variable region of a heavy chain antibody (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.

[0018] In one embodiment, the aforementioned extracellular antigen binding domain is the heavy chain variable region of an anti-BCMA heavy chain antibody.

[0019] In one embodiment, the aforementioned 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Preferably, the extracellular antigen binding domain comprises a 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.

[0028] 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 (as shown in SEQ ID NO:75) domain and the (AP)6 (as shown in SEQ ID NO:76).

[0029] 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, CD3ζ, DAP10, CD79, FcεR1γ, FcRγ or Fcβ / γ full length protein is used as an adapter molecule.

[0030] Preferably, the aforementioned intracellular signaling domain is the intracellular signaling domain of DAP12, 41BB or CD28. More preferably, the aforementioned intracellular signaling domain is the intracellular signaling domain of DAP12.

[0031] Preferably, the hinge domain is connected to the NKp30 transmembrane domain via a NKp30 extracellular stalk region.

[0032] Preferably, the 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 T2A self-truncating peptide. For example, the nucleic acid sequence of the FcεR1γ adapter molecule may be located at the 5' end of the 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 intracellular signaling domain and the nucleic acid sequence of the natural killer protein 30 transmembrane domain or between the nucleic acid sequence of the FcεR1γ adapter molecule and the nucleic acid sequence of the natural killer protein 30 transmembrane domain.

[0033] The hinge domain is connected to the NKp30 transmembrane domain via a NKp30 extracellular stalk region.

[0034] 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 the hinge domain of IgG4. 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 a single-chain variable region fragment of FMC63.

[0035] 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 variable region fragment.

[0036] 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 multiple, 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 variable region fragment.

[0037] Preferably, in the aforementioned isolated nucleic acid molecule, when the aforementioned extracellular antigen binding domain comprises the heavy chain variable region of the anti-CD22 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 multiple, a higher proportion of Tscm, and a better cytotoxic ability. More preferably, the heavy chain variable region comprising the anti-CD22 antibody is an m971 single-chain variable region fragment.

[0038] To achieve the above-mentioned purpose, the present invention further provides a plasmid, which comprises the above-mentioned isolated nucleic acid molecule, and the vector 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, intracellular signaling domain, 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, intracellular signaling domain, T2A self-truncating peptide, extracellular antigen binding domain, hinge domain, NKp30 transmembrane domain and NKp30 cytoplasmic domain, 3'LTR.

[0039] 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.

[0040] Preferably, in the aforementioned method for preparing chimeric antigen receptor-cells, the aforementioned "introduction" is introduction by transduction using lentivirus.

[0041] Preferably, the aforementioned nucleated cells are leukocytes. Preferably, the aforementioned nucleated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

[0042] To achieve the aforementioned object, the present invention further provides an isolated cell containing the isolated nucleic acid molecule as described above.

[0043] Preferably, the aforementioned separated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

[0044] 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.

[0045] 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 has therapeutic effects. In one embodiment, the autoimmune disease is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, systemic sclerosis or multiple sclerosis.

[0046] The advantage of the present invention is that by transferring the nucleic acid sequences of the NKp30 transmembrane domain, NKp30 cytoplasmic domain and intracellular signal transduction 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 both in vivo and in vitro, and will not over-activate T cells while significantly killing cancer cells, and show persistence in re-challenge experiments; 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

[0047] Figure 1 is a schematic diagram of the pLAS5w vector;

[0048] Figure 2 Schematic diagram of plasmids of Examples 1-1A to 1-2A and Comparative Example 1A;

[0049] Figure 3 Schematic diagram of the cell membrane protein structure of CAR-T cells in Examples 1-1C, 1-2C and Control Example 1C;

[0050] Figures 4A to 4D The flow cytometry results of the T cell subset distribution of the CAR-T cells of Example 1-1C, 1-2C, Control Example 1C and the untransduced group;

[0051] Figure 5A , Figure 5B , Figure 5C The real-time results of cell toxicity comparison of CAR-T cells of Examples 1-1C and 1-2C, CAR-T cells of Control Example 1C, T cells of the untransduced group, and target cells at E:T ratios of 10:1, 3:1, and 1:1 in Test Example 5 are shown respectively;

[0052] Figure 6Schematic diagram of the plasmids of Example 2A and Comparative Example 2A;

[0053] Figure 7 Schematic diagram of the cell membrane protein structure of CAR-T cells in Example 2C and Control Example 2C;

[0054] Fig. 8A , Figure 8B They are the flow cytometry results of the CAR-T cells of Example 2C in Test Example 8 expressing anti-mesothelin antibodies and NKp30;

[0055] Figure 8C , Fig.8D The flow cytometry results of the CAR-T cells of the control example 2C in the test example 8 for the expression of anti-mesothelin antibody and NKp30 are respectively

[0056] Fig. 9A , 9B 9C are flow cytometric analysis results of T cell subset distribution in Example 2C, Control Example 2C and the non-transduced group in Test Example 9 on the 7th day;

[0057] Fig. 10A , Fig. 10B , Fig. 10C , Fig. 10D The real-time results of cell toxicity comparison of the CAR-T cells of Example 2C in Test Example 10, the CAR-T cells of Control Example 2C, the T cells of the untransduced group, and the target cells at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1, respectively;

[0058] Fig.11A The in vivo anti-tumor effects 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 E:T ratios of 3:1 and 1:1;

[0059] Fig. 11B The logarithmic bioluminescent imaging signals of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C, the T cells of the untransduced group, and the saline group at E:T ratios of 3:1 and 1:1 in Test Example 12;

[0060] Fig. 12A The results of the in vivo anti-tumor re-challenge test of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C and the saline group in Test Example 12 at E:T ratios of 3:1 and 1:1;

[0061] Fig. 12BThe logarithmic bioluminescent imaging signals of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C and the target cells in the in vivo anti-tumor re-challenge at E:T ratios of 3:1 and 1:1 in Test Example 12, * indicates p value <0.05, ** indicates p value <0.01;

[0062] Fig.13A is the ratio of T cells in the peripheral blood of each group of mice in Test Example 12;

[0063] Fig. 13B The ratio of CAR-T cells in T cells of each group of mice in Test Example 12;

[0064] Fig.14 Schematic diagram of the plasmids of Example 3A, Comparative Example 3-1A and Comparative Example 3-2A;

[0065] Fig.15 Schematic diagram of the cell membrane protein structure of CAR-T cells in Example 3C and Control Example 3-1C and Control Example 3-2C;

[0066] Fig.16A , Fig. 16B They are the flow cytometry results of the CAR-T cells of Example 3C in Test Example 15 expressing anti-CD22 receptor and NKp30;

[0067] Fig. 16C , Fig.16D They are the flow cytometry results of the CAR-T cells of the control example 3-1C in the test example 15 expressing anti-CD22 receptor and NKp30;

[0068] Fig.17A , Fig. 17B , Fig. 17C and Fig.17D They are the flow cytometric analysis results of T cell subset distribution on day 7 in Example 3C, Control Example 3-1C, Control Example 3-2C and the non-transduced group in Test Example 16;

[0069] Fig.18A and Fig.18B The real-time results of the cytotoxicity comparison of the CAR-T cells of Example 3C, the CAR-T cells of Control Example 3-1C, Control Example 3-2C, and the T cells of the untransduced group in Test Example 17 at E:T ratios of 3:1 and 1:1;

[0070] Fig.19 The comparison results of cell toxicity analyzed by flow cytometry at E:T ratios of 3:1 and 1:1 for the CAR-T cells of Example 3C, the CAR-T cells of Control Example 3-1C, Control Example 3-2C, and the T cells of the untransduced group in Test Example 18;

[0071] Fig. 20 Schematic diagram of the plasmids of Example 4A and Comparative Example 4A;

[0072] Fig.21 Schematic diagram of the cell membrane protein structure of CAR-T cells in Example 4C and Control Example 4C;

[0073] Fig.22A , Fig. 22B They are the flow cytometry results of the CAR-T cells of Example 4C in Test Example 21 expressing anti-CD19 receptor and NKp30;

[0074] Fig. 22C , Fig.22D These are the flow cytometry results of the CAR-T cells of Control Example 4C in Test Example 21 expressing anti-CD19 receptor and NKp30 respectively;

[0075] Fig.23A , Fig. 23B and Fig.23C They are the flow cytometric analysis results of T cell subset distribution on day 7 in Example 4C, Control Example 4C and the non-transduced group in Test Example 22;

[0076] Fig.24 The results of the real-time cytotoxicity test of the CAR-T cells of Example 4C, the CAR-T cells of Control Example 4C, the T cells of the untransduced group, the target cells and the full lysis group in Test Example 23 at an E:T ratio of 1:1 are compared;

[0077] Fig.25 The results are compared for the cold light killing of the CAR-T cells of Example 4C, the CAR-T cells of Control Example 4C, and the T cells of the untransduced group at E:T ratios of 3:1 and 1:1. DETAILED DESCRIPTION

[0078] The invention will be further illustrated by the following examples, which are not intended to limit the present invention disclosed above. Those skilled in the art may make slight improvements and modifications without departing from the scope of the present invention.

[0079] 1. Preparation and testing of CAR-T cells with intracellular signaling domains and NKp30 fragments

[0080] Example 1-1: Nucleic acid molecules having the DAP12 intracellular signaling domain and the complete fragment of NKp30

[0081] First, the 3' end of the full-length FcεR1γ nucleic acid sequence (as shown in SEQ ID NO: 77) was connected to the 5' end of the nucleic acid sequence of the intracellular signaling domain of DAP12 (SEQ ID NO: 78) to obtain a connected nucleic acid molecule fragment 1, and then the connected nucleic acid molecule fragment 1 was connected to the NKp30 complete fragment (including the immunoglobulin-like domain) sequence (SEQ ID NO: 79) through the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO: 80) to obtain the nucleic acid molecule having the DAP12 intracellular signaling domain and the NKp30 complete fragment of Example 1-1 (as shown in SEQ ID NO: 81). The NKp30 complete fragment includes an immunoglobulin-like domain, a stalk region, a NKp30 transmembrane domain (Tm), and a NKp30 cytoplasmic domain. The nucleic acid molecule having the DAP12 intracellular signaling domain and the complete NKp30 fragment of Example 1-1 has the full-length FcεR1γ nucleic acid sequence, the DAP12 intracellular signaling domain nucleic acid sequence, the T2A self-truncated peptide nucleic acid sequence and the complete NKp30 fragment from the 5' end to the 3' end.

[0082] Example 1-2 Nucleic acid molecules having 41BB intracellular signaling domain and NKp30 complete fragment

[0083] The preparation method of Example 1-2 is similar to the preparation method of Example 1-1, except that the intracellular signaling domain used is the 41BB intracellular signaling domain (as shown in SEQ ID NO:82), and the obtained nucleic acid molecule having the 41BB intracellular signaling domain and the complete fragment of NKp30 of Example 1-2 is shown in SEQ ID NO:83.

[0084] Control Example 1: Modified wild-type NKp30 nucleic acid molecule

[0085] First, the 3' end of a full-length FcεR1γ nucleic acid sequence (as shown in SEQ ID NO: 77) was connected to the 5' end of the aforementioned NKp30 complete fragment (as shown in SEQ ID NO: 79) through the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO: 80) to obtain the modified wild-type NKp30 nucleic acid molecule of Control Example 1 (as shown in SEQ ID NO: 84). The modified wild-type NKp30 nucleic acid molecule of Control Example 1 has the full-length FcεR1γ nucleic acid sequence, the T2A self-truncating peptide nucleic acid sequence and the NKp30 complete fragment from the 5' end to the 3' end.

[0086] Example 1-1A, 1-2A and Comparative Example 1A: Construction of a plasmid having the nucleic acid molecule of Example 1-1, 1-2 or Comparative Example 1

[0087] 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, 1-2 and Comparative Example 1 were grafted into the pLAS5w vector using BstBI and EcoRI restriction enzymes to obtain the plasmids of Examples 1-1A and 1-1B and Comparative Example 1A, respectively. Figure 2 shown.

[0088] Example 1-1B, 1-2B and Control Example 1B: Preparation of Lentivirus with Nucleic Acid Molecules of Example 1-1, 1-2 or Control Example 1

[0089] 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, 1-2B and Control Example 1B. Specifically, 50 μg of one of the plasmids of Examples 1-1A and 1-2A or the plasmid of Control Example 1A containing the target gene was used as the transfer plasmid; 45 μg of pCMV deltaR8.91 was used as the packaging plasmid (purchased from Academia Sinica); and 5 μg of pMD.G was used as the envelope plasmid (purchased from Academia Sinica). The above plasmids 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 7HEK293 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 supernatant containing the first lentiviral particles was collected and supplemented with fresh Opti-MEM culture medium containing 1 mM sodium pyruvate for 24 hours. The supernatant containing the lentiviral particles was collected again. The supernatant containing the lentiviral particles was collected and centrifuged at 1500 rpm for 10 minutes at 4°C to remove cell debris. The supernatant after centrifugation was then filtered through a 0.45 μm filter membrane (Membrane-Solutions). The two collected and filtered supernatants were mixed together. Lenti-X Concentration Reagent (Takara Bio) was then used for 3:1 concentration, i.e. the volume ratio of the filtered upper layer to Lenti-X was 3:1. Specifically, the mixture of the upper layer and Lenti-X Concentration 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 concentrated using 2 mL X-VIVO TM 15 culture medium (Lonza) was resuspended to obtain the concentrated lentivirus of Examples 1-1B and 1-2B and the lentivirus of Control Example 1B, and stored at -80°C for future use.

[0090] Test Example 1: Testing of the lentiviral titer of Example 1-1B, Example 1-2B, and Control Example 1B

[0091] The lentiviral titer was determined by transducing 3-fold serial dilutions of the lentiviral sample to 4 × 10 4 The assay was performed using Jurkat cells (purchased from BCRC, Cat. No. 60424), a human acute leukemia T cell. The lentivirus sample used in this test example 1 was the concentrated lentivirus of Example 1-1B, 1-2B, 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 4Jurkat cells / 100 μL culture medium / well were added to the dish, and lentivirus transduction was performed for 72 hours. After washing the excess lentivirus, the expression of NKp30 was detected using an anti-NKp30 antibody (BioLegend, Cat. No. 325228) labeled with BV421 fluorescent dye at a concentration of 1 μg / mL, and the expression of the target protein was analyzed by flow cytometry (Sony, SA3800) to measure the transduction efficacy 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 lentivirus of the nucleic acid molecule having an intracellular signaling domain and a complete fragment of NKp30 (including an immunoglobulin-like domain) was calculated using the following formula, and the effective titer of the lentivirus obtained was used to determine the amount of lentivirus used in the preparation of CAR-T cells.

[0092] Lentivirus effective titer (TU / mL) = (percentage of cells expressing NKp30) × 4 × 10 4 (Number of cells) × 20 × dilution factor

[0093] The experimental results are shown in Table 1 below:

[0094] Table 1: Effective titer of lentivirus of Example 1-1B, 1-2B and Control Example 1B (transducing unit, TU / mL)

[0095] Group Example 1-1B Example 1-2B Comparative Example 1B Lentivirus effective titer <![CDATA[>1×10 9 ]]> <![CDATA[>1×10 9 ]]> <![CDATA[>1×10 9 ]]>

[0096] If the effective titer of lentivirus is greater than or equal to 1×10 6 TU / mL, when preparing CAR-T cells, using a smaller volume of lentivirus can smoothly transduce T cells into CAR-T cells, and can make the transduced T cells grow well. Therefore, according to the results of Table 1 above, the lentiviruses of Examples 1-1B, 1-2B and Control Example 1B all meet this standard, which is conducive to further preparing CAR-T cells.

[0097] Example 1-1C, 1-2C, Control Example 1C: Preparation of CAR-T cells with nucleic acid molecules of Example 1-1, 1-2 or Control Example 1

[0098] Fresh blood was collected from healthy donors with their consent. On day 0, human peripheral blood mononuclear cells (PBMC) were collected using SepMate centrifuge tubes (STEMCELL, Cat. No. 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 fluid (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 separated PBMC cells were detected for CD3 in PBMC using anti-CD3 antibodies (Biolegend, Cat. No. 300441) and flow cytometry. + The proportion of T cells and calculated CD3 + The number of T cells. Take CD3 + Magnetic beads with anti-CD3 / CD28 antibodies on the surface, which are three times the number of T cells, were added to PBMC cells and incubated at room temperature for 30 to 60 minutes to allow the magnetic beads with anti-CD3 / CD28 antibodies on the surface to bind to the 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, the T cell and magnetic bead complex formed by antigen-antibody binding was obtained. The T cell and magnetic bead complex was 2×10 5 The 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. TM15 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 24 hours by adding one of the lentivirus of Examples 1-1B, 1-2B, or Control Example 1B with a viral infection dose MOI (multiplicity of infection) of 5 to obtain the CAR-T cells and magnetic bead complexes of Examples 1-1C, 1-2C, and Control Example 1C, and the T cell group without lentivirus treatment is used as the control group (untransduced group). Among them, the MOI here = viral titer (TU / mL) × viral volume (mL) / total number of T cells. On the third day, the suspension of CAR-T cells and magnetic bead complexes was removed from the magnetic stand to obtain the CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C. The structural schematics of each group are shown as follows. Figure 3 The obtained CAR-T cells were transferred to X-VIVO containing 200 IU / mL of recombinant human IL-2 (R&D, 202-GMP-01M). TM 15% medium in a new culture flask, and cultured under the same conditions as the transduction step, allowing the CAR-T cells to proliferate until day 7. Finally, the number of CAR-T cells in the non-transduced group and Examples 1-1C, 1-2C, and Control Example 1C 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.

[0099] Test Example 2: Proliferation test of CAR-T cells in Example 1-1C, Example 1-2C, and Control Example 1C

[0100] The proliferation rate of the CAR-T cells of Example 1-1C, 1-2C and Control Example 1C was calculated by calculating the total number of cells at a specific time point during the preparation of the CAR-T cells of Example 1-1C, 1-2C and Control Example 1C. Specifically, on the third day of the preparation of the CAR-T cells, the CAR-T cells of Example 1-1C, 1-2C and Control Example 1C were cultured at a concentration of 1×10 6 The cells were seeded at a density of 10 cells / mL in a 12-well plate. After removing the magnetic beads, the cell viability and cell density were measured, including staining the cells with acridine orange / propidium iodide (AO / PI) and automatically quantifying the cell number using an automatic cell counter (Luna Automated Cell Counter, Logos Biosystems). The remaining cells were subcultured at 2×10 5 Up to 5×10 5The cells were seeded in a culture dish at a density of 10 cells / mL to expand the cells. The number of cells was measured on days 0 and 7 of preparing CAR-T cells to calculate the cell expansion rate. The experimental results are shown in Table 2 below. Among them, the expansion rate of CAR-T cells in Examples 1-1C, 1-2C and Control Example 1C is the proliferation multiple calculated after standardization with the number of cells on day 0.

[0101] Table 2: Expansion folds of CAR-T cells of Examples 1-1C, 1-2C and Control Example 1C on Day 7

[0102]

[0103] 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 2 above, the CAR-T cells of Examples 1-1C, 1-2C and Control Example 1C all have good T cell expansion multiples. In both Example 1-1C and Example 1-2C, the expansion effect of the CAR-T cells of Example 1-1C with the DAP12 intracellular signaling domain as the intracellular signaling domain is better than that of the CAR-T of Example 1-2C with the 41BB intracellular signaling domain as the intracellular signaling domain.

[0104] Test Example 3: Expression detection of CAR-T cells in Example 1-1C, 1-2C and Control Example 1C; Transduction rate of lentivirus in Example 1-1B, 1-2B and Control Example 1B

[0105] The expression of NKp30 in the CAR-T cells of Examples 1-1C, 1-2C and Control Example 1C was detected using an anti-NKp30 antibody (BioLegend, 325228) labeled with BV421 fluorescent dye and a flow cytometer. The percentage of cells expressing NKp30 compared to the total number of cells was used as the transduction rate of the lentivirus of Examples 1-1B, 1-2B and Control Example 1B. The experimental results are shown in Table 3 below.

[0106] Table 3: Transduction rate of lentivirus of Examples 1-1B, 1-2B and Control Example 1B

[0107] Group Example 1-1B Example 1-2B Comparative Example 1B Transduction rate 96％ 88％ 40％

[0108] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial to the functional test of the CAR-T cells after subsequent amplification. Therefore, according to the results of Table 3 above, it can be concluded that the lentiviruses of Example 1-1B and Example 1-2B have excellent transduction rates, and the transduction rate of the lentivirus of Example 1-1B with DAP12 as the intracellular signaling domain is better than that of the lentivirus of Example 1-2B with 41BB as the intracellular signaling domain, and both are better than the lentivirus of Control Example 1B.

[0109] Test Example 4: T cell phenotype analysis of CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C

[0110] T cell typing analysis was performed on the CAR-T cells of Example 1-1C, 1-2C, and Control Example 1C on the 7th day after activation using 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 identify the distribution (%) of each T cell subset according to Annals of Oncology Volume 32, Issue 11, Pages 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 (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, 1-2C and Control Example 1C is shown in Table 4 below. The results of Examples 1-1C and 1-2C and the flow cytometry results of the T cell subset distribution of Control Example 1C and the non-transduced group are shown in Table 4, respectively. Figure 4A , 4B , 4C and 4D. Among them, T cells expressing CD45RA and CCR7 are Tscm, and T cells expressing CD45RA but not CCR7 are T EMRA (or T EFF), T cells that do not express CCR7 and CD45RA are T EM , those that express CCR7 but not CD45RA are T CM .

[0111] Table 4: Percentage of Tscm in CAR-T cells of Example 1-1C, 1-2C, Control Example 1C and non-transduced group

[0112] Group Example 1-1C Example 1-2C Comparative Example 1C Non-transduced group (control group) %Tscm 66.7％ 66.9％ 56.2％ 53.7％

[0113] If the percentage of stem cell-like memory T cells (Tscm) in CAR-T cells is above 40%, the high percentage of Tscm can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Figures 4A to 4D It can be seen that Examples 1-1C and 1-2C of the modified wild-type NKp30 CAR-T cells having an intracellular signaling domain both have a higher proportion of Tscm, which is higher than the CAR-T cells of the control example 1C and the untransduced activated T cells. Therefore, it can be expected that the CAR-T cells of Examples 1-1C and 1-2C can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0114] Test Example 5: Real-time cytotoxicity assay (RTCA) using xCELLigence

[0115] 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% fetal bovine serum (FBS) and cultured in an environment of 5% CO2 and 37°C for subsequent xCELLigence real-time cytotoxicity assays.

[0116] In order to test the anti-tumor activity of the CAR-T cells in Examples 1-1C, 1-2 and Control Example 1C, the HeLa cell line described in the previous paragraph was used as a target cell for a cytotoxicity test. The effector cells used in this test example were the CAR-T cells in Examples 1-1C, 1-2C and Control Example 1C.

[0117] 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 about 90 hours: First, the aforementioned HeLa cells were used as target cells (T) at a rate of 1.2×10 4 The density of cells / mL was 1:1, and after being seeded in a background-corrected culture dish, background impedance measurement was performed for 22 hours to allow the target cells to attach. Then, effector cells (E) were added to the culture dish at effector-to-target ratios (E:T ratio) of 10:1, 3:1, and 1:1, and co-cultured to make the final volume of each well 200 μL. The effector cells used in this test example are the CAR-T cells of Examples 1-1C, 1-2, and Control Example 1C, and untransduced activated T cells are used as the control group (untransduced group). The cell index (CI) was detected every 15 minutes for at least 72 hours. The original RTCA impedance data was normalized with the cell index data before the addition of effector cells. The aforementioned cell index is a dimensionless parameter, which is obtained by measuring the relative change in electrical impedance to represent the cell state. For example, when cells do not exist or are not attached to the electrode, the CI is zero; under the same physiological conditions, the more cells are attached to the electrode, the greater the CI value. In addition, changes in cell status, such as changes in cell morphology or cell adhesion, will lead to changes in CI. Figures 5A to 5C shown.

[0118] The results of poisoning for 24 hours are shown in Table 5 below.

[0119] Table 5: CAR-T cell toxicity test results of Examples 1-1C, 1-2C and Control Example 1C

[0120] Group Example 1-1C Example 1-2C Comparative Example 1C Specific cleavage (E:T = 10:1) 100％ 75％ 50％

[0121] According to Table 5 and Figures 5A to 5CThe results show that the cytotoxicity of the CAR-T cells of Example 1-1C and Example 1-2C is better than that of the control example 1C. And the cytotoxicity of the CAR-T cells of Example 1-1C reaches 100% at E:T ratios of 10:1, 3:1 and 1:1 at 24 hours. Since the cytotoxicity of CAR-T cells is >50%, it will have sufficient cytotoxicity when the patient's autologous cells are used to prepare CAR-T in the future. According to the results of Table 5 above, it can be seen that the CAR-T cells of Examples 1-1C and 1-2C have good cytotoxicity, and the CAR-T of Example 1-1C into which the intracellular signaling domain of DAP12 is transferred has a cytotoxicity better than the CAR-T cells of Example 1-2C into which the intracellular signaling domain of 41BB is transferred.

[0122] In summary, the lentivirus of Examples 1-1B and 1-2B of the present invention, because the control example and the embodiment have an intracellular signaling domain and a complete NKp30 fragment containing a NKp30 transmembrane domain, all have excellent transduction rates, and the CAR-T cells obtained after transduction have excellent CAR-T cell proliferation rates and excellent cytotoxicity, and have a high proportion of Tscm, and it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. It can be seen that when preparing CAR-T cells, if the nucleic acid sequence of the intracellular signaling domain is transferred, such as the intracellular signaling domain of DAP12 or 41BB, it can bring excellent cytotoxic effects to the prepared CAR-T cells, and is particularly suitable for CAR-T therapy.

[0123] 2. Preparation and testing of anti-mesothelin-NKp30 CAR-T cells with intracellular signaling domains

[0124] Example 2: Nucleic acid molecules having an intracellular signaling domain, a single-chain variable region fragment of an anti-mesothelin antibody, and a NKp30 fragment without an immunoglobulin-like domain

[0125] The 3' end of the nucleic acid sequence of the single-chain variable region fragment SS1 of the anti-mesothelin antibody (as shown in SEQ ID NO:85) is connected to the 5' end of a NKp30 fragment (as shown in SEQ ID NO:86) without an immunoglobulin-like domain through a nucleic acid sequence with a hinge domain of (G4S)3 (as shown in SEQ ID NO:66). The nucleic acid sequence of the aforementioned NKp30 fragment without an immunoglobulin-like domain contains an extracellular stalk region (stalk) and a NKp30 transmembrane domain and a NKp30 cytoplasmic domain, but does not contain an immunoglobulin-like domain. Afterwards, the 3' end of the nucleic acid sequence of the aforementioned NKp30 fragment without an immunoglobulin-like domain is connected to the 5' end of the nucleic acid sequence of the intracellular signaling domain of DAP12 (SEQ ID NO:78) to obtain a connected nucleic acid molecule fragment 2. Then, the 5' end of the connected nucleic acid molecule fragment 2 was connected to the 3' end of the full-length FcεR1γ nucleic acid sequence (as shown in SEQ ID NO: 77) through the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO: 80), to obtain the nucleic acid molecule of Example 2 having the intracellular signaling domain, the single-chain variable region fragment of the anti-mesothelin antibody, and the NKp30 fragment without the immunoglobulin-like domain, as shown in SEQ ID NO: 87. The nucleic acid molecule of Example 2 from the 5' end to the 3' end respectively comprises the nucleic acid sequence of the full-length FcεR1γ, the nucleic acid sequence of the T2A self-truncating peptide, the nucleic acid sequence of SS1, the nucleic acid sequence of the (G4S)3 hinge domain, the nucleic acid sequence of the NKp30 fragment without the immunoglobulin-like domain, and the nucleic acid sequence of the DAP12 intracellular signaling domain.

[0126] Control Example 2 Nucleic acid molecule having an intracellular signaling domain, a single-chain variable region fragment of an anti-mesothelin antibody and not containing any NKp30 fragment

[0127] The 3' end of the nucleic acid sequence of the anti-mesothelin single-chain variable region fragment SS1 (as shown in SEQ ID NO: 85) is connected to the nucleic acid sequence of the CD8 hinge domain (as shown in SEQ ID NO: 67)

[0128] The 5' end of the nucleic acid sequence of the CD8 transmembrane domain (as shown in SEQ ID NO: 88) was connected, and the 5' end of the nucleic acid molecule of the 4-1BB intracellular signaling domain (SEQ ID NO: 82) was further connected to the 3' end of the nucleic acid sequence of the CD8 transmembrane domain, and then the 3' end of the nucleic acid sequence of the 4-1BB intracellular signaling domain was connected to the 5' end of the nucleic acid sequence of the CD3 ζ signaling domain (SEQ ID NO: 89) to obtain the nucleic acid molecule of Control Example 2 (as shown in SEQ ID NO: 90). The nucleic acid molecule of Control Example 2 is respectively the nucleic acid sequence of SS1, the nucleic acid sequence of the CD8 hinge domain, the nucleic acid sequence of the CD8 transmembrane domain, the nucleic acid sequence of the 4-1BB intracellular signaling domain and the nucleic acid sequence of the CD3 ζ signaling domain from the 5' end to the 3' end.

[0129] Example 2A, Control Example 2A: Construction of plasmids with nucleic acid molecules of Example 2 and Control Example 2

[0130] The preparation method of Example 2A is similar to the preparation methods of Examples 1-1A and 1-2A, except that Example 2A uses restriction enzymes to insert the single-chain variable region fragment of Example 2 having an intracellular signaling domain, an anti-mesothelin antibody, and the NKp30 fragment without the immunoglobulin-like domain into the aforementioned pLAS5w vector. In contrast, in Control Example 2A, GenScript was commissioned 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 resulting anti-mesothelin-NKp30 plasmid having an intracellular signaling domain of Example 2A and the plasmid of Control Example 2A are as follows: Figure 6 shown.

[0131] Example 2B, Control Example 2B: Preparation of lentivirus with nucleic acid molecules of Example 2 and Control Example 2

[0132] The preparation method of Example 2B is similar to the preparation methods of Examples 1-1B and 1-2B, except that the preparation of 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.

[0133] Test Example 6: Testing of the lentiviral titer of Example 2B and Control Example 2B

[0134] The test method of Test Example 6 is similar to that of Test Example 1, except that biotinylated human mesothelin protein, His, Avitag (MSN-H82E9, ACRO Biosystems) is used to identify anti-mesothelin receptors, and phycoerythrin (PE)-labeled streptavidin (Invitrogen) diluted 500 times is used to detect the expression of anti-mesothelin receptors of the lentivirus of Example 2B and Control Example 2B. The test results are shown in Table 6 below:

[0135] Table 6: Effective titer of lentivirus in Example 2B and Control Example 2B

[0136] Group Example 2B Comparative Example 2B Lentivirus effective titer (TU / mL) <![CDATA[2×10 7 ]]> <![CDATA[3.9×10 7 ]]>

[0137] If the effective titer of lentivirus is greater than or equal to 1×10 6 TU / mL, when preparing CAR-T cells, a smaller volume of lentivirus can be used to successfully transduce T cells into CAR-T cells, and the transduced T cells can grow well. Therefore, according to the results of Table 6 above, the lentiviruses of Example 2B and Control Example 2B meet this standard, which is conducive to the subsequent preparation of CAR-T cells.

[0138] Example 2C: Anti-mesothelin-NKp30 CAR-T cells with intracellular signaling domain

[0139] The method for preparing the anti-mesothelin-NKp30 CAR-T cells with intracellular signaling domains of Example 2C is similar to the method for preparing the CAR-T cells of Examples 1-1C and 1-2C, except that the T cells in this example are transduced with the lentivirus of Example 2B, and the MOI during lentiviral transduction is 2. The CAR-T cells of Control Example 2C are transduced with the lentivirus of Control Example 2B, and the MOI during lentiviral transduction is 2. The schematic diagram of the structure of the prepared CAR-T cells of Example 2C and Control Example 2C is shown in FIG. Figure 7 As shown, the CD3ζ in Example 2C is inherent in T cells. Since the nucleic acid sequence transferred into the CAR-T cells of Example 2C and Control Example 2C has FcεR1γ, they are multi-chain CAR-T cells.

[0140] Test Example 7: Proliferation test of CAR-T cells of Example 2C, Control Example 2C and T cells of the non-transduced group

[0141] The test method of Test Example 6 is similar to that of Test Example 2, except that this test example detects the expansion rate of the CAR-T cells of Example 2C, and uses untransduced activated T cells as a control group. The results are shown in Table 7 below.

[0142] Table 7: Expansion folds of CAR-T cells of Example 2C and Control Example 2C and T cells of the untransduced group on day 7

[0143]

[0144] 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 7 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.

[0145] Test Example 8: Performance detection of CAR-T cells in Example 2C and Control Example 2C; Transduction rate of lentivirus in Example 2B and Control Example 2B

[0146] The test method of Test Example 8 is similar to that of Test Example 3, except that this test example uses biotinylated human mesothelin protein, His, Avitag (MSN-H82E9, ACRO Biosystems) to identify anti-mesothelin receptors, and uses 500-fold diluted PE-labeled streptavidin (Invitrogen) to detect the expression of anti-mesothelin receptors in CAR-T cells of Example 2C and Control Example 2C. The test results of Example 2C and Control Example 2C are as follows: Fig. 8A , 8B The results of flow cytometry are shown in Figures 8C and 8D. The percentage 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 8 below.

[0147] Table 8: Transduction rate of lentivirus of Example 2B and Control Example 2B

[0148] Group Example 2B Comparative Example 2B Transduction rate 70% to 100% 60% to 80%

[0149] 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. 8A The results show that the lentivirus of Example 2B has a better transduction rate than that of Control Example 2B.

[0150] Test Example 9: T cell phenotype analysis of CAR-T cells of Example 2C, Control Example 2C and T cells of the untransduced group

[0151] The test method of Test Example 9 is similar to that of Test Example 4, except that this test example is for the 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 9 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 9 below. Fig. 9A , 9B As shown in Figure 9C, among them, T cells expressing CD45RA and CCR7 are Tscm.

[0152] Table 9: Tscm percentage in CAR-T cells of Example 2C, Control Example 2C, and T cells of the untransduced group

[0153] Group Example 2C Comparative Example 2C Untransduced group %Tscm 62％ 57％ 52％

[0154] 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. 9A , 9B From Figure 9C, it can be seen that Example 2C having an intracellular signaling domain has a higher proportion of Tscm%, which is better than the CAR-T cells of the control example 2C and the activated T cells of the non-transduced group. Therefore, it can be expected that the CAR-T cells of Example 2C can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0155] Test Example 10: Real-time Cytotoxicity Assay Using xCELLigence

[0156] The experimental method of Test Example 10 is similar to that of Test Example 5, except that in this test example, the CAR-T cells of Example 2C and Control Example 2C are used as effector cells, and the aforementioned HeLa cell line is used as target cells. The target cells are seeded in a background-corrected culture plate (E-plate 96well, Agilent Technologies, 300600910) and cultured overnight. After the target cells are attached for impedance measurement for 18 hours, the effector cells are added to the culture plate at an E:T ratio of 3:1, 1:1, 0.3:1, and 0.1:1 for testing. The results of the toxicity experiments are shown as follows: Fig. 10A , 10B , 10C and 10D.

[0157] The results of the 24-hour poisoning experiment are shown in Table 10 below.

[0158] Table 10: Real-time cytotoxicity test results of CAR-T cells in Example 2C and Control Example 2C

[0159] Group Example 2C Comparative Example 2C Specific cleavage (E:T = 1:1) 65％ >60％

[0160] According to Table 10 and Figures 10A to 10D The results show that the cytotoxicity of the CAR-T cells in Example 2C is better than that of the control example 2C of the traditional single-chain CAR-T cells. Especially when the E:T ratio is 0.3:1 and 0.1:1, the cytotoxicity of Example 2C is more obvious than that of the control example 2C, and the standardized cell index value is greatly reduced, indicating that the target cells are greatly poisoned. In addition, if the cytotoxicity of CAR-T cells is >50%, it will have sufficient cytotoxicity when the patient cells are used to prepare CAR-T in the future. According to the results of Table 10 above, it can be seen that the CAR-T cells in Example 2C have good cytotoxicity.

[0161] Test Example 11: Cold light poisoning test

[0162] SKOV3 ovarian cancer cells (hereinafter referred to as SKOV3 cells) (purchased from ATCC, Cat. No. HTB-77) were cultured in 10% FBS McCoy 5A Medium (Gibco, 16600082), and SKOV3 cells with high mesothelin expression were screened by flow cytometry using an anti-mesothelin antibody labeled with green fluorescent protein. The PLL-CMV-rFluc-T2A-GFP-mPGK Lenti-Labeler lentiviral vector (System Biosciences) was then transduced into the aforementioned SKOV3 cells with high mesothelin expression, and a single stable expressing cell line was screened to obtain a GFP / Luc cell line expressing both green fluorescent protein (GFP) and luminescent enzyme. + SKOV3 cells (SKOV3-mLE).

[0163] In order to test the CAR-T cells of Example 2C, Control Example 2C and the activated T cells of the non-transduced group, the anti-tumor activity of the cells prepared on the 7th day was detected, and the ovarian cancer SKOV3 cells expressing mesothelin and luminescence enzyme were used as target cells to perform a cytotoxicity test. The effector cells used in this test example were the CAR-T cells of Example 2C, and the non-transduced T cells were used as the control group (non-transduced group). Specifically, the effector cells and the target cells were co-cultured in a 96-well plate at an E:T ratio of 3:1, cultured at 37°C and 5% carbon dioxide for 48 hours, and three replicates were performed.

[0164] The luminescence produced by the luminescence enzyme expressed by the surviving cancer cells was measured using the luminescence enzyme test 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:

[0165] Cell-specific lysis % = 100% × (number of experimentally lysed target cells – number of spontaneously lysed target cells) ÷ (number of maximum lysed target cells – number of spontaneously lysed target cells)

[0166] in,

[0167] The number of target cells lysed in the experiment = the number of target cells lysed in the group co-cultured with effector cells

[0168] 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 in RPMI-1640 medium containing 10% FBS at 37°C and 5% carbon dioxide for 48 hours

[0169] The maximum number of target cells lysed = the number of target cells lysed after adding 100 μl (microliters) of 1% TritonX-100 to the group without effector cells

[0170] The experimental results are shown in Table 11 below:

[0171] Table 11: Results of the cold light toxicity test of the CAR-T cells of Example 2C, Control Example 2C and the T cells of the non-transduced group (control group) (specific lysis %)

[0172] Group Example 2C Comparative Example 2C Non-transduced group (control group) Specific cleavage (E:T=3:1) 90％ 60％ About 0

[0173] When the cytotoxicity of CAR-T cells is >50%, there will be sufficient cytotoxicity when CAR-T cells are subsequently prepared using patient cells. According to the results in Table 11, the CAR-T cells of Example 2C are significantly better than the cytotoxicity of the control example 2C of the traditional single-chain CAR-T cells after being co-cultured with the target cells for 48 hours. Therefore, it can be inferred that the CAR-T cells of Example 2C have sufficient cytotoxicity when CAR-T cells are subsequently prepared using patient cells.

[0174] Test Example 12: Animal Experiment

[0175] First, 40 B2m gene-deficient female mice (NOD.Cg-Prkdc) aged 5 to 10 weeks were provided. scid Iq tm1Wjl B2m em1Narl / YckNarl (abbreviated as ASID-B2m KO)), the mice were divided into 8 groups (including a spare mouse group, as a tumor re-challenge positive control group), each group of 5, and kept in a sterile environment. The animal care and handling procedures of the experiment were in accordance with the experimental methods approved by the Institutional Animal Care and Use Committee (IACUC). Since the CAR-T cells of the present invention are derived from human T cells, when xenogeneic human T cells are implanted into immunodeficient experimental mice, it is expected that the mice will develop graft versus host disease (GvHD). However, previous experiments have shown that if the gene of beta-2-microglobulin (B2m) in immunodeficient mice is deleted, GvHD can be significantly slowed down, so this experiment uses immunodeficient mice with B2m gene deletion.

[0176] 5×10 SKOV3-mLE cells in Test Example 11 were taken 6Each cell was suspended in 100 μL PBS and then mixed with an equal volume of Matrigel (BD Bioscience) to obtain a SKOV3-mLE cell injection mixture. On day 0, the SKOV3-mLE cell injection mixture was subcutaneously inoculated into the right side of the body of 35 mice. Five mice that were not inoculated with SKOV3-mLE cells were also kept in reserve. From day 10, the bioluminescence imaging (BLI) signal was measured weekly using an in vivo imaging system (IVIS) (Perkin Elmer) until day 80. The measurement time was 80 days in total. If a mouse was sacrificed before day 80, it would be sacrificed in advance because it met the humane sacrifice criteria, and the signal of the mouse sacrificed in advance would not be measured until day 80. Among them, on the 21st day after implantation of SKOV3-mLE cells (as target cells), the other 7 groups of mice except those not inoculated with SKOV3-mLE cells were injected with saline (control group), or injected with CAR-T cells of Example 2C or CAR-T cells of Control Example 2 or untransduced T cells (the above as effector cells, E) from the tail artery, wherein E:T=1:1 or 3:1. On the 55th day, the aforementioned SKOV3-mLE cell injection mixture was implanted into the other side of the body of the remaining surviving mice and the spare group mice, i.e., the left side, for tumor re-challenge test. In the re-challenge test stage, a total of 22 mice were injected with SKOV3-mLE cells, of which 5 spare mice were given cancer cells for the first time. In this test example, the logarithmic scale after quantification of BLI signal represents the tumor volume in vivo of the mouse, and T cells in the peripheral blood of the mouse are taken for CAR expression analysis and T cell phenotype analysis, wherein the CAR expression analysis is as described in Test Example 8, and the phenotype analysis method is as described in Test Example 9. The experimental data were expressed as mean ± standard deviation and were analyzed statistically with Tukey's post hoc test for single-factor independent variance.

[0177] The experimental results are shown in Tables 12 and 13. Fig.11A , 11B In Table 12, "Day -4" is 4 days before the injection of effector cells, and "Day +10", "Day +17", "Day +24" and "Day +31" are 10 days, 17 days, 24 days and 31 days after the injection of effector cells, respectively.

[0178] Table 12: Results of in vivo cytotoxicity test [log(*E+11) photons / second] of Example 2C (abbreviated as E2C) and Control Example 2C (abbreviated as CE2C) evaluated by animal tumor BLI intensity

[0179]

[0180] ns means not statistically significant; * means p < 0.05, ** means p < 0.01, *** means p < 0.005

[0181] Table 13: Data of in vitro testing of the characteristics and functions of effector cells after preparing effector CAR-T cells and before injection into mice (Control Example 2C and Example 2C are abbreviated as CE2C and E2C, respectively)

[0182]

[0183] like Fig.11A As shown, 10 days after the injection of a higher dose (E:T=3:1) of CAR-T cells from Example 2C group (Day 31 of the total test period), the tumor volume can be reduced. According to the visual observation of BLI, in the mice given a high dose of CAR-T cells from Example 2C group, 3 / 5 mice can detect the BLI area. Compared with other effector cell groups, the BLI detectable area showed a smaller area. Observing the mice in each group as a whole, the group given a high dose of CAR-T from Example 2C, after conversion to a logarithmic scale, its BLI signal was also significantly lower than that of all other groups (as shown in Table 12 and Fig. 11B shown).

[0184] like Fig.11A As shown, after the 24th day of CAR-T cell treatment (the 45th day of the total experimental period), the BLI signal in mice administered with a high dose of CAR-T cells of Example 2C could no longer be detected visually (<10 8 Photons / second). A significant reduction in the detectable signal in the BLI area was also observed in mice treated with two doses of CAR-T cells from Control Example 2C or low-dose (E:T=1:1) CAR-T cells from Example 2C, but the BLI signals of these mice treated in different groups did not reach the level of visual undetectability. The BLI values ​​of mice treated with CAR-T cells from Example 2C and Control Example 2C were lower than those of their respective UTD control groups; and the BLI values ​​of mice treated with high-dose CAR-T cells from Example 2C were also significantly lower than those of other groups, including other groups treated with CAR-T cells (i.e., Control Example 2C and low-dose CAR-T cells from Example 2C). These differences in BLI detectable areas persisted until 31 days after treatment (day 52 of the total trial period), before some mice developed life-threatening GvHD (e.g. Fig.11A , Fig. 11B , as shown in Table 12).

[0185] In addition, Fig.11A Tumor elimination (signal reduction) was observed in the Example 2C treatment group, but GvHD was not immediately induced in the Example 2C treatment group, where the control Example 2C group, which was administered with traditional single-chain CAR-T, immediately induced GvHD in mice. Although the symptoms of GvHD have been significantly alleviated or delayed by using ASID-B2m KO mice, signs of GvHD are still obvious for mice treated with traditional single-chain CAR-T cells (Control Example 2C). This GvHD may be attributed to the phenomenon of overproliferation of human CAR-T cells and attacking host cells due to the lack of human cancer cells as target cells. Before the re-challenge test with SKOV3-mLE cancer cells, 3 mice treated with CAR-T from the Control Example 2C group (accounting for 3 / 10 of the total number of Control Example 2C groups) were sacrificed due to GvHD symptoms (such as Fig.11A Most of the surviving control 2C CAR-T treated mice (6 out of 7) were sacrificed 11 days after tumor re-challenge and also due to GvHD before the CAR-T efficacy could be evaluated (as shown in Figure 2C). Fig. 12A As shown). Fig.13A , Fig. 13B During the entire experiment, mice treated with CAR-T cells of control example 2C, with CD3+ cells as T cells, had significantly higher numbers of total T cells in peripheral blood and CAR-T in T cells than those of example 2C, which may indicate that the design of traditional single-chain CAR-T may tend to produce overactivated CAR-T cells. According to Table 13, the CAR-T cells of example 2C have a Tscm ratio that is not inferior to that of the untransduced group, and it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy; and the CAR-T cells of example 2C in Table 13 are based on the literature Galli E. et al. Br J Haematol. 2023 Nov; 203 (4): 564-570, because the proportion of CD8+T cells in T cells is higher than 50%, it will have excellent cancer cell killing efficacy.

[0186] The low-dose CAR-T cell-treated group of Example 2C showed an anti-tumor response to tumor re-challenge (eg Fig. 12A , 12BAs shown). Re-infusion of SKOV3-mLE cells resulted in GvHD symptoms in most mice treated with CAR-T cells, except for those treated with low-dose CAR-T cells of Example 2C. In mice treated with CAR-T cells of Example 2C group, the BLI signal was also reduced to a level below visual detection, indicating that Example 2C had a significant anti-tumor effect, and this value was also statistically lower than the control group of the saline-treated group 18 days after tumor re-challenge.

[0187] According to the above results, the lentivirus of Example 2B of the present invention has an excellent transduction rate because it has an intracellular signaling domain and contains a NKp30 transmembrane domain (a NKp30 fragment without an immunoglobulin-like domain), and the CAR-T cells obtained after transduction have excellent cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm, and it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. It has also been verified in animal experiments that it has an excellent effect of killing tumor cells, while reducing graft-versus-host disease, avoiding excessive activation of traditional single-chain CAR-T cells, and causing cytokine release syndrome (CRS) with fatal potential. Overactivated CAR-T cells are also prone to exhaustion and reduce their persistence in the body. According to the content of the document Korell F.et al, Med.2022Aug 12; 3(8):538-564, the efficacy cannot be sustained or may even increase the possibility of recurrence. From the above, it can be inferred that when preparing CAR-T cells, if the nucleic acid sequence of the intracellular signaling domain is simultaneously introduced into the T cells, such as the intracellular signaling domain of DAP12, it can bring excellent cytotoxic effect to the prepared CAR-T cells.

[0188] 3. Preparation and testing of anti-CD22-NKp30 CAR-T cells with intracellular signaling domains

[0189] Example 3: Nucleic acid molecules having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD22 antibody, and a NKp30 fragment without an immunoglobulin-like domain

[0190] The 3' end of the nucleic acid sequence with full-length FcεR1γ (as shown in SEQ ID NO:77) was connected to the 5' end of the nucleic acid sequence of DAP12 intracellular signaling domain (SEQ ID NO:78) to obtain a connected nucleic acid molecule fragment 1, and the 3' end of the connected nucleic acid molecule fragment 1 was then connected to the 5' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody (as shown in SEQ ID NO:91) through the nucleic acid sequence of the T2A self-truncating peptide, and the 5' end of the nucleic acid sequence of the CD28 hinge domain (as shown in SEQ ID NO:68) was further connected to the 3' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody, and finally the nucleic acid sequence of the NKp30 fragment without immunoglobulin-like domain (as shown in SEQ ID NO: NO:86), and connected to the 3' end of the nucleic acid sequence of the CD28 hinge domain to obtain the nucleic acid molecule of Example 3 having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD22 antibody, and NKp30, as shown in SEQ ID NO:92. The nucleic acid molecule of Example 3 comprises, from the 5' end to the 3' end, the nucleic acid sequence of the full-length FcεR1γ, the nucleic acid sequence of the DAP12 intracellular signaling domain, the nucleic acid sequence of the T2A self-truncated peptide, the nucleic acid sequence of the m971 single-chain variable region fragment, the nucleic acid sequence of the CD28 hinge domain, and the nucleic acid sequence of the NKp30 fragment without an immunoglobulin-like domain (NKp30 without Ig-like domain).

[0191] Control Example 3-1 Nucleic acid molecule having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD22 antibody and not containing any NKp30 fragment

[0192] The preparation method of Control Example 3-1 is similar to the preparation method of Control Example 2, except that the single-chain variable region fragment used in Control Example 3-1 is not SS1 of the single-chain variable region fragment of the anti-mesothelin antibody in Control Example 2, but the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody (as shown in SEQ ID NO: 91). The nucleic acid sequence of the nucleic acid molecule of Control Example 3-1 is shown in SEQ ID NO: 93.

[0193] Control Example 3-2 Nucleic acid molecules having no intracellular signaling domain and having a single-chain variable region fragment of an anti-CD22 antibody and a NKp30 fragment without an immunoglobulin-like domain

[0194] The 3' end of the nucleic acid sequence of the full-length FcεR1γ (as shown in SEQ ID NO: 77) was connected to the 5' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody (as shown in SEQ ID NO: 91) via the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO: 80), and then the 5' end of the nucleic acid sequence of the CD28 hinge domain (as shown in SEQ ID NO: 68) was further connected to the 3' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the aforementioned anti-CD22 antibody. Finally, the 5' end of the nucleic acid sequence of the aforementioned NKp30 fragment without an immunoglobulin-like domain (as shown in SEQ ID NO: 86) was connected to the 3' end of the nucleic acid sequence of the CD28 hinge domain to obtain the anti-CD22-NKp30 nucleic acid molecule without an intracellular signaling domain of Control Example 3-2, as shown in SEQ ID NO: 94. The nucleic acid molecule of control example 3-2 comprises the nucleic acid sequence of the full-length FcεR1γ, the nucleic acid sequence of the T2A self-truncated peptide, the nucleic acid sequence of the m971 single-chain variable region fragment, the nucleic acid sequence of the CD28 hinge domain, and the nucleic acid sequence of the NKp30 fragment without the immunoglobulin-like domain (NKp30 without Ig-like domain) from the 5' end to the 3' end.

[0195] Example 3A, Reference Example 3-1A, Reference Example 3-2A: Construction of plasmids with nucleic acid molecules of Example 3, Reference Example 3-1, Reference Example 3-2

[0196] The preparation method of Example 3A is similar to the preparation methods of Example 1-1A and Example 1-2A, except that Example 3A utilizes HpaI and EcoRI restriction enzymes to insert the nucleic acid molecule of Example 3 into the aforementioned pLAS5w vector.

[0197] In control example 3-1A, GenScript was commissioned to use NheI and EcoRI restriction enzymes to insert the nucleic acid molecule of control example 3-1 into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39). In control example 3-2A, HpaI and EcoRI restriction enzymes were used to insert the nucleic acid molecule of control example 3-2 into the aforementioned pLAS5w vector. The plasmids obtained in Example 3A, control example 3-1A, and control example 3-2A are as follows: Fig.14 shown.

[0198] Example 3B, Control Example 3-1B, Control Example 3-2B: Preparation of lentivirus with nucleic acid molecules of Example 3, Control Example 3-1, Control Example 3-2

[0199] The preparation method of Example 3B is similar to the preparation methods of Examples 1-1B and 1-2B, except that the preparation of Example 3B uses the plasmid of Example 3A as the transfer plasmid. The preparation of the lentivirus of Control Example 3-1B and Control Example 3-2B uses the plasmid of Control Example 3-1A and Control Example 3-2A as the transfer plasmid, respectively.

[0200] Test Example 13: Testing of the lentivirus titer of Example 3B

[0201] The test method of Test Example 13 is similar to that of Test Example 1, except that biotinylated human CD22, FcTag, (Sino Biological. Cat: 11958-H41H-B) is used to identify the anti-CD22 receptor, and 500-fold diluted PE-labeled streptavidin (Invitrogen) is used to detect the expression of the anti-CD22 receptor of the lentivirus of Example 3B and Control Example 3-1B. The test results are shown in Table 14 below:

[0202] Table 14: Effective titer of lentivirus in Example 3B and Control Example 3-1B

[0203] Group Example 3B Comparative Example 3-1B Lentivirus effective titer (TU / mL) <![CDATA[1.3×10 7 ]]> <![CDATA[1.2×10 7 ]]>

[0204] If the effective titer of lentivirus is greater than or equal to 1×10 6 TU / mL, when preparing CAR-T cells, a smaller volume of lentivirus can be used to successfully transduce T cells into CAR-T cells, and the transduced T cells can grow well. Therefore, according to the results of Table 14 above, the lentivirus of Example 3B meets this standard, which is conducive to the subsequent preparation of CAR-T cells.

[0205] Example 3C: Preparation of CAR-T cells with nucleic acid molecules of Example 3, Control Example 3-1, and Control Example 3-2

[0206] The preparation method of the CAR-T cells of Example 3C is similar to the preparation method of Examples 1-1C and 1-2C, except that the T cells in this example are transduced with the lentivirus of Example 3B, and the MOI during the lentivirus transduction is 3. The CAR-T cells of Control Examples 3-1C and 3-2C are transduced with the lentivirus of Control Examples 3-1B and 3-2B, and the MOI during the lentivirus transduction is 3. The schematic diagram of the structure of the prepared CAR-T cells of Example 3C and Control Example 2C is shown in FIG. Fig.15 shown.

[0207] Test Example 14: Proliferation test of CAR-T cells of Example 3C, Control Example 3-1C, Control Example 3-2C and T cells of the non-transduced group

[0208] The test method of Test Example 14 is similar to that of Test Example 2, except that this test example detects the expansion rate of CAR-T cells in Example 3C and Control Example 3-1C and Control Example 3-2C, and additionally uses untransduced activated T cells as a control group. The results are shown in Table 15 below.

[0209] Table 15: Expansion folds of CAR-T cells of Example 3C, Control Examples 3-1C, 3-2C, and T cells of the non-transduced group on day 7

[0210]

[0211] 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 15 above, it can be seen that the CAR-T cells of Example 3C have a good T cell expansion multiple and can be used for subsequent T cell functional tests.

[0212] Test Example 15: Expression detection of CAR-T cells in Example 3C and Control Example 3-1C; Transduction rate of lentivirus in Example 3B and Control Example 3-1B

[0213] The test method of Test Example 15 is similar to that of Test Example 3, except that this test example uses biotinylated human CD22 protein, Fc Tag, (Sino Biological. Cat: 11958-H41H-B) to identify anti-CD22 receptors, and uses 500-fold diluted PE-labeled streptavidin (Invitrogen) to detect the expression of anti-CD22 receptors of CAR-T cells in Example 3C and Control Example 3-1C. The test results of Example 3C and Control Example 3-1C are as follows: Fig.16A , 16B The flow cytometry results are shown in Table 16, 16C, and 16D. The percentage of cells expressing anti-CD22 receptors compared to the total number of cells was used as the transduction rate of the lentivirus of Example 3B or Control Example 3-1B. The experimental results are shown in Table 16.

[0214] Table 16: Transduction rate of lentivirus of Example 3B and Control Example 3-1B

[0215] Group Example 3B Comparative Example 3-1B Transduction rate 40% to 80% 30% to 75%

[0216] 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 show that the lentivirus of Example 3B has a good transduction rate.

[0217] Test Example 16: T cell phenotype analysis of CAR-T cells of Example 3C

[0218] The test method of Test Example 16 is similar to that of Test Example 4, except that this test example is for the CAR-T cells of Example 3C, Control Example 3-1C, and Control Example 3-2C, and the cells are tested on the 7th day of preparation. The ratio of Tscm in the CAR-T cells of Example 3C, Control Example 3-1C, 3-2C and the T cells of the untransduced group (control group) is shown in Table 17 below; the results of Example 3C and the flow cytometer results of Control Example 3-1C, Control Example 3-2C and the distribution of untransduced T cell subsets are shown in Table 17 below. Fig.17A , 17B , 17C and 17D, among which, T cells expressing CD45RA and CCR7 are Tscm.

[0219] Table 17: Percentage of Tscm in CAR-T cells of Example 3C, Control Example 3-1C, Control Example 3-2C, and T cells in the untransduced group

[0220] Group Example 3C Comparative Example 3-1C Comparative Example 3-2C Non-transduced group (control group) %Tscm 70.5％ 68.9％ 74.5％ 71.6％

[0221] 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.17A , 17B , 17C and 17D, it can be seen that Example 3C has a higher proportion of Tscm, which is slightly better than the traditional single-chain CAR-T cells of Control Example 3-1C, 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.

[0222] Test Example 17: Real-time Cytotoxicity Assay Using xCELLigence

[0223] First, a human CD22 cDNA fragment was cloned from the Raji human lymphoma cell line (purchased from Elabscience, Cat. No. CL-0189). The truncated CD22 cDNA fragment (as shown in SEQ ID NO: 95) was inserted into the pLAS5w.Ppuro vector (purchased from the RNAi Core of Academia Sinica, product number C6-8-39) to obtain a vector containing truncated CD22. The aforementioned vector containing truncated CD22 was transfected into the SKOV3 human ovarian cancer cell line using Polyjet (SignaGen Laboratories). The transfected SKOV3 cells were cultured in McCoy 5AMedium (Gibco) medium containing 2 μg / mL puromycin and 10% FBS to select SKOV3 cell lines that are resistant to puromycin and express CD22. Finally, the expression of CD22 in the cells was confirmed again using flow cytometry. The SKOV3 cell line expressing CD22 was used as the target cell for subsequent cytotoxicity experiments using the xCELLigence system.

[0224] The test method of Test Example 17 is similar to that of Test Example 5, except that the CAR-T cells of Example 3C, Control Example 3-1C, and Control Example 3-2C are used as effector cells in this test example, and the aforementioned SKOV3 cell line expressing CD22 is used as the target cell. The target cells are planted in a background-corrected culture dish, and the background impedance is measured for 20 hours. After the target cells are attached, the effector cells are added to the culture dish at an E:T ratio of 3:1 and 1:1 for testing. The results of the toxicity experiments are as follows: Fig.18A and 18B shown.

[0225] according to Fig.18A , 18B The results show that the CAR-T cell killing ability of Example 3C is better than that of the traditional single-chain CAR-T cell control example 3-1C and the control example 3-2C without the intracellular signal transduction domain. When the killing ability of CAR-T cells is >50%, it will have sufficient killing ability when the patient cells are used to prepare CAR-T cells in the future. Fig.18A , 18B It can be seen from the results that the CAR-T cells of Example 3C have a good cytotoxic effect and are superior to the CAR-T cells of Control Examples 3-1C and 3-2C.

[0226] Test Example 18: Human lymphoma cell cytotoxicity test analyzed by flow cytometry

[0227] In order to test the anti-tumor activity of the anti-CD22-NKp30 CAR-T cells with intracellular signaling domains of Example 3C, Daudi human lymphoma cells (hereinafter referred to as Daudi cells) were cultured with CellTrace TM Violet calibration, cultured in RPMI-1640 medium (Gibco) containing 10% FBS for subsequent flow cytometry analysis of toxicity test. The effector cells used in this test example are the CAR-T cells of Example 3C and Control Example 3-1C and Control Example 3-2C, and untransduced T cells are used as a control group. Specifically, the target cells were inoculated at 1.2×10 4 The cells 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) 1:1 and 3:1. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours, and the test was repeated three times. Next, the 96-well plate was centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells in each well were washed with 200μL PBS containing 2% fetal calf serum (FCS). After removing the PBS used for washing, the cells were collected with 300μL flow buffer (PBS containing 2% FCS), and 15μL counting beads (CountBright TM Plus Absolute Counting Beads, Invitrogen, C36995), 3000 microbeads were collected by flow cytometry to calculate the number of target cells. The cell killing rate was calculated by the following formula:

[0228] 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%

[0229] The experimental results are as follows Fig.19 And E:T=3:1 as shown in Table 18 below.

[0230] Table 18: Human lymphoma cell cytotoxicity test results analyzed by flow cytometry for Example 3C and Comparative Example 3-1C and Comparative Example 3-2C

[0231] Group Example 3C Comparative Example 3-1C Comparative Example 3-2C Specific cleavage (E:T=3:1) 85％ 77％ 14％

[0232] according to Fig.19As shown in Table 18, when E:T is 1:1, the cytotoxic effect of the CAR-T cells of Example 3C is 71%, which is much better than 45% of the control example 3-1C of the traditional single-chain CAR-T cells, 5% of the control example 3-2C without the intracellular signal transduction domain, and 5% of the control group (non-transduced group); when E:T is 3:1, the cytotoxic effect of the CAR-T cells of Example 3C is 85%, which is better than 77% of the control example 3-1C of the traditional single-chain CAR-T cells, 14% of the control example 3-2C without the intracellular signal transduction domain, and 27% of the control group (non-transduced group). It can be seen from the comparison results of Example 3C and Control Example 3-2C that the addition of the intracellular signal transduction domain can significantly increase the cancer cell cytotoxic effect.

[0233] According to the above results, the lentivirus of Example 3B of the present invention has an intracellular signal transduction domain and contains a NKp30 transmembrane domain (a NKp30 fragment without an immunoglobulin-like domain) with excellent transduction rate, and the CAR-T cells obtained after transduction have excellent cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm, and it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. It can be inferred that when preparing CAR-T cells, if the nucleic acid sequence of the intracellular signal transduction domain is transferred, such as the intracellular signal transduction domain of DAP12, it can bring excellent cytotoxic effects to the prepared CAR-T cells, and is particularly suitable for CAR-T therapy.

[0234] 4. Preparation and testing of anti-CD19-NKp30 CAR-T cells with intracellular signaling domains

[0235] Example 4: Nucleic acid molecules having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD19 antibody, and a NKp30 fragment without an immunoglobulin-like domain

[0236] The preparation method of Example 4 is similar to the preparation method of Example 3, except that the single-chain variable region fragment used in Example 4 is not m971, which is a single-chain variable region fragment of the anti-CD22 antibody in Example 3, but FMC63, which is a single-chain variable region fragment of the anti-CD19 antibody (as shown in SEQ ID NO: 96), and the hinge domain selected is the hinge domain of human IgG4 (as shown in SEQ ID NO: 69). The nucleic acid sequence of the nucleic acid molecule of Example 4 is shown in SEQ ID NO: 97. From the 5' end to the 3' end, the nucleic acid sequence of the nucleic acid molecule of Example 4 is respectively the nucleic acid sequence of the full-length FcεR1γ, the nucleic acid sequence of the intracellular signaling domain of DAP12, the nucleic acid sequence of the T2A self-truncated peptide, the nucleic acid sequence of the FMC63 single-chain variable region fragment, the nucleic acid sequence of the IgG4 hinge domain, and the nucleic acid sequence of the NKp30 fragment without the immunoglobulin-like domain (NKp30 without Ig-likedomain).

[0237] Control Example 4: Nucleic acid molecule having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD19 antibody, and not containing any NKp30 fragment

[0238] The preparation method of Control Example 4 is similar to the preparation method of Control Example 2, except that the single-chain variable region fragment used in Control Example 4 is not SS1 of the single-chain variable region fragment of the anti-mesothelin antibody in Control Example 2, but the nucleic acid sequence of the single-chain variable region fragment FMC63 of the anti-CD19 antibody (as shown in SEQ ID NO: 96). The nucleic acid sequence of the nucleic acid molecule of Control Example 4 is shown in SEQ ID NO: 98.

[0239] Example 4A: Construction of plasmids with nucleic acid molecules of Example 4 and Control Example 4

[0240] The preparation method of Example 4A is similar to the preparation method of Example 1-1A and Example 1-2A, except that Example 4A utilizes HpaI and EcoR1 restriction enzymes to insert the anti-CD19-NKp30 nucleic acid molecule having an intracellular signaling domain of Example 4 into the aforementioned pLAS5w vector.

[0241] In control example 4A, GenScript was commissioned to use NheI and EcoR restriction enzymes to insert the nucleic acid molecule of control example 4 into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39). Fig. 20 shown.

[0242] Example 4B, Control Example 4B: Preparation of a Lentivirus with the Nucleic Acid Molecules of Example 4 or Control Example 4

[0243] The preparation method of Example 4B is similar to the preparation methods of Examples 1-1B and 1-2B, except that the preparation of Example 4B uses the plasmid of Example 4A as the transfer plasmid, while the preparation of the lentivirus of Control Example 4B uses the plasmid of Control Example 4A as the transfer plasmid.

[0244] Test Example 19: Testing of the lentiviral titer of Example 4B and Control Example 4B

[0245] The test method of Test Example 19 is similar to that of Test Example 1, except that biotinylated human CD19, FcTag (ACROBiosystems, CD9-H8259) is used to identify the anti-CD19 receptor, and 500-fold diluted PE-labeled streptavidin (Invitrogen) is used to detect the expression of the anti-CD19 receptor of the lentivirus of Example 4B and Control Example 4B. The test results are shown in Table 19 below:

[0246] Table 19: Effective titer of lentivirus of Example 4B and Control Example 4B (transduction unit, TU / mL)

[0247] Group Example 4B Comparative Example 4B Lentivirus effective titer <![CDATA[1×10 7 Up to 1×10 8 ]]> <![CDATA[2×10 6 Up to 3×10 6 ]]>

[0248] If the effective titer of lentivirus is greater than or equal to 1×10 6 TU / mL, when preparing CAR-T cells, a smaller volume of lentivirus can be used to successfully transduce T cells into CAR-T cells, and the transduced T cells can grow well. Therefore, according to the results of Table 19 above, the lentivirus of Example 4B meets this standard, which is conducive to the subsequent preparation of CAR-T cells.

[0249] Example 4C, Control Example 4C: Preparation of CAR-T cells with nucleic acid molecules of Example 4 and Control Example 4

[0250] The preparation method of the CAR-T cells of Example 4C is similar to the preparation methods of Examples 1-1C and 1-2C, except that the T cells in this example are transduced with the lentivirus of Example 4B, and the MOI during the lentivirus transduction is 2. The CAR-T cells of Control Example 4C are transduced with the lentivirus of Control Example 4C, and the MOI during the lentivirus transduction is 2. The schematic diagram of the structure of the prepared CAR-T cells of Example 4C and Control Example 4C is shown in FIG. Fig.21 shown.

[0251] Test Example 20: Proliferation test of CAR-T cells in Example 4C and Control Example 4C

[0252] The test method of this test example is similar to that of test example 2, except that this test example detects the expansion rate of the CAR-T cells of Example 4C and Control Example 4C, and additionally uses untransduced activated T cells as a control group (untransduced group). The results are shown in Table 20 below.

[0253] Table 20: Expansion folds of CAR-T cells of Example 4C and Control Example 4C on day 7

[0254]

[0255] 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 20 above, it can be seen that the CAR-T cells of Example 4C have a good T cell expansion multiple and can be used for subsequent T cell functional tests.

[0256] Test Example 21: Expression detection of CAR-T cells in Example 4C and Control Example 4C; Transduction rate of Example 4B and Control Example 4C

[0257] The test method of Test Example 21 is similar to that of Test Example 3, except that this test example uses biotinylated human CD19, Fc Tag (ACROBiosystems, CD9-H8259) to identify the anti-CD19 receptor, and uses 500-fold diluted PE-labeled streptavidin (Invitrogen) to detect the expression of the anti-CD19 receptor of the CAR-T cells of Example 4C and Control Example 4C. The test results of Example 4C and Control Example 4C are as follows: Fig.22A , 22B , 22C and 22D. The percentage of cells expressing anti-CD19 receptors compared to the total number of cells was used as the transduction rate of the lentivirus of Example 4B or Control Example 4B. The experimental results are shown in Table 21 below.

[0258] Table 21: Transduction rate of lentivirus of Example 4B and Control Example 4B

[0259] Group Example 4B Comparative Example 4B Transduction rate 30% to 50% 30% to 85%

[0260] 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.22A The results show that the lentivirus of Example 4B has a good transduction rate.

[0261] Test Example 22: T cell phenotype analysis of the CAR-T cells of Example 4C

[0262] The test method of Test Example 22 is similar to that of Test Example 4, except that this test example is for the CAR-T cells of Example 4C, and the cells on the 7th day of preparation are tested. The ratio of Tscm in the CAR-T cells of Example 4C, Control Example 4C and T cells of the non-transduced group is shown in Table 22 below; the results of Example 4C and the flow cytometer results of the distribution of T cell subsets of Control Example 4C and non-transduced T cells are shown in Table 22 below. Fig.23A , 23B As shown in Figure 23C, among them, T cells expressing CD45RA and CCR7 are Tscm.

[0263] Table 22: Tscm percentage in CAR-T cells of Example 4C, Control Example 4C, and T cells of the untransduced group

[0264] Group Example 4C Comparative Example 4C Untransduced group %Tscm 71.2％ 71.8％ 78.3％

[0265] 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.23A , 23B From Figures 23C and 23C, it can be seen that Example 4C has a Tscm higher than 40% and is similar to traditional single-chain CAR-T cells. Therefore, it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0266] Test Example 23: Real-time Cytotoxicity Assay Using xCELLigence

[0267] A human CD19 cDNA fragment was cloned from the Raji human lymphoma cell line. A truncated CD19 cDNA fragment (SEQ ID NO: 99) was amplified using PCR. The truncated CD19 cDNA fragment (1-342 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 CD19 cDNA fragment was inserted into the pLAS5w.Ppuro vector (purchased from the RNAi Core of Academia Sinica, product number C6-8-39) having a puromycin resistance gene using the HpaI and NheI restriction enzyme sites to obtain a vector containing truncated CD19. The vector containing 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) containing 2 μg / mL puromycin and 10% FBS to screen out drug-resistant SKOV3 cell lines that also expressed 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 subsequent cytotoxicity assay of the xCELLigence system.

[0268] The test method of this test example is similar to that of test example 5, except that in this test example, the CAR-T cells of Example 4C and Control Example 4C are used as effector cells, and the aforementioned SKOV3 cell line expressing CD19 is used as target cells. The target cells are planted in a culture dish with background correction, and background impedance measurement is performed for 20 hours. After the target cells are attached, the effector cells are added to the culture dish at an E:T ratio of 1:1 for testing. In addition, 100 μl (microliters) of 1% Triton X-100 is added to the culture dish containing 2×10 4 The target cells were cultured in a medium containing 10 cells / mL of target cells as the complete lysis group. The results of the toxicity experiments were as follows: Fig.24 The results of the poisoning test for 48 hours are shown in Table 23.

[0269] Table 23: Cytotoxicity test results of Example 4C and Control Example 4C

[0270] Group Example 4C Comparative Example 4C Specific cleavage 92％ 84.4％

[0271] When the cytotoxicity of CAR-T cells is >50%, there will be sufficient cytotoxicity when the patient's cells are used to prepare CAR-T cells in the future. Fig.24From the results in Table 23, it can be seen that the CAR-T of Example 4C can achieve a cancer cell cytotoxic effect of more than 80% after co-culture with target cells for 48 hours, and is slightly better than the CAR-T cell cytotoxic effect of the control example 4C of the traditional single-chain CAR-T cell. Therefore, it can be inferred that the CAR-T cells of Example 4C have sufficient cytotoxic ability when the patient cells are used to prepare CAR-T in the future.

[0272] Test Example 24 Cold Light Poisoning Test

[0273] The test method of Test Example 24 is similar to that of Test Example 18, except that the CAR-T cells of Example 4C and Control Example 4C are used as effector cells, and the Raji cell line expressing green fluorescent protein and luminescent enzyme is used as target cell. The effector cells and target cells are tested at an E:T ratio of 1:1 and 3:1, respectively. The co-culture time of the effector cells and target cells is 24 hours. The results of the toxicity test are as follows: Fig.25 shown.

[0274] The experimental results are as follows Fig.25 As shown, regardless of the E:T ratio of 1:1 or 3:1, the cytotoxic effect of the CAR-T cells of Example 4C was greater than 50%, and was significantly better than the cytotoxic effect of the single-chain CAR-T cells of Control Example 4C.

[0275] According to the above results, the lentivirus of Example 4B of the present invention has an excellent transduction rate due to the intracellular signal transduction domain and the NKp30 fragment containing the NKp30 transmembrane domain without the immunoglobulin-like domain, and the CAR-T cells obtained after transduction have excellent CAR-T cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm, and it can be expected that it can reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. It can be inferred that when preparing CAR-T cells, if the nucleic acid sequence of the intracellular signal transduction domain is transferred, such as the intracellular signal transduction domain of DAP12, it can bring excellent cytotoxicity to the prepared CAR-T cells, and is particularly suitable for CAR-T therapy.

[0276] In summary, if the genes of intracellular signaling domains, such as DAP12 intracellular signaling domain, 41BB intracellular signaling domain or CD28 intracellular signaling domain, and NKp30 transmembrane domain and NKp30 cytoplasmic domain are transferred into T cells, the T cells will have NKp30 receptor complexes and become multi-chain CAR-T cells, and they will have excellent cancer cell killing ability in both in vitro and in vivo experiments.

[0277] 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) Extracellular antigen binding domain; (b) Hinge domain; (c) Natural Killer Protein 30 (NKp30) transmembrane domain; and (d) natural killer protein 30 (NKp30) cytoplasmic domain, and (e) Intracellular signaling domain.

2. The isolated nucleic acid molecule of claim 1, wherein The extracellular antigen binding domain comprises a heavy chain variable region.

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

4. 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.

5. The isolated nucleic acid molecule of claim 1, wherein The intracellular signaling domain is the intracellular signaling domain of DAP12, 41BB or CD28.

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

7. 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, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-CD22 antibody and the hinge domain is the hinge domain of CD28, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-CD19 antibody and the hinge domain is the hinge domain of IgG4.

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

9. A plasmid comprising the isolated nucleic acid molecule according to any one of claims 1 to 8, wherein the plasmid is a DNA plasmid or an RNA plasmid.

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

11. The method for preparing a chimeric antigen receptor-cell according to claim 10, wherein the introduction is by transduction using a lentivirus.

12. An isolated cell comprising the isolated nucleic acid molecule of any one of claims 1 to 8.

13. The isolated cell of claim 12, wherein The separated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

14. Use of the isolated cells of claim 12 or 13 for the preparation of a medicament for treating or slowing down cancer.

15. Use of the isolated cells according to claim 12 or 13, 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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