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

Abstract

A nucleic acid molecule comprising an intracellular signaling domain and a NKp30 transmembrane domain, and a chimeric antigen receptor comprising the nucleic acid molecule. The nucleic acid molecule comprises nucleic acid sequences of the following segments: (a) an extracellular antigen binding domain; (b) a hinge domain; (c) a NKp30 transmembrane domain; (d) a NKp30 cytoplasmic domain; and (e) an intracellular signaling domain. By transferring the nucleic acid sequences of the intracellular signaling domain, the NKp30 transmembrane domain, and the NKp30 cytoplasmic domain into T cells, the T cells are endowed with NKp30 receptor complexes and become multichain CAR-T cells which can form a stable immune synapse with cancer cells and then have an excellent cancer cell killing capability.

Description

Nucleic acid molecule with intracellular signaling domain and natural killer protein 30 transmembrane domain and chimeric antigen receptor containing the same Technical Field

[0001] The present invention relates to a nucleic acid molecule for preparing a chimeric antigen receptor, in particular a nucleic acid molecule for preparing a chimeric antigen receptor with excellent cancer cell cytotoxicity, 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 through a multi-stage process, typically starting as a mild precancerous lesion and progressing to a malignant tumor, which then metastasizes. These changes are not caused by a single factor but rather by the interaction of a person's genetic makeup and external factors.

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

[0004] Cancer not only causes physical and mental suffering to patients, but also places severe emotional and financial strain on their families. However, the high-pressure environment of modern society makes it difficult to completely eliminate negative external factors, and the number of people suffering from cancer continues to increase year by year.

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

[0006] Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a novel CAR-T cell. When the modified T cells are injected back into the human body, they can recognize and kill tumor cells expressing specific antigens.

[0008] To achieve the aforementioned objectives, the present invention provides an isolated nucleic acid molecule comprising the following nucleic acid sequence:

[0009] (a) Extracellular antigen-binding domain;

[0010] (b) Hinge domain;

[0011] (c) Natural Killer Protein 30 (NKp30) transmembrane domain;

[0012] (d) natural killer protein 30 (NKp30) cytoplasmic domain; and

[0013] (e) Intracellular signaling domain.

[0014] The present invention transfers the genes encoding the NKp30 transmembrane domain, NKp30 cytoplasmic domain, and intracellular signaling domain into T cells, allowing the T cells to possess the NKp30 receptor complex, thereby becoming multi-chain CAR-T cells. These cells can form stable immune synapses with cancer cells and possess excellent cancer cell-killing capabilities.

[0015] Preferably, the extracellular antigen binding domain is the NKp30 extracellular domain.

[0016] Preferably, the extracellular antigen-binding domain comprises a heavy chain variable region.

[0017] Preferably, the extracellular antigen-binding domain comprises: an anti-mesothelin antibody heavy chain variable region, an anti-CD22 antibody heavy chain variable region, an anti-CD19 antibody heavy chain variable region, an anti-B cell maturation antigen (BCMA) antibody heavy chain variable region, an anti-CD123 antibody heavy chain variable region, an anti-GPRC5D (G protein–coupled receptor, class C, group 5, member D) antibody heavy chain variable region, or an anti-TSHR (thyroid-stimulating hormone receptor, TSH receptor) antibody heavy chain variable region.

[0018] In one embodiment, the heavy chain variable region of the extracellular antigen-binding domain may be derived from 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.

[0019] In one embodiment, the extracellular antigen-binding domain is the heavy chain variable region of an anti-BCMA heavy chain antibody.

[0020] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of a first anti-BCMA heavy chain antibody and a heavy chain variable region of a second anti-BCMA heavy chain antibody, which are tandemly linked by G4S, as 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; and 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; and CDRH3 is shown in the amino acid sequence of SEQ ID NO: 9.

[0021] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-CD19 antibody, wherein the heavy chain variable region of the anti-CD19 antibody comprises the complementarity determining regions: CDRH1 is represented by the amino acid sequence of SEQ ID NO: 10; CDRH2 is represented by the amino acid sequence of SEQ ID NO: 11; and CDRH3 is represented by the amino acid sequence of SEQ ID NO: 12. In one embodiment, the extracellular antigen-binding domain further comprises a light chain variable region of an anti-CD19 antibody, wherein the light chain variable region of the anti-CD19 antibody comprises the following CDRs: CDRL1 is represented by the amino acid sequence of SEQ ID NO: 13; CDRL2 is represented by the amino acid sequence of SEQ ID NO: 14; and CDRL3 is represented by the amino acid sequence of SEQ ID NO: 15. In one embodiment, the extracellular antigen-binding domain is an FMC63 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO: 16.

[0022] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-mesothelin antibody, wherein the heavy chain variable region of the anti-mesothelin antibody comprises the following CDRs: CDRH1 is represented by the amino acid sequence of SEQ ID NO: 17; CDRH2 is represented by the amino acid sequence of SEQ ID NO: 18; and CDRH3 is represented by the amino acid sequence of SEQ ID NO: 19. In one embodiment, the extracellular antigen-binding domain further comprises a light chain variable region of an anti-mesothelin antibody, wherein the light chain variable region of the anti-mesothelin antibody comprises the following CDRs: CDRL1 is represented by the amino acid sequence of SEQ ID NO: 20; CDRL2 is represented by the amino acid sequence of SEQ ID NO: 21; and CDRL3 is represented by the amino acid sequence of SEQ ID NO: 22. In one embodiment, the extracellular antigen-binding domain is an SS1 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO: 23.

[0023] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-BCMA antibody, wherein the heavy chain variable region of the anti-BCMA antibody comprises the following CDRs: CDRH1 is represented by the amino acid sequence of SEQ ID NO: 24; CDRH2 is represented by the amino acid sequence of SEQ ID NO: 25; and CDRH3 is represented by the amino acid sequence of SEQ ID NO: 26. In one embodiment, the 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 represented by the amino acid sequence of SEQ ID NO: 27; CDRL2 is represented by the amino acid sequence of SEQ ID NO: 28; and CDRL3 is represented by the amino acid sequence of SEQ ID NO: 29. In one embodiment, the extracellular antigen-binding domain is a J6M0 single-chain variable region fragment, as represented by 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 represented by the amino acid sequence of SEQ ID NO: 31; CDRH2 is represented by the amino acid sequence of SEQ ID NO: 32; and CDRH3 is represented by the amino acid sequence of SEQ ID NO: 33. In one embodiment, the 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 represented by the amino acid sequence of SEQ ID NO: 34; CDRL2 is represented by the amino acid sequence of SEQ ID NO: 35; and CDRL3 is represented by the amino acid sequence of SEQ ID NO: 36. In one embodiment, the extracellular antigen-binding domain is a BB2121 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO: 37.

[0024] In one 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 represented by the amino acid sequence of SEQ ID NO:38; CDRH2 is represented by the amino acid sequence of SEQ ID NO:39; and CDRH3 is represented by the amino acid sequence of SEQ ID NO:40. 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 represented by the amino acid sequence of SEQ ID NO:41; CDRL2 is represented by the amino acid sequence of SEQ ID NO:42; and CDRL3 is represented by the amino acid sequence of SEQ ID NO:43. In one embodiment, the extracellular antigen-binding domain is an m971 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO:44. 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 represented by the amino acid sequence of SEQ ID NO: 45; CDRH2 is represented by the amino acid sequence of SEQ ID NO: 46; and CDRH3 is represented by the amino acid sequence of SEQ ID NO: 47. 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 represented by the amino acid sequence of SEQ ID NO: 48; CDRL2 is represented by the amino acid sequence of SEQ ID NO: 49; and CDRL3 is represented by the amino acid sequence of SEQ ID NO: 50. In one embodiment, the extracellular antigen-binding domain is an LL2 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO: 51. In another embodiment, the extracellular antigen-binding domain comprises the 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 set forth in the amino acid sequence of SEQ ID NO:52; CDRH2 is set forth in the amino acid sequence of SEQ ID NO:53; and CDRH3 is set forth in the amino acid sequence of SEQ ID NO:54. In one embodiment, the extracellular antigen-binding domain further comprises the 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 set forth in the amino acid sequence of SEQ ID NO:55; CDRL2 is set forth in the amino acid sequence of SEQ ID NO:56; and CDRL3 is set forth 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 set forth in the amino acid sequence of SEQ ID NO:58.In another embodiment, the extracellular antigen-binding domain comprises the 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 set forth in the amino acid sequence of SEQ ID NO: 59; CDRH2 is set forth in the amino acid sequence of SEQ ID NO: 60; and CDRH3 is set forth in the amino acid sequence of SEQ ID NO: 61. In one embodiment, the extracellular antigen-binding domain further comprises the 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 set forth in the amino acid sequence of SEQ ID NO: 62; CDRL2 is set forth in the amino acid sequence of SEQ ID NO: 63; and CDRL3 is set forth 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 set forth in the amino acid sequence of SEQ ID NO: 65.

[0025] Preferably, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-CD19 antibody, which may be an FMC63 single chain variable region fragment.

[0026] Preferably, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-mesothelin antibody, which may be an SS1 single chain variable region fragment.

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

[0028] Preferably, the extracellular antigen binding domain comprises the heavy chain variable region of an anti-CD22 antibody, which may be an m971 single chain variable region fragment, an LL2 single chain variable region fragment, a G5 / 44 single chain variable region fragment or an RFB4 single chain variable region fragment.

[0029] 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).

[0030] Preferably, the isolated nucleic acid molecule further comprises a nucleic acid sequence for an adapter gene. More preferably, the adapter molecule is CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / γ. According to the present invention, the full length of CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / γ is used as the adapter molecule.

[0031] Preferably, the intracellular signaling domain is the intracellular signaling domain of DAP12, 41BB or CD28. More preferably, the intracellular signaling domain is the intracellular signaling domain of DAP12.

[0032] Preferably, the hinge domain and the NKp30 transmembrane domain are connected via an NKp30 extracellular stalk region.

[0033] Preferably, the isolated nucleic acid molecule further comprises a nucleic acid sequence for the CD3ζ signaling domain, an FcεR1γ adaptor molecule, or a T2A self-truncating peptide. For example, the FcεR1γ adaptor molecule nucleic acid sequence may be located at the 5' end of the isolated nucleic acid molecule; and the T2A self-truncating peptide nucleic acid sequence may be located between the intracellular signaling domain nucleic acid sequence and the NK protein 30 transmembrane domain nucleic acid sequence, or between the FcεR1γ adaptor molecule nucleic acid sequence and the NK protein 30 transmembrane domain nucleic acid sequence.

[0034] The hinge domain is connected to the NKp30 transmembrane domain via an NKp30 extracellular stalk region.

[0035] Preferably, in the aforementioned isolated nucleic acid molecule, when the aforementioned 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. When this extracellular antigen binding domain and hinge domain are combined, the resulting lentivirus can have an excellent transduction rate, and the resulting CAR-T cells have a better expansion rate, a higher proportion of stem cell-like memory T cells (Tscm), and better cytotoxicity. More preferably, the heavy chain variable region of the anti-CD19 antibody is an FMC63 single-chain variable region fragment.

[0036] Preferably, in the aforementioned isolated nucleic acid molecule, when the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-mesothelin antibody, the hinge domain is a (G4S)3 hinge domain. This combination of the extracellular antigen-binding domain and the hinge domain can result in a superior transduction efficiency of the resulting lentivirus, and the resulting CAR-T cells exhibit a better expansion rate, a higher proportion of Tscm, and superior cytotoxicity. More preferably, the heavy chain variable region of the anti-mesothelin antibody is an SS1 single-chain variable region fragment.

[0037] Preferably, in the aforementioned isolated nucleic acid molecule, when the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-BCMA antibody, the hinge domain is the hinge domain of CD28. This combination of the extracellular antigen-binding domain and the hinge domain can result in a superior transduction efficiency of the resulting lentivirus, and the resulting CAR-T cells have a better expansion rate, a higher proportion of Tscm, and superior cytotoxicity. More preferably, the heavy chain variable region of the anti-BCMA antibody is a J6M0 single-chain variable region fragment.

[0038] Preferably, in the aforementioned isolated nucleic acid molecule, when the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-CD22 antibody, the hinge domain is the hinge domain of CD28. This combination of the extracellular antigen-binding domain and the hinge domain can result in a superior transduction efficiency of the resulting lentivirus, and the resulting CAR-T cells exhibit a better expansion rate, a higher proportion of Tscm, and superior cytotoxicity. More preferably, the heavy chain variable region comprising the anti-CD22 antibody is the m971 single-chain variable region fragment.

[0039] To achieve the aforementioned objectives, the present invention further provides a plasmid comprising the isolated nucleic acid molecule described above, wherein the vector is a DNA plasmid or an RNA plasmid. According to the present invention, the nucleic acid sequences on the plasmid are arranged from 5' to 3' in the following order: FcεR1γ adaptor 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 nucleic acid sequences on the plasmid are arranged from 5' to 3' in the following order: 5' LTR, promoter, FcεR1γ adaptor molecule, intracellular signaling domain, T2A self-truncating peptide, extracellular antigen-binding domain, hinge domain, NKp30 transmembrane domain, NKp30 cytoplasmic domain, and 3' LTR.

[0040] To achieve the aforementioned objectives, the present invention further provides a method for preparing a chimeric antigen receptor-cell, comprising introducing the aforementioned isolated nucleic acid molecule into a nucleated cell.

[0041] Preferably, in the aforementioned method for preparing chimeric antigen receptor-cells, the aforementioned "introduction" is introduced by transduction using lentivirus.

[0042] Preferably, the nucleated cells are leukocytes. Preferably, the nucleated cells are T cells, natural killer cells, natural killer T cells or adipose-derived stem cells.

[0043] To achieve the aforementioned objectives, the present invention further provides an isolated cell containing the isolated nucleic acid molecule as described above.

[0044] Preferably, the aforementioned separated cells are T cells, natural killer cells, natural killer T cells or adipose stem cells.

[0045] To achieve the aforementioned objectives, the present invention further provides a use of the aforementioned isolated cells for preparing a medicament for treating or slowing the progression of cancer. For example, the medicament can treat 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.

[0046] To achieve the aforementioned objectives, the present invention further provides a use of the aforementioned isolated cells for preparing a medicament for treating or alleviating an autoimmune disease. The autoimmune disease is a disease associated with an autoimmune response for which CD19 CAR-T cells are therapeutically effective. In one embodiment, the autoimmune disease is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, systemic sclerosis, or multiple sclerosis.

[0047] The advantage of this invention lies in the transfer of nucleic acid sequences encoding the NKp30 transmembrane domain, NKp30 cytoplasmic domain, and intracellular signaling domain into T cells, allowing them to harbor the NKp30 receptor complex, creating multi-chain CAR-T cells capable of forming stable immune synapses with cancer cells and exhibiting excellent cancer cell-killing capabilities. The resulting CAR-T cells exhibit excellent cytotoxicity both in vivo and in vitro, significantly killing cancer cells without overactivating T cells, and exhibiting persistence in rechallenge experiments. Furthermore, they possess a high percentage of Tscm, which is expected to reduce the cytokine release syndrome and neurotoxicity associated with CAR-T cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a schematic diagram of the pLAS5w vector;

[0049] FIG2 is a schematic diagram of the plasmids of Examples 1-1A to 1-2A and Comparative Example 1A;

[0050] FIG3 is a schematic diagram of the cell membrane protein structure of CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C;

[0051] Figures 4A to 4D are flow cytometry results of T cell subset distribution of CAR-T cells of Examples 1-1C, 1-2C, Control Example 1C, and a non-transduced group;

[0052] Figures 5A, 5B, and 5C are the real-time results of cytotoxicity comparisons of the CAR-T cells of Examples 1-1C and 1-2C, the CAR-T cells of Control Example 1C, the T cells of the untransduced group, and the target cells in Test Example 5 at E:T ratios of 10:1, 3:1, and 1:1, respectively;

[0053] FIG6 is a schematic diagram of the plasmids of Example 2A and Comparative Example 2A;

[0054] FIG7 is a schematic diagram of the cell membrane protein structure of CAR-T cells in Example 2C and Control Example 2C;

[0055] FIG8A and FIG8B are flow cytometry results of the expression of anti-mesothelin antibodies and NKp30 by the CAR-T cells of Example 2C in Test Example 8, respectively;

[0056] FIG8C and FIG8D are flow cytometry results of the expression of anti-mesothelin antibodies and NKp30 by CAR-T cells of control example 2C in test example 8, respectively.

[0057] 9A, 9B, and 9C are flow cytometric analysis results of T cell subset distributions in Example 2C, Control Example 2C, and the non-transduced group in Test Example 9 on day 7;

[0058] Figures 10A, 10B, 10C, and 10D are real-time results of cytotoxicity comparisons 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 target cells in Test Example 10 at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1, respectively;

[0059] FIG11A shows 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;

[0060] FIG11B shows the logarithmic bioluminescence 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 in Test Example 12 at E:T ratios of 3:1 and 1:1;

[0061] FIG12A shows the results of an 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 at E:T ratios of 3:1 and 1:1 in Test Example 12;

[0062] FIG12B shows the logarithmic bioluminescence imaging signals of the CAR-T cells of Example 2C, the CAR-T cells of Control Example 2C, and the target cells in Test Example 12 after in vivo anti-tumor re-challenge at E:T ratios of 3:1 and 1:1, with * indicating a p-value <0.05 and ** indicating a p-value <0.01;

[0063] FIG13A shows the ratio of T cells in the peripheral blood of mice in each group of Test Example 12;

[0064] FIG13B shows the proportion of CAR-T cells in T cells of each group of mice in Test Example 12;

[0065] Figure 14 is a schematic diagram of the plasmids of Example 3A and Comparative Examples 3-1A and 3-2A;

[0066] Figure 15 is a schematic diagram of the cell membrane protein structure of CAR-T cells in Example 3C and Control Examples 3-1C and 3-2C;

[0067] FIG16A and FIG16B are flow cytometry results of the CAR-T cells of Example 3C in Test Example 15 expressing anti-CD22 receptor and NKp30, respectively;

[0068] Figures 16C and 16D are flow cytometry results of the CAR-T cells expressing anti-CD22 receptor and NKp30 in Control Example 3-1C in Test Example 15, respectively;

[0069] Figures 17A, 17B, 17C, and 17D are the flow cytometric analysis results of T cell subset distributions in Example 3C, Control Example 3-1C, Control Example 3-2C, and the non-transduced group in Test Example 16 on day 7;

[0070] Figures 18A and 18B are real-time results of cytotoxicity comparisons of the CAR-T cells of Example 3C, the CAR-T cells of Control Example 3-1C, the CAR-T cells of 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;

[0071] FIG19 is a flow cytotoxicity comparison result 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 18 at E:T ratios of 3:1 and 1:1;

[0072] Figure 20 is a schematic diagram of the plasmids of Example 4A and Comparative Example 4A;

[0073] FIG21 is a schematic diagram of the cell membrane protein structure of CAR-T cells of Example 4C and Control Example 4C;

[0074] FIG22A and FIG22B are flow cytometry results of the CAR-T cells of Example 4C in Test Example 21 expressing anti-CD19 receptor and NKp30, respectively;

[0075] FIG22C and FIG22D are flow cytometry results of the CAR-T cells expressing anti-CD19 receptor and NKp30 in Control Example 4C of Test Example 21, respectively;

[0076] FIG23A , FIG23B , and FIG23C are flow cytometric analysis results of T cell subset distributions in Example 4C, Control Example 4C, and the non-transduced group in Test Example 22 on day 7, respectively;

[0077] FIG24 is a comparison of the results of a real-time cytotoxicity test of the CAR-T cells of Example 4C, the CAR-T cells of Control Example 4C, T cells of the untransduced group, target cells, and the full lysis group at an E:T ratio of 1:1 in Test Example 23;

[0078] FIG25 is a comparison of the cold light toxicity results 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

[0079] The present 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.

[0080] 1. Preparation and testing of CAR-T cells with intracellular signaling domains and NKp30 fragments

[0081] Example 1-1: Nucleic acid molecules containing the DAP12 intracellular signaling domain and the complete NKp30 fragment

[0082] First, the 3' end of the full-length FcεR1γ nucleic acid sequence (as shown in SEQ ID NO:77) was ligated to the 5' end of the DAP12 intracellular signaling domain nucleic acid sequence (SEQ ID NO:78) to produce a ligated nucleic acid molecule fragment 1. This ligated nucleic acid molecule fragment 1 was then ligated to the NKp30 complete fragment (including the immunoglobulin-like domain) sequence (SEQ ID NO:79) via the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO:80) to produce the nucleic acid molecule of Example 1-1 comprising the DAP12 intracellular signaling domain and the NKp30 complete fragment (as shown in SEQ ID NO:81). The NKp30 complete fragment comprises the immunoglobulin-like domain, the stalk region, the NKp30 transmembrane domain (Tm), and the NKp30 cytoplasmic domain. The nucleic acid molecule having the DAP12 intracellular signaling domain and the complete NKp30 fragment of Example 1-1 comprises the full-length FcεR1γ nucleic acid sequence, the DAP12 intracellular signaling domain nucleic acid sequence, the T2A self-truncating peptide nucleic acid sequence, and the complete NKp30 fragment from the 5' end to the 3' end.

[0083] Example 1-2 Nucleic acid molecules having the 41BB intracellular signaling domain and the complete NKp30 fragment

[0084] The preparation method of Example 1-2 is similar to that of Example 1-1, except that the intracellular signaling domain used is the 41BB intracellular signaling domain (as shown in SEQ ID NO: 82). The resulting nucleic acid molecule having the 41BB intracellular signaling domain and the complete NKp30 fragment of Example 1-2 is shown in SEQ ID NO: 83.

[0085] Control Example 1: Modified wild-type NKp30 nucleic acid molecule

[0086] First, the 3' end of a full-length FcεR1γ nucleic acid sequence (as shown in SEQ ID NO: 77) was ligated to the 5' end of the aforementioned complete NKp30 fragment (as shown in SEQ ID NO: 79) via the nucleic acid sequence of a 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 consisted, from the 5' end to the 3' end, of the full-length FcεR1γ nucleic acid sequence, the nucleic acid sequence of the T2A self-truncating peptide, and the complete NKp30 fragment.

[0087] Example 1-1A, 1-2A and Comparative Example 1A: Construction of a plasmid containing the nucleic acid molecule of Example 1-1, 1-2 or Comparative Example 1

[0088] The pLAS5w.Ppuro vector (purchased from the Academia Sinica RNAi Core, product number C6-8-39), which contains a TAR chimeric 5'LTR nucleic acid sequence and is driven by an RSV promoter, was depleted of the hPGK promoter and PAC gene (commissioned by GenScript), resulting in the pLAS5w vector, as shown in Figure 1. The nucleic acid molecules of Examples 1-1, 1-2, and Control Example 1 were then ligated into the pLAS5w vector using the BstBI and EcoRI restriction enzymes to generate the plasmids of Examples 1-1A and 1-1B, and Control Example 1A, respectively, as shown in Figure 2.

[0089] 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

[0090] Lentiviral particles of Examples 1-1B, 1-2B, and Control Example 1B were prepared by transfecting the target gene into human embryonic kidney HEK293 cells (ATCC, CRL-3216) using PolyJet transfection reagent (SignaGen Laboratories, SL100688). Specifically, 50 μg of one of the target gene-containing plasmids from Examples 1-1A and 1-2A, or the plasmid from Control Example 1A, 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). These plasmids were incubated with 300 μL of PolyJet transfection reagent in 10 mL of serum-free DMEM medium for 15 minutes to produce a PolyJet / DNA mixture. After adding the Polyjet / DNA mixture to 100 mL of DMEM medium containing 10% FBS, add the 9×10 7 HEK293 cells were cultured in T875 flasks. After 12 to 18 hours of culture, the medium was replaced with 100 ml of Opti-MEM (Gibco, 31985070) containing 1 mM sodium pyruvate (Sartorius). 16 hours after transfection, the medium was replaced with fresh Opti-MEM. 48 hours after transfection, the supernatant containing the lentiviral particles was collected and supplemented with fresh Opti-MEM 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 was then filtered through a 0.45 μm filter (Membrane-Solutions). The two supernatants were combined and filtered. Lenti-X Concentration Reagent (Takara Bio) was then used for concentration at a 3:1 ratio, resulting in a 3:1 volume ratio of the filtered supernatant to Lenti-X. Specifically, the supernatant and Lenti-X concentration reagent mixture was mixed overnight at 4°C by tumbling, then centrifuged at 1500 rpm for 45 minutes at 4°C. The supernatant was removed, and the lower pellet was resuspended in 2 mL of X-VIVO™ 15 medium (Lonza) to obtain the concentrated lentiviruses of Examples 1-1B and 1-2B, and the lentivirus of Control Example 1B. The concentrated lentiviruses were then stored at -80°C until use.

[0091] Test Example 1: Testing of the lentiviral titer of Example 1-1B, Example 1-2B, and Control Example 1B

[0092] The lentiviral titer was determined by transducing a 3-fold serial dilution of the lentiviral sample to 4 × 104 Individual human acute leukemia T cell Jurkat cells (purchased from BCRC, Cat.No.60424) were used for the assay. Among them, the lentivirus samples used in this test example 1 are the concentrated lentiviruses of Examples 1-1B, 1-2B, and Control Example 1B. 50 μL of the lentivirus sample was added to 100 μL of X-VivoTM15 culture medium, and serially diluted 3-fold with the aforementioned culture medium. This dilution step was repeated until it was diluted 6561 times to obtain multiple groups of serially diluted lentivirus samples. Then, 50 μL of each group of serially diluted lentivirus samples was taken and added to a 96-well plate (U bottom), in which each well of the plate contained 4×10 4 Jurkat cells / 100 μL culture medium / plate well were added, and lentivirus was transduced for 72 hours. After washing away excess lentivirus, NKp30 expression was detected using an anti-NKp30 antibody (BioLegend, Cat. No. 325228) labeled with BV421 fluorescent dye at a concentration of 1 μg / mL. The expression of the target protein was analyzed using a flow cytometer (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. The effective titer of the lentivirus containing nucleic acid molecules with intracellular signaling domains and complete NKp30 fragments (including immunoglobulin-like domains) was calculated using the following formula, and the obtained effective titer of the lentivirus was used to determine the amount of lentivirus used in the preparation of CAR-T cells.

[0093] Lentivirus effective titer (TU / mL) = (percentage of cells expressing NKp30) × 4 × 10 4 The experimental results of (cell number) × 20 × dilution factor are shown in Table 1 below:

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

[0095] 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 successfully transduce T cells into CAR-T cells and enable good growth of the transduced T cells. Therefore, according to the results in Table 1 above, the lentiviruses of Examples 1-1B, 1-2B, and Control Example 1B all meet this standard, which is conducive to further preparation of CAR-T cells.

[0096] Example 1-1C, 1-2C, Control Example 1C: Preparation of CAR-T cells with the nucleic acid molecules of Example 1-1, 1-2 or Control Example 1

[0097] Fresh blood was collected from healthy donors with their consent. On day 0, human peripheral blood mononuclear cells (PBMCs) were collected using SepMate centrifuge tubes (STEMCELL, Cat. No. 86450). Briefly, the blood sample was diluted with the same volume of PBS buffer containing 2% FBS as the fresh blood sample. The diluted sample was then added dropwise to the SepMate centrifuge tube along the tube wall. The SepMate tube was held vertically and the blood sample was mixed with the density gradient centrifugation medium (Ficoll) in the SepMate. The tube was centrifuged 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 for 8 minutes at room temperature. The lower layer was removed as the PBMCs. Next, T cells were isolated from the collected peripheral blood mononuclear cells (PBMCs) using magnetic beads containing anti-CD3 / CD28 antibodies on their surface (the magnetic beads were CTS (Cell Therapy Systems) Dynabeads CD3 / CD28 (Gibco, Cat. No. 40203D)) and DynaMag-Spin (purchased from Thermo). The isolated PBMC cells were detected for CD3 expression in the PBMCs using anti-CD3 antibodies (Biolegend, Cat. No. 300441) and flow cytometry. + The proportion of T cells and CD3 + The number of T cells. Take CD3 + Magnetic beads containing anti-CD3 / CD28 antibodies on the surface of 3 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 containing 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 from PBMCs. The T cell and magnetic bead complex formed by antigen-antibody binding was obtained. The T cell and magnetic bead complex was 2×10 5The density of cells / mL was dispersed in X-VIVOTM15 medium (Lonza) containing 200 IU / mL recombinant human IL-2 (R&D Systems) and cultured at 37°C and 5% carbon dioxide for 24 hours to activate T cells. The activated T cells were then transduced for 24 hours by adding one of the lentivirus of Example 1-1B, 1-2B, or Control Example 1B with a viral infection dose MOI (multiplicity of infection) of 5 to obtain the CAR-T cell and magnetic bead complexes of Example 1-1C, 1-2C, and Control Example 1C, and the T cell group without lentivirus treatment was used as the control group (untransduced group). Wherein, MOI here = viral titer (TU / mL) × viral volume (mL) / total number of T cells. On the third day, the suspension of the CAR-T cell and magnetic bead complex was removed by a magnetic stand to obtain the CAR-T cells of Example 1-1C, 1-2C, and Control Example 1C. The schematic diagram of the structure of each group is shown in Figure 3. The obtained CAR-T cells were transferred to a new culture flask containing 200 IU / mL recombinant human IL-2 (R&D, 202-GMP-01M) in X-VIVOTM15 medium and cultured under the same conditions as the transduction step, allowing the CAR-T cells to proliferate until day 7. Finally, the cell numbers of the untransduced group and the CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C were 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.

[0098] Test Example 2: Proliferation test of CAR-T cells in Example 1-1C, Example 1-2C, and Control Example 1C

[0099] The proliferation rate of the CAR-T cells of Examples 1-1C, 1-2C and Control Example 1C was calculated by counting the total cell number at a specific time point during the preparation of the CAR-T cells of Examples 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 Examples 1-1C, 1-2C and Control Example 1C were inoculated at a concentration of 1×10 6 Cells were seeded at a density of 10 cells / mL in a 12-well plate. After removing the magnetic beads, cell viability and cell density were measured by staining the cells with Acridine Orange / Propidium Iodide (AO / PI) and quantifying the cell number using an automated cell counter (Luna Automated Cell Counter, Logos Biosystems). The remaining cells were subcultured at a density of 2×10 5 to 5×10 5Cells were seeded at a density of 10 cells / mL in a culture dish and allowed to expand. Cell number was measured on days 0 and 7 of CAR-T cell preparation to calculate the cell expansion rate. The experimental results are shown in Table 2 below. The expansion rate of CAR-T cells in Examples 1-1C, 1-2C, and Control Example 1C is the proliferation fold normalized to the cell number on day 0.

[0100] Table 2: Expansion folds of CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C on day 7

[0101] If the expansion factor can reach 5 times or more, it will be beneficial for subsequent T cell functional testing. Therefore, according to the results in Table 2 above, the CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C all have good T cell expansion factors. In both Example 1-1C and Example 1-2C groups, the expansion effect of the CAR-T cells of Example 1-1C using the DAP12 intracellular signaling domain as the intracellular signaling domain is better than that of the CAR-T of Example 1-2C using the 41BB intracellular signaling domain as the intracellular signaling domain.

[0102] Test Example 3: Expression Detection of CAR-T Cells in Examples 1-1C, 1-2C, and Control Example 1C; Transduction Rate of Lentivirus in Examples 1-1B, 1-2B, and Control Example 1B

[0103] NKp30 expression in the CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C was measured using an anti-NKp30 antibody (BioLegend, 325228) labeled with BV421 fluorescent dye and flow cytometry. The percentage of cells expressing NKp30 relative to the total cell count was used as the transduction efficiency of the lentiviruses in Examples 1-1B, 1-2B, and Control Example 1B. The experimental results are shown in Table 3 below.

[0104] Table 3: Transduction efficiency of lentivirus of Examples 1-1B, 1-2B and Control Example 1B

[0105] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial for the functional testing of the subsequent CAR-T cells after expansion. Therefore, according to the results in Table 3 above, it can be concluded that the lentiviruses of Examples 1-1B and 1-2B both 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.

[0106] Test Example 4: T cell phenotype analysis of CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C

[0107] T cell typing analysis was performed on the CAR-T cells of Example 1-1C, 1-2C, and Control Example 1C on day 7 after activation using antibody fluorescence labeling and flow cytometry (Sony T cell surface markers were detected using an ELISA (BioLegend, SA3800) with SA3800 software. Antibodies used included CD3-FITC (BioLegend, 300406), CD4-PE-Cy7 (BioLegend, 300512), CD8-AF700 (Beckman, B76279), CD95-APC (BioLegend, 305612), CD45RA-PE (BioLegend, 304108), and CCR7-BV421 (BioLegend, 353208). A fluorescence-minus-one (FMO) control group containing all fluorochromes except CCR7-BV421 and CD45RA-PE was also established. In this test, untransduced activated T cells served as the control group (untransduced 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 (T EMRA ) (or called effector T cells (effector T cells, T EFF ), and the proportion of Tscm in the CAR-T cells of Examples 1-1C, 1-2C and Control Example 1C is shown in Table 4 below. The flow cytometry results of the T cell subset distribution of Examples 1-1C and 1-2C and Control Example 1C and the untransduced group are shown in Figures 4A, 4B, 4C and 4D, respectively. 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 .

[0108] Table 4: Percentage of Tscm in CAR-T cells of Examples 1-1C, 1-2C, Control Example 1C, and the untransduced group

[0109] If the percentage of stem cell-like memory T cells (Tscm) in CAR-T cells is 40% or greater, the high Tscm ratio can reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As shown in Table 4 and Figures 4A to 4D above, Examples 1-1C and 1-2C of CAR-T cells modified with wild-type NKp30 containing an intracellular signaling domain both have high Tscm ratios, both higher than those of the CAR-T cells of Control Example 1C and untransduced activated T cells. Therefore, it is expected that the CAR-T cells of Examples 1-1C and 1-2C can reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0110] Test Example 5: Real-time cytotoxicity assay (RTCA) using xCELLigence

[0111] 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 supplemented with 10% fetal bovine serum (FBS) in an environment of 5% CO2 and 37°C for subsequent xCELLigence real-time cytotoxicity assays.

[0112] In order to test the anti-tumor activity of the CAR-T cells in Examples 1-1C, 1-2 and Control Example 1C, a cytotoxicity test was performed using the HeLa cell line described in the previous paragraph as a target cell. The effector cells used in this test example were the CAR-T cells in Examples 1-1C, 1-2C and Control Example 1C.

[0113] The xCELLigence RTCA system (Agilent Technologies, RTCA SP) was placed in a 37°C, 5% CO2 cell culture incubator and connected to an external analysis and control unit via a cable. RTCA Software Pro software was used to control and monitor the instrument in real time, including real-time data display and analysis. Specifically, the xCELLigence RTCA system was used to continuously monitor the cytotoxicity of tumor cells for approximately 90 hours: First, the aforementioned HeLa cells were used as target cells (T) at a rate of 1.2×10 4 The cells were plated at a density of 100 μL / mL and seeded in a background-corrected culture dish. Background impedance measurement was performed for 22 hours to allow target cells to attach. Effector cells (E) were then added to the culture dish at effector-to-target ratios (E:T ratios) of 10:1, 3:1, and 1:1, so that the final volume of each well was 200 μL. The effector cells used in this test example were the CAR-T cells of Examples 1-1C, 1-2, and Control Example 1C. Untransduced activated T cells were also used as a control group (untransduced group). The cell index (CI) was monitored every 15 minutes for at least 72 hours. The original RTCA impedance data was normalized to the cell index data before the addition of effector cells. The aforementioned cell index is a dimensionless parameter that is obtained by measuring the relative change in electrical impedance to represent the cell state. For example, when cells are absent or unattached to the electrode, the CI is zero. Under the same physiological conditions, the greater the number of cells attached to the electrode, the greater the CI. Furthermore, changes in cell status, such as changes in cell morphology or cell adhesion, can also lead to changes in the CI, as shown in Figures 5A to 5C.

[0114] The results of poisoning for 24 hours are shown in Table 5 below.

[0115] Table 5: Results of CAR-T cell cytotoxicity tests in Examples 1-1C, 1-2C, and Control Example 1C

[0116] The results in Table 5 and Figures 5A to 5C show that the cytotoxicity of the CAR-T cells of Examples 1-1C and 1-2C was superior to that of the control 1C. Furthermore, the cytotoxicity of the CAR-T cells of Example 1-1C reached 100% at 24-hour E:T ratios of 10:1, 3:1, and 1:1. Since CAR-T cells with a cytotoxicity >50% are expected to have sufficient cytotoxicity when subsequently using the patient's autologous cells to generate CAR-T cells, the results in Table 5 indicate that both the CAR-T cells of Examples 1-1C and 1-2C exhibited excellent cytotoxicity, and that the CAR-T cells of Example 1-1C, which had the intracellular signaling domain of DAP12, exhibited even greater cytotoxicity than the CAR-T cells of Example 1-2C, which had the intracellular signaling domain of 41BB.

[0117] In summary, the lentiviruses of Examples 1-1B and 1-2B of the present invention, because they contain an intracellular signaling domain and an intact NKp30 fragment containing the NKp30 transmembrane domain, all have excellent transduction rates compared to the control and examples. The CAR-T cells obtained after transduction have excellent CAR-T cell expansion rates and excellent cytotoxicity, and even have a high proportion of Tscm. This is expected to reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. This shows that when preparing CAR-T cells, the introduction of a nucleic acid sequence containing an intracellular signaling domain, such as the intracellular signaling domain of DAP12 or 41BB, can have an excellent cytotoxic effect on the resulting CAR-T cells and is particularly suitable for CAR-T therapy.

[0118] 2. Preparation and testing of anti-mesothelin-NKp30 CAR-T cells with intracellular signaling domains

[0119] Example 2: Nucleic acid molecules comprising an intracellular signaling domain, a single-chain variable region fragment of an anti-mesothelin antibody, and an NKp30 fragment lacking an immunoglobulin-like domain

[0120] The 3' end of the nucleic acid sequence of the single-chain variable region fragment SS1 of an anti-mesothelin antibody (as shown in SEQ ID NO:85) was ligated to the 5' end of a NKp30 fragment lacking an immunoglobulin-like domain (as shown in SEQ ID NO:86) via a nucleic acid sequence with a (G4S)3 hinge domain (as shown in SEQ ID NO:66). The nucleic acid sequence of the NKp30 fragment lacking an immunoglobulin-like domain comprises an extracellular stalk region, the NKp30 transmembrane domain, and the NKp30 cytoplasmic domain, but lacks an immunoglobulin-like domain. Subsequently, the 3' end of the nucleic acid sequence of the NKp30 fragment lacking an immunoglobulin-like domain was ligated to the 5' end of the nucleic acid sequence of the intracellular signaling domain of DAP12 (SEQ ID NO:78), yielding a ligated nucleic acid molecule fragment 2. The 5' end of this ligated nucleic acid molecule fragment 2 was then ligated to the 3' end of the full-length FcεR1γ nucleic acid sequence (shown in SEQ ID NO: 77) via the nucleic acid sequence of the T2A self-truncating peptide (SEQ ID NO: 80), yielding the nucleic acid molecule of Example 2 comprising the intracellular signaling domain, the single-chain variable region fragment of an anti-mesothelin antibody, and the NKp30 fragment without the immunoglobulin-like domain, as shown in SEQ ID NO: 87. From the 5' end to the 3' end, the nucleic acid molecule of Example 2 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.

[0121] Control Example 2: Nucleic acid molecules having an intracellular signaling domain, a single-chain variable region fragment of an anti-mesothelin antibody, and not containing any NKp30 fragment

[0122] 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) was linked to the 5' end of the nucleic acid sequence of the CD8 transmembrane domain (as shown in SEQ ID NO:88) via the nucleic acid sequence of the CD8 hinge domain (as shown in SEQ ID NO:67). The 3' end of the nucleic acid sequence of the CD8 transmembrane domain was further linked to the 5' end of the nucleic acid molecule of the 4-1BB intracellular signaling domain (SEQ ID NO:82). The 3' end of the nucleic acid sequence of the 4-1BB intracellular signaling domain was then linked to the 5' end of the nucleic acid sequence of the CD3 zeta 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 comprises, from 5' to 3' end, 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 zeta signaling domain.

[0123] Example 2A, Control Example 2A: Construction of plasmids with nucleic acid molecules of Example 2 and Control Example 2

[0124] The preparation method of Example 2A is similar to that of Examples 1-1A and 1-2A, except that Example 2A utilizes restriction enzymes to introduce the single-chain variable region fragment of Example 2, containing an intracellular signaling domain and an anti-mesothelin antibody, and the NKp30 fragment lacking the immunoglobulin-like domain into the aforementioned pLAS5w vector. In contrast, Control Example 2A commissioned GenScript to utilize the HpaI and EcoRI restriction enzymes to introduce the nucleic acid molecule of Control Example 2 into the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, product number C6-8-39). The resulting anti-mesothelin-NKp30 plasmid containing an intracellular signaling domain of Example 2A and the plasmid of Control Example 2A are shown in Figure 6 .

[0125] Example 2B, Control Example 2B: Preparation of lentivirus with nucleic acid molecules of Example 2 and Control Example 2

[0126] The preparation method of Example 2B is similar to that 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.

[0127] Test Example 6: Testing of Lentivirus Titer in Example 2B and Control Example 2B

[0128] The experimental method for Test Example 6 was similar to that of Test Example 1, except that biotinylated human mesothelin protein, His, Avitag (MSN-H82E9, ACRO Biosystems), was used to identify the anti-mesothelin receptor. Phycoerythrin (PE)-labeled streptavidin (Invitrogen) diluted 500-fold was used to detect the expression of the anti-mesothelin receptor in the lentiviruses of Example 2B and Control Example 2B. The test results are shown in Table 6 below:

[0129] Table 6: Effective titers of lentiviruses in Example 2B and Control Example 2B

[0130] 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 achieve good growth of the transduced T cells. Therefore, according to the results in Table 6 above, the lentiviruses of Example 2B and Control Example 2B both meet this standard, which is beneficial for subsequent use in preparing CAR-T cells.

[0131] Example 2C: Anti-mesothelin-NKp30 CAR-T cells with intracellular signaling domains

[0132] The method for preparing the anti-mesothelin-NKp30 CAR-T cells with an intracellular signaling domain in Example 2C is similar to the method for preparing the CAR-T cells in Examples 1-1C and 1-2C, except that the T cells in this example are transduced using the lentivirus of Example 2B, and the MOI during lentiviral transduction is 2. The CAR-T cells of Control Example 2C are transduced using 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 Figure 7, wherein 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 contains FcεR1γ, they are multi-chain CAR-T cells.

[0133] Test Example 7: Proliferation test of CAR-T cells of Example 2C, Control Example 2C, and T cells of the untransduced group

[0134] The experimental method of Test Example 6 is similar to that of Test Example 2, except that this test example tests 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.

[0135] 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

[0136] Since an expansion multiple of 5 or more will be beneficial for subsequent T cell functional testing, according to the results in Table 7 above, the CAR-T cells of Example 2C have a good T cell expansion multiple and are superior to the CAR-T cells of Control Example 2C.

[0137] 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

[0138] The experimental 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 the anti-mesothelin receptor. 500-fold diluted PE-labeled streptavidin (Invitrogen) is used to detect the expression of the anti-mesothelin receptor in the CAR-T cells of Example 2C and Control Example 2C. The test results of Example 2C and Control Example 2C are shown in the flow cytometry results of Figures 8A, 8B and 8C, 8D, respectively. The percentage of cells expressing the anti-mesothelin receptor compared to the total number of cells is used as the transduction efficiency of the lentivirus of Example 2B or Control Example 2B. The experimental results are shown in Table 8 below.

[0139] Table 8: Transduction efficiency of lentivirus of Example 2B and Control Example 2B

[0140] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial for the subsequent functional testing of the CAR-T cells after expansion. According to the results of Table 9 and Figure 8A above, it can be seen that the lentivirus of Example 2B has a better transduction rate than that of Control Example 2B.

[0141] Test Example 9: T cell phenotype analysis of CAR-T cells of Example 2C, Control Example 2C, and T cells of the untransduced group

[0142] The experimental method of Test Example 9 is similar to that of Test Example 4, except that this test example tests the CAR-T cells of Example 2C on day 7 of preparation. The proportion of Tscm in the CAR-T cells of Example 2C and Control Example 2C and in the T cells of the untransduced group is shown in Table 9 below; the results of Example 2C and the flow cytometry results of the distribution of T cell subsets of Control Example 2C and the untransduced group are shown in Figures 9A, 9B, and 9C, respectively. Among them, T cells expressing CD45RA and CCR7 are Tscm.

[0143] Table 9: Percentage of Tscm in CAR-T cells of Example 2C, Control Example 2C, and T cells of the untransduced group

[0144] CAR-T cells containing stem cell-like memory T cells exceeding 40% can further reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As shown in Table 9 and Figures 9A, 9B, and 9C, Example 2C, which contains an intracellular signaling domain, exhibits a higher percentage of Tscm%, compared to the CAR-T cells of Control Example 2C and the activated T cells of the untransduced group. Therefore, it is expected that the CAR-T cells of Example 2C can reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0145] Test Example 10: Real-time Cytotoxicity Assay Using xCELLigence

[0146] 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 96 well, Agilent Technologies, 300600910) and cultured overnight. After the target cells are allowed to attach and impedance measurement is performed for 18 hours, the effector cells are then added to the culture plate at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1 for testing. The results of the toxicity experiments are shown in Figures 10A, 10B, 10C, and 10D, respectively.

[0147] The results of the 24-hour poisoning experiment are shown in Table 10 below.

[0148] Table 10: Real-time cytotoxicity test results of CAR-T cells in Example 2C and Control Example 2C

[0149] The results in Table 10 and Figures 10A to 10D demonstrate that the cytotoxicity of the CAR-T cells in Example 2C is superior to that of the conventional single-chain CAR-T cell control Example 2C. In particular, at E:T ratios of 0.3:1 and 0.1:1, the cytotoxicity of Example 2C is significantly superior to that of the control Example 2C, with significant reductions in standardized cell markers, indicating significant cytotoxicity of target cells. Furthermore, a cytotoxicity of CAR-T cells exceeding 50% would provide sufficient cytotoxicity for subsequent preparation of CAR-T cells using patient cells. The results in Table 10 demonstrate that the CAR-T cells in Example 2C exhibit excellent cytotoxicity.

[0150] Test Example 11: Cold Light Poisoning Test

[0151] SKOV3 ovarian cancer cells (hereinafter referred to as SKOV3 cells) (purchased from ATCC, Cat. No. HTB-77) were cultured in McCoy 5A Medium (Gibco, 16600082) supplemented with 10% FBS. SKOV3 cells expressing high levels of mesothelin were selected using a flow cytometer 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 mesothelin-expressing SKOV3 cells. A single stable expressing cell line was then selected to obtain a GFP / Luc cell line expressing both green fluorescent protein (GFP) and luciferase. + SKOV3 cells (SKOV3-mLE).

[0152] To test the CAR-T cells of Example 2C, Control Example 2C, and activated T cells from the untransduced group, anti-tumor activity was tested on cells prepared on day 7. Cytotoxicity assays were performed using ovarian cancer SKOV3 cells expressing mesothelin and luminescence enzymes as target cells. The effector cells used in this test example were the CAR-T cells of Example 2C, and untransduced T cells served as the control group (untransduced group). Specifically, the effector cells and target cells were co-cultured in a 96-well plate at an E:T ratio of 3:1, incubated at 37°C, 5% carbon dioxide for 48 hours, and tested in triplicate.

[0153] The luminescence produced by the luminescence enzyme expressed by viable cancer cells was measured using the Luminescence Enzyme Assay System Reagent Kit (Promega, E1501) to estimate the relative number of viable cancer cells not lysed by CAR-T cells. The cell-specific lysis ratio was calculated using the following formula:

[0154] Specific cell lysis % = 100% × (number of experimentally lysed target cells – number of spontaneously lysed target cells) ÷ (maximum number of lysed target cells – number of spontaneously lysed target cells)

[0155] in,

[0156] The number of target cells lysed in the experiment = the number of target cells lysed in the group co-cultured with effector cells

[0157] The number of spontaneously lysed target cells = the number of lysed target cells in the group without effector cells, i.e., 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.

[0158] The maximum number of target cells lysed = the number of target cells lysed after adding 100 μl (microliters) of 1% Triton X-100 to the group without effector cells

[0159] The experimental results are shown in Table 11 below:

[0160] Table 11: Results of the luminescence cytotoxicity test of the CAR-T cells of Example 2C, Control Example 2C, and T cells of the untransduced group (control group) (specific lysis %)

[0161] When the cytotoxicity of CAR-T cells is greater than 50%, sufficient cytotoxicity will be achieved when CAR-T cells are subsequently prepared using patient cells. According to the results in Table 11, the CAR-T cells of Example 2C have a significantly better cytotoxic effect than the CAR-T cells of Control Example 2C, which are traditional single-chain CAR-T cells, after being co-cultured with 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.

[0162] Test Example 12: Animal Experiment

[0163] First, 40 B2m gene-deficient female mice (NOD.Cg-Prkdc) aged 5 to 10 weeks were provided. scid Il2rg 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 mice, and housed in a sterile environment. The experimental animal care and handling procedures were in accordance with the experimental methods approved by the Institutional Animal Care and Use Committee (IACUC). Because 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 beta-2 microglobulin (B2m) gene is deleted in immunodeficient mice, GvHD can be significantly alleviated. Therefore, this experiment used immunodeficient mice with B2m gene deletion.

[0164] 5×10 SKOV3-mLE cells in Test Example 11 were taken 6Each cell was suspended in 100 μL of PBS and then mixed with an equal volume of Matrigel (BD Bioscience) to create the SKOV3-mLE cell injection mixture. On day 0, this SKOV3-mLE cell injection mixture was subcutaneously inoculated into the right flank of 35 mice. Five additional mice were kept as a reserve and not inoculated with SKOV3-mLE cells. Bioluminescence imaging (BLI) signals were measured weekly from day 10 until day 80 using an In Vivo Imaging System (IVIS) (Perkin Elmer). Mice sacrificed before day 80 were considered humane and were not monitored until day 80. On day 21 after implantation of SKOV3-mLE cells (as target cells), the other seven groups of mice, except those not inoculated with SKOV3-mLE cells, were injected via the tail artery with saline (control group), CAR-T cells from Example 2C, CAR-T cells from Control Example 2, or untransduced T cells (each serving as effector cells, E), with an E:T ratio of 1:1 or 3:1. On day 55, the SKOV3-mLE cell injection mixture was implanted into the left flank of the remaining surviving mice and the spare group of mice for tumor rechallenge. During the rechallenge phase, a total of 22 mice were injected with SKOV3-mLE cells, including five spare mice that were given cancer cells for the first time. In this test, the logarithmic scale of the BLI signal was used to represent the in vivo tumor volume. T cells in the peripheral blood of the mice were also collected for CAR expression analysis and T cell phenotyping. The CAR expression analysis was performed as described in Test Example 8, and the phenotyping analysis was performed as described in Test Example 9. The experimental data were expressed as mean ± standard deviation and were statistically analyzed with Tukey's post hoc test for single-factor independent variance.

[0165] The experimental results are shown in Tables 12 and 13 and Figures 11A, 11B, 12A, 12B, 13A, and 13B. In Table 12, "Day -4" represents 4 days before effector cell injection, and "Day +10," "Day +17," "Day +24," and "Day +31" represent 10 days, 17 days, 24 days, and 31 days after effector cell injection, respectively.

[0166] Table 12: Results of in vivo cytotoxicity assays of Example 2C (abbreviated as E2C) and Control Example 2C (abbreviated as CE2C) using BLI intensity in animal tumors [log(*E+11) photons / second]

[0167] ns means not statistically significant; * means p < 0.05, ** means p < 0.01, *** means p < 0.005

[0168] Table 13: Data from in vitro testing of effector cell characteristics and functions after preparation of effector CAR-T cells and before administration to mice (Control Example 2C and Example 2C are abbreviated as CE2C and E2C, respectively)

[0169] As shown in Figure 11A, 10 days after infusion (day 31 of the total experimental period) of a higher dose (E:T = 3:1) of CAR-T cells from Example 2C, tumor volume was reduced. Based on visual observations of BLI, in mice administered with the high dose of CAR-T cells from Example 2C, 3 / 5 mice had detectable BLI areas. Compared to the other effector cell groups, the BLI detectable area was smaller. Observing the overall mice population, the BLI signal of the group administered with the high dose of CAR-T cells from Example 2C was significantly lower than that of all other groups after conversion to a logarithmic scale (as shown in Table 12 and Figure 11B).

[0170] As shown in FIG11A , after 24 days of CAR-T cell treatment (day 45 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 BLI detectable signal was also observed in mice treated with either dose of the control 2C CAR-T cells or the low-dose (E:T = 1:1) CAR-T cells of Example 2C. However, the BLI signal in these mice treated with the different groups did not reach the point of being visually undetectable. The BLI values ​​of mice treated with both the Example 2C and control 2C CAR-T cells were lower than those of their respective UTD control groups. Furthermore, the BLI values ​​of mice treated with the high-dose CAR-T cells of Example 2C were significantly lower than those of the other groups, including those treated with other CAR-T cells (i.e., the control 2C and low-dose CAR-T cells of Example 2C). These differences in the BLI detectable area persisted until 31 days after treatment (day 52 of the total trial period), before some mice developed life-threatening GvHD (as shown in Figures 11A and 11B, and Table 12).

[0171] Furthermore, tumor elimination (signal reduction) can be observed in Figure 11A , but GvHD did not immediately develop in the Example 2C-treated group. In contrast, the Control Example 2C group, which was treated with conventional single-chain CAR-T cells, immediately developed GvHD in mice. Although GvHD symptoms were significantly alleviated or delayed in ASID-B2m KO mice, signs of GvHD remained evident in mice treated with conventional single-chain CAR-T cells (Control Example 2C). This GvHD may be attributed to the overproliferation of human CAR-T cells, which attack host cells due to the lack of human cancer cells as target cells. Three mice (3 / 10 of the Control Example 2C group) treated with CAR-T cells from the Control Example 2C group succumbed to GvHD symptoms before rechallenge with SKOV3-mLE cancer cells (as shown in Figure 11A ). Most of the surviving mice treated with CAR-T cells from Control Example 2C (6 out of 7) were sacrificed 11 days after tumor re-challenge due to GvHD before the efficacy of CAR-T cells could be evaluated (as shown in Figure 12A). According to Figures 13A and 13B, throughout the entire experiment, the number of total T cells in the peripheral blood and CAR-T cells in the mice treated with CAR-T cells from Control Example 2C, with CD3+ cells as T cells, was significantly higher than that in Example 2C, which may indicate that the design of traditional single-chain CAR-T cells may tend to produce over-activated 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 non-transduced 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 will have excellent cancer cell killing efficacy according to 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%.

[0172] The low-dose CAR-T cell-treated group of Example 2C exhibited an anti-tumor response upon tumor re-challenge (as shown in Figures 12A and 12B). Re-infusion of SKOV3-mLE cells resulted in GvHD symptoms in most mice treated with CAR-T cells, except those treated with low-dose CAR-T cells of Example 2C. In mice treated with CAR-T cells from Example 2C, the BLI signal decreased to below visually detectable levels, indicating a significant anti-tumor effect. Even 18 days after tumor re-challenge, this value was statistically lower than that of the saline-treated control group.

[0173] According to the above results, the lentivirus of Example 2B of the present invention has an excellent transduction rate due to its intracellular signaling domain and NKp30 transmembrane domain (NKp30 fragment without 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 and avoiding the overactivation of traditional single-chain CAR-T cells, which may cause fatal cytokine release syndrome (CRS). Overactivated CAR-T cells are also prone to exhaustion and reduce their persistence in the body. According to the content of the literature Korell F. et al, Med. 2022 Aug 12; 3 (8): 538-564, the therapeutic effect 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 a nucleic acid sequence of an intracellular signaling domain, such as the intracellular signaling domain of DAP12, is simultaneously introduced into T cells, it can bring about excellent cytotoxic effects on the prepared CAR-T cells.

[0174] 3. Preparation and testing of anti-CD22-NKp30 CAR-T cells with intracellular signaling domains

[0175] Example 3: Nucleic acid molecules comprising an intracellular signaling domain, a single-chain variable region fragment of an anti-CD22 antibody, and an NKp30 fragment lacking an immunoglobulin-like domain

[0176] The 3' end of the nucleic acid sequence containing the full-length FcεR1γ (as shown in SEQ ID NO: 77) was ligated to the 5' end of the nucleic acid sequence of the DAP12 intracellular signaling domain (SEQ ID NO: 78) to obtain a ligated nucleic acid molecule fragment 1. The 3' end of the ligated nucleic acid molecule fragment 1 was then ligated 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). The 5' end of the nucleic acid sequence of the CD28 hinge domain (as shown in SEQ ID NO: 68) was further ligated to the 3' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody. Finally, the nucleic acid sequence of the NKp30 fragment without the immunoglobulin-like domain (as shown in SEQ ID NO: 91) was ligated to the 5' end of the nucleic acid sequence of the CD28 hinge domain (as shown in SEQ ID NO: 68). The 5' end of the nucleic acid sequence of Example 3 (shown in SEQ ID NO: 86) was ligated 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-truncating 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).

[0177] 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

[0178] The preparation method of Control Example 3-1 is similar to that of Control Example 2, except that the single-chain variable region fragment used in Control Example 3-1 is not SS1, the single-chain variable region fragment of the anti-mesothelin antibody used in Control Example 2, but rather the nucleic acid sequence of m971, the single-chain variable region fragment of the anti-CD22 antibody (as shown in SEQ ID NO: 91). The nucleic acid sequence of the nucleic acid molecule obtained in Control Example 3-1 is shown in SEQ ID NO: 93.

[0179] 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

[0180] The 3' end of the nucleic acid sequence of the full-length FcεR1γ (as shown in SEQ ID NO:77) was linked 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). The 5' end of the nucleic acid sequence of the CD28 hinge domain (as shown in SEQ ID NO:68) was further linked 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 NKp30 fragment without the immunoglobulin-like domain (as shown in SEQ ID NO:86) was linked to the 3' end of the nucleic acid sequence of the CD28 hinge domain to obtain the anti-CD22-NKp30 nucleic acid molecule without the 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, from the 5' end to the 3' end, 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).

[0181] Example 3A, Control Example 3-1A, Control Example 3-2A: Construction of plasmids with nucleic acid molecules of Example 3, Control Example 3-1, Control Example 3-2

[0182] The preparation method of Example 3A is similar to that 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.

[0183] In Control Example 3-1A, GenScript was commissioned to use the NheI and EcoRI restriction enzymes to insert the nucleic acid molecule from Control Example 3-1 into the pLAS5w.Ppuro vector (purchased from the Academia Sinica RNAi Core, product number C6-8-39). In Control Example 3-2A, the nucleic acid molecule from Control Example 3-2 was inserted into the aforementioned pLAS5w vector using the HpaI and EcoRI restriction enzymes. The resulting plasmids for Example 3A, Control Example 3-1A, and Control Example 3-2A are shown in Figure 14.

[0184] 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

[0185] The preparation method of Example 3B is similar to that of Examples 1-1B and 1-2B, except that the plasmid of Example 3A was used as the transfer plasmid in Example 3B. Lentiviruses of Control Examples 3-1B and 3-2B were prepared using the plasmids of Control Examples 3-1A and 3-2A, respectively, as the transfer plasmids.

[0186] Test Example 13: Testing of the Lentivirus Titer of Example 3B

[0187] The experimental method for Test Example 13 was similar to that of Test Example 1, except that a biotinylated human CD22 Fc Tag (Sino Biological. Cat: 11958-H41H-B) was used to identify the anti-CD22 receptor. 500-fold diluted PE-labeled streptavidin (Invitrogen) was used to detect the expression of the anti-CD22 receptor of the lentiviruses in Example 3B and Control Example 3-1B. The test results are shown in Table 14 below:

[0188] Table 14: Effective titers of lentiviruses in Example 3B and Control Example 3-1B

[0189] 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 ensure good growth of the transduced T cells. Therefore, according to the results in Table 14 above, the lentivirus of Example 3B meets this standard and is suitable for subsequent preparation of CAR-T cells.

[0190] Example 3C: Preparation of CAR-T cells with nucleic acid molecules of Example 3, Control Example 3-1, and Control Example 3-2

[0191] The preparation method of the CAR-T cells of Example 3C is similar to that 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 lentiviral 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 lentiviral 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 Figure 15.

[0192] 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 untransduced group

[0193] Test Example 14 was conducted in a similar manner to Test Example 2, except that the expansion rate of the CAR-T cells from Example 3C and Control Examples 3-1C and 3-2C was measured. Untransduced activated T cells served as a control group. The results are shown in Table 15 below.

[0194] Table 15: Expansion folds of CAR-T cells from Example 3C, Control Examples 3-1C, 3-2C, and T cells from the untransduced group on day 7

[0195] 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 testing.

[0196] 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

[0197] The experimental 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 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 CAR-T cells of Example 3C and Control Example 3-1C. The test results of Example 3C and Control Example 3-1C are shown in the flow cytometry results of Figures 16A, 16B, 16C, and 16D, respectively. The number of cells expressing the anti-CD22 receptor is used as a percentage of the total number of cells as the transduction rate of the lentivirus of Example 3B or Control Example 3-1B. The experimental results are shown in Table 16 below.

[0198] Table 16: Transduction efficiency of lentivirus of Example 3B and Control Example 3-1B

[0199] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial for the functional testing of the subsequent CAR-T cells after expansion. Therefore, according to the results of Table 16 and Figure 16A above, it can be seen that the lentivirus of Example 3B has a good transduction rate.

[0200] Test Example 16: T cell phenotype analysis of the CAR-T cells of Example 3C

[0201] The experimental 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 proportion 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 cytometry results of Control Example 3-1C, Control Example 3-2C and the distribution of untransduced T cell subsets are shown in Figures 17A, 17B, 17C and 17D, respectively, among which T cells expressing CD45RA and CCR7 are Tscm.

[0202] 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

[0203] CAR-T cells containing stem cell-like memory T cells exceeding 40% can further reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As shown in Table 17 and Figures 17A, 17B, 17C, and 17D, Example 3C exhibits a higher proportion of Tscm, slightly superior to the traditional single-chain CAR-T cell of Control Example 3-1C, and similar to activated T cells in the untransduced group. Therefore, it is expected to reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0204] Test Example 17: Real-time Cytotoxicity Assay Using xCELLigence

[0205] 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 (shown as SEQ ID NO: 95) was inserted into the pLAS5w.Ppuro vector (purchased from the Academia Sinica RNAi Core, Cat. No. C6-8-39) to generate a vector containing the truncated CD22. This vector containing the truncated CD22 was transfected into the SKOV3 human ovarian cancer cell line using a Polyjet (SignaGen Laboratories). The transfected SKOV3 cells were cultured in McCoy 5A Medium (Gibco) supplemented with 2 μg / mL puromycin and 10% FBS to select for SKOV3 cell lines that were puromycin-resistant and expressed CD22. Finally, CD22 expression in the cells was confirmed using flow cytometry. The SKOV3 cell line expressing CD22 was used as the target cell for subsequent cytotoxicity experiments using the xCELLigence system.

[0206] The experimental 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 seeded in a background-corrected culture dish 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 3:1 and 1:1 for testing. The results of the toxicity experiments are shown in Figures 18A and 18B, respectively.

[0207] The results in Figures 18A and 18B show that the cytotoxicity of the CAR-T cells in Example 3C is superior to that of the conventional single-chain CAR-T cell control example 3-1C and the control example 3-2C, which lacks an intracellular signaling domain. When the cytotoxicity of CAR-T cells is >50%, sufficient cytotoxicity is expected when CAR-T cells are subsequently prepared using patient cells. The results in Figures 18A and 18B demonstrate that the CAR-T cells in Example 3C exhibit excellent cytotoxicity and are superior to those in the control examples 3-1C and 3-2C.

[0208] Test Example 18: Human lymphoma cell cytotoxicity assay using flow cytometry analysis

[0209] In order to test the anti-tumor activity of the anti-CD22-NKp30 CAR-T cells with intracellular signaling domains in Example 3C, Daudi human lymphoma cells (hereinafter referred to as Daudi cells) were cultured with CellTrace TM Violet standardization, 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 cultured at 1.2×10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate, and effector cells were added to adjust the effector cell to target cell ratio (E:T) to 1:1 and 3:1. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours, and the experiment was repeated three times. Next, the 96-well plate was centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells in each well were washed with 200μL of PBS containing 2% fetal calf serum (FCS). After removing the PBS used for washing, the cells were collected with 300μL of flow buffer (PBS containing 2% FCS), and 15μL of counting beads (CountBright TMAfter using Absolute Counting Beads (Invitrogen, C36995), 3,000 beads were collected by flow cytometry to calculate the number of target cells. The cytotoxicity rate was then calculated using the following formula:

[0210] Cytotoxicity rate (%) = (number of target cells in the group without effector cells added - number of target cells after co-culture with effector cells) ÷ number of target cells in the group without effector cells added × 100%

[0211] The experimental results are shown in FIG19 and Table 18 below for E:T=3:1.

[0212] Table 18: Human lymphoma cell cytotoxicity test results analyzed by flow cytometry for Example 3C and Control Examples 3-1C and 3-2C

[0213] According to the results in Figure 19 and Table 18, at an E:T ratio of 1:1, the cytotoxicity of the CAR-T cells of Example 3C was 71%, significantly superior to the 45% of the control example 3-1C for conventional single-chain CAR-T cells, the 5% of the control example 3-2C without an intracellular signaling domain, and the 5% of the control group (untransduced group). At an E:T ratio of 3:1, the cytotoxicity of the CAR-T cells of Example 3C was 85%, superior to the 77% of the control example 3-1C for conventional single-chain CAR-T cells, the 14% of the control example 3-2C without an intracellular signaling domain, and the 27% of the control group (untransduced group). The comparison of the results of Example 3C with the control example 3-2C shows that the addition of the intracellular signaling domain significantly enhances the cancer cell cytotoxicity.

[0214] Based on the above results, it can be seen that the lentivirus of Example 3B of the present invention has an excellent transduction rate due to its intracellular signaling domain and the NKp30 transmembrane domain (NKp30 fragment without the immunoglobulin-like domain). The CAR-T cells obtained after transduction have excellent cell proliferation rate and excellent cytotoxicity, and have a high proportion of Tscm. It is 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 a nucleic acid sequence of an intracellular signaling domain is transferred, such as the intracellular signaling domain of DAP12, it can bring excellent cytotoxic effects to the prepared CAR-T cells and is particularly suitable for CAR-T therapy.

[0215] IV. Preparation and testing of anti-CD19-NKp30 CAR-T cells with intracellular signaling domains

[0216] Example 4: Nucleic acid molecules comprising an intracellular signaling domain, a single-chain variable region fragment of an anti-CD19 antibody, and an NKp30 fragment lacking an immunoglobulin-like domain

[0217] The preparation method of Example 4 is similar to that of Example 3, except that the single-chain variable region fragment used in Example 4 is not m971, the single-chain variable region fragment of the anti-CD22 antibody in Example 3, but rather FMC63, the single-chain variable region fragment of the anti-CD19 antibody (shown in SEQ ID NO:96), and the hinge domain selected is the hinge domain of human IgG4 (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 DAP12 intracellular signaling domain, the nucleic acid sequence of the T2A self-truncating 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-like domain).

[0218] Control Example 4: Nucleic acid molecules having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD19 antibody, and no NKp30 fragment

[0219] The preparation method of Control Example 4 is similar to that of Control Example 2, except that the single-chain variable region fragment used in Control Example 4 is not SS1, the single-chain variable region fragment of the anti-mesothelin antibody used in Control Example 2, but rather the nucleic acid sequence of FMC63, the single-chain variable region fragment of the anti-CD19 antibody (as shown in SEQ ID NO: 96). The nucleic acid sequence of the nucleic acid molecule obtained in Control Example 4 is shown in SEQ ID NO: 98.

[0220] Example 4A: Construction of a plasmid containing the nucleic acid molecules of Example 4 and Control Example 4

[0221] The preparation method of Example 4A is similar to that 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.

[0222] For Control Example 4A, GenScript was commissioned to insert the nucleic acid molecule from Control Example 4 into the pLAS5w.Ppuro vector (purchased from the Academia Sinica RNAi Core, product number C6-8-39) using the NheI and EcoR restriction enzymes. The plasmids of Example 4 and Control Example 4A, prepared as described above, are shown in Figure 20.

[0223] Example 4B, Control Example 4B: Preparation of Lentivirus with Nucleic Acid Molecules of Example 4 or Control Example 4

[0224] The preparation method of Example 4B is similar to that 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.

[0225] Test Example 19: Testing of Lentivirus Titer in Example 4B and Control Example 4B

[0226] The experimental method for Test Example 19 was similar to that of Test Example 1, except that biotinylated human CD19 Fc Tag (ACROBiosystems, CD9-H8259) was used to identify the anti-CD19 receptor, and 500-fold diluted PE-labeled streptavidin (Invitrogen) was 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:

[0227] Table 19: Effective titer of lentivirus of Example 4B and Control Example 4B (transduction units, TU / mL)

[0228] 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 ensure good growth of the transduced T cells. Therefore, according to the results in Table 19 above, the lentivirus of Example 4B meets this standard and is suitable for subsequent preparation of CAR-T cells.

[0229] Example 4C, Control Example 4C: Preparation of CAR-T cells with nucleic acid molecules of Example 4 and Control Example 4

[0230] 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 lentiviral transduction is 2. The CAR-T cells of Control Example 4C are transduced with the lentivirus of Control Example 4C, and the MOI during lentiviral transduction is 2. Schematic diagrams of the structures of the prepared CAR-T cells of Example 4C and Control Example 4C are shown in Figure 21.

[0231] Test Example 20: Proliferation test of CAR-T cells in Example 4C and Control Example 4C

[0232] This test example was conducted in a similar manner to Test Example 2, except that this test example examined the expansion rates of the CAR-T cells of Example 4C and Control Example 4C, and also included untransduced activated T cells as a control group (untransduced group). The results are shown in Table 20 below.

[0233] Table 20: Expansion folds of CAR-T cells of Example 4C and Control Example 4C on day 7

[0234] 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 testing.

[0235] 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

[0236] The experimental 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 500-fold diluted PE-labeled streptavidin (Invitrogen) is used 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 shown in the flow cytometry results of Figures 22A, 22B, 22C, and 22D, respectively. The number of cells expressing the anti-CD19 receptor is used as a percentage of the total number of cells as the transduction rate of the lentivirus of Example 4B or Control Example 4B. The experimental results are shown in Table 21 below.

[0237] Table 21: Transduction efficiency of lentivirus of Example 4B and Control Example 4B

[0238] If the transduction rate of the prepared CAR-T cells is greater than 25%, it will be beneficial for the subsequent functional testing of the CAR-T cells after expansion. Therefore, according to the results of Table 21 and Figure 22A above, it can be seen that the lentivirus of Example 4B has a good transduction rate.

[0239] Test Example 22: T cell phenotype analysis of the CAR-T cells of Example 4C

[0240] The experimental method of Test Example 22 is similar to that of Test Example 4, except that this test example tests the CAR-T cells of Example 4C on day 7 of preparation. The proportion of Tscm in the CAR-T cells of Example 4C, Control Example 4C, and T cells in the untransduced group is shown in Table 22 below; the results of Example 4C and the flow cytometry results of the distribution of T cell subsets of Control Example 4C and untransduced T cells are shown in Figures 23A, 23B, and 23C, respectively. Among them, T cells expressing CD45RA and CCR7 are Tscm.

[0241] Table 22: Percentage of Tscm in CAR-T cells of Example 4C, Control Example 4C, and T cells in the untransduced group

[0242] CAR-T cells containing stem cell-like memory T cells exceeding 40% can further reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As shown in Table 22 and Figures 23A, 23B, and 23C, Example 4C exhibits a Tscm exceeding 40%, similar to that of conventional single-chain CAR-T cells. Therefore, it is expected to reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy.

[0243] Test Example 23: Real-time Cytotoxicity Assay Using xCELLigence

[0244] 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 (amino acids 1-342) contains the extracellular and transmembrane domains but lacks the intracellular and cytoplasmic signaling domains. The truncated CD19 cDNA fragment was inserted into the pLAS5w.Ppuro vector (purchased from the Academia Sinica RNAi Core, product number C6-8-39) containing the puromycin resistance gene using the HpaI and NheI restriction enzyme sites to generate a vector containing the truncated CD19. The vector containing the truncated CD19 was transfected into the SKOV3 human ovarian cancer cell line using a Polyjet (SignaGen Laboratories, SL100688). Transfected SKOV3 cells were cultured in McCoy's 5A Medium (Gibco) supplemented with 2 μg / mL puromycin and 10% FBS to identify drug-resistant SKOV3 cell lines that also expressed CD19. Finally, CD19 expression was confirmed by flow cytometry. SKOV3 cells expressing CD19 were subsequently used as target cells in cytotoxicity assays using the xCELLigence system.

[0245] The experimental method of this test example is similar to that of Test Example 5, except that this test example uses the CAR-T cells of Example 4C and Control Example 4C as effector cells, and the aforementioned SKOV3 cell line expressing CD19 as target cells. The target cells are seeded 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 500 cells / mL of target cells as the complete lysis group. The results of the toxicity test are shown in Figure 24. The results of the toxicity test for 48 hours are shown in Table 23.

[0246] Table 23: Cytotoxicity test results of Example 4C and Control Example 4C

[0247] When the cytotoxicity of CAR-T cells is >50%, sufficient cytotoxicity will be achieved when CAR-T cells are subsequently prepared using patient cells. According to the results in Figure 24 and Table 23, the CAR-T cells of Example 4C can achieve a cancer cell cytotoxicity effect exceeding 80% after being co-cultured with target cells for 48 hours, and the cytotoxicity is slightly better than that of the CAR-T cells of the control example 4C of the traditional single-chain CAR-T cells. Therefore, it can be inferred that the CAR-T cells of Example 4C have sufficient cytotoxicity when CAR-T cells are subsequently prepared using patient cells.

[0248] Test Example 24 Cold Light Poisoning Test

[0249] The experimental 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 the 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 experiment are shown in Figure 25.

[0250] The experimental results are shown in FIG25 . Regardless of whether the E:T ratio is 1:1 or 3:1, the cytotoxic effect of the CAR-T cells of Example 4C is greater than 50%, and is significantly better than the cytotoxic effect of the single-chain CAR-T cells of Control Example 4C.

[0251] Based on the above results, it can be seen that the lentivirus of Example 4B of the present invention has excellent transduction efficiency due to its intracellular signaling domain and the NKp30 fragment without immunoglobulin-like domain containing the NKp30 transmembrane domain. The CAR-T cells obtained after transduction have excellent CAR-T cell expansion rate and excellent cytotoxicity, and even have a high proportion of Tscm. It is 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 a nucleic acid sequence containing an intracellular signaling domain is transferred, such as the intracellular signaling domain of DAP12, it can bring excellent cytotoxicity to the prepared CAR-T cells and is particularly suitable for CAR-T therapy.

[0252] In summary, if the genes for intracellular signaling domains, such as the DAP12 intracellular signaling domain, the 41BB intracellular signaling domain, or the CD28 intracellular signaling domain, and the NKp30 transmembrane domain and NKp30 cytoplasmic domain are transferred into T cells, the T cells will possess the NKp30 receptor complex and become multi-chain CAR-T cells. These cells will have excellent cancer cell cytotoxicity in both in vitro and in vivo experiments.

[0253] 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 are only specific embodiments of the present invention and are 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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