Nucleic acid molecules having an intracellular signaling domain and a transmembrane domain of natural killer protein 30, and chimeric antigen receptors containing the same.

Abstract

The present invention provides a nucleic acid molecule having an intracellular signaling domain and a transmembrane domain of NKp30, and a chimeric antigen receptor containing the same. The nucleic acid molecule includes a nucleic acid sequence of a fragment comprising (a) an extracellular antigen-binding domain, (b) a hinge domain, (c) a transmembrane domain of NKp30, (d) a cytoplasmic domain of NKp30, and (e) an intracellular signaling domain. By transduction of the nucleic acid sequences of the intracellular signaling domain, the transmembrane domain of NKp30, and the cytoplasmic domain of NKp30 into T cells, the T cells acquire an NKp30 receptor complex and become heavy chain CAR-T cells, which can form stable immune synapses with cancer cells and have excellent cancer cell-toxic activity.

Description

[Technical Field]

[0001] This invention relates to nucleic acid molecules for producing chimeric antigen receptors, and more particularly to nucleic acid molecules for producing chimeric antigen receptors having excellent cancer cell-toxic activity. It also relates to chimeric antigen receptors containing such molecules and their applications. [Background technology]

[0002] Cancer is caused by the transformation of normal cells into tumor cells through a multi-step process. Typically, a relatively mild precancerous lesion progresses from the outset to a malignant tumor, and metastasis occurs. This transformation is not due to a single factor, but rather to the combined effect of an individual's genes and external factors.

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

[0004] Cancer not only causes physical and mental suffering to patients, but also brings serious emotional and economic stress to their families. However, in the high-stress environment of modern society, it is difficult to completely eliminate negative external factors, and the number of people diagnosed with cancer is increasing year by year.

[0005] Therefore, there is still a pressing need for effective ways to treat cancer. [Overview of the project] [Problems that the invention aims to solve]

[0006] To overcome the shortcomings of the prior art, the advantage of the present invention lies in providing novel CAR-T cells that, after being administered to the human body, can identify and kill tumor cells representing specific antigens. [Means for solving the problem]

[0007] To achieve the above objective, the present invention (a) Extracellular antigen-binding domain, (b) Hinge domain and (c) The transmembrane domain of Natural Killer Protein 30 (NKp30), (d) The cytoplasmic domain of natural killer protein 30 (NKp30) and (e) Provide an isolated nucleic acid molecule containing a nucleic acid sequence of a fragment with an intracelular signaling domain. This invention involves transferring the genes for the transmembrane domain, cytoplasmic domain, and intracellular signaling domain of NKp30 into T cells. As a result, T cells become equipped with an NKp30 receptor complex, transforming into heavy chain CAR-T cells that can form stable immune synapses with cancer cells and possess excellent cancer cell-toxicity.

[0008] Preferably, the extracellular antigen-binding domain is the NKp30 extracellular domain.

[0009] Preferably, the extracellular antigen-binding domain includes a heavy chain variable region.

[0010] Preferably, the extracellular antigen-binding domain comprises the heavy-chain variable region of an anti-mesothelin antibody, the heavy-chain variable region of an anti-CD22 antibody, the heavy-chain variable region of an anti-CD19 antibody, the heavy-chain variable region of an anti-B-cell maturation antigen (BCMA) antibody, the heavy-chain variable region of an anti-CD123 antibody, the heavy-chain variable region of an anti-GPRC5D (G protein-coupled receptor, class C, group 5, member D) antibody, or the heavy-chain variable region of an anti-TSHR (thyroid-stimulating hormone receptor) antibody.

[0011] In one embodiment, the heavy-chain variable region of the extracellular antigen-binding domain can be derived from heavy-chain variable regions of heavy-chain antibodies (VHH) or single-chain variable fragments (scFv). Here, the heavy-chain variable region of the heavy-chain antibody is the single-chain binding region of a heavy-chain antibody derived from a camelid.

[0012] In one embodiment, the extracellular antigen-binding domain is the heavy-chain variable region of an anti-BCMA heavy-chain antibody.

[0013] In one embodiment, the extracellular antigen-binding domain includes the heavy-chain variable region of a first anti-BCMA heavy-chain antibody and the heavy-chain variable region of a second anti-BCMA heavy-chain antibody serialized by G4S, as shown by the amino acid sequence of SEQ ID NO:1. Here, the heavy-chain variable region of the first anti-BCMA heavy-chain antibody is shown by 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 by the amino acid sequence of SEQ ID NO:3. Here, the heavy-chain variable region of the first anti-BCMA heavy-chain antibody includes complementarity-determining regions (CDRs), CDRH1 is shown by the amino acid sequence of SEQ ID NO:4, CDRH2 is shown by the amino acid sequence of SEQ ID NO:5, CDRH3 is shown by the amino acid sequence of SEQ ID NO:6. On the other hand, the heavy-chain variable region of the second anti-BCMA heavy-chain antibody includes the following CDRs, CDRH1 is shown by the amino acid sequence of SEQ ID NO:7, CDRH2 is shown by the amino acid sequence of SEQ ID NO:8, CDRH3 is shown by the amino acid sequence of SEQ ID NO:9.

[0014] In one embodiment, the extracellular antigen-binding domain includes the heavy-chain variable region of an anti-CD19 antibody, and the heavy-chain variable region of the anti-CD19 antibody includes complementarity-determining regions. CDRH1 is shown by the amino acid sequence of SEQ ID NO:10, CDRH2 is shown by the amino acid sequence of SEQ ID NO:11, and CDRH3 is shown by the amino acid sequence of SEQ ID NO:12. In one embodiment, the extracellular antigen-binding domain further includes the light-chain variable region of the anti-CD19 antibody, and the light-chain variable region of the anti-CD19 antibody includes the following CDRs. CDRL1 is shown by the amino acid sequence of SEQ ID NO:13, CDRL2 is shown by the amino acid sequence of SEQ ID NO:14, and CDRL3 is shown 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 shown by the amino acid sequence of SEQ ID NO:16.

[0015] In one embodiment, the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-mesothelin antibody, the heavy chain variable region of the anti-mesothelin antibody comprises the following CDRs, where CDRH1 is represented by the amino acid sequence of SEQ ID NO:17, CDRH2 by the amino acid sequence of SEQ ID NO:18, and CDRH3 by the amino acid sequence of SEQ ID NO:19. In one embodiment, the extracellular antigen-binding domain further comprises the light chain variable region of an anti-mesothelin antibody, the light chain variable region of the anti-mesothelin antibody comprises the following CDRs, where CDRL1 is represented by the amino acid sequence of SEQ ID NO:20, CDRL2 by the amino acid sequence of SEQ ID NO:21, and CDRL3 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.

[0016] In one embodiment, the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-BCMA antibody, the heavy chain variable region of the anti-BCMA antibody comprising the following CDRs, where CDRH1 is represented by the amino acid sequence of SEQ ID NO:24, CDRH2 by the amino acid sequence of SEQ ID NO:25, and CDRH3 by the amino acid sequence of SEQ ID NO:26. In another embodiment, the extracellular antigen-binding domain further comprises the light chain variable region of an anti-BCMA antibody, the light chain variable region of the anti-BCMA antibody comprising the following CDRs, where CDRL1 is represented by the amino acid sequence of SEQ ID NO:27, CDRL2 by the amino acid sequence of SEQ ID NO:28, and CDRL3 by the amino acid sequence of SEQ ID NO:29. In yet another 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, where CDRH1 is represented by the amino acid sequence of SEQ ID NO:31, CDRH2 by the amino acid sequence of SEQ ID NO:32, and CDRH3 by the amino acid sequence of SEQ ID NO:33. In one embodiment, the extracellular antigen-binding domain further comprises the light chain variable region of the anti-BCMA antibody, where the light chain variable region of the anti-BCMA antibody comprises the following CDRs, where CDRL1 is represented by the amino acid sequence of SEQ ID NO:34, CDRL2 by the amino acid sequence of SEQ ID NO:35, and CDRL3 by the amino acid sequence of SEQ ID NO:36. In one embodiment, the extracellular antigen-binding domain is a BB 2121 single-chain variable region fragment, as represented by the amino acid sequence of SEQ ID NO:37.

[0017] In one embodiment, the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of the anti-CD22 antibody comprising the following CDRs, where CDRH1 is represented by the amino acid sequence of SEQ ID NO:38, CDRH2 by the amino acid sequence of SEQ ID NO:39, and CDRH3 by the amino acid sequence of SEQ ID NO:40. In another embodiment, the extracellular antigen-binding domain further comprises the light chain variable region of an anti-CD22 antibody, the light chain variable region of the anti-CD22 antibody comprising the following CDRs, where CDRL1 is represented by the amino acid sequence of SEQ ID NO:41, CDRL2 by the amino acid sequence of SEQ ID NO:42, and CDRL3 by the amino acid sequence of SEQ ID NO:43. In yet another embodiment, the extracellular antigen-binding domain is an m971 single-chain variable region fragment, as represented by the amino acid sequence of SEQID NO:44. In another embodiment, the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of the anti-CD22 antibody comprising the following CDRs, where CDRH1 is represented by the amino acid sequence of SEQ ID NO:45, CDRH2 by the amino acid sequence of SEQ ID NO:46, and CDRH3 by the amino acid sequence of SEQ ID NO:47. In one embodiment, the extracellular antigen-binding domain further comprises the light chain variable region of an anti-CD22 antibody, the light chain variable region of the anti-CD22 antibody comprising the following CDRs, where CDRL1 is represented by the amino acid sequence of SEQ ID NO:48, CDRL2 by the amino acid sequence of SEQ ID NO:49, and CDRL3 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 a heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of the anti-CD22 antibody comprises the following CDRs, where CDRH1 is represented by the amino acid sequence of SEQ ID NO: 52, CDRH2 is represented by the amino acid sequence of SEQ ID NO: 53, and CDRH3 is represented by 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, the light chain variable region of the anti-CD22 antibody comprising the following CDRs, CDRL1 represented by the amino acid sequence of SEQ ID NO: 55, CDRL2 represented by the amino acid sequence of SEQ ID NO: 56, and CDRL3 represented by the amino acid sequence of SEQ ID NO: 57. In one embodiment, the extracellular antigen-binding domain is a G 5 / 44 single-chain variable region fragment, as represented by 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, the heavy chain variable region of the anti-CD22 antibody comprising the following CDRs, CDRH1 represented by the amino acid sequence of SEQ ID NO: 59, CDRH2 represented by the amino acid sequence of SEQ ID NO: 60, and CDRH3 represented by the amino acid sequence of SEQ ID NO: 61. In one embodiment, the extracellular antigen-binding domain further comprises a light chain variable region of an anti-CD22 antibody, the light chain variable region of the anti-CD22 antibody comprising the following CDRs, where CDRL1 is shown by the amino acid sequence of SEQ ID NO:62, CDRL2 is shown by the amino acid sequence of SEQ ID NO:63, and CDRL3 is shown by the amino acid sequence of SEQ ID NO:64. In one embodiment, the extracellular antigen-binding domain is an RFB 4 single-chain variable region fragment, as shown by the amino acid sequence of SEQ ID NO:65.

[0018] Preferably, the extracellular antigen-binding domain includes the heavy chain variable region of the anti-CD19 antibody and may also be an FMC63 single-chain variable region fragment.

[0019] Preferably, the extracellular antigen-binding domain includes the heavy chain variable region of the anti-mesothelin antibody and may also be an SS1 single-chain variable region fragment.

[0020] Preferably, the extracellular antigen-binding domain includes the heavy chain variable region of the anti-BCMA antibody and may be a J6M0 single-chain variable region fragment or a BB 2121 single-chain variable region fragment.

[0021] Preferably, the extracellular antigen-binding domain includes the heavy chain variable region of the anti-CD22 antibody and 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.

[0022] Preferably, the hinge domain is selected from the group consisting of (G4S)3 (indicated by SEQ ID NO: 66), the hinge domain of CD (indicated by SEQ ID NO: 67), the hinge domain of CD28 (indicated by SEQ ID NO: 68), the hinge domain of IgG4 (indicated by SEQ ID NO: 69), EAAAKGGGGS (indicated by SEQ ID NO: 70), (EAAAK)3 (indicated by SEQ ID NO: 71), the hinge domain of GST (indicated by SEQ ID NO: 72), the hinge domain of EAAAK-GS (indicated by SEQ ID NO: 73), the hinge domain of IgD (indicated by SEQ ID NO: 74), IgG4-CH3 (indicated by SEQ ID NO: 75), and (AP)6 (indicated by SEQ ID NO: 76).

[0023] Preferably, the isolated nucleic acid molecule further comprises the nucleic acid sequence of the adapter gene. More preferably, the adapter molecule may be CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / γ. According to the present invention, full-length proteins of CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / r are used as the adapter molecule.

[0024] 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.

[0025] Preferably, the hinge domain and the transmembrane domain of NKp30 are connected via the NKp30 cell stalk region.

[0026] Preferably, the isolated nucleic acid molecule further comprises a nucleic acid sequence of the CD3ζ signaling domain, an FcεR1γ adapter molecule, or a T2A self-cleaving peptide. For example, the nucleic acid sequence of the FcεR1γ adapter molecule may be located at the 5' end of the isolated nucleic acid molecule, while the nucleic acid sequence of the T2A self-cleaving peptide may be located between the nucleic acid sequence of the intracellular signaling domain and the nucleic acid sequence of the transmembrane domain of the natural killer protein 30, or between the nucleic acid sequence of the FcεR1γ adapter molecule and the nucleic acid sequence of the transmembrane domain of the natural killer protein 30.

[0027] The hinge domain and the transmembrane domain of NKp30 are connected via the NKp30 cell stalk region.

[0028] Preferably, in the isolated nucleic acid molecule, the extracellular antigen-binding domain includes the heavy chain variable region of an anti-CD19 antibody, and the hinge domain is the hinge domain of IgG4. Combining the extracellular antigen-binding domain and the hinge domain in this way allows the resulting lentivirus to have an excellent transduction rate, and the resulting CAR-T cells have better amplification factors, a higher proportion of stem cell-like memory T cells (Tscm), and superior cytotoxicity. More preferably, the heavy chain variable region of the anti-CD19 antibody is an FMC63 single-chain variable region fragment.

[0029] Preferably, in the isolated nucleic acid molecule, the extracellular antigen-binding domain includes the heavy chain variable region of an anti-mesothelin antibody, and the hinge domain is the (G4S)3 hinge domain. Combining the extracellular antigen-binding domain and the hinge domain in this way allows the resulting lentivirus to have a superior transduction rate, and the resulting CAR-T cells to have better amplification factors, a higher Tscm ratio, and superior cytotoxicity. More preferably, the heavy chain variable region of the anti-mesothelin antibody is an SS1 single-chain variable region fragment.

[0030] Preferably, in the isolated nucleic acid molecule, the extracellular antigen-binding domain includes the heavy chain variable region of an anti-BCMA antibody, and the hinge domain is the CD28 hinge domain. Combining the extracellular antigen-binding domain and the hinge domain in this way allows the resulting lentivirus to have a superior transduction rate, and the resulting CAR-T cells to have better amplification factors, a higher Tscm ratio, and superior cytotoxicity. More preferably, the heavy chain variable region of the anti-BCMA antibody is a J6M0 single-chain variable region fragment.

[0031] Preferably, in the isolated nucleic acid molecule, the extracellular antigen-binding domain contains the heavy chain variable region of an anti-CD22 antibody, and the hinge domain is the hinge domain of CD28. Combining the extracellular antigen-binding domain and the hinge domain in this way allows the resulting lentivirus to have a superior transduction rate, and the resulting CAR-T cells to have better amplification factors, a higher Tscm ratio, and superior cytotoxicity. More preferably, the heavy chain variable region containing the anti-CD22 antibody is an m971 single-chain variable region fragment.

[0032] To achieve the above objective, the present invention further provides a plasmid comprising the isolated nucleic acid molecule, wherein the carrier is a DNA plasmid or an RNA plasmid. According to the present invention, the nucleic acid sequence in the plasmid is, in 5' to 3' sequence order, an FcεR1γ adapter molecule, an intracellular signaling domain, a T2A self-cleaving peptide, an extracellular antigen-binding domain, a hinge domain, a transmembrane domain of NKp30, and a cytoplasmic domain of NKp30. In one embodiment, the nucleic acid sequence in the plasmid is, in 5' to 3' sequence order, a 5'LTR, a promoter, an FcεR1γ adapter molecule, an intracellular signaling domain, a T2A self-cleaving peptide, an extracellular antigen-binding domain, a hinge domain, a transmembrane domain of NKp30, and a cytoplasmic domain of NKp30, and a 3'LTR.

[0033] To achieve the above objective, the present invention further provides a method for producing chimeric antigen receptor cells, which includes introducing the aforementioned isolated nucleic acid molecules into nucleated cells.

[0034] Preferably, the method for producing the chimeric antigen receptor-cells, wherein the "introduction" is performed by transduction of lentiwill.

[0035] 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.

[0036] To achieve the above objective, the present invention further provides isolated cells containing the isolated nucleic acid molecules described above.

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

[0038] To achieve the above objectives, the present invention further provides uses of the isolated cells described above for preparing pharmaceuticals for treating or mitigating cancer. For example, they can be used to 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.

[0039] To achieve the above objectives, the present invention further provides uses of the isolated cells described above for preparing pharmaceuticals for treating or alleviating autoimmune diseases. The autoimmune diseases are diseases related to autoimmune reactions in which CD19CAR-T has a therapeutic effect. In one embodiment, the autoimmune diseases are systemic lupus erythematosus (SLE), idiopathic inframmatory myositis, systemic sclerosis, or multiple sclerosis.

[0040] The advantage of this invention is that by transferring the nucleic acid sequences of the transmembrane domain, cytoplasmic domain, and intracellular signaling domain of NKp30 into T cells, the T cells acquire an NKp30 receptor complex, becoming heavy chain CAR-T cells that can form stable immune synapses with cancer cells and possess excellent cancer cell toxicity. Furthermore, the resulting CAR-T cells have excellent cytotoxicity both in vivo and ex vivo, significantly killing cancer cells without excessively activating T cells, showing durability in rechallenge trials, and possessing a high percentage of Tscm, which is expected to reduce cytokine release syndrome and neurotoxicity associated with CAR-T cell therapy. [Brief explanation of the drawing]

[0041] [Figure 1] Figure 1 is a schematic diagram of the pLAS5w carrier. [Figure 2] Figure 2 shows schematic diagrams of the plasmids for Examples 1-1A to 1-2A and control example 1A. [Figure 3] Figure 3 is a schematic diagram of the cell membrane protein structure of CAR-T cells in Examples 1-1C, 1-2C, and control example 1C. [Figure 4A-4D] Figures 4A-4D show flow cytometry results regarding the T cell subpopulation distribution of CAR-T cells and non-transduction groups in Examples 1-1C, 1-2C, and control example 1C. [Figures 5A-5C]Figures 5A, 5B, and 5C show real-time results of cell elimination comparisons in Test Example 5, where the E:T ratios of CAR-T cells in Examples 1-1C and 1-2C, CAR-T cells in Control Example 1C, T cells in the non-transduction group, and target cells were 10:1, 3:1, and 1:1, respectively. [Figure 6] Figure 6 is a schematic diagram of the plasmids of Example 2A and Control Example 2A. [Figure 7] Figure 7 is a schematic diagram of the cell membrane protein structure of CAR-T cells in Example 2C and Control Example 2C. [Figures 8A-8B] Figures 8A and 8B show the flow cytometry results for the expression of anti-mesothelin antibody and NKp30 in CAR-T cells of Example 2C in Test Example 8, respectively. [Figure 8C-8D] Figures 8C and 8D show the flow cytometry results for anti-mesothelin antibody and NKp30 expression in CAR-T cells of control example 2C in test example 8, respectively. [Figures 9A-9C] Figures 9A, 9B, and 9C show the flow cytometry results for the T cell subpopulation distribution on day 7 of Example 2C, Control Example 2C, and the non-transduction group in Test Example 9, respectively. [Figure 10A-10D] Figures 10A, 10B, 10C, and 10D show the real-time results of cell elimination comparisons in Test Example 10, where the E:T ratios of CAR-T cells in Example 2C, CAR-T cells in Control Example 2C, T cells in the non-transduction group, and target cells were 3:1, 1:1, 0.3:1, and 0.1:1, respectively. [Figure 11A] Figure 11A shows the in vivo antitumor effect in Test Example 12, where the E:T ratios of CAR-T cells in Example 2C, CAR-T cells in Control Example 2C, and T cells in the non-transduction group were 3:1 and 1:1, respectively. [Figure 11B] Figure 11B shows the logarithmic bioluminescence imaging signals for CAR-T cells from Example 2C, CAR-T cells from control example 2C, T cells from the non-transduction group, and the saline group in Test Example 12, where the E:T ratios are 3:1 and 1:1, respectively. [Figure 12A]Figure 12A shows the results of a re-challenge study of in vivo antitumor cells in Test Example 12, where the E:T ratios for CAR-T cells in Example 2C, CAR-T cells in Control Example 2C, and the saline group were 3:1 and 1:1, respectively. [Figure 12B] Figure 12B shows the logarithmic bioluminescence imaging signals of re-challenge of in vivo antitumor cells in Test Example 12, where the E:T ratios of CAR-T cells in Example 2C, CAR-T cells in Control Example 2C, and target cells were 3:1 and 1:1, respectively. * indicates a p-value < 0.05, and * indicates a p-value < 0.01. [Figure 13A] Figure 13A shows the percentage of T cells in the peripheral blood of mice in each group of Test Example 12. [Figure 13B] Figure 13B shows the percentage of CAR-T cells in the T cells of each mouse group in Test Example 12. [Figure 14] Figure 14 shows schematic diagrams of the plasmids for Example 3A and control examples 3-1A and 3-2A. [Figure 15] 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. [Figures 16A-16B] Figures 16A and 16B show the flow cytometry results for the expression of anti-CD22 receptor and NKp30 in CAR-T cells of Example 3C in Test Example 15, respectively. [Figures 16C-16D] Figures 16C and 16D show the flow cytometry results for the expression of anti-CD22 receptor and NKp30 in CAR-T cells of control example 3-1C in test example 15, respectively. [Figures 17A-17D] Figures 17A, 17B, 17C, and 17D show the flow cytometry results of the T cell subpopulation distribution on day 7 for Example 3C, Control Example 3-1C, Control Example 3-2C, and the non-transduction group in Test Example 16, respectively. [Figure 18] Figure 18 shows the real-time results of a cell elimination comparison in Test Example 17, where the E:T ratios of CAR-T cells in Example 3C, control example 3-1C, control example 3-2C, and non-transduction group T cells were 3:1 and 1:1, respectively. [Figure 19]Figure 19 shows the cell toxicity comparison results analyzed by flow cytometer with E:T ratios of 3:1 and 1:1 for CAR-T cells from Example 3C, control example 3-1C, control example 3-2C, and the non-transduction group in Test Example 18. [Figure 20] Figure 20 is a schematic diagram of the plasmids of Example 4A and Control Example 4A. [Figure 21] Figure 21 is a schematic diagram of the cell membrane protein structure of CAR-T cells in Example 4C and Control Example 4C. [Figures 22A-22B] Figures 22A and 22B show the flow cytometry results for the expression of anti-CD19 receptor and NKp30 in CAR-T cells of Example 4C in Test Example 21, respectively. [Figures 22C-22D] Figures 22C and 22D show the flow cytometry results for the expression of anti-CD19 receptor and NKp30 in CAR-T cells of Example 4C in Test Example 21, respectively. [Figures 23A-23C] Figures 23A, 23B, and 23C show the flow cytometry results of the T cell subpopulation distribution on day 7 for Example 4C, Control Example 4C, and the non-transduction group in Test Example 22, respectively. [Figure 24] Figure 24 shows the comparative results of a real-time cytotoxicity assay in Test Example 23, where the E:T ratio was 1:1 for CAR-T cells in Example 4C, CAR-T cells in Control Example 4C, T cells in the non-transduction group, target cells, and the complete degradation group. [Figure 25] Figure 25 shows the results of cold light sterilization, where the E:T ratios of CAR-T cells in Example 4C, CAR-T cells in Control Example 4C, and T cells in the non-transduction group were 3:1 and 1:1, respectively. [Modes for carrying out the invention]

[0042] The present invention will be further illustrated by the following examples, which are not limited to those disclosed prior to the present invention. Those skilled in the art can make minor improvements and variations without departing from the scope of the present invention.

[0043] 1. Production and testing of CAR-T cells possessing intracellular signaling domains and NKp30 fragments.

[0044] Example 1-1: Nucleic acid molecule containing the DAP12 intracellular signaling domain and the complete fragment of NKp30

[0045] First, the 3' end of the full-length FcεR1γ nucleic acid sequence (indicated by SEQ ID NO: 77) was joined to the 5' end of the intracellular signaling domain nucleic acid sequence of DAP12 (SEQ ID NO: 78) to obtain the joined nucleic acid molecule fragment 1. This joined nucleic acid molecule fragment 1 and the sequence of the complete fragment of NKp30 (including the immunoglobulin-like domain) (SEQ ID NO: 79) were joined by the nucleic acid sequence of the T2A self-cleaving peptide (SEQ ID NO: 80) to obtain the nucleic acid molecule (indicated by SEQ ID NO: 81) having the DAP12 intracellular signaling domain and the complete fragment of NKp30 as in Example 1-1. Here, the complete fragment of NKp30 includes an immunoglobulin-like domain, a stalk region, a transmembrane domain (Tm) of NKp30, and a cytoplasmic domain of NKp30. On the other hand, the nucleic acid molecule in Example 1-1, which has the DAP12 intracellular signaling domain and the complete fragment of NKp30, consists of the full-length nucleic acid sequence of FcεR1γ, the nucleic acid sequence of the DAP12 intracellular signaling domain, the nucleic acid sequence of the T2A self-cleaving peptide, and the complete fragment of NKp30, in that order from the 5' end to the 3' end.

[0046] Examples 1-2 Nucleic acid molecules containing the 41BB intracellular signaling domain and the complete fragment of NKp30

[0047] The manufacturing method for Example 1-2 is similar to that of Example 1-1, except that the intracellular signaling domain used is the 41BB intracellular signaling domain (indicated by SEQ ID NO: 82), and the nucleic acid molecule containing the complete fragment of the 41BB intracellular signaling domain and NKp30 obtained in Example 1-2 is indicated by SEQ ID NO: 83.

[0048] Control example 1: Mutant wild-type NKp30 nucleic acid molecule

[0049] First, the 3' end of the full-length FcεR1γ nucleic acid sequence (indicated by SEQ ID NO: 77) was joined to the nucleic acid sequence of the T2A self-cleaving peptide (SEQ ID NO: 80) and the 5' end of the complete fragment of NKp30 (indicated by SEQ ID NO: 79) to obtain the mutant wild-type NKp30 nucleic acid molecule of control example 1 (indicated by SEQ ID NO: 84). The mutant wild-type NKp30 nucleic acid molecule of example 1 then consists of the full-length FcεR1γ nucleic acid sequence, the T2A self-cleaving peptide nucleic acid sequence, and the complete fragment of NKp30, in that order from the 5' end to the 3' end.

[0050] Examples 1-1A, 1-2A, and Control Example 1A: Construction of plasmids containing nucleic acid molecules from Examples 1-1, 1-2, or Control Example 1.

[0051] As shown in Figure 1, the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, catalog number C6-8-39) driven by an RSV promoter containing a TAR chimeric 5'LTR nucleic acid sequence was modified to remove the hPGK promoter and PAC gene (by requesting GenScript to perform this modification) to obtain the pLAS5w vector. Furthermore, as shown in Figure 2, the nucleic acid molecules of Examples 1-1, 1-2, and Control Example 1 were conjugated to the pLAS5w vector using BstBI and EcoRI restriction enzymes to obtain plasmids of Examples 1-1A, 1-1B, and Control Example 1A, respectively.

[0052] Examples 1-1B, 1-2B, and Control Example 1B: Production of lentiwills containing nucleic acid molecules of Examples 1-1, 1-2, or Control Example 1

[0053] Human embryonic kidney cells (HEK293 cells, CRL-3216) were transfected with the target gene using PolyJet transfection reagent (SignaGen Laboratories, SL 100688) to produce lentiwill particles for Examples 1-1B, 1-2B, and control example 1B. Specifically, one of the plasmids from Examples 1-1A and 1-2A, or the plasmid from Example 1A, containing 50 micrograms (μg) of the target gene, was used as a transfer plasmid. 45 μg of pCMV deltaR8.91 was used as a packaging plasmid (purchased from Academia Sinica), and 5 μg of pMD.G was used as an envelope plasmid (purchased from Academia Sinica). These plasmids were cultured with 300 microliters (μL) of PolyJet transfection reagent in 10 ml (mL) of serum-free DMEM medium for 15 minutes to obtain a Polyjet / DNA mixture. After adding the Polyjet / DNA mixture to 100 mL of DMEM medium containing 10% FBS, 9 × 10 7100 HEK293 cells were added dropwise to T875 culture bottles pre-seed with these cells. After 12-18 hours of incubation, the medium was replaced with 100 ml of Opti-MEM (Gibco, 31985070) containing 1 mM sodium pyruvate (Sartorius). After 16 hours of transfection, the medium was replaced with fresh Opti-MEM, and after 48 hours of transfection, the upper layer containing the first lenticular particles was collected, supplemented with fresh Opti-MEM medium containing 1 mM sodium pyruvate, incubated for 24 hours, and the upper layer containing lenticular particles was collected again. Cell debris was removed from the collected supernatant containing lenticular particles by centrifugation at 1500 rpm for 10 minutes at 4°C, and the resulting upper layer was permeated through a 0.45 μm filter membrane (Membrane-Solutions). The upper layers collected and filtered twice were mixed. Furthermore, the solution was concentrated using Lenti-X concentration reagent (Takara Bio) in a 3:1 ratio, i.e., the volume ratio of the filtered upper layer to Lenti-X was 3:1. Specifically, the supernatant and the mixture of Lenti-X concentration reagent were inverted and mixed at 4°C, left overnight, then centrifuged at 1500 rpm for 45 minutes at 4°C, the supernatant was removed, and the lower layer was collected in 2 mL of X-VIVO2. TM Fresh medium (Lonza) was suspended, and the lentivirs of Examples 1-1B, 1-2B, and control Example 1B were obtained after concentration and stored at -80°C for reserve.

[0054] Test Example 1: Testing of lenthwill titer in Examples 1-1B, 1-2B, and Control Example 1B

[0055] Lentivir titer was determined by measuring 4 × 10⁶ of a 3-fold sequence-diluted lentivir sample. 4 The measurement was performed by transducing the lentivir sample into human acute leukemia T cells (Jurkat cells, purchased from BCRC, Cat. No. 60424). The lentivir samples used in this study example 1 were the enriched lentivir samples from Examples 1-1B, 1-2B, and control example 1B. 50 μL of lentivir sample was mixed with 100 μL of X-Vivo. TMThe sample was added to culture medium 15, and then the sequence was diluted 3-fold with the same medium. This dilution process was repeated until the sample was diluted 6561-fold, yielding multiple groups of sequence-diluted lentinewill samples. Next, 50 μL of each sequence-diluted lentinewill sample was taken and added to a 96-well plate (U-shaped bottom), with 4 × 10⁶ samples placed in each well of the plate. 4 A well plate containing 100 μL of Jurkat cells in 100 μL of medium was used. After adding lentivir, lentivir transduction was performed for 72 hours. After washing away excess lentivir, NKp30 expression was detected using a 1 μg / mL anti-NKp30 antibody (BioLegend, Cat. No. 325228) labeled with BV421 fluorescent dye. The expression level of the target protein was analyzed using a flow cytometer (Sony, SA 3800) to measure the effect of lentivir transduction. Otherwise, Jurkat cells that had not been transdulated with lentivir were used as the negative control group. Here, the lentivir titer of nucleic acid molecules containing the intracellular signaling domain and the complete fragment of NKp30 (including the immunoglobulin-like domain) was calculated using the following formula, and the amount of lentivir to be used when producing CAR-T cells was determined based on the obtained lentivir titer.

[0056] Lentivir titer (TU / mL) = (percentage of cells expressing NKp30) × 4 × 10 4 (Number of cells) × 20 × Dilution factor

[0057] The experimental results are shown in Table 1 below. Table 1: Lentenwill titers (transducing units, TU / mL) of Examples 1-1B, 1-2B, and Control Example 1B.

[0058] The lentiwill titer is 1 × 10 6When it is above TU / mL, when manufacturing CAR-T cells, a small volume of lentivirus can be used to smoothly transduce T cells into CAR-T cells and the transduced T cells can grow well. Therefore, based on the results in Table 1 above, the lentiviruses of Example 1-1B, 1-2B and Comparative Example 1B all meet this criterion and are further advantageous for the production of CAR-T cells.

[0059] Example 1-1C, 1-2C, Comparative Example 1C: Production of CAR-T cells having the nucleic acid molecules of Example 1-1, 1-2 or Comparative Example 1

[0060] Fresh blood was collected after obtaining consent from a healthy donor. On day 0, human peripheral blood mononuclear cells (PBMC) were collected using a SepMate centrifuge tube (STEMCELL, Cat. No. 86450). Briefly, the blood sample was diluted with PBS buffer containing 2% FBS in the same volume as the fresh blood sample, and then the diluted sample was dropped along the tube wall into the SepMate centrifuge tube. The SepMate tube was held vertically and the blood sample was mixed with the density gradient centrifugation fluid (ficoll) in SepMate. Centrifugation was performed 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 further centrifuged at 300×g for 8 minutes at room temperature. Taking the lower layer part, it became PBMC. Next, magnetic beads containing anti-CD3 / CD28 antibodies on the surface (the magnetic beads are CTS (Cell Therapy Systems) Dynabeads CD3 / CD28 (Gibco, Cat. No. 40203D)) were used to separate T cells from the collected peripheral blood mononuclear cells (PBMCs) by DynaMag-Spin (purchased from Thermo). The separated PBMC cells were used with an anti-CD3 antibody (Biolegend, Cat. No. 300441) to detect the proportion of CD3 + T cells in PBMC by flow cytometry and at the same time, calculate the number of CD3 + T cells. CD3 +Three times the number of T cells are taken in magnetic beads containing anti-CD3 / CD28 antibodies on their surface, added to PBMC cells, and cultured at room temperature for 30-60 minutes to allow the magnetic beads containing anti-CD3 / CD28 antibodies to bind to the T cells. Then, CD3-positive T cells are selected using a magnet (DynaMag-Spin;Thermo), achieving the objective of removing B cells, NK cells, and mononuclear cells from the PBMCs. After this, a complex of T cells and magnetic beads formed by the binding of antigen and antibody can be obtained. The T cell and bead complex is then subjected to X-VIVO2 containing 200 IU / mL of human recombinant IL-2 (R&D Systems). TM 2 × 10⁵ units in 15 medium (Lonza) 5 The cells were dispersed at a density of cells / mL and cultured at 37°C in 5% carbon dioxide for 24 hours to activate the T cells. The activated T cells were transductioned via one of Examples 1-1B or 1-2B, which had a viral infection dose MOI (multiplicity of infection) of 5, or by transduction with the lentivirus of control Example 1B for 24 hours to obtain CAR-T cell complexes of Examples 1-1C, 1-2C, and control Example 1C. The T cell group not treated with lentivirus was designated as the control group (non-transduction group). Here, MOI = viral titer (TU / mL) × viral volume (mL) / total number of T cells. On the third day, the suspension of CAR-T cell complexes was removed using a magnet to remove the magnetic beads, and CAR-T cells of Examples 1-1C, 1-2C, and control Example 1C were obtained. Schematic diagrams of the structures of each group are shown in Figure 3. The obtained CAR-T cells were then subjected to X-VIVO2 containing 200 IU / mL of human IL-2 recombinant (R&D, 202-GMP-01M). TM The cells were transferred to a new culture vial of 15 medium and cultured under the same conditions as the transduction process, and the CAR-T cells were grown until day 7. Finally, the number of CAR-T cells was calculated for the non-transduction group and for Examples 1-1C, 1-2C, and control example 1C. Additional medium was added every 1-3 days during the CAR-T culture period, and the cell density was increased to 1.0 × 10⁶. 6 The solution was subsequently adjusted to cells / mL.

[0061] Test Example 2: CAR-T cell proliferation test of Example 1-1C, Example 1-2C, and control example 1C

[0062] The proliferation rates of CAR-T cells in Examples 1-1C, 1-2C, and Control Example 1C were calculated by determining the total number of cells from a specific point in time during the period in which the CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C were produced. Specifically, on the third day after the production of the CAR-T cells, 1 × 10⁶ CAR-T cells from Examples 1-1C, 1-2C, and Control Example 1C were placed in a 12-well plate. 6 Cells were seeded at a density of cells / mL, and after removing the magnetic beads, cell viability and cell density were measured. The cells were stained with acridine orange / propidium iodide (AO / PI), and the cell count was automatically quantified using an automated cell counter (Luna Automated Cell Counter, Logos Biosystems). The remaining cells were subcultured to a density of 2 × 10⁶ cells. 5 ~5×10 5 Cells were seeded into culture plates at a density of cells / mL to amplify them. The number of cells was measured on day 0 and day 7 when CAR-T cells were produced, and the cell amplification rate was calculated. The experimental results are shown in Table 2 below. Here, the amplification rates of CAR-T cells in Examples 1-1C, 1-2C, and control example 1C are the proliferation multipliers calculated after normalizing the number of cells on day 0.

[0063] Table 2: Amplification factor of CAR-T cells on day 7 in Examples 1-1C, 1-2C, and control example 1C

[0064] Since an amplification factor of 5 times or more is advantageous for subsequent T cell functionality tests, as can be seen from the results in Table 2 above, the CAR-T cells of Examples 1-1C, 1-2C, and control example 1C all exhibited good T cell amplification factors. On the other hand, in the two groups of Examples 1-1C and 1-2C, the CAR-T cell amplification effect of Example 1-1C, which uses the DAP12 intracellular signaling domain as its intracellular signaling domain, was superior to that of the CAR-T cells of Example 1-2C, which uses the 41BB intracellular signaling domain as its intracellular signaling domain.

[0065] Test Example 3: Detection of CAR-T cell expression in Examples 1-1C, 1-2C and control example 1C, and transduction rate of lentivirus in Examples 1-1B, 1-2B and control example 1B.

[0066] NKp30 expression in CAR-T cells of Examples 1-1C, 1-2C, and Control Example 1C was detected by flow cytometry using an anti-NKp30 antibody (BioLegend, 325228) labeled with BV421 fluorescent dye. Subsequently, the percentage of cells expressing NKp30 relative to the total number of cells was defined as the lentiviral transduction rate for Examples 1-1B, 1-2B, and Control Example 1B, and the experimental results are shown in Table 3 below.

[0067] Table 3: Transduction rates of lentilwill in Examples 1-1B, 1-2B, and Control Example 1B

[0068] A transduction rate exceeding 25% for producing CAR-T cells is advantageous for subsequent functional testing of amplified CAR-T cells. Therefore, from the results in Table 3 above, both the lentiwills of Example 1-1B and Example 1-2B had excellent transduction rates. The lentiwill transduction rate of Example 1-1B, which uses DAP12 as its intracellular signaling domain, was superior to that of the lentiwill of Example 1-2B, which uses 41BB as its intracellular signaling domain, and both were superior to the lentiwill of control Example 1B.

[0069] Test Example 4: T cell phenotypic analysis of CAR-T cells from Examples 1-1C, 1-2C, and control example 1C.

[0070] T cell typing analysis was performed on CAR-T cells 7 days after activation during the manufacturing process of Examples 1-1C, 1-2C, and control example 1C, using antibody fluorescence calibration and flow cytometry (Sony Biotechnology, SA 3800). T cell surface markers were detected using 3800 software. The antibodies used were CD3-FITC (BioLegend, 300406), CD4-PE-Cy7 (BioLegend, 300512), CD8-AF700 (Beckman, B76279), CD95-APC (BioLegend, 305612), CD45RA-PE (BioLegend, 304108), and CCR7-BV421 (BioLegend, 353208). A control group containing all fluorescent dyes except CCR7-BV421 and CD45RA-PE (FMO - fluorescence-minus-one) was established. In this study, untransductioned activated T cells were used as the control group (non-transduction group). The staining results obtained by the above method are based on Annals of Oncology Volume 32, Issue 11, Pages 1366-1380, and refer to Tscm, central memory T cell (T). CM ), effector memory T cell (T EM ), terminally differentiated effector memory T cell (T EMRA )(or effector T cell, T EFF The distribution (%) of each subpopulation of T cells was determined, and the percentage of Tscm in CAR-T cells for Examples 1-1C, 1-2C, and control example 1C is shown in Table 4 below. The results for Examples 1-1C, 1-2C, and the flow cytometry results for the T cell subpopulation distribution of control example 1C and the non-transduction group are shown in Figures 4A, 4B, 4C, and 4D, respectively. Of these, T cells expressing CD45RA and CCR7 were Tscm, and T cells expressing CD45RA but not CCR7 were T EMRA (orTEFF )T cells that do not express CCR7 and do not express CD45RA are T EM Therefore, T cells that expressed CCR7 but did not express CD45RA are T CM That was the case.

[0071] Table 4: Percentage of Tscm of CAR-T cells in Examples 1-1C, 1-2C, Control Example 1C, and the non-transduction group.

[0072] When the proportion of stem cell-like memory T cells (Tscm) in CAR-T cells is 40% or higher, a high proportion of Tscm can reduce cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy. As can be seen from Table 4 and Figures 4A to 4D above, Examples 1-1C and 1-2C, which are CAR-T cells of mutant wild-type NKp30 with an intracellular signaling domain, both have a relatively high proportion of Tscm, and both are higher than the CAR-T cells and untransductioned activated T cells of control example 1C. Therefore, the CAR-T cells of Examples 1-1C and 1-2C are expected to reduce cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy.

[0073] Test Example 5: Real-time cytotoxicity assay (RTCA) using xCELLigence

[0074] HeLa cervical cancer cells (hereinafter abbreviated as HeLa cells) are mesenteric-positive cells. HeLa cells (purchased from Elabscience, CL-0101) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), under conditions of 5% CO2 and 37°C, and used for subsequent real-time cytotoxicity assays of xCELLigence.

[0075] To test the antitumor activity of CAR-T cells from Examples 1-1C, 1-2, and Control Example 1C, cytotoxicity assays were performed using the HeLa cell lines described in the preamble as target cells. The effector cells used in these tests were the CAR-T cells from Examples 1-1C, 1-2C, and Control Example 1C.

[0076] The xCELLigence RTCA system (Agilent Technologies, RTCA SP) was placed in a 37°C, 5% carbon dioxide cell culture box and connected via cable to the interface of an external analysis and control unit. Real-time control and instrument monitoring, including real-time data display and analysis functions, were performed using RTCA Software Pro. Specifically, the xCELLigence RTCA system was used to continuously monitor the tumor cell killing effect for approximately 90 hours. First, the aforementioned HeLa cells were used as target cells (T) and 1.2 × 10⁶ cells were used. 4After seeding at a cell / mL density in a background-corrected culture plate, background impedance measurement was performed for 22 hours. Target cells were then attached, and effector cells (E) were added to the culture plate at effector-to-target ratios (E:T ratio) of 10:1, 3:1, and 1:1, until the final volume per well reached 200 μL. The effector cells used in this study were CAR-T cells from Examples 1-1C, 1-2, and control example 1C, and untransductioned activated T cells were used as the control group (non-transduction group). Cell index (CI) was detected every 15 minutes and detected for at least 72 hours. The original RTCA impedance data is a standardized version of the cell index data before the addition of effector cells. The cell index is a dimensionless parameter obtained by measuring the relative change in electrical impedance and indicates the cell state. For example, if no cells are present or not attached to the electrode, the CI is zero. On the other hand, under the same physiological conditions, the CI value increases as the number of cells attached to the electrode increases. Furthermore, changes in the cellular state, such as changes in cell morphology or cell adhesion, all cause changes in CI, as shown in Figures 5A-5C.

[0077] The results of the 24-hour killing spree are shown in Table 5 below. Table 5: CAR-T cell toxicity assay results for Examples 1-1C, 1-2C, and control example 1C

[0078] As can be seen from the results in Table 5 and Figures 5A-5C, the CAR-T cell toxicity of Examples 1-1C and 1-2C was superior to that of the control example 1C. Furthermore, the 24-hour toxic effect of CAR-T cells in Examples 1-1C reached 100% when the E:T ratio was 10:1, 3:1, and 1:1. When the toxicity of CAR-T cells was >50%, it was found that they had sufficient toxicity when subsequently used to produce CAR-T cells from the patient's own cells. From the results in Table 5 above, it was found that the CAR-T cells of Examples 1-1C and 1-2C all had good cytotoxicity, and the CAR-T cells of Example 1-1C, which had the intracellular signaling domain of DAP12 transdulated, had superior cytotoxicity to the CAR-T cells of Example 1-2C, which had the intracellular signaling domain of 41BB transdulated.

[0079] Based on the above, the lentiviruses of Examples 1-1B and 1-2B of the present invention possess an intracellular signaling domain and, in both the control example and the example, the complete fragment of NKp30 containing the transmembrane domain of NKp30 exhibited excellent transduction rates. The CAR-T cells obtained through this transduction exhibited excellent CAR-T cell amplification rates and superior cytotoxicity, and had a higher proportion of Tscm, thus reducing cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy. It was found that transduction of nucleic acid sequences of intracellular signaling domains, such as DAP12 or 41BB, during the production of CAR-T cells resulted in excellent cytotoxic effects in the produced CAR-T cells, making them particularly suitable for CAR-T therapy.

[0080] 2. Production and testing of CAR-T cells possessing the anti-mesothelin-NKp30 intracellular signaling domain.

[0081] Example 2: Nucleic acid molecule having an NKp30 fragment that does not contain an intracellular signaling domain, a single-chain variable region fragment of an anti-mesothelin antibody, or an immunoglobulin-like domain.

[0082] The 3' end of the nucleic acid sequence of the single-chain variable region fragment SS1 of the anti-mesothelin antibody (indicated by SEQ ID NO: 85) was ligated to the 5' end of an NKp30 fragment (indicated by SEQ ID NO: 86) that does not contain an immunoglobulin-like domain, via the nucleic acid sequence of the hinge domain (G4S)3 (indicated by SEQ ID NO: 66). Here, the nucleic acid sequence of the NKp30 fragment that does not contain an immunoglobulin-like domain itself contains the extracellular stalk region, the transmembrane domain of NKp30, and the cytoplasmic domain of NKp30, but does not contain an immunoglobulin-like domain. Subsequently, the 3' end of the nucleic acid sequence of the NKp30 fragment that does not contain 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) to obtain the ligated nucleic acid molecular fragment 2. Next, the 5' end of this ligated nucleic acid molecule fragment 2 and the 3' end of the full-length FcεR1γ nucleic acid sequence (indicated by SEQ ID NO: 77) were ligated via the T2A self-cleaving peptide nucleic acid sequence (SEQ ID NO: 80), and as shown in SEQ ID NO: 87, nucleic acid molecules of the intracellular signaling domain, the single-chain variable region fragment containing the anti-mesothelin antibody, and the NKp30 fragment without the immunoglobulin-like domain were obtained in Example 2. The nucleic acid molecule of Example 2 consists of the full-length FcεR1γ nucleic acid sequence, the T2A self-cleaving peptide nucleic acid sequence, the SS1 nucleic acid sequence, the (G4S)3 hinge domain nucleic acid sequence, the NKp30 fragment without the immunoglobulin-like domain nucleic acid sequence, and the DAP12 intracellular signaling domain nucleic acid sequence, from the 3' end to the 5' end, respectively.

[0083] Control example 2: A nucleic acid molecule containing an intracellular signaling domain and a single-chain variable region fragment of an anti-mesothelin antibody, but without any NKp30 fragments.

[0084] The 3' end of the nucleic acid sequence (SEQ ID NO: 85) of the anti-mesothelin single-chain variable region fragment SS1 was ligated to the 5' end of the nucleic acid sequence of the CD8 transmembrane domain (SEQ ID NO: 88) via the nucleic acid sequence of the CD8 hinge domain (SEQ ID NO: 67). The 5' end of the nucleic acid molecule of the 4-1BB intracellular signaling domain (SEQ ID NO: 82) was then ligated to the 3' end of the nucleic acid sequence of the CD8 transmembrane domain. Subsequently, the 3' end of the nucleic acid sequence of the 4-1BB intracellular signaling domain was ligated to the 5' end of the nucleic acid sequence of the CD3ζ signaling domain (SEQ ID NO: 89) to obtain the nucleic acid molecule of control example 2 (SEQ ID NO: 90). The nucleic acid molecule in control example 2 consists of the SS1 nucleic acid sequence, the CD8 hinge domain nucleic acid sequence, the CD8 transmembrane domain nucleic acid sequence, the 4-1BB intracellular signaling domain nucleic acid sequence, and the CD3ζ signaling domain nucleic acid sequence, respectively, from the 3' end to the 5' end.

[0085] Example 2A, Control Example 2A: Construction of plasmids containing nucleic acid molecules from Example 2 and Control Example 2.

[0086] The manufacturing method for Example 2A is the same as that for Examples 1-1A and 1-2A, except that an NKp30 fragment having the intracellular signaling domain of Example 2 and a single-chain variable region fragment of an anti-mesothelin antibody, but without the immunoglobulin-like domain, is conjugated to the pLAS5w vector using restriction enzymes. On the other hand, for control example 2A, GenScript was asked to conjugate the nucleic acid molecule of control example 2 to pLAS5w.Ppuro (purchased from Academia Sinica RNAi Core, catalog number C6-8-39) using HpaI and EcoRI restriction enzymes. The obtained plasmid of Example 2A having the anti-mesothelin-NKp30 intracellular signaling domain and the plasmid of control example 2A are shown in Figure 6.

[0087] Example 2B, Control Example 2B: Production of lentinewills containing nucleic acid molecules from Example 2 and Control Example 2

[0088] The manufacturing method for Example 2B is the same as that for Examples 1-1B and 1-2B, except that the plasmid used in Example 2B is a transfer plasmid. On the other hand, the lentiwill for control example 2B uses the plasmid from control example 2A as the transfer plasmid.

[0089] Test Example 6: Test of the lenthwill titer of Example 2B

[0090] The test method for Test Example 6 is the same as that for Test Example 1, except that the mesothelin receptor is identified using biotinylated human mesothelin protein, His, and Avitag (MSN-H82E9, ACRO Biosystems), and the expression of the mesothelin receptor in lentiwill from Example 2B and Control Example 2B is detected using streptavidin (Invitrogen) labeled with 500-fold diluted phycoerythrin (PE). The test results are shown in Table 6 below. Table 6: Example 2B and Lentenwill titers for Example 2B

[0091] If the lentiwill titer is 1 × 106 When the TU / mL level is above this, a small volume of lentiwill can be used to smoothly transduce T cells into CAR-T cells during CAR-T cell production, and the growth of T cells after transduction can be improved. Therefore, based on the results in Table 6 above, all lentiwills in Example 2B and Control Example 2B meet this criterion and are advantageous for subsequent CAR-T cell production.

[0092] Example 2C: CAR-T cells possessing the anti-mesothelin-NKp30 intracellular signaling domain

[0093] The method for producing CAR-T cells having the anti-mesothelin-NKp30 intracellular signaling domain in Example 2C is the same as the methods for producing CAR-T cells in Examples 1-1C and 1-2C, except that the T cells in this example are transductioned using the lentiwill of Example 2B, and the MOI at the time of lentiwill transduction is 2. On the other hand, the CAR-T cells of Control Example 2C are transductioned with the lentiwill of Control Example 2B, and the MOI at the time of lentiwill transduction is 2. Schematic diagrams of the structures of the obtained CAR-T cells of Example 2C and Control Example 2C are shown in Figure 7, and the CD3ζ in Example 2C is specific to T cells. Here, the nucleic acid sequences transductioned into the CAR-T cells of Example 2C and Control Example 2C contain FcεR1γ, thus they are multi-chain CAR-T cells.

[0094] Test Example 7: Proliferation test of CAR-T cells and non-transduction T cells from Example 2C, Example 2C.

[0095] The test method for Test Example 6 is the same as that for Test Example 2, except that this test example detects the amplification rate of CAR-T cells from Example 2C and uses untransductioned activated T cells as the control group. The results obtained are shown in Table 7 below.

[0096] Table 7: Amplification factor of CAR-T cells and non-transduction T cells in Example 2C and Control Example 2C on day 7

[0097] Since an amplification factor of 5 times or more is advantageous for subsequent T cell functionality tests, as can be seen from the results in Table 7 above, the CAR-T cells of Example 2C have a good T cell amplification factor and are superior to the CAR-T cells of Control Example 2C.

[0098] Test Example 8: Detection of CAR-T cell expression in Example 2C and Control Example 2C, and transduction rate of lentivirus in Example 2B and Control Example 2B.

[0099] The test method for Test Example 8 is the same as that of Test Example 3, except that this test example identifies the mesothelin receptor using biotinylated human mesothelin protein, His, and Avitag (MSN-H82E9, ACRO Biosystems), and detects the expression of the mesothelin receptor in CAR-T cells of Example 2C and Control Example 2C using 500-fold diluted PE-labeled streptavidin (Invitrogen). The test results for Example 2C and Control Example 2C are shown in the flow cytometer results in Figures 8A, 8B, 8C, and 8D, respectively. The percentage of cells expressing the anti-mesothelin receptor relative to the total number of cells is defined as the lentivir transduction rate for Example 2B or Control Example 2B, and the experimental results are shown in Table 8 below.

[0100] Table 8: Transduction rates of lentwill in Example 2B and Control Example 2B

[0101] A transduction rate exceeding 25% for producing CAR-T cells is advantageous for subsequent functional testing of amplified CAR-T cells. From the results in Table 9 and Figure 8A above, it was found that the lentiwill of Example 2B and the control example 2B had a favorable transduction rate.

[0102] Test Example 9: T cell phenotypic analysis of CAR-T cells and non-transduction group T cells from Example 2C and control example 2C.

[0103] The test method for Test Example 9 is the same as that for Test Example 4, except that this test example examines CAR-T cells from Example 2C on day 7 after production. The proportion of Tscm in CAR-T cells from Example 2C and control Example 2C, and in T cells from the non-transduction group, is shown in Table 9 below. The results for Example 2C and the flow cytometry results for the subpopulation distribution of control Example 2C and non-transduction T cells are shown in Figures 9A, 9B, and 9C, respectively, where T cells expressing CD45RA and CCR7 are Tscm.

[0104] Table 9: Percentage of Tscm in CAR-T cells and non-transduction T cells in Example 2C and Control Example 2C.

[0105] If the proportion of stem cell-like memory T cells in CAR-T cells is 40% or higher, cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy can be further reduced. From Table 9 and Figures 9A, 9B, and 9C above, it can be seen that Example 2C, which has an intracellular signaling domain, has a relatively high Tscm% and is superior to the CAR-T cells of control Example 2C and the activated T cells of the non-transduction group. Therefore, the CAR-T cells of Example 2C are expected to reduce cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy.

[0106] Test Example 10: Real-time cytotoxicity assay using xCELLigence

[0107] The test method for Test Example 10 is the same as that for Test Example 5, except that this test example uses CAR-T cells from Example 2C and Control Example 2C as effector cells, the HeLa cell line is used as the target cell, the target cells are seeded on a background-corrected culture plate (E-plate 96 well, Agilent Technologies, 300600910), cultured overnight, and then the target cells are allowed to adhere and impedance measurements are performed for 18 hours. Finally, effector cells are added to the culture plate in E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1 for testing. The results of the toxicity assays are shown in Figures 10A, 10B, 10C, and 10C, 10D, respectively.

[0108] On the other hand, the results of the 24-hour toxicity assay are shown in Table 10 below.

[0109] Table 10: Real-time toxicity assay results of CAR-T cells in Example 2C and Control Example 2C

[0110] The results in Table 10 and Figures 10A-10D show that the CAR-T cell toxicity of Example 2C was superior to that of the conventional single-chain CAR-T cell control example 2C. In particular, when the E:T ratio was 0.3:1 and 0.1:1, the killing effect of Example 2C was superior to that of control example 2C, with a significant decrease in the normalized cell index value, indicating that the target cells were significantly killed. Furthermore, if the CAR-T cell toxicity is >50%, it can be used to produce CAR-T cells from the patient's cells afterward, and the results in Table 10 above show that all CAR-T cells in Example 2C have good cell-killing effects.

[0111] Test Example 11: Cold Phototoxicity Assay

[0112] SKOV3 ovarian cancer cells (hereinafter abbreviated as SKOV3 cells) (purchased from ATCC, Cat. No. HTB-77) were cultured in 10% FBS McCoy 5A Medium (Gibco, 16600082) and screened for SKOV3 cells expressing high levels of mesothelin using a green fluorescent protein-labeled anti-mesothelin antibody via flow cell solarization. Then, the PLL-CMV-rFluc-T2A-GFP-mPGK Lenti-Labeller lentiwill vector (System Biosciences) was screened and transduction was performed into the aforementioned SKOV3 cells expressing high levels of mesothelin. By screening for cell lines that stably express the vector as a single entity, it was determined that GFP / Luc, which simultaneously expresses green fluorescent protein (GFP) and cold-light enzyme, was identified. + We obtained SKOV3 cells (SKOV3-mLE).

[0113] To test the CAR-T cells of Example 2C and Control Example 2C, and the activated T cells of the non-transduction group, antitumor activity was detected in cells 7 days after production. Cytotoxicity assays were performed using ovarian cancer SKOV3 cells expressing mesothelin and cold-light enzyme as target cells. The effector cells used in this test were the CAR-T cells of Example 2C, and non-transduction T cells were used as the control group (non-transduction group). Specifically, effector cells and target cells were cultured in a 3:1 E:T ratio in a 96-well plate, incubated at 37°C and 5% carbon dioxide for 48 hours, and the test was repeated three times.

[0114] The cold light generated after the action of cold-light enzymes expressed by surviving cancer cells was measured using a cold-light enzyme test system reagent group (Promega, E 1501), and the relative number of surviving cancer cells not lysed by CAR-T cells was estimated. The site-specific lysis ratio of cells was then calculated using the following formula. Specific cell lysis = 100% × (Number of target cells lysed experimentally - Number of target cells spontaneously lysed) ÷ (Maximum number of target cells lysed - Number of target cells spontaneously lysed) Note that the number of target cells lysed in the experiment = the number of target cells lysed in the group cultured with effector cells. The number of target cells that spontaneously lyse = the number of target cells that lyse in the group without effector cells, i.e., the number of cells that lyse after culturing target cells in RPMI-1640 medium containing 10% FBS and culturing at 37°C and 5% carbon dioxide for 48 hours. The maximum number of target cells to be lysed = the number of target cells lysed after adding 100 μL (microliters) of 1% TritonX-100 to the group without effector cells.

[0115] The experimental results are shown in Table 11 below. Table 11: Results of cold phototoxicity assays (site-specific lysis %) for CAR-T cells and non-transduction group (control group) of Example 2C and Control Example 2C.

[0116] If the CAR-T cells have a toxic activity of 50% or more, they can have sufficient toxic activity when subsequently used to produce CAR-T cells in the patient's cells. As can be seen from the results in Table 11, the CAR-T cells of Example 2C, after being cultured with target cells for 48 hours, clearly exhibited superior CAR-T cell-killing activity compared to the control example 2C of conventional single-chain CAR-T cells. Therefore, it can be inferred that the CAR-T cells of Example 2C have sufficient toxic activity when subsequently used to produce CAR-T cells in the patient's cells.

[0117] Test Example 12: Animal Experiments

[0118] First, female mice with B2m gene deficiency and immunodeficiency (NOD.Cg-Prkdc) aged 5-10 weeks. scid Il2rg tm1Wjl B2m em1NarlForty YckNarl mice (abbreviated as ASID-B2m KO) were provided and divided into eight groups of five mice each (including a reserve mouse group and a tumor re-challenge positive control group). The mice were housed in a sterile environment, and the care and handling procedures for the experimental animals conformed to experimental methods approved by the Institutional Animal Care and Use Committee (IACUC). Since the CAR-T cells of this invention are derived from human T cells, it is expected that transplanting heterologous human T cells into immunodeficient experimental mice will cause graft-versus-host disease (GvHD) in the mice. However, previous experiments have shown that GvHD is significantly alleviated when the β-2-microglobulin (B2m) gene is deleted in immunodeficient mice. Therefore, this study used immunodeficient mice with B2m gene deficiency.

[0119] 5 × 10⁻⁶ SKOV3-mLE cells from Test Example 11 6Individual cells were isolated, suspended in 100 μL of PBS, and then mixed with an equal volume of Matrigel (BD Bioscience) to obtain the SKOV3-mLE cell injection mixture. On day 0, the SKOV3-mLE cell injection mixture was subcutaneously transplanted into the right side of the bodies of 35 mice, and 5 mice that had not been inoculated with SKOV3-mLE cells were set aside. From day 10, the bioluminescence imaging (BLI) signal was measured weekly using an In Vivo Imaging System (IVIS) (Perkin Elmer) until day 80, for a total of 80 days. Mice that met the humane sacrificial criteria within 80 days were sacrificed early, and the signal was not measured for mice that were sacrificed early until day 80. Here, on day 21 after transplantation with SKOV3-mLE cells (target cells), seven other groups of mice that had not been inoculated with SKOV3-mLE cells were either injected with saline via the tail artery (control group) or injected with CAR-T cells from Example 2C or CAR-T cells from Control Example 2 or untransductioned T cells (all referred to as effector cells, E). Here, E:T = 1:1 or 3:1. On day 55, the SKOV3-mLE cell injection mixture was transplanted into the contralateral side, i.e., the left side, of the bodies of the remaining surviving mice and the reserve group mice, and a tumor rechallenge test was performed. In the rechallenge test stage, a total of 22 mice were injected with SKOV3-mLE cells, of which five reserve mice received cancer cells for the first time. In this example, the in vivo tumor volume of mice is shown on a logarithmic scale quantifying the BLI signal. T cells are collected from the peripheral blood of mice, and CAR expression levels and T cell phenotypic analysis are performed. The CAR expression analysis is described in Example 8, and the method for phenotypic analysis is described in Example 9. Experimental data are expressed as mean ± standard deviation, and statistical analysis is performed based on the number of unique single-factor mutations in Tukey's post-hoc test.

[0120] The experimental results are shown in Tables 12 and 13 and Figures 11A, 11B, 12A, 12B, 13A, and 13B below. In Table 12, "-4 days" refers to 4 days before the administration of effector cells, while "+10 days," "+17 days," "+24 days," and "+31 days" refer to 10, 17, 24, and 31 days after the administration of effector cells, respectively.

[0121] Table 12 In vivo cytotoxicity assay [log( * The results of evaluating Example 2C (abbreviated as E2C) and Control Example 2C (abbreviated as CE2C) in [E+11) photons / second]

[0122] ns indicates that the result is not statistically significant; * represents p<0.05, ** represents p<0.01, and *** represents p<0.005.

[0123] Table 13: Data from in vitro studies on the characteristics and function of effector cells (CAR-T) after their production and before administration to mice (Control example 2C and Example 2C are abbreviated as CE2C and E2C, respectively).

[0124] As shown in Figure 11A, tumor volume was reduced 10 days after injection of CAR-T cells from the Example 2C group at a high dose (E:T=3:1) (31st day of the total study period). Visual observation of BLI revealed that in 3 / 5 of the mice administered with the high dose of CAR-T cells from the Example 2C group, the BLI area was smaller compared to other groups of different effector cells. When observing the mice in each group as a whole, the group administered with the high dose of CAR-T cells from Example 2C had significantly lower BLI signals than all other groups (shown in Table 12 and Figure 11B) after converting their BLI signals to a logarithmic scale.

[0125] As shown in Figure 11A, in mice treated with CAR-T cells and administered a high dose of Example 2C CAR-T cells on day 24 (day 45 of the total study period), the BLI signal was no longer visually detectable (<108 (photons / second). In mice treated with CAR-T cells from control example 2C at two different doses or from CAR-T cells from example 2C at a low dose (E:T=1:1), a clear decrease in the detectable signal in the BLI region was observed; however, the BLI signal in mice treated with these different groups was not undetectable to the naked eye. Mice treated with CAR-T cells from example 2C and control example 2C had lower BLI values ​​than their respective UTD control groups, while mice treated with CAR-T cells from the high-dose example 2C group had significantly lower BLI values ​​than other groups treated with CAR-T cells (i.e., CAR-T cells from control example 2C and low-dose example 2C). These differences in the detectable BLI region persisted until day 31 of treatment (day 52 of the total study period) (i.e., just before some mice developed life-threatening GvHD (shown in Figures 11A, 11B, and Table 12)).

[0126] Furthermore, tumor removal (signal reduction) can be observed in Figure 11A, but the group treated with Example 2C did not immediately develop GvHD, whereas the control group 2C, treated with conventional single-chain CAR-T cells, immediately developed GvHD in mice. While the use of ASID-B2mKO mice clearly reduced or delayed the symptoms of GvHD, signs of GvHD remained evident in mice treated with conventional single-chain CAR-T cells (Example 2C). This GvHD may be caused by overproliferated human CAR-T cells, and because there is a shortage of target cells, such as human cancer cells, the cells attack host cells. Before the rechallenge trial using SKOV3-mLE cancer cells, three mice treated with CAR-T cells from the control group 2C (representing 3 / 10 of the total number of mice in the control group 2C) died from GvHD symptoms (shown in Figure 11A). In the control group 2C, most of the mice treated with CAR-T cells survived (6 out of 7 mice), but within 11 days of tumor rechallenge, they succumbed to GvHD when the CAR-T effect could not be evaluated (shown in Figure 12A). On the other hand, as shown in Figures 13A and 13B, throughout the entire test process, the mice treated with CAR-T cells from control group 2C, using CD3+ cells as T cells, showed significantly higher total T cell counts in their peripheral blood and CAR-T cell counts compared to 2C. This suggests that the conventional single-chain CAR-T design may tend to produce overly activated CAR-T cells. As can be seen from Table 13, the CAR-T cells of Example 2C have a Tscm ratio that is not inferior to that of the non-transduction group, and are expected to reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. Furthermore, as described in the literature Galli E. et al. BrJ Haematol. 2023 Nov;203(4):564-570, the CAR-T cells of Example 2C in Table 13 have a CD8+ T cell ratio of over 50%, indicating excellent therapeutic efficacy in killing cancer cells.

[0127] The group treated with low-dose CAR-T cells from Example 2C showed an antitumor effect in tumor rechallenge (shown in Figures 12A and 12B). Except for the mice treated with low-dose CAR-T cells from Example 2C, most of the mice treated with CAR-T cells developed GvHD upon reinjection of SKOV3-mLE cells. Even in mice administered with CAR-T cells from the Example 2C group, by weakening the BLI signal to below a visually detectable level, Example 2C had a significantly greater antitumor effect, and at 18 days after tumor rechallenge, it was statistically lower than the control group treated with saline.

[0128] As can be seen from the results above, the lentiwill of Example 2B of the present invention has an intracellular signaling domain and contains the transmembrane domain of NKp30 (NKp30 fragment that does not contain an immunoglobulin-like domain), and therefore has an excellent transduction rate. Furthermore, the CAR-T cells obtained by transduction have an excellent cell amplification rate and excellent cytotoxicity, and have a higher proportion of Tscm, so it is expected that cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy can be reduced. In animal experiments, it was verified that it has an excellent tumor cell killing effect, reduces the incidence of graft-versus-host disease, and can avoid the development of cytokine release syndrome (CRS), which can be fatal due to excessive activation of conventional single-chain CAR-T cells. Excessively activated CAR-T cells are also prone to decay and their endurance in the body is reduced, and according to the literature Korell F. et al, Med. 2022 Aug 12;3(8):538-564, this may result in the therapeutic effect not being sustained or an increased possibility of recurrence. As can be inferred from the above, when producing CAR-T cells, simultaneously introducing nucleic acid sequences of intracellular signaling domains, such as the intracellular signaling domain of DAP12, into T cells can result in superior cell-killing effects in the produced CAR-T cells.

[0129] III. Production and testing of CAR-T cells containing an intracellular signaling domain and anti-CD22-NKp30

[0130] Example 3: Nucleic acid molecule having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD22 antibody, and an NKp30 fragment that does not contain an immunoglobulin-like domain.

[0131] The 3' end of a nucleic acid sequence containing the full length of FcεR1γ (indicated by SEQ ID NO: 77) is ligated to the 5' end of a nucleic acid sequence of the DAP12 intracellular signaling domain (SEQ ID NO: 78) to obtain nucleic acid molecular fragment 1. The 3' end of this nucleic acid molecular fragment 1 is then ligated to the 5' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody (indicated by SEQ ID NO: 91) via the nucleic acid sequence of the T2A self-cleaving peptide (SEQ ID NO: 80). Furthermore, the 5' end of the nucleic acid sequence of the hinge domain of CD28 (indicated by SEQ ID NO: 68) is 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 5' end of the nucleic acid sequence of the NKp30 fragment that does not contain the immunoglobulin-like domain (indicated by SEQ ID NO: 86) is ligated to the 3' end of the nucleic acid sequence of the CD28 hinge domain. As indicated by NO:92, a nucleic acid molecule having the intracellular signaling domain of Example 3, a single-chain variable region fragment of the anti-CD22 antibody, and NKp30 was obtained. The nucleic acid molecule of Example 3 consists of the full-length nucleic acid sequence of FcεR1γ, the nucleic acid sequence of the DAP12 intracellular signaling domain, the nucleic acid sequence of the T2A self-cleaving peptide, the nucleic acid sequence of the m971 single-chain variable region fragment, the nucleic acid sequence of the CD28 hinge domain, and the nucleic acid sequence of the NKp30 fragment without an immunoglobulin-like domain (NKp30 without Ig-like domain), from the 3' end to the 5' end, respectively.

[0132] Control Example 3-1: A nucleic acid molecule possessing an intracellular signaling domain and a single-chain variable region fragment of an anti-CD22 antibody, but containing no NKp30 fragments.

[0133] The manufacturing method for Control Example 3-1 is the same as that for 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 (indicated by SEQ ID NO: 91) of the single-chain variable region fragment m971 of the anti-CD22 antibody. The nucleic acid sequence of the obtained nucleic acid molecule of Control Example 3-1 is shown by SEQ ID NO: 93.

[0134] Control Example 3-2: A nucleic acid molecule lacking an intracellular signaling domain and containing a single-chain variable region fragment of an anti-CD22 antibody and an NKp30 fragment lacking an immunoglobulin-like domain.

[0135] The 3' end of the full-length FcεR1γ nucleic acid sequence (indicated by SEQ ID NO: 77) and the 5' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody (indicated by SEQ ID NO: 91) were linked via the nucleic acid sequence of the T2A self-cleaving peptide (SEQ ID NO: 80). Then, the 5' end of the nucleic acid sequence of the CD28 hinge domain (indicated by SEQ ID NO: 68) was linked to the 3' end of the nucleic acid sequence of the single-chain variable region fragment m971 of the anti-CD22 antibody. Finally, the 5' end of the nucleic acid sequence of the NKp30 fragment that does not contain the immunoglobulin-like domain (indicated by SEQ ID NO: 86) was linked to the 3' end of the nucleic acid sequence of the CD28 hinge domain, resulting in the anti-CD22-NKp30 nucleic acid molecule lacking the intracellular signaling domain of control example 3-2, as shown by SEQ ID NO: 94. The nucleic acid molecules in control example 3-2 consist of the full-length FcεR1γ nucleic acid sequence from the 3' end to the 5' end, the T2A self-cleaving peptide nucleic acid sequence, the m971 single-chain variable region fragment nucleic acid sequence, the CD28 hinge domain nucleic acid sequence, and the NKp30 fragment without an immunoglobulin-like domain (NKp30 without Ig-like domain) nucleic acid sequence, respectively.

[0136] Example 3A, Control Example 3-1A, Control Example 3-2A: Construction of plasmids containing nucleic acid molecules from Example 3, Control Example 3-1, and Control Example 3-2.

[0137] The manufacturing method for Example 3A is the same as that for Examples 1-1A and 1-2A, except that Example 3A uses HpaI and EcoRI restriction enzymes to conjugate the nucleic acid molecule of Example 3 to the pLAS5w vector.

[0138] For control example 3-1A, GenScript was instructed to conjugate the nucleic acid molecule of control example 3-1 to the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, catalog number C6-8-39) using NheI and EcoRI restriction enzymes. For control example 3-2A, the nucleic acid molecule of control example 3-2 was conjugated to the aforementioned pLAS5w vector using HpaI and EcoRI restriction enzymes. The obtained plasmids of Example 3A, control example 3-1A, and control example 3-2A are shown in Figure 14.

[0139] Example 3B, Control Example 3-1B, Control Example 3-2B: Production of lentiwills containing nucleic acid molecules from Example 3, Control Example 3-1, and Control Example 3-2.

[0140] The manufacturing method for Example 3B is the same as that for Examples 1-1B and 1-2B, except that the plasmid used in Example 3B is a transfer plasmid. On the other hand, the lentiwills for Control Example 3-1B and Control Example 3-2B were manufactured using the plasmids from Control Example 3-1A and Control Example 3-2A, respectively, as transfer plasmids.

[0141] Test Example 13: Test of Lentiwill titer of Example 3B

[0142] The test method for Test Example 13 is the same as that for Test Example 1, except that the anti-CD22 receptor is identified using biotinylated human CD22, Fc Tag (Sino Biological.Cat:1958-H41H-B), and the expression of the anti-CD22 receptor in lentivirus from Example 3B and control example 3-1B is detected using 500-fold diluted PE-labeled streptavidin (Invitrogen). The test results are shown in Table 14 below.

[0143] Table 14: Lentiwill titers of Example 3B and Control Example 3-1B

[0144] The lentiwill titer is 1 × 10 6 When the TU / mL level is above this, a smaller volume of lentiwill can be used when producing CAR-T cells, allowing for smooth transduction of T cells into CAR-T cells and promoting good growth of T cells after transduction. Therefore, based on the results in Table 14 above, the lentiwill of Example 3B meets this criterion and is advantageous for subsequent CAR-T cell production.

[0145] Example 3C: Production of CAR-T cells containing nucleic acid molecules from Example 3, Control Example 3-1, and Control Example 3-2

[0146] The method for producing CAR-T cells in Example 3C is the same as the production methods in Examples 1-1C and 1-2C, except that the T cells in this example are transdulated with the lentiwill from Example 3B, and the MOI at the time of lentiwill transduction is 3. On the other hand, the CAR-T cells of control examples 3-1C and 3-2C are transdulated with the lentiwill from control examples 3-1B and 3-2B, and the MOI at the time of lentiwill transduction is 3. Schematic structural diagrams of the CAR-T cells obtained in Example 3C and Example 2C are shown in Figure 15.

[0147] Test Example 14: Proliferation study of CAR-T cells and non-transduction T cells in Example 3C, Control Example 3-1C, and Control Example 3-2C.

[0148] The test method for Test Example 14 is the same as that of Test Example 2, except that this test example detects the amplification rate of CAR-T cells in Example 3C and control examples 3-1C and 3-2C, and uses untransductioned activated T cells as the control group. The results obtained are shown in Table 15 below.

[0149] Table 15: Amplification factor of CAR-T cells and non-transduction T cells in Example 3C, Control Examples 3-1C and 3-2C on day 7.

[0150] Since an amplification factor of 5 or more is advantageous for subsequent T cell functionality tests, as can be seen from the results in Table 15 above, the CAR-T cells of Example 3C have a good T cell amplification factor and can be used for subsequent T cell functionality tests.

[0151] Test Example 15: Detection of CAR-T cell expression in Example 3C and control example 3-1C, and the transduction rate of lentivirus in Example 3B and control example 3-1B.

[0152] The test method for Test Example 15 is the same as that of Test Example 3, except that this test example identifies the anti-CD22 receptor using biotinylated human CD22 protein, Fc Tag, (Sino Biological.Cat:1158-H41H-B), and detects the expression of the anti-CD22 receptor in CAR-T cells of Example 3C and control Example 3-1C using 500-fold diluted PE-labeled streptavidin (Invitrogen). The test results for Example 3C and Example 3-1C are shown in the flow cytometer results in Figures 16A, 16B, 16C, and 16D, respectively. The percentage of cells expressing the anti-CD22 receptor relative to the total number of cells is then used as the lentivir transduction rate for Example 3B or control Example 3-1B, and the experimental results are shown in Table 16 below.

[0153] Table 16: Transduction rates of lentwill in Example 3B and Control Example 3-1B

[0154] A transduction rate exceeding 25% for producing CAR-T cells is advantageous for subsequent functional testing of amplified CAR-T cells. As can be seen from the results in Table 16 and Figure 16A above, the lentiwill of Example 3B has a good transduction rate.

[0155] Test Example 16: T cell phenotypic analysis of CAR-T cells from Example 3C

[0156] The test method for Test Example 16 is the same as that of Test Example 4, except that this test example detects CAR-T cells from Example 3C, Control Example 3-1C, and Control Example 3-2C on cells 7 days after production. The proportion of Tscm in CAR-T cells from Example 3C, Control Example 3-1C, and 3-2C, and in T cells from the non-transduction group (control group) is shown in Table 17 below. The results for Example 3C and the flow cytometry results for the subpopulation distribution of control example 3-1C, control example 3-2C, and non-transduction T cells are shown in Figures 17A, 17B, 17C, and 17D, respectively, where T cells expressing CD45RA and CCR7 are Tscm.

[0157] Table 17: Percentage of Tscm in CAR-T cells and non-transduction T cells in Example 3C, Control Example 3-1C, and Control Example 3-2C.

[0158] When the percentage of stem cell-like memory T cells in CAR-T cells is 40% or higher, cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy can be further reduced. As can be seen from Table 17 and Figures 17A, 17B, 17C, and 17D above, Example 3C has a high percentage of Tscm, is slightly superior to the conventional single-chain control example 3-1C CAR-T cells, and is similar to the activated T cells in the non-transduction group, so it is expected to reduce cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy.

[0159] Test Example 17: Real-time cytotoxicity assay using xCELLigence

[0160] First, human CD22 cDNA fragments were selected and propagated from the Raji human lymphoma cell line (purchased from Elabscience, Cat. No. CL-0189). The cleaved CD22 cDNA fragments (indicated by SEQ ID NO: 95) were bound to the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, catalog number C6-8-39) to obtain a vector containing cleaved CD22. The vector containing cleaved CD22 was transfected into the SKOV3 human ovarian cancer cell line using Polyjet (SignaGen Laboratories). The transfected SKOV3 cells were cultured in McCoy 5 A Medium (Gibco) medium containing 2 μg / mL promycin and 10% FBS, and SKOV3 cell lines that produced purmycin resistance and simultaneously expressed CD22 were screened. Finally, the expression of CD22 in the cells was reconfirmed using a flow cytometer. SKOV3 cell lines expressing CD22 were used as target cells for subsequent cytotoxicity assays using the xCELLigence system.

[0161] The test method for Test Example 17 is the same as that for Test Example 5, except that this test example uses CAR-T cells from Example 3C, Control Example 3-1C, and Control Example 3-2C as effector cells, and the SKOV3 cell line expressing CD22 as the target cells. The target cells are seeded on a culture plate with a background correction, background impedance is measured for 20 hours to allow the target cells to adhere, and then the effector cells are added to the culture plate in E:T ratios of 3:1 and 1:1 for testing. The toxicity assay results are shown in Figures 18A and 18B, respectively.

[0162] As can be seen from the results in Figures 18A and 18B, the CAR-T toxicity of Example 3C is superior to that of conventional single-chain CAR-T cells, control example 3-1C, and control example 3-2C, which lacks an intracellular signaling domain. If the CAR-T cell toxicity is >50%, then when CAR-T cells are subsequently produced in patient cells, they can have sufficient toxicity. As can be seen from the results in Figures 18A and 18B, the CAR-T cells of Example 3C have a good cytotoxic effect and are superior to the CAR-T cells of control examples 3-1C and 3-2C.

[0163] Test Example 18: Human Lymphoma Cytotoxicity Assay by Flow Cytometry Analysis

[0164] To test the antitumor activity of anti-CD22-NKp30CAR-T cells with an intracellular signaling domain in Example 3C, this test example used Daudi human lymphoma cells (hereinafter abbreviated as Daudi cells) in CellTrace. TM. Cells were standardized with Violet, cultured in RPMI-1640 medium (Gibco) containing 10% FBS, and used in subsequent toxicity assays by flow cytometry. The effector cells used in this study were CAR-T cells from Example 3C and control examples 3-1C and 3-2C, with untransductioned T cells used as the control group. Specifically, 1.2 × 10⁶ target cells were used. 4 Cells were seeded in a 96-well plate at a cell density of 10 cells / well, effector cells were added, and the ratio of effector cells to target cells (E:T) was adjusted to 1:1, 3:1 or less. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours, and the test was repeated three times. Next, the 96-well plate was centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells in each well were further washed with 200 μL of PBS containing 2% fetal calf serum (FCS). After removing the washing PBS, the cells were collected in 300 μL of liquid buffer (PBS containing 2% FCS) and 15 μL of counting beads (CountBright). TMAfter adding Plus Absolute Counting Beads (Invitrogen, C36995), the number of target cells was counted to collect 3000 beads using a flow cytometer. The cell elimination rate was calculated using the following formula.

[0165] Cell killing rate = (Number of target cells in the group without effector cells - Number of target cells after adding effector cells and culturing together) ÷ Number of target cells in the group without effector cells × 100%

[0166] The experimental results are shown in Figure 19 and Table 18 below (E:T=3:1). Table 18: Results of human lymphoma cytotoxicity assays by flow cytometry analysis of Example 3C and control examples 3-1C and 3-2C.

[0167] As can be seen from the results in Figure 19 and Table 18, when the E:T ratio was 1:1, the cell-killing effect of CAR-T cells in Example 3C was 71%, which is far superior to the conventional single-chain CAR-T cell control example 3-1C (45%), the control example 3-2C (5%) lacking an intracellular signaling domain, and the control group (non-transduction group) (5%). When the E:T ratio was 3:1, the cell-killing effect of CAR-T cells in Example 3C was 85%, which is superior to the conventional single-chain CAR-T cell control example 3-1C (77%), the control example 3-2C (14%) lacking an intracellular signaling domain, and the control group (non-transduction group) (27%). Here, as can be seen from the comparison results between Example 3C and control example 3-2C, increasing the intracellular signaling domain can clearly increase the cancer cell-killing effect.

[0168] As can be seen from the results above, the lentiwill of Example 3B of the present invention has an excellent transduction rate due to containing an intracellular signaling domain and the transmembrane domain of NKp30 (an NKp30 fragment that does not contain an immunoglobulin-like domain), and the CAR-T cells obtained by transduction have an excellent cell amplification rate and excellent cytotoxicity, have a higher proportion of Tscm, and are expected to reduce cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As can be inferred from this, when producing CAR-T cells, transduction of nucleic acid sequences of intracellular signaling domains, such as the intracellular signaling domain of DAP12, can result in excellent cytotoxic effects in the produced CAR-T cells, making them particularly suitable for CAR-T therapy.

[0169] IV. Production and testing of anti-CD19-NKp30CAR-T cells possessing intracellular signaling domains.

[0170] Example 4: Nucleic acid molecule having an intracellular signaling domain, a single-chain variable region fragment of an anti-CD19 antibody, and an NKp30 fragment that does not contain an immunoglobulin-like domain.

[0171] The manufacturing method for Example 4 is the same as that for Example 3, except that the single-chain variable region fragment used in Example 4 is FMC63 (indicated by SEQ ID NO: 96), a single-chain variable region fragment of the anti-CD19 antibody, instead of m971, a single-chain variable region fragment of the anti-CD22 antibody, as used in Example 3, and the selected hinge domain is the hinge domain of human IgG4 (indicated by SEQ ID NO: 69). The nucleic acid sequence of the nucleic acid molecule in Example 4 is shown by SEQ ID NO: 97. The nucleic acid molecule in Example 4 consists of the full-length nucleic acid sequence of FcεR1γ, the nucleic acid sequence of the DAP12 intracellular signaling domain, the nucleic acid sequence of the T2A self-cleaving 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 an immunoglobulin-like domain (NKp30 without Ig-like domain), from the 3' end to the 5' end.

[0172] Control example 4: A nucleic acid molecule containing an intracellular signaling domain and a single-chain variable region fragment of an anti-CD19 antibody, but without any NKp30 fragments.

[0173] The manufacturing method for Control Example 4 is the same as that for Control Example 2, except that the single-chain variable region fragment used in Control Example 4 was the nucleic acid sequence (shown as SEQ ID NO: 96) of the single-chain variable region fragment FMC63 of the anti-CD19 antibody, rather than SS1 of the single-chain variable region fragment of the anti-mesothelin antibody used in Control Example 2. The nucleic acid sequence of the obtained nucleic acid molecule for Control Example 4 is shown as SEQ ID NO: 98.

[0174] Example 4A: Construction of plasmids containing nucleic acid molecules from Example 4 and Control Example 4

[0175] The manufacturing method for Example 4A is the same as that for Examples 1-1A and 1-2A, except that Example 4A uses HpaI and EcoR1 restriction enzymes to bind and access the anti-CD19-NKp30 nucleic acid molecule having the intracellular signaling domain of Example 4 to the pLAS5w vector.

[0176] For control example 4A, GenScript was instructed to conjugate the nucleic acid molecule of control example 4 to the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core, catalog number C6-8-39) using NheI and EcoR restriction enzymes. Figure 20 shows the plasmid of example 4 and the plasmid of control example 4A obtained using the above manufacturing method.

[0177] Example 4B, Control Example 4B: Production of lenticular molecules of Example 4 or Control Example 4

[0178] The manufacturing method for Example 4B is the same as that for Examples 1-1B and 1-2B, except that the plasmid used in Example 4A is a transfer plasmid. On the other hand, the lentiwill for control example 4B was manufactured using the plasmid from control example 4A as a transfer plasmid.

[0179] Test Example 19: Example 4B, Test of Lentiwill Titer of Example 4B

[0180] The test method for Test Example 19 is the same as that for Test Example 1, except that the anti-CD19 receptor is identified using biotinylated human CD19 and Fc Tag (ACRObiosystems, CD9-H 8259), and the expression of the anti-CD19 receptor in lentiwill from Example 4B and control Example 4B is detected using 500-fold diluted PE-labeled streptavidin (Invitrogen). The test results are shown in Table 19 below.

[0181] Table 19: Lentiwill titers (transduction units, TU / mL) of Example 4B and Control Example 4B

[0182] The lentiwill titer is 1 × 10 6When the TU / mL level is above this, a smaller volume of lentiwill can be used when producing CAR-T cells, allowing for smooth transduction of T cells into CAR-T cells and promoting good growth of T cells after transduction. Therefore, as can be seen from the results in Table 19 above, the lentiwill of Example 4B meets this criterion and is advantageous for subsequent CAR-T cell production.

[0183] Example 4C, Control Example 4C: Production of CAR-T cells containing nucleic acid molecules from Example 4 and Control Example 4.

[0184] The method for producing CAR-T cells in Example 4C is the same as the production methods in Examples 1-1C and 1-2C, except that the T cells in this example are transdulated with the lentiwill of Example 4B, and the MOI during the transduction of lentiwill is 2. On the other hand, the CAR-T cells of Control Example 4C are transdulated with the lentiwill of Control Example 4C, and the MOI during the transduction of lentiwill is 2. Schematic diagrams of the structures of the obtained CAR-T cells of Example 4C and Control Example 4C are shown in Figure 21.

[0185] Test Example 20: CAR-T cell proliferation test of Example 4C and control example 4C

[0186] The test method in this example is the same as in Example 2, except that this example detects the amplification rate of CAR-T cells in Example 4C and Control Example 4C, and that untransdulated active T cells are designated as the control group (non-transduction group). The results obtained are shown in Table 20 below.

[0187] Table 20: Amplification factor of CAR-T cells on day 7 in Example 4C and Control Example 4C

[0188] Since an amplification factor of 5 or more is advantageous for subsequent T cell functionality tests, as can be seen from the results in Table 20 above, the CAR-T cells of Example 4C have a good T cell amplification factor and can be used for subsequent T cell functionality tests.

[0189] Test Example 21: Detection of CAR-T cell expression in Example 4C and Control Example 4C, and conductivity of Example 4B and Control Example 4C

[0190] The test method for Test Example 21 is the same as that of Test Example 3, except that this test example identifies the anti-CD19 receptor using biotinylated human CD19 and Fc Tag (ACROBIosystems, CD9-H 8259), and detects the expression of the anti-CD19 receptor in CAR-T cells of Example 4C and Control Example 4C using 500-fold diluted PE-labeled streptavidin (Invitrogen). The test results for Example 4C and Control Example 4C are shown in the flow cytometer results in Figures 22A, 22B, 22C, and 22D, respectively. The percentage of cells expressing the anti-CD19 receptor relative to the total number of cells is then expressed as the lentivir transduction rate for Example 4B or Control Example 4B, and the experimental results are shown in Table 21 below.

[0191] Table 21: Transduction rates of lentwill in Example 4B and Control Example 4B

[0192] A transduction rate exceeding 25% for producing CAR-T cells is advantageous for subsequent functional testing of amplified CAR-T cells. As can be seen from the results in Table 21 and Figure 22A above, the lentiwill of Example 4B has a good transduction rate.

[0193] Test Example 22: T cell phenotypic analysis of CAR-T cells from Example 4C

[0194] The test method for Test Example 22 is the same as that for Test Example 4, except that this test example uses CAR-T cells from Example 4C, specifically cells 7 days after production, for detection. The proportion of Tscm in CAR-T cells from Example 4C, control Example 4C, and the T cells from the non-transduction group is shown in Table 22 below. The results for Example 4C and the flow cytometry results for the subpopulation distribution of T cells in Example 4C and the non-transduction group are shown in Figures 23A, 23B, and 23C, respectively, where T cells expressing CD45RA and CCR7 are Tscm.

[0195] Table 22: Percentage of Tscm in CAR-T cells and non-transduction T cells in Example 4C and Control Example 4C.

[0196] If the proportion of stem cell-like memory T cells in CAR-T cells is 40% or higher, cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy can be further reduced. As can be seen from Table 22 and Figures 23A, 23B, and 23C above, Example 4C has more than 40% Tscm and is similar to conventional single-chain CAR-T cells, so it is expected to reduce cytokine release syndrome and neurotoxicity induced by CAR-T cell therapy.

[0197] Test Example 23: Real-time cytotoxicity assay using xCELLigence

[0198] Human CD19 cDNA fragments were selected and propagated from the Raji human lymphoma cell line. The cleaved CD19 cDNA fragment (SEQ ID NO: 99) was amplified using PCR. Here, the cleaved CD19 cDNA fragment (1-342 amino acids) refers to a fragment that has an extracellular domain and a transmembrane domain but lacks an intracellular domain and a cytoplasmic signaling domain. The cleaved CD19 cDNA fragment was bound to a pLAS5w.Ppuro vector (purchased from RNAi Core, Academia Sinica, catalog number C6-8-39) containing a primycin drug resistance gene using HpaI and NheI restriction enzyme sites to obtain a vector containing the cleaved CD19. The vector containing the cleaved CD19 was transfected into the SKOV3 human ovarian cancer cell line using Polyjet (SignaGen Laboratories, SL 100688). Transfected SKOV3 cells were cultured in McCoy 5A Medium (Gibco) medium containing 2 μg / mL promycin and 10% FBS, and SKOV3 cell lines exhibiting drug resistance and co-expressing CD19 were screened. Finally, CD19 expression in the cells was reconfirmed using flow cytometry. SKOV3 cell lines expressing CD19 were used as target cells for subsequent cytotoxicity assays using the xCELLigence system.

[0199] The test method for this example involved using CAR-T cells from Example 4C and Control Example 4C as effector cells, and the SKOV3 cell line expressing CD19 as the target cells. The target cells were seeded into a culture plate with corrected background, background impedance was measured for 20 hours, and after the target cells had attached, the effector cells were added to the culture plate in a 1:1 E:T ratio for testing. Furthermore, 100 μL (microliters) of 1% Triton X-100 was added in 2 × 10⁻¹⁶ units. 4The procedure is the same as in Test Example 5, except that cells / mL are added to the culture medium of the target cells to form a completely lysated group. The results of the toxicity assays are shown in Figure 24. On the other hand, the results of the toxicity assay performed for 48 hours are shown in Table 23.

[0200] Table 23: Cytotoxicity assay results for Example 4C and Control Example 4C

[0201] If CAR-T cells have a toxicity of 50% or more, they can have sufficient toxicity when subsequently used to produce CAR-T cells from the patient's cells. As can be seen from the results in Figure 24 and Table 23, the CAR-T cells of Example 4C achieved a cancer cell killing effect of 80% or more after being cultured with target cells for 48 hours, and this is slightly better than the CAR-T cell killing effect of the control example 4C of conventional single-chain CAR-T cells. Therefore, it can be inferred that the CAR-T cells of Example 4C have sufficient toxicity when subsequently used to produce CAR-T cells from the patient's cells.

[0202] Test Example 24: Cold Phototoxicity Assay

[0203] The test method for Test Example 24 is the same as that for Test Example 18, except that this test example uses CAR-T cells from Example 4C and Control Example 4C as effector cells, and the Raji cell line expressing the aforementioned green fluorescent protein and cold-light enzyme as target cells, and the effector cells and target cells are tested in E:T ratios of 1:1 and 3:1, respectively, and the culture time with the effector cells and target cells is 24 hours. The toxicity assay results are shown in Figure 25.

[0204] As shown in Figure 25, the experimental results indicate that, regardless of whether the E:T ratio was 1:1 or 3:1, the CAR-T cell killing effect of Example 4C exceeded 50%, and was clearly superior to the killing effect of the single-chain CAR-T cells of the control Example 4C.

[0205] As can be seen from the results above, the lentiwill of Example 4B of the present invention has an excellent transduction rate because it contains an NKp30 fragment that includes an intracellular signaling domain and a transmembrane domain of NKp30 but does not contain an immunoglobulin-like domain. Furthermore, the CAR-T cells obtained by transduction have an excellent CAR-T cell amplification rate and excellent cytotoxicity, and are expected to have a higher Tscm ratio, thereby reducing cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. As can be inferred from this, when producing CAR-T cells, transduction of nucleic acid sequences of intracellular signaling domains, such as the intracellular signaling domain of DAP12, can result in excellent cytotoxic effects in the produced CAR-T cells, making them particularly suitable for CAR-T therapy.

[0206] As can be seen from the above, when genes for intracellular signaling domains such as the DAP12 intracellular signaling domain, the 41BB intracellular signaling domain, or the CD28 intracellular signaling domain, as well as the transmembrane domain and cytoplasmic domain of NKp30, are transductioned into T cells, the T cells acquire the NKp30 receptor complex, become heavy chain CAR-T cells, and exhibit excellent cancer cell toxicity, regardless of whether the study is conducted in vitro or in vivo.

[0207] The specific examples described above have provided further details regarding the objectives, technical proposals, and beneficial effects of the present invention. However, these are merely specific examples of the present invention and do not limit it. It should be understood that any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the present invention should all be included within the scope of protection of the present invention.

Claims

1. (a) Extracellular antigen-binding domain, (b) Hinge domain and (c) The transmembrane domain of Natural Killer Protein 30 (NKp30), (d) The cytoplasmic domain of natural killer protein 30 (NKp30) and (e) An isolated nucleic acid molecule characterized by containing a nucleic acid sequence of a fragment with an intracellular signaling domain.

2. The isolated nucleic acid molecule according to claim 1, characterized in that the extracellular antigen-binding domain includes a heavy chain variable region.

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

4. The aforementioned hinge domain is (G ４ S) ３ CD8 hinge domain, CD28 hinge domain, IgG4 hinge domain, EAAAKGGGGGS, (EAAAAK) ３ , the hinge domain of GST, the hinge domain of EAAAK-GS, the hinge domain of IgD, the IgG4-CH3 domain, and (AP) ６ The isolated nucleic acid molecule according to claim 1, characterized in that it is selected from the group consisting of the following.

5. The isolated nucleic acid molecule according to claim 1, characterized in that the intracellular signaling domain is the intracellular signaling domain of DAP12, 41BB, or CD28.

6. The isolated nucleic acid molecule according to claim 1, characterized in that the hinge domain and the transmembrane domain of NKp30 are connected via the NKp30 cell stalk structure (stalk) region.

7. The extracellular antigen-binding domain comprises the heavy chain variable region of the anti-mesothelin antibody, and the hinge domain is (G ４ S) ３ The isolated nucleic acid molecule according to claim 1, characterized in that the extracellular antigen-binding domain includes the heavy chain variable region of an anti-CD22 antibody, the hinge domain is the hinge domain of CD28, the extracellular antigen-binding domain includes 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 according to claim 1, further comprising the nucleic acid sequence of an adapter gene.

9. A plasmid comprising an 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 producing chimeric antigen receptor cells, characterized by comprising introducing an isolated nucleic acid molecule according to any one of claims 1 to 8 into nucleated cells.

11. The method for producing chimeric antigen receptor cells according to claim 10, characterized in that the introduction is performed by transduction of lentiwill.

12. An isolated cell characterized by containing an isolated nucleic acid molecule according to any one of claims 1 to 8.

13. The isolated cells according to claim 12, characterized in that the isolated cells are T cells, natural killer cells, natural killer T cells, or adipose-derived stem cells.

14. The use of the isolated cells according to claim 12 or 13, characterized by manufacturing a pharmaceutical product for treating or alleviating cancer.

15. The use of the isolated cells according to claim 12 or 13, characterized by manufacturing a pharmaceutical product for treating or alleviating an autoimmune disease, which is systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, systemic sclerosis, or multiple sclerosis.

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