Isolated nucleic acid molecule containing nkp30 transmembrane domain and chimeric antigen receptor containing the same

Abstract

To provide an isolated nucleic acid molecule containing an NKp30 transmembrane domain, and to provide a chimeric antigen receptor containing the same.SOLUTION: An isolated nucleic acid molecule containing an NKp30 transmembrane domain comprises: nucleic acid sequences encoding (a) an extracellular antigen-binding domain comprising a heavy chain variable region; (b) a hinge domain; (c) an NKp30 transmembrane domain; and (d) an NKp30 cytoplasmic domain. By introducing the nucleic acid sequences of the NKp30 transmembrane domain and the NKp30 cytoplasmic domain in combination with the extracellular antigen-binding domain into a T cell, the resulting CAR-T cell becomes a multi-chain CAR-T cell containing an NKp30 receptor complex. Therefore, the resulting CAR-T cell forms stable immune synapses with cancer cells and exhibits excellent cytotoxicity against cancer cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a nucleic acid molecule for preparing a chimeric antigen receptor (CAR), in particular, to a nucleic acid molecule for preparing a CAR that exhibits excellent cytotoxicity against cancer cells. The present invention also relates to a CAR containing the nucleic acid molecule and applications of the CAR containing the nucleic acid molecule. [Background technology]

[0002] Cancer is caused by the multi-step transformation of normal cells into tumor cells, usually resulting from the gradual progression of mild precancerous lesions into malignant tumors and metastasis. This transformation is not caused by a single factor, but rather occurs as a result of the interaction between an individual's genetic factors and external factors.

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

[0004] Cancer not only causes physical and mental pain to patients, but also causes severe mental and financial stress to their families. However, in the high-stress environment of modern society, it is difficult to completely eliminate harmful external factors, and the number of cancer patients continues to increase year by year.

[0005] Thus, there remains an urgent need for effective cancer treatments. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] CAR T therapy beyond cancer: the evolution of a living drug,Daniel J.Baker et al.,Nature,vol.619,pp.707-715,2023 Summary of the Invention [Problem to be solved by the invention]

[0007] To overcome the problems of the prior art, the present invention has the objective or advantage of providing a novel chimeric antigen receptor (CAR) design for CAR-T cells, which when introduced into the human body, engineered T cells bearing a specific antigen binding domain can identify tumor cells expressing that antigen and kill the tumor cells by cytotoxicity. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, the present invention provides an isolated nucleic acid molecule having a nucleic acid sequence encoding (a) an extracellular antigen-binding domain having a heavy chain variable region, (b) a hinge domain, (c) a natural killer protein 30 (NKp30) transmembrane domain, and (d) an NKp30 cytoplasmic domain.

[0009] In the present invention, the nucleic acid sequence of the NKp30 transmembrane domain and the NKp30 cytoplasmic domain are introduced into T cells to cause the T cells to express an NKp30 receptor complex, thereby forming multi-chain CAR-T cells. The CAR-T cells then form a stable immune synapse with cancer cells and exert excellent cytotoxicity against the cancer cells.

[0010] Preferably, the extracellular antigen-binding domain comprises the heavy chain variable region of an anti-mesothelin (MSLN) 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-BCMA (B cell maturation antigen) 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 of the invention, the extracellular antigen-binding domain may be selected from a heavy chain variable region derived from a heavy chain antibody (VHH) or a single chain variable fragment (scFv), the heavy chain variable region derived from a heavy chain antibody may be derived from a single chain binding region of a camelid heavy chain antibody.

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

[0013] In one embodiment of the invention, the extracellular antigen binding domain may comprise a first heavy chain variable region of an anti-BCMA heavy chain antibody and a second heavy chain variable region of an anti-BCMA heavy chain antibody, the first heavy chain variable region and the second heavy chain variable region being linked by a G4S peptide encoding the amino acid sequence of SEQ ID NO:1. The first heavy chain variable region of the anti-BCMA heavy chain antibody encodes the amino acid sequence of SEQ ID NO:2 and the second heavy chain variable region of the anti-BCMA heavy chain antibody encodes the amino acid sequence of SEQ ID NO:3. The first heavy chain variable region of the anti-BCMA heavy chain antibody has the complementarity determining regions (CDRs) shown below: CDRH1 encoding the amino acid sequence of SEQ ID NO:4, CDRH2 encoding the amino acid sequence of SEQ ID NO:5 and CDRH3 encoding the amino acid sequence of SEQ ID NO:6. The second heavy chain variable region of the anti-BCMA heavy chain antibody has the CDRs shown below. CDRH1 encodes the amino acid sequence of SEQ ID NO:7, CDRH2 encodes the amino acid sequence of SEQ ID NO:8, and CDRH3 encodes the amino acid sequence of SEQ ID NO:9.

[0014] In one embodiment of the present invention, the extracellular antigen-binding domain may comprise a heavy chain variable region of an anti-CD19 antibody. The heavy chain variable region of the anti-CD19 antibody has the following CDRs: CDRH1 encoding the amino acid sequence of SEQ ID NO:10, CDRH2 encoding the amino acid sequence of SEQ ID NO:11, and CDRH3 encoding the amino acid sequence of SEQ ID NO:12. In one embodiment of the present invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-CD19 antibody, which has the following CDRs: CDRL1 encoding the amino acid sequence of SEQ ID NO:13, CDRL2 encoding the amino acid sequence of SEQ ID NO:14, and CDRL3 encoding the amino acid sequence of SEQ ID NO:15. In one embodiment of the present invention, the extracellular antigen-binding domain may be FMC63scFv encoding the amino acid sequence of SEQ ID NO:16.

[0015] In one embodiment of the invention, the extracellular antigen-binding domain may comprise a heavy chain variable region of an anti-mesothelin antibody, which has the following CDRs: CDRH1 encoding the amino acid sequence of SEQ ID NO:17, CDRH2 encoding the amino acid sequence of SEQ ID NO:18, and CDRH3 encoding the amino acid sequence of SEQ ID NO:19. In one embodiment of the invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-mesothelin antibody, which has the following CDRs: CDRL1 encoding the amino acid sequence of SEQ ID NO:20, CDRL2 encoding the amino acid sequence of SEQ ID NO:21, and CDRL3 encoding the amino acid sequence of SEQ ID NO:22. In one embodiment of the invention, the extracellular antigen-binding domain may be SS1scFv encoding the amino acid sequence of SEQ ID NO:23.

[0016] In one embodiment of the invention, the extracellular antigen binding domain may comprise a heavy chain variable region of an anti-BCMA antibody, the heavy chain variable region of the anti-BCMA antibody having the CDRs shown below: CDRH1 encoding the amino acid sequence of SEQ ID NO:24, CDRH2 encoding the amino acid sequence of SEQ ID NO:25, and CDRH3 encoding the amino acid sequence of SEQ ID NO:26. In one embodiment of the invention, the extracellular antigen binding domain may further comprise a light chain variable region of an anti-BCMA antibody, the light chain variable region of the anti-BCMA antibody having the CDRs shown below: CDRL1 encoding the amino acid sequence of SEQ ID NO:27, CDRL2 encoding the amino acid sequence of SEQ ID NO:28, and CDRL3 encoding the amino acid sequence of SEQ ID NO:29. In one embodiment of the invention, the extracellular antigen binding domain may be J6M0scFv encoding the amino acid sequence of SEQ ID NO:30. In another embodiment of the invention, the heavy chain variable region of the anti-BCMA antibody has the CDRs shown below. CDRH1 encodes the amino acid sequence of SEQ ID NO:31, CDRH2 encodes the amino acid sequence of SEQ ID NO:32 and CDRH3 encodes the amino acid sequence of SEQ ID NO:33. In one embodiment of the invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-BCMA antibody, the light chain variable region of the anti-BCMA antibody having the following CDRs: CDRL1 encodes the amino acid sequence of SEQ ID NO:34, CDRL2 encodes the amino acid sequence of SEQ ID NO:35 and CDRL3 encodes the amino acid sequence of SEQ ID NO:36. In one embodiment of the invention, the extracellular antigen-binding domain may be BB2121scFv encoding the amino acid sequence of SEQ ID NO:37.

[0017] In one embodiment of the invention, the extracellular antigen-binding domain may comprise a heavy chain variable region of an anti-CD22 antibody, which has the following CDRs: CDRH1 encoding the amino acid sequence of SEQ ID NO:38, CDRH2 encoding the amino acid sequence of SEQ ID NO:39, and CDRH3 encoding the amino acid sequence of SEQ ID NO:40. In one embodiment of the invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-CD22 antibody, which has the following CDRs: CDRL1 encoding the amino acid sequence of SEQ ID NO:41, CDRL2 encoding the amino acid sequence of SEQ ID NO:42, and CDRL3 encoding the amino acid sequence of SEQ ID NO:43. In one embodiment of the invention, the extracellular antigen-binding domain may be m971scFv encoding the amino acid sequence of SEQ ID NO:44. In another embodiment of the invention, the extracellular antigen-binding domain may comprise a heavy chain variable region of an anti-CD22 antibody, the heavy chain variable region of the anti-CD22 antibody having the following CDRs: CDRH1 encoding the amino acid sequence of SEQ ID NO:45, CDRH2 encoding the amino acid sequence of SEQ ID NO:46, and CDRH3 encoding the amino acid sequence of SEQ ID NO:47. In one embodiment of the invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-CD22 antibody, the light chain variable region of the anti-CD22 antibody having the following CDRs: CDRL1 encoding the amino acid sequence of SEQ ID NO:48, CDRL2 encoding the amino acid sequence of SEQ ID NO:49, and CDRL3 encoding the amino acid sequence of SEQ ID NO:50. In one embodiment of the invention, the extracellular antigen-binding domain may be LL2scFv encoding the amino acid sequence of SEQ ID NO:51. In another embodiment of the invention, the extracellular antigen-binding domain may comprise the heavy chain variable region of an anti-CD22 antibody, which has the CDRs shown below.CDRH1 encodes the amino acid sequence of SEQ ID NO:52, CDRH2 encodes the amino acid sequence of SEQ ID NO:53, and CDRH3 encodes the amino acid sequence of SEQ ID NO:54. In one embodiment of the invention, the extracellular antigen-binding domain may further comprise a light chain variable region of an anti-CD22 antibody, which has the CDRs shown below: CDRL1 encodes the amino acid sequence of SEQ ID NO:55, CDRL2 encodes the amino acid sequence of SEQ ID NO:56, and CDRL3 encodes the amino acid sequence of SEQ ID NO:57. In one embodiment of the invention, the extracellular antigen-binding domain may be a G5 / 44scFv encoding the amino acid sequence of SEQ ID NO:58. In another embodiment of the invention, the extracellular antigen-binding domain may further comprise a heavy chain variable region of an anti-CD22 antibody, which has the CDRs shown below: CDRH1 encodes the amino acid sequence of SEQ ID NO:59, CDRH2 encodes the amino acid sequence of SEQ ID NO:60, and CDRH3 encodes the amino acid sequence of SEQ ID NO:61. In one embodiment of the invention, the extracellular antigen-binding domain may comprise a light chain variable region of an anti-CD22 antibody, which has the following CDRs: CDRL1 encodes the amino acid sequence of SEQ ID NO:62, CDRL2 encodes the amino acid sequence of SEQ ID NO:63, and CDRL3 encodes the amino acid sequence of SEQ ID NO:64. In one embodiment of the invention, the extracellular antigen-binding domain may be RFB4scFv encoding the amino acid sequence of SEQ ID NO:65.

[0018] Preferably, the extracellular antigen-binding domain may have a heavy chain variable region of an anti-CD19 antibody, and the extracellular antigen-binding domain may be FMC63scFv.

[0019] Preferably, the extracellular antigen-binding domain may have a heavy chain variable region of an anti-mesothelin antibody, and the extracellular antigen-binding domain may be SS1scFv.

[0020] Preferably, the extracellular antigen-binding domain may have a heavy chain variable region of an anti-BCMA antibody, and the extracellular antigen-binding domain may be J6M0scFv or BB2121scFv.

[0021] Preferably, the extracellular antigen-binding domain may have a heavy chain variable region of an anti-CD22 antibody, and the extracellular antigen-binding domain may be m971scFv, LL2scFv, G5 / 44scFv, or RFB4scFv.

[0022] Preferably, said hinge domain may be selected from the group consisting of (G4S)3 (shown as SEQ ID NO:66), CD8 hinge domain (shown as SEQ ID NO:67), CD28 hinge domain (shown as SEQ ID NO:68), IgG4 hinge domain (shown as SEQ ID NO:69), EAAAKGGGGS (shown as SEQ ID NO:70), (EAAAK)3 (shown as SEQ ID NO:71), GST hinge domain (shown as SEQ ID NO:72), EAAAK-GS hinge domain (shown as SEQ ID NO:73), IgD hinge domain (shown as SEQ ID NO:74), IgG4-CH3 domain (shown as SEQ ID NO:75) and (AP)6 (shown as SEQ ID NO:76).

[0023] Preferably, the isolated nucleic acid molecule may further comprise a nucleic acid sequence encoding an adaptor. More preferably, the adaptor may be CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / γ. The present invention employs full-length proteins of CD3ζ, DAP10, CD79, FcεR1γ, FcRγ, or Fcβ / γ as the adaptor.

[0024] Preferably, the isolated nucleic acid molecule may further comprise a nucleic acid sequence encoding the signal transduction domain of CD3ζ, a nucleic acid sequence encoding an FcεR1γ adaptor, or a nucleic acid sequence encoding a T2A self-cleaving peptide. For example, the nucleic acid sequence encoding an FcεR1γ adaptor may be located at the 5' end of the isolated nucleic acid molecule, and the nucleic acid sequence encoding a T2A self-cleaving peptide may be located between the nucleic acid sequence encoding the FcεR1γ adaptor and the nucleic acid sequence encoding the extracellular antigen-binding domain.

[0025] Preferably, the hinge domain and the NKp30 transmembrane domain are linked by the NKp30 stalk domain.

[0026] Preferably, in the isolated nucleic acid molecule, when the extracellular antigen binding domain has the heavy chain variable region of anti-CD19 antibody and the hinge domain is (EAAAK)3, the specific combination of the extracellular antigen binding domain and the hinge domain results in a lentivirus with good transduction efficiency. The CAR-T (CAR-T cell) produced using this specific combination proliferates faster, has a higher proportion of stem cell-like memory T cells (Tscm cells), and shows stronger cytotoxicity. More preferably, the heavy chain variable region of the anti-CD19 antibody may be a fragment of FMC63scFv.

[0027] Preferably, in the isolated nucleic acid molecule, when the extracellular antigen binding domain has the heavy chain variable region of anti-mesothelin antibody and the hinge domain is (G4S)3, this specific combination of the extracellular antigen binding domain and the hinge domain can produce a lentivirus with good transduction efficiency. The CAR-T produced using this specific combination grows faster, has a higher proportion of Tscm cells, and shows stronger cytotoxicity. More preferably, the heavy chain variable region of the anti-mesothelin antibody can be a fragment of SS1scFv.

[0028] Preferably, in the isolated nucleic acid molecule, when the extracellular antigen binding domain has the heavy chain variable region of anti-BCMA antibody and the hinge domain is the hinge of CD28, the specific combination of the extracellular antigen binding domain and the hinge domain can produce a lentivirus with good transduction efficiency.The CAR-T produced by using this specific combination grows faster, has a higher proportion of Tscm cells, and shows stronger cytotoxicity.More preferably, the heavy chain variable region of the anti-BCMA antibody can be a fragment of J6M0scFv.

[0029] In order to achieve the above object, the present invention further provides a plasmid comprising the above isolated nucleic acid molecule, which may be a DNA plasmid or an RNA plasmid. According to the present invention, the order of the nucleic acid fragment from the 5' end to the 3' end of the plasmid may be FcεR1γ adaptor, T2A self-cleaving peptide, extracellular antigen binding domain, hinge domain, NKp30 transmembrane domain, and NKp30 cytoplasmic domain. In one embodiment of the present invention, the order of the nucleic acid fragment from the 5' end to the 3' end of the plasmid may be 5'LTR, promoter, FcεR1γ adaptor, T2A self-cleaving peptide, extracellular antigen binding domain, hinge domain, NKp30 transmembrane domain, NKp30 cytoplasmic domain, and 3'LTR.

[0030] In order to achieve the above-mentioned object, the present invention further provides a method for preparing a chimeric antigen receptor cell, specifically a chimeric antigen receptor gene-modified cell, which comprises the step of introducing the above-mentioned isolated nucleic acid molecule into a nucleated cell.

[0031] Preferably, in the method for preparing a chimeric antigen receptor cell, the step of introducing is accomplished by lentiviral transduction.

[0032] Preferably, the nucleated cells may be white blood cells.

[0033] Preferably, the nucleated cells may be T cells.

[0034] To achieve the above object, the present invention further provides an isolated cell comprising the above isolated nucleic acid molecule.

[0035] Preferably, the isolated cells may comprise T cells, natural killer cells, natural killer T cells or adipose derived stem cells (ADSCs).

[0036] In order to achieve the above object, the present invention further provides the use of the isolated cells for preparing a medicament for treating or alleviating cancer. That is, the present invention further provides a pharmaceutical composition for treating or alleviating cancer, comprising an effective amount of the isolated cells. For example, the cancer includes, but is not limited to, B-cell acute lymphoblastic leukemia, B-cell lymphoma, Leydig cell tumor, ovarian cancer, acute myeloid leukemia, mesothelioma, colon cancer, lung cancer, pancreatic cancer, thyroid cancer, and / or multiple myeloma. Specifically, the use refers to a method of utilizing the isolated cells for preparing a medicament for treating or alleviating cancer.

[0037] In order to achieve the above object, the present invention further provides a method for treating or alleviating cancer, the method comprising administering to a subject in need thereof a drug comprising an effective amount of the above-mentioned isolated cells.

[0038] In order to achieve the above object, the present invention further provides a use of the isolated cells for preparing a drug for treating or alleviating an autoimmune disease. That is, the present invention further provides a pharmaceutical composition for treating or alleviating an autoimmune disease, the pharmaceutical composition comprising an effective amount of the isolated cells, and the autoimmune disease may be a disease related to an autoimmune reaction treatable by CD19CAR-T cells. In one embodiment of the present invention, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy, systemic sclerosis, or multiple sclerosis. Specifically, the use refers to a method of utilizing the isolated cells for preparing a drug for treating or alleviating an autoimmune disease.

[0039] In order to achieve the above object, the present invention further provides a method for treating or alleviating an autoimmune disease, the method comprising administering to a subject in need thereof a drug comprising an effective amount of the above-mentioned isolated cells.

[0040] The advantages of the present invention are described below. In the present invention, a nucleic acid sequence of the NKp30 transmembrane domain and an NKp30 cytoplasmic domain are introduced into T cells to express a complex containing a component of the NKp30 receptor in the T cells, resulting in a multi-chain CAR-T cell. Such CAR-T cells are expected to be able to effectively kill cancer cells by forming a stable immune synapse with the cancer cells. Not only do these CAR-T cells significantly reduce the number of live cancer cells, but their antitumor effect is sustained, preventing the recurrence of cancer cells and the growth of tumors. In addition, the obtained CAR-T cells are expected to possess a high proportion of Tscm cells, reducing the cytokine release syndrome and neurotoxicity caused by such treatment.

[0041] Other objects, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram of the pLAS5w vector. [Diagram 2] FIG. 2 is a schematic diagram of the plasmids of Examples 1-1A to 1-5A and Comparative Example 1A. [Diagram 3] FIG. 3 is a schematic diagram of the cell membrane protein structure for the CAR-T cells of Examples 1-1C to 1-5C and Comparative Example 1C. [Figure 4]Figures 4A and 4B are diagrams showing the expression of anti-CD19 receptors and NKp30 receptors of CAR-T of Examples 1-4C, respectively, as determined by flow cytometry analysis in Test Example 3. Figures 4C and 4D are diagrams showing the expression of anti-CD19 receptors and NKp30 receptors of CAR-T of Examples 1-5C, respectively, as determined by flow cytometry analysis in Test Example 3. Figures 4E and 4F are diagrams showing the expression of anti-CD19 receptors and NKp30 receptors of CAR-T of Comparative Example 1C, respectively, as determined by flow cytometry analysis in Test Example 3. [Diagram 5] 5A, 5B, and 5C are diagrams showing the distribution of T cell subpopulations of CAR-T in Examples 1-5C, Comparative Example 1C, and the non-transduced group analyzed by flow cytometry in Test Example 4, respectively. [Figure 6] FIG. 6 shows the cytotoxicity rates of CAR-T cells of Examples 1-5C and Comparative Example 1C and T cells of the non-transduced group against luciferase-expressing Raji cells when the E / T ratio was 3:1 in Test Example 5, determined using a luciferase killing assay. [Figure 7] Figure 7A shows the real-time change in normalized cell index signal, which represents the number of viable CD19-expressing cancer cells, determined using a real-time cytotoxicity assay in response to CAR-T cells of Examples 1-5C and Comparative Example 1C and T cells of the non-transduced group at an E / T ratio of 0.1 to 1 in Test Example 6. Figure 7B shows the cytotoxicity rate of CAR-T cells of Examples 1-5C and Comparative Example 1C and T cells of the non-transduced group against CD19-expressing SKOV3 cells 72 hours after treatment at an E / T ratio of 0.1:1 in Test Example 6. [Figure 8] FIG. 8 is a schematic diagram of the plasmids of Example 2A and Comparative Example 2A. [Figure 9] Figures 9A and 9B are schematic diagrams of the cell membrane protein structures of the CAR-T cells of Example 2C and Comparative Example 2C, respectively. [Figure 10]Figures 10A and 10B are diagrams showing the expression of anti-mesothelin receptor and NKp30 receptor of CAR-T of Example 2C, respectively, as determined by flow cytometry analysis in Test Example 8. Figures 10C and 10D are diagrams showing the expression of anti-mesothelin receptor and NKp30 receptor of CAR-T of Comparative Example 2C, respectively, as determined by cytometry analysis in Test Example 8. [Figure 11] 11A, 11B, and 11C are diagrams showing the distribution of T cell subpopulations of CAR-T in Example 2C, Comparative Example 2C, and the non-transduced group analyzed by flow cytometry in Test Example 10, respectively. [Figure 12] Figures 12A, 12B, and 12C show real-time changes in normalized cell index signals, representing the number of live HeLa cells, in response to CAR-T cells of Example 2C and Comparative Example 2C and T cells of the non-transduced group at E / T ratios of 3:1, 1:1, and 0.3:1, respectively, in Test Example 11. [Figure 13] Figures 13A, 13B, and 13C are figures showing the original, the first repetition, and the second repetition, respectively, in Test Example 12, when fresh live Hela cancer cells were repeatedly administered once a day to the CAR-T cells of Example 2C and Comparative Example 2C and the T cells of the non-transduced group at an E / T ratio of 1:1, and show the cytotoxicity of CAR-T as shown by the real-time change in the normalized cell index signal representing the number of live Hela cells. [Figure 14] FIG. 14 is a schematic diagram of the plasmids of Example 3A and Comparative Example 3A. [Figure 15] FIG. 15 is a schematic diagram of the cell membrane protein structure for the CAR-T cells of Example 3C and Comparative Example 3C. [Figure 16] Figures 16A and 16B are diagrams showing the expression of anti-BCMA receptors and NKp30 receptors of CAR-T of Example 3C as determined by flow cytometry analysis in Test Example 15. Figure 16C is a diagram showing the expression of anti-BCMA receptors of CAR-T of Comparative Example 3C as determined by flow cytometry analysis in Test Example 15. [Figure 17] Figures 17A, 17B, and 17C show the distribution of T cell subpopulations of CAR-T in Example 3C, Comparative Example 3C, and the non-transduced group analyzed by flow cytometry in Test Example 16, respectively. [Figure 18] FIG. 18 is a graph showing the cytotoxicity rates of CAR-T in Example 3C, Comparative Example 3C, and the non-transduced group against BCMA-expressing SKOV3 cancer cells analyzed by flow cytometry in Test Example 17. [Figure 19] 19A and 19B show real-time changes in normalized cell index signal, representing the number of live BCMA-expressing SKOV3 cancer cells, in response to CAR-T cells of Example 3C and Comparative Example 3C and T cells of the non-transduced group at E / T ratios of 0.3:1 and 0.1:1, respectively, in Example 18. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The following describes the implementation and effects of the present invention by showing several embodiments and test examples. Those skilled in the art can easily understand the advantages and effects of the present invention according to the contents of the specification. Various improvements and modifications can be made to implement or apply the present invention without departing from the scope of the invention.

[0044] 1. Generation and characterization of anti-CD19-NKp30 expressing CAR-T <Examples 1-1 to 1-5: Anti-CD19-NKp30 Nucleic Acid Molecular Design> First, a nucleic acid sequence encoding the full-length protein of FcεR1γ adaptor (shown as SEQ ID NO:77) and a nucleic acid sequence encoding FMC63scFv capable of targeting CD19 (shown as SEQ ID NO:78) were prepared. The above two nucleic acid sequences were linked to obtain a linked sequence, and then the 3' end of the linked sequence was linked to the 5' end of a nucleic acid sequence of NKp30 without immunoglobulin-like domain (shown as SEQ ID NO:79). Here, the nucleic acid sequence of NKp30 without immunoglobulin-like domain includes the NKp30 stalk domain, the NKp30 transmembrane domain and the NKp30 cytoplasmic domain. Using this method, the anti-CD19-NKp30 nucleic acid molecules of Examples 1-1 to 1-5 were obtained. More specifically, the nucleic acid sequences of FcεR1γ and FMC63 were linked to each other via a nucleic acid sequence encoding a T2A self-cleaving peptide (shown as SEQ ID NO:80), and the nucleic acid sequence of NKp30 not containing an immunoglobulin-like domain and the nucleic acid sequence of FMC63 were linked to each other via a nucleic acid sequence of a different hinge domain. The nucleic acid sequences of the hinge domains used in Examples 1-1 to 1-5 were (G4S)3 (shown as SEQ ID NO:66), the hinge domain of CD28 (shown as SEQ ID NO:68), the hinge domain of CD8 (shown as SEQ ID NO:67), the hinge domain of IgG4 (shown as SEQ ID NO:69), and (EAAAK)3 (shown as SEQ ID NO:71), respectively, and the hinge domain of CD28, the hinge domain of CD8, and the hinge domain of IgG4 were of human origin. The nucleic acid sequences of the nucleic acid molecules of Examples 1-1 to 1-5 are set forth as SEQ ID NOs: 81 to 85, respectively.

[0045] Comparative Example 1 does not have NKp30 that does not contain the immunoglobulin-like domain, and was prepared based on the following method. The 3' end of the nucleic acid sequence encoding FMC63 was linked to a nucleic acid sequence encoding the transmembrane domain of CD8 (shown as SEQ ID NO:86), and FMC63 and the transmembrane domain of CD8 were linked by the hinge domain of CD8. Then, the 3' end of the nucleic acid sequence encoding the transmembrane domain of CD8 was further linked to a nucleic acid sequence encoding 4-1BB (shown as SEQ ID NO:87) and a nucleic acid sequence encoding the signal transduction domain of CD3ζ (shown as SEQ ID NO:88). The nucleic acid sequence of the nucleic acid molecule of Comparative Example 1 is shown as SEQ ID NO:89.

[0046] The hinge domains of Examples 1-1 to 1-5 (abbreviated as E1-1 to E1-5) and Comparative Example 1 (abbreviated as CE1) are also shown in Table 1.

[0047] Table 1: Hinge domains of Examples 1-1 to 1-5 and Comparative Example 1 TIFF2025059072000001.tif37165

[0048] <Examples 1-1A to 1-5A: Construction of anti-CD19-NKp30 plasmid> As shown in Figure 1, the pLAS5w.Ppuro vector, which has the nucleotide sequence of TAR chimeric 5'LTR and is driven by RSV promoter, was purchased from Academia Sinica RNAi Core Facility (product number: C6-8-39). After deleting the PAC gene and hPGK promoter of the pLAS5w.Ppuro vector (performed by GenScript), the pLAS5w vector was obtained. The nucleic acid molecules of Examples 1-1 to 1-5 and Comparative Example 1 were ligated into the pLAS5w vector using restriction enzymes HpaI and EcoRI, respectively, to obtain the anti-CD19-NKp30 plasmids of Examples 1-1A to 1-5A and the plasmid of Comparative Example 1A shown in Figure 2.

[0049] <Examples 1-1B to 1-5B: Preparation of anti-CD19-NKp30 lentivirus> The lentiviral particles of Examples 1-1B to 1-5B and Comparative Example 1B were produced by introducing a target gene into human embryonic kidney cells 293 (HEK293 cells, ATCC, CRL-3216) using Polyjet transfection reagent (purchased from SignaGen Laboratories, SL100688). Specifically, 50 μg of the plasmids of Examples 1-1A to 1-5A or Comparative Example 1A containing the target gene were used as the transferable plasmid, 45 μg of pCMV deltaR8.91 (purchased from Academia Sinica) were used as the packaging plasmid, and 5 μg of pMD.G (purchased from Academia Sinica) were used as the envelope plasmid. The transferable plasmid, packaging plasmid, and envelope plasmid were then cultured in 10 mL of serum-free DMEM (Dulbecco's modified Eagle's medium) using 300 μL of Polyjet transfection reagent for 15 minutes to obtain a Polyjet / DNA mixture. The Polyjet / DNA mixture was then added to 100 mL of DMEM containing 10% FBS (fetal bovine serum), and then diluted with 9 × 10 7The solution was added dropwise to a T875 flask seeded with HEK293 cells. After 12 to 18 hours of transfection (e.g., 16 hours of transfection), the medium was replaced with 100 mL of Opti-MEM (purchased from Gibco, 31985070) containing 1 mM sodium pyruvate (purchased from Sartorius). After another 48 hours of transfection, the supernatant containing the lentivirus particles was collected as the first batch of lentivirus. The transfection culture medium was then supplemented with fresh Opti-MEM containing 1 mM sodium pyruvate. After another 24 hours, the supernatant containing the lentivirus particles was again collected as the second batch of lentivirus. The supernatants containing the first and second batches of lentivirus were separately centrifuged at 1500 rpm at 4°C for 10 minutes to remove cell debris. The supernatants were then filtered through a 0.45 μm filter (purchased from Membrane Solutions) and combined to obtain a filtered supernatant. The filtered supernatant was then concentrated with Lenti-X reagent (purchased from Takara Bio Inc.) at a ratio of 3:1, i.e., the volume ratio of the filtered supernatant to the Lenti-X reagent was 3:1. Specifically, the filtered supernatant and the Lenti-X reagent were mixed by rotating up and down overnight at 4°C, and then the mixture was centrifuged at 1500 rpm at 4°C for 45 minutes. The supernatant was carefully removed, and the pellet was then centrifuged with X-VIVO. TM The concentrated lentiviruses were resuspended in 2 mL of 15 medium (purchased from Lonza) to obtain concentrated lentiviruses of Examples 1-1B to 1-5B and concentrated lentivirus of Comparative Example 1B. These concentrated lentiviruses were usually stored at -80°C until use.

[0050] <Test Example 1: Titer measurement of anti-CD19-NKp30 lentiviruses of Examples 1-1B to 1-5B> 4×10 4Jurkat cells (human acute leukemia T cells) were transduced with 3-fold serially diluted lentivirus samples to measure viral titers. Here, the lentivirus samples used in Test Example 1 were the concentrated lentiviruses of Examples 1-1B to 1-5B and the concentrated lentivirus of Comparative Example 1B. Specifically, in order to dilute each lentivirus concentration 3-fold, 50 μL of each lentivirus sample was added to X-VIVO TM 15 medium and incubate until the final dilution of the lowest lentivirus titer standard reaches a 6561-fold dilution of the original concentrated lentivirus. TM By repeating the addition of 15 medium, multiple groups of reconstituted diluted lentivirus samples were obtained. Next, 50 μL of each reconstituted diluted lentivirus sample was added to a 96-well round-bottom plate, and 4 × 10 4 Jurkat cells at a cell density of 4 × 10 5 The cells were cultured at 100 / mL. Lentiviral transduction was performed for 72 hours. To detect successfully transduced Jurkat cells, 200ng of Biotinylated Human CD19, Fc tag (purchased from ACROBiosystems, CD9-H8259) was employed to target anti-CD19 receptor, and 500-fold diluted streptavidin-conjugated phycoerythrin (PE) (purchased from Invitrogen, 12-4317-87) was employed to detect the expression of anti-CD19 receptor. The transduction efficiency of lentivirus was determined by analyzing the expression of target protein using a flow cytometer (purchased from Sony, SA3800). Non-transduced Jurkat cells were used as a negative control. The functional viral titer of lentivirus with anti-CD19-NKp30 was calculated by the following formula, and the experimental results are shown in Table 2. The obtained functional viral titer was then used to determine the appropriate amount of lentivirus to be applied to the production of CAR-T cells.

[0051] Functional titer of lentivirus [transducing units (TU) / mL] = (percentage of cells expressing anti-CD19 receptor) x 4 x 10 4 (pieces) x 20 x dilution ratio

[0052] Table 2: Functional viral titers of lentiviruses of Examples 1-1B to 1-5B (abbreviated as E1-1B to E1-5B) and Comparative Example 1B (abbreviated as CE1B) TIFF2025059072000002.tif38165

[0053] Functional virus titer is 1×10 6 TU / mL or more, T cells could be successfully transduced and a lower amount of lentivirus could be used to transduce CAR-T cells, while a lower amount of lentivirus used for transduction also tended to correlate with better growth of T cells for CAR-T production. Therefore, according to the results in Table 2, Example 1-1B, Example 1-4B, Example 1-5B and Comparative Example 1B met the above criteria, which is of further benefit to the preparation of CAR-T cells. Furthermore, Table 2 shows that the functional virus titer of Example 1-4B with IgG4 hinge domain is closest to the functional virus titer of Comparative Example 1B.

[0054] <Examples 1-1C, 1-4C, and 1-5C: Preparation of CAR-T cells expressing anti-CD19-NKp30> Fresh peripheral blood samples were collected from healthy volunteers with informed consent. On day 0, human peripheral blood mononuclear cells (PBMCs) were isolated using SepMate centrifuge tubes (purchased from STEMCELL, 86450). Briefly, fresh blood samples were diluted with an equal volume of PBS buffer containing 2% FBS to obtain diluted blood samples. Then, the diluted blood samples were separately added dropwise along the wall of the SepMate centrifuge tubes while keeping the SepMate centrifuge tubes in a vertical position so that the diluted blood samples and the Ficoll density gradient centrifuge solution were well mixed in the SepMate centrifuge tubes. The SepMate centrifuge tubes containing this mixture were then centrifuged at 1200 x g for 20 min at room temperature. The supernatant (rich in mononuclear cells) was then gently poured into a new tube for collection and repeatedly washed with PBS buffer containing 2% FBS, followed by centrifugation at 300 x g for 8 min at room temperature, and finally, the pelleted PBMC cells from each wash were collected. Next, T cells were isolated from the collected PBMCs using Dynabeads coated with anti-CD3 / CD28 antibodies (i.e., CTS (Cell Therapy Systems) Dynabeads CD3 / CD28 (purchased from Gibco, 40203D); hereafter referred to as CD3 / CD28 Dynabeads) in combination with DynaMag-Spin (purchased from Thermo). + The percentage of T cells was measured by anti-CD3 antibody (Biolegend, 300441) detection and flow cytometry analysis, and CD3 T cells were counted by total cell counting of PBMCs. + The amount of T cells was calculated. Next, CD3 / CD28 Dynabeads were cultured at a bead / cell ratio of 3:1 (i.e., CD3 + The CD3 / CD28 Dynabeads were mixed with PBMCs at 3x the number of beads per T cell. The mixture of CD3 / CD28 Dynabeads and PBMCs was then incubated at room temperature for 30-60 minutes to allow the CD3 / CD28 Dynabeads to bind to the T cells. Next, B cells were removed using DynaMag-Spin, and NK cells and monocytes were simultaneously isolated from CD3-positive T cells (CD3 +T cells) were selected. This resulted in the formation of a complex between CD3 / CD28 Dynabeads and T cells formed by antigen-antibody binding. The cells were then cultured at a density of 2 × 10 5 The complexes of CD3 / CD28 Dynabeads and T cells were incubated in X-VIVO containing 200 IU / mL of recombinant human IL-2 (purchased from R&D Systems). TM After dispersing in 15 medium (purchased from Lonza), the cells were cultured at 37°C and 5% CO2 for 24 hours to activate the T cells. The next day, the lentiviruses of Examples 1-1B, 1-4B, and 1-5B and Comparative Example 1B were added to the activated T cells, respectively, and left for 48 hours at a multiplicity of infection (MOI) of 2 to perform lentiviral transduction. This resulted in the complexes of CD3 / CD28 Dynabeads and CAR-T cells of Examples 1-1C, 1-4C, and 1-5C and Comparative Example 1C. The T cells that were not transduced using lentivirus were designated as the non-transduced group. Here, MOI = [viral titer (TU / mL) x viral dose (mL)] / total number of T cells. On the third day, the CD3 / CD28 Dynabeads were detached from the complexes of the CD3 / CD28 Dynabeads and the CAR-T cells containing anti-CD19-NKp30 of Examples 1-1C, 1-4C, and 1-5C, and the CAR-T cells of Comparative Example 1C were obtained. The structural schematic diagram of each group is shown in FIG. 3. In the figure, the nucleic acid sequence after transduction of Examples 1-1C, 1-4C, and 1-5C has FcεR1γ, so the CAR-T cells of Examples 1-1C, 1-4C, and 1-5C are called multi-chain CAR-T cells, and the nucleic acid sequence after transduction of Comparative Example 1C does not have FcεR1γ, so the CAR-T cells of Comparative Example 1C are called single-chain CAR-T cells. The obtained CAR-T cells were incubated with X-VIVO containing 200 IU / mL of recombinant human IL-2. TM After transferring to a new flask with 15 medium, the cells were cultured under the same conditions used in the viral transduction step above for cell expansion. After expanding the CAR-T cells for a total of 7 days (another 4 days after removal of the CD3 / CD28 Dynabeads), the cell numbers of the non-transduced group and the CAR-T cells containing anti-CD19-NKp30 were measured. From day 4 to day 7 of the CAR-T cell culture period, the cell density was increased to 1 × 10 6To maintain the cell density at approximately 50 cells / mL, additional medium was added every 1 to 3 days.

[0055] <Test Example 2: Cell proliferation rate of CAR-T cells in Examples 1-1C, 1-4C, and 1-5C, and Comparative Example 1C> The cell proliferation rate of the CAR-T cells of Examples 1-1C, 1-4C, and 1-5C was obtained by calculating the total cell number at a specific time point during the preparation of the CAR-T cells containing anti-CD19-NKp30 of Examples 1-1C, 1-4C, and 1-5C. Specifically, on the third day during the preparation of the CAR-T cells, after removing the CD3 / CD28 Dynabeads, the cell density was 1×10 6 After seeding at 12 well plates at 10 cells / mL, CAR-T cells containing anti-CD19-NKp30 were quantified by staining the cells with acridine orange / propidium iodide (AO / PI). CAR-T cell viability, number, and density were automatically determined using an automated cell counter (Luna automated cell counter, purchased from Logos Biosystems). A known amount of CAR-T cells was then subcultured to a cell density of 2 × 10 5 / mL to 5 x 10 5 The cells were seeded on a cell culture plate at 100 / mL and cell proliferation was carried out. The number of CAR-T cells was measured on days 0 and 7 using the same method, and the cell proliferation rate, i.e., the fold increase in cell proliferation, shown in Table 3 was calculated. Here, the cell proliferation rate of CAR-T cells containing anti-CD19-NKp30 was calculated by dividing the number of cells measured on day 7 by the number of cells measured on day 0.

[0056] Table 3: Fold cell proliferation for CAR-T cells of Examples 1-1C, 1-4C, and 1-5C (abbreviated as E1-1C, E1-4C, and E1-5C) and Comparative Example 1C (abbreviated as CE1C) TIFF2025059072000003.tif38165

[0057] If the cell proliferation rate was greater than 5-fold (between day 0 and day 7), it was beneficial for the subsequent T cell functional test. Therefore, according to the results in Table 3, for the CAR-T cells of Example 1-1C, Example 1-4C, and Example 1-5C, the fold of cell proliferation met the above criteria, and the CAR-T cells of Example 1-1C having (G4S)3 as the hinge domain had the highest cell proliferation rate, significantly exceeding the CAR-T cells of Comparative Example 1C.

[0058] <Test Example 3: Results of CAR-positive expression of CAR-T cells in Examples 1-1C, 1-4C, and 1-5C and Comparative Example 1C showing transduction efficiency of lentiviruses in Examples 1-1B, 1-4B, and 1-5B and Comparative Example 1B>

[0059] Biotinylated Human CD19, Fc tag (purchased from ACROBiosystems, CD9-H8259) 100μg / mL and 500-fold diluted streptavidin-conjugated phycoerythrin (PE) (purchased from Invitrogen, 12-4317-87) reconstituted in ice-cold PBS containing 2% FBS were used to detect the expression of anti-CD19 receptors on the CAR-T cells of Examples 1-1C, 1-4C, and 1-5C and Comparative Example 1C according to the manufacturer's instructions. NKp30 expression was also detected by adding 5μL of anti-NKp30 antibody (purchased from Biolegend) conjugated with BV421 fluorescent dye reconstituted in the same buffer. Flow cytometric analysis of anti-CD19 receptor and NKp30 receptor expression for CAR-T cells of Examples 1-4C is shown in Figures 4A and 4B, respectively, flow cytometric analysis of anti-CD19 receptor and NKp30 receptor expression for CAR-T cells of Examples 1-5C is shown in Figures 4C and 4D, respectively, and flow cytometric analysis of anti-CD19 receptor and NKp30 receptor expression for CAR-T cells of Comparative Example 1C is shown in Figures 4E and 4F, respectively. For each group, lentiviral transduction efficiency was determined based on the percentage of cells that expressed anti-CD19 receptor, as listed in Table 4.

[0060] Table 4: Lentiviral transduction efficiency of Examples 1-1B, 1-4B and 1-5B (abbreviated as E1-1B, E1-4B and E1-5B) and Comparative Example 1B (abbreviated as CE1B) TIFF2025059072000004.tif39165

[0061] A transduction efficiency of more than 25% during the preparation of CAR-T cells was beneficial for the subsequent functional testing of the expanded CAR-T cells. Thus, according to the results in Table 4, the lentivirus of Example 1-4B having an IgG4 hinge domain and the lentivirus of Example 1-5B having an (EAAAK)3 hinge domain both showed transduction efficiencies that met the above criteria.

[0062] <Test Example 4: T cell subpopulation analysis of CAR-T cells of Examples 1-1C, 1-4C, and 1-5C and Comparative Example 1C> During the preparation of CAR-T cells with anti-CD19-NKp30 in Examples 1-1C, 1-4C and 1-5, T cell subpopulations of CAR-T cells on day 7 were evaluated by detecting the cell surface markers of fluorescently labeled T cells with a flow cytometer equipped with SA3800 software (purchased from Sony Biotechnology) and antibodies, where the antibodies used were CD3-FITC (purchased from Biolegend, 300406), CD4-PE-Cy7 (purchased from Biolegend, 300512), CD8-AF700 (purchased from Beckman, B76279), CD95-APC (purchased from Biolegend, 305612), CD45RA-PE (purchased from Biolegend, 304108) and CCR7-BV421 (purchased from Biolegend, 353208). For flow cytometry analysis, samples were stained with all the above fluorescently labeled antibodies except for CCR7-BV421 and CD45RA-PE, since a Fluorescence Minus One (FMO) control group was also included. In this test example, non-transduced activated T cells were used as a control group (non-transduced group). Using the staining results obtained by the above method, each T cell subpopulation (stem cell-like memory T cells (Tscm), central memory T cells (Tscm), and IL-16-16T cells) were identified according to Annals of Oncology, Vol. 32, No. 11, pp. 1366-1380. CM ), effector memory T cells (T EM ) or terminally differentiated effector memory T cells [T EMRA ;Effector T cells (T EFF The distribution (expressed as a percentage) of Tscm in CAR-T cells of Examples 1-1C, 1-4C, and 1-5C and Comparative Example 1C is shown in Table 5. The distribution of T cell subpopulations analyzed by flow cytometry for Examples 1-5C, Comparative Example 1C, and the non-transduced group is shown in Figures 5A, 5B, and 5C, respectively. Here, T cells that simultaneously express CD45RA and CCR7 are considered as Tscm, and T cells that express CD45RA but not CCR7 are considered as Tscm. EMRA (T EFFT cells that do not express CCR7 and CD45RA are considered to be T EM T cells that express CCR7 but not CD45RA are considered T CM It was considered that.

[0063] Table 5: Percentage of Tscm for CAR-T cells of Examples 1-1C, 1-4C and 1-5C (abbreviated as E1-1C, E1-4C and E1-5C) and Comparative Example 1C (abbreviated as CE1C) and for T cells of the non-transduced group (abbreviated as UTD). TIFF2025059072000005.tif47165

[0064] CAR-T with a high Tscm ratio reduced the cytokine release syndrome and neurotoxicity caused by such cell therapy, so the desired range of Tscm ratio of CAR-T cells is more than 40%. According to the results of Table 5 and Figures 5A to 5C, Example 1-1C (with (G4S)3 hinge domain), Example 1-4C (with IgG4 hinge domain) and Example 1-5C (with (EAAAK)3 hinge domain) all have a high Tscm ratio, which is comparable to the conventional single-chain CAR-T cells of Comparative Example 1C. Therefore, it is expected that the CAR-T cells of Examples 1-1C, 1-4C and 1-5C can reduce the cytokine release syndrome and neurotoxicity caused by such treatment.

[0065] <Test Example 5: Cytotoxicity of CAR-T cells of Examples 1-4C and 1-5C determined based on luciferase killing assay>

[0066] To establish luciferase-expressing Raji cell lines, the lentiviral vector PLL-CMV-rFluc-T2A-GFP-mPGK Lenti-Labeler (purchased from System Biosciences) was transduced into Raji human lymphoma cell lines (purchased from Elabscience, EP-CL-0189, hereafter referred to as Raji cells). Then, to exclude single cell lines capable of stable expression, we selected GFP / Luc, which can simultaneously express green fluorescent protein (GFP) and firefly luciferase enzyme. + Raji cells were obtained. Raji cells constitutively express CD19, and therefore GFP / Luc + Raji cells were cultured in RPMI-1640 medium (purchased from Gibco) containing 10% FBS and employed as target cells for the anti-CD19 CAR-T luciferase killing assay.

[0067] To determine the antitumor activity of CAR-T cells prepared according to the 7-day production method with anti-CD19-NKp30 CAR expression in Examples 1-4C and 1-5C, the above GFP / Luc + Raji cells were used as target cells, and luciferase killing assay was performed. The effector cells used in this test example were the CAR-T cells of Examples 1-4C and 1-5C and Comparative Example 1C, and non-transduced T cells were used as the non-transduced group (control group). Specifically, the effector cells and target cells were co-cultured in a 96-well microplate at 37°C and 5% CO2 for 24 hours with an effector / target ratio (E / T ratio) of 3:1. The experiment was performed in triplicate.

[0068] The firefly luciferase activity exhibited by live luciferase-expressing Raji cells was quantified using a luciferase assay system (purchased from Promega, E1501) by supplying luciferin to generate a bioluminescence reaction. The relative number of viable cancer cells not lysed by CAR-T cells can be evaluated based on luciferase detection. Thus, the results of specific cell lysis rate were calculated using the following formula:

[0069] Specific cell lysis rate (%) = 100% x [(number of target cells lysed by treatment) - (number of target cells spontaneously lysed)] / [(maximum number of target cells lysed) - (number of target cells spontaneously lysed)] Wherein, "number of target cells lysed by treatment" = number of lysed target cells obtained from the group in which target cells were co-cultured with CAR-T cells (i.e., effector cells) "Number of spontaneously lysed target cells" = number of lysed target cells obtained from the effector cell group, i.e., the group without CAR-T cells. Simply GFP / Luc + The number of cells lysed by culturing Raji cells in RPMI-1640 medium containing 10% FBS at 37° C. under 5% CO 2 for 24 hours is shown. "Maximum number of lysed target cells" = number of lysed target cells obtained after adding 100 μL of 1% Triton X-100 to the group without effector cells

[0070] The experimental results are shown in Table 6. Here, Figure 6 shows the cytotoxicity rates as indicated by the specific cytolysis rates of the CAR-T cells of Examples 1-5C, the CAR-T cells of Comparative Example 1C, and the T cells of the non-transduced group.

[0071] Table 6: Results of luciferase killing assay (specific cell lysis rate) of CAR-T cells of Examples 1-4C and 1-5C (abbreviated as E1-4C and E1-5C) and Comparative Example 1C (abbreviated as CE1C), and T cells of the non-transduced group (abbreviated as UTD). TIFF2025059072000006.tif47165

[0072] According to the results of Table 6 and Figure 6, the cytotoxicity of the CAR-T cells of Examples 1-4C and 1-5C was both better than that of the non-transduced group, and the cytotoxicity of the CAR-T cells of Examples 1-5C was much better than that of the CAR-T cells of Comparative Example 1C. Furthermore, if the cytotoxicity of the CAR-T cells under documented is more than 50%, it is expected that the CAR-Ts made using the patient's cells will also show sufficient cytotoxicity. According to the results of Table 6, the CAR-T cells of Examples 1-4C and 1-5C both showed cytotoxicity that met the >50% criteria, and the cytotoxicity of the CAR-T cells of Examples 1-5C (with (EAAAK)3 hinge domain) was much better than that of the conventional single-chain CAR-T cells of Comparative Example 1C.

[0073] <Test Example 6: Real-time cytotoxicity assay (RTCA) for comparison of Examples 1-5C and Comparative Example 1C against CD19-expressing adherent cancer cells>

[0074] A human CD19 cDNA fragment was cloned from Raji cells (purchased from Elabscience, CL-0189). A cDNA fragment encoding a truncated CD19 (shown as SEQ ID NO:90) was amplified by PCR. Here, the truncated CD19 cDNA fragment (amino acids 1-342) refers to a cDNA fragment containing the extracellular and transmembrane domains but lacking the intracellular and cytoplasmic signaling domains. The truncated CD19 cDNA fragment was ligated into pLAS5w.Ppuro vector containing anti-puromycin gene (purchased from Academia Sinica RNAi Core Facility, product number: C6-8-39) using the restriction enzyme sites HpaI and NheI, and then the resulting product was introduced into SKOV3 cells (human ovarian cancer cell line) by applying Polyjet transfection reagent (purchased from SignaGen Laboratories, SL100688). The CD19-depleted transfected SKOV3 cells were cultured in McCoy's 5A medium (purchased from Gibco, 16600082) containing 2 μg / mL puromycin and 10% FBS to select SKOV3 cell lines that were puromycin resistant and simultaneously expressed CD19. Finally, the expression of CD19 was reconfirmed by flow cytometry analysis. The CD19-expressing SKOV3 cell lines were used as target cells for real-time cytotoxicity assays performed using the xCELLigence system (hereinafter referred to as the xCELLigence RTCA system).

[0075] The xCELLigence RTCA system (purchased from Agilent Technologies, RTCA SP) was installed in a cell culture machine under 37°C and 5% CO2, and cabled to the interface of the external analysis unit and control unit. In addition, the RTCA Software Pro software has the function of controlling and monitoring the instrument in real time, including displaying and analyzing real-time data. Specifically, the xCELLigence RTCA system was used to continuously monitor the changes in the cytotoxicity of the therapeutic cells against the tumor cells for 90 hours. McCoy5A medium containing 10% FBS was added to each well of a 96-well microplate (E-plate, Agilent Technologies, 300600910) to culture the cancer cells. After measuring the back impedance of the medium for a short time, SKOV3 cells expressing the above-mentioned deleted CD19 were used as target cells, and the cell density of 2 × 10 4The cells were seeded on the microplate at 100 μL / mL and left overnight to ensure that the target cells (T) attached. To allow the cancer cells to grow sufficiently, the back impedance measurement was performed for a total of 29 hours. At this time, the effector cells (E) were added to the microplate and co-cultured so that the ratio of effector cells to target cells (E / T ratio) was 0.1:1 and the final volume of each well was 200 μL. The effector cells used in this test example were the CAR-T cells of Example 1-5C and Comparative Example 1C. In addition, non-transduced T cells were used as the non-transduced group (control group), and target cells treated with 100 μL of 1% Triton X-100 were used as the total lysis group. The cell index (CI) was measured every 15 minutes for at least 72 hours. The raw RTCA back impedance data was normalized using the cell index value measured before the addition of the effector cells. The cell index is a dimensionless parameter and is obtained by measuring the relative change in electrical impedance that indicates the state of the cells. For example, if there are no cells present or no cells attached to the electrode, the cell index will be zero. In the same physiological condition, the more cells that are attached to the electrode, the higher the cell index value. However, the cell index may change due to changes in cell conditions, such as changes in cell morphology or cell adhesion.

[0076] The experimental results are shown in Figures 7A and 7B, which show that the cytotoxicity of the CAR-T cells of Example 1-5C (having an (EAAAK)3 hinge domain) was significantly better than that of the CAR-T cells of Comparative Example 1C and the T cells of the non-transduced group, and was more than three times that of the CAR-T cells of Comparative Example 1C, demonstrating excellent cytotoxicity.

[0077] In summary, the lentiviruses of Examples 1-5C (with the (EAAAK)3 hinge domain) had reasonable transduction efficiency, and the CAR-T cells of Examples 1-5C proliferated well and showed good cytotoxicity, with a high proportion of Tscm, potentially leading to reduced cytokine release syndrome and neurotoxicity caused by such treatment. Thus, the (EAAAK)3 hinge domain was the optimal hinge domain to match the anti-CD19 receptor.

[0078] 2. Generation and characterization of CAR-T cells expressing anti-mesothelin-NKp30 Example 2: Anti-mesothelin-NKp30 nucleic acid molecule design The preparation method of Example 2 was the same as that of Example 1-1, except that the single-chain variable fragment used in Example 2 was a nucleic acid sequence (shown as SEQ ID NO:91) encoding SS1scFv capable of targeting mesothelin, instead of FMC63scFv for anti-CD19 used in the preparation method of Example 1-1, and the hinge domain used was (G4S)3. The nucleic acid sequence of the nucleic acid molecule of Example 2 is shown as SEQ ID NO:92.

[0079] The preparation method of Comparative Example 2 not containing NKp30 was the same as that of Comparative Example 1, except that the single chain variable fragment used in Comparative Example 2 was a nucleic acid sequence encoding SS1scFv capable of targeting mesothelin, instead of FMC63scFv for anti-CD19 used in the preparation method of Comparative Example 1. The nucleic acid sequence of the nucleic acid molecule of Comparative Example 2 is shown as SEQ ID NO:93.

[0080] Example 2A: Construction of anti-mesothelin-NKp30 plasmid Preparation method A of Example 2 was the same as the preparation method of the plasmid of Example 1-1A, except that the anti-mesothelin-NKp30 nucleic acid molecule of Example 2 was ligated to the above-mentioned pLAS5w vector using the restriction enzymes BstBI and EcoRI. Meanwhile, the nucleic acid molecule of Comparative Example 2 was ligated to the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core Facility, product number: C6-8-39) using the restriction enzymes HpaI and EcoRI to obtain the plasmid of Comparative Example 2A (performed by GenScript). The prepared anti-mesothelin-NKp30 plasmid of Example 2A and the plasmid of Comparative Example 2A are shown in FIG. 8.

[0081] Example 2B: Construction of anti-mesothelin-NKp30 lentivirus Preparation method B of Example 2 was the same as preparation method B of Example 1-1, except that the plasmid of Example 2A was used as the transferable plasmid of Example 2B. On the other hand, the plasmid of Comparative Example 2A was used as the transferable plasmid for the lentivirus of Comparative Example 2B.

[0082] <Test Example 7: Measurement of the titer of anti-mesothelin-NKp30 lentivirus of Example 2B> The method of Test Example 7 was the same as that of Test Example 1, except that Biotinylated Human Mesothelin, His, Avitag (purchased from ACROBiosystems, MSN-H82E9) was used to target the anti-mesothelin receptor, and 500-fold diluted streptavidin-conjugated PE (purchased from Invitrogen, 12-4317-87) was used to detect the expression of the anti-mesothelin receptor. The results of Test Example 7 are shown in Table 7.

[0083] Table 7: Functional viral titers of lentiviruses of Example 2B (abbreviated as E2B) and Comparative Example 2B (abbreviated as CE2B) TIFF2025059072000007.tif38165

[0084] Functional virus titer is 1×10 6TU / mL or more, T cells could be successfully transduced and lower amounts of lentivirus could be used to transduce CAR-T cells, while lower doses of lentivirus used for transduction also tended to correlate with better growth of T cells for CAR-T generation. Thus, according to the results in Table 7, Example 2B (with (G4S)3 hinge domain) met the above criteria, which provides additional benefits for the preparation of CAR-T cells.

[0085] Example 2C: Preparation of CAR-T cells expressing anti-mesothelin-NKp30 The method for preparing CAR-T cells comprising anti-mesothelin-NKp30 of Example 2C was the same as that of Example 1-1C, except that T cells were transduced with the lentivirus of Example 2B, and the MOI of lentiviral transduction was 3. Meanwhile, the CAR-T cells of Comparative Example 2C were prepared by transduction with the lentivirus of Comparative Example 2B, and the MOI of lentiviral transduction was 3. Schematic structures of the CAR-T cells comprising anti-mesothelin-NKp30 of Example 2C and the CAR-T cells of Comparative Example 2C are shown in Figures 9A and 9B, respectively.

[0086] <Test Example 8: Cell proliferation rate of CAR-T cells containing anti-mesothelin-NKp30> The method of Test Example 8 was the same as that of Test Example 2, except that the cell proliferation rate of CAR-T cells containing anti-mesothelin-NKp30 of Example 2C was detected. The results are shown in Table 8.

[0087] Table 8: Fold cell proliferation of CAR-T cells of Example 2C (abbreviated as E2C) and Comparative Example 2C (abbreviated as CE2C) TIFF2025059072000008.tif38165

[0088] If the cell proliferation rate was more than 5 times, it was beneficial for the subsequent T cell functional test. Therefore, according to the results in Table 8, for the CAR-T cells of Example 2C, the cell proliferation rate meets the above criteria and is considered to be greater than that of the CAR-T cells of Comparative Example 2C.

[0089] <Test Example 9: Results of CAR-T cell CAR-positive expression in Example 2C and Comparative Example 2C showing transduction efficiency of lentiviruses in Example 2B and Comparative Example 2B> The method of Test Example 9 was the same as that of Test Example 3, except that Biotinylated Human Mesothelin, His, Avitag (purchased from ACROBiosystems, MSN-H82E9) was used to target the anti-mesothelin receptor, and 500-fold diluted streptavidin-conjugated PE (purchased from Invitrogen, 12-4317-87) was used to detect the expression of anti-mesothelin receptor and NKp30 receptor in the CAR-T cells of Example 2C and Comparative Example 2C. The flow cytometry analysis results of Example 2C and Comparative Example 2C are shown in Figures 10A, 10B, 10C, and 10D, respectively. For Example 2B or Comparative Example 2B, the transduction efficiency of the lentivirus was determined based on the percentage of cells that expressed the anti-mesothelin receptor, as listed in Table 9.

[0090] Table 9: Lentiviral transduction efficiency of Example 2B (abbreviated as E2B) and Comparative Example 2B (abbreviated as CE2B) TIFF2025059072000009.tif38165

[0091] If the transduction efficiency during the preparation of CAR-T cells was higher than 25%, it was beneficial for the subsequent functional testing of the expanded CAR-T cells. Therefore, according to the results in Table 9 and Figure 10A, the lentivirus of Example 2B showed a transduction efficiency that met the above criteria.

[0092] <Test Example 10: T cell subpopulation analysis of CAR-T cells of Example 2C> The method of Test Example 10 was the same as that of Test Example 4, except that the analysis was performed on the 7th day after the preparation of CAR-T cells containing anti-mesothelin-NKp30 of Example 2C. The percentages of Tscm in the CAR-T cells of Example 2C and Comparative Example 2C and the T cells of the non-transduced group are shown in Table 10, and the distribution of the subpopulations of T cells of Example 2C, Comparative Example 2C and the non-transduced group analyzed by flow cytometry are shown in Figures 11A, 11B and 11C, respectively. Here, T cells that simultaneously express CD45RA and CCR7 were considered as Tscm.

[0093] Table 10: Percentage of Tscm in Example 2C (abbreviated as E2C) and Comparative Example 2C (abbreviated as CE2C), and percentage of Tscm in activated T cells in the non-transduced group (abbreviated as UTD) TIFF2025059072000010.tif47165

[0094] When the proportion of Tscm in CAR-T cells is higher than 40%, it can potentially reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. According to the results of Table 10 and Figure 11A, Figure 11B and Figure 11C, Example 2C (with (G4S)3 hinge domain) has a high proportion of Tscm, which is higher than that in the conventional single-chain CAR-T cells of Comparative Example 2C and is equivalent to that in the activated T cells of the non-transduced group. Therefore, it is expected that the CAR-T cells of Example 2C can potentially reduce the cytokine release syndrome and neurotoxicity caused by such treatment.

[0095] <Test Example 11: Real-time cytotoxicity assay for analysis of Example 2C and Comparative Example 2C against HeLa cancer cells> The Hela cervical cancer cell line (hereafter referred to as Hela cells) is a mesothelin-positive cell line. Hela cells (purchased from Elabscience, CL-0101) were cultured in DMEM containing 10% FBS at 37°C under 5% CO2 and then used as target cells for real-time cytotoxicity assays performed on the xCELLigence system.

[0096] The method of Test Example 11 was the same as that of Test Example 6, except that the effector cells were the CAR-T cells of Example 2C or Comparative Example 2C, and the target cells were the HeLa cells. The cell density was 1.2×10 4 Target cells at 100 / mL were seeded on a 96-well microplate with the impedance behind corrected, and then cultured overnight for 18 hours to ensure that the target cells adhered. Effector cells were then added to the microplate at E / T ratios of 3:1, 1:1, and 0.3:1. In Test Example 11, the total lysis group was not used as a control group.

[0097] The experimental results with different E / T ratios are shown in Figures 12A to 12C. 58 hours after the start of the experiment, i.e., after a co-culture period of 40 hours, the CAR-T cells of Example 2C were able to kill 100% of HeLa cells at all three E / T ratios. Meanwhile, the cytotoxicity of the CAR-T cells of Example 2C was comparable to that of the single-chain CAR-T cells of Comparative Example 2C.

[0098] <Test Example 12: Readministration test of real-time cytotoxicity assay using the xCELLigence system> Test Example 12 aimed to confirm whether effector cells could exhibit sustained cytotoxicity when continuously restimulated by target cells. DMEM containing 10% FBS was added to each well of a 96-well microplate (E-plate, Agilent Technologies, 300600910). After briefly measuring the back impedance of the medium, HeLa cells (purchased from Elabscience, CL-0101) were used as target cells and cultured in a 96-well microplate containing DMEM containing 10% FBS. The target cells were cultured at a cell density of 1.2 x 10 in a state corrected for back impedance. 4Target cells were seeded at 100x10 cells / mL in 96-well microplates and left to grow overnight to allow attachment of target cells. Back-impedance measurements were performed for 18 hours to allow sufficient cancer cell growth, at which time effector cells were added to the microplates at an E / T ratio of 1:1. For tumor rechallenge assays, HeLa cells were refed daily (2.4x10 cells / mL on days 2 and 3, respectively). 4 Hela cells). In Test Example 12, the CAR-T cells of Example 2C and Comparative Example 2C were used as effector cells, and non-transduced T cells were used as a non-transduced group (control group). The cell index was measured every 15 minutes for at least 72 hours, and the raw RTCA transverse impedance data was normalized using the cell index value measured before the addition of the effector cells.

[0099] The real-time cytotoxicity results of CAR-T against Hela cancer cells on day 1 and the subsequent cancer cell re-administration on days 2 and 3 are shown in Figures 13A, 13B, and 13C, respectively. The cytotoxicity of Example 2C was higher than that of Comparative Example 2C both before and after the cancer cell re-administration. Therefore, it was found that Example 2C not only exhibits superior cytotoxicity against cancer cells, but also has a sustained cytotoxic effect. Therefore, Test Example 12 confirmed that the CAR-T cells of Example 2C have both a higher cytotoxicity and a sustained cytotoxicity than the CAR-T cells of Comparative Example 2C.

[0100] In summary, the lentivirus of Example 2B (with the (G4S)3 hinge domain) had sufficient transduction efficiency, and the CAR-T cells of Example 2C proliferated well, exhibited potent and sustained cytotoxicity, and possessed a high proportion of Tscm, potentially leading to reduced cytokine release syndrome and neurotoxicity caused by such treatment. Therefore, the (G4S)3 hinge domain was a suitable hinge domain to match the anti-mesothelin receptor.

[0101] 3. Generation and characterization of CAR-T cells expressing anti-BCMA-NKp30 Example 3: Anti-BCMA-NKp30 nucleic acid molecule design The preparation method of Example 3 was the same as that of Example 1-1, except that the single chain variable fragment used in Example 3 was a nucleic acid sequence (shown as SEQ ID NO:94) encoding J6M0scFv capable of targeting BCMA, instead of FMC63scFv for anti-CD19 used in the preparation method of Example 1-1, and the hinge domain used was the hinge domain of CD28. The nucleic acid sequence of the nucleic acid molecule of Example 3 is shown as SEQ ID NO:95.

[0102] The preparation method of Comparative Example 3 not containing NKp30 was the same as that of Comparative Example 1, except that the single chain variable fragment used in Comparative Example 3 was a nucleic acid sequence encoding J6M0scFv capable of targeting BCMA, instead of FMC63scFv for anti-CD19 used in the preparation method of Comparative Example 1. The nucleic acid sequence of the nucleic acid molecule of Comparative Example 3 is shown as SEQ ID NO:96.

[0103] Example 3A: Construction of anti-BCMA-NKp30 plasmid The preparation method of Example 3A was the same as the preparation method of the plasmid of Example 1-1A, except that the anti-BCMA-NKp30 nucleic acid molecule of Example 3 was ligated to the above-mentioned pLAS5w vector with the restriction enzymes NheI and EcoRI. Meanwhile, the nucleic acid molecule of Comparative Example 3 was ligated to the pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core Facility, product number: C6-8-39) with the restriction enzymes HpaI and EcoRI to obtain the plasmid of Comparative Example 3A (performed by GenScript). The prepared anti-BCMA-NKp30 plasmid of Example 3A and the plasmid of Comparative Example 3A are shown in FIG. 14.

[0104] Example 3B: Preparation of anti-BCMA-NKp30 lentivirus The preparation method of Example 3B was the same as that of Examples 1-2B, except that the plasmid of Example 3A was used as the transferable plasmid of Example 3B. Meanwhile, the plasmid of Comparative Example 3A was used as the transferable plasmid for the lentivirus of Comparative Example 3B.

[0105] <Test Example 13: Titer measurement of anti-BCMA-NKp30 lentivirus of Example 3B> The method of Test Example 13 was the same as that of Test Example 1, except that Biotinylated BCMA Protein, His, Avitag (His&AVI Tag, purchased from Sino Biological, Cat: 10620-H40H-B) was used to target the anti-BCMA receptor, and 500-fold diluted streptavidin-conjugated PE (purchased from Invitrogen, 12-4317-87) was used to detect the expression of the anti-BCMA receptor. The results of Test Example 13 are shown in Table 11.

[0106] Table 11: Functional viral titers of lentiviruses of Example 3B (abbreviated as E3B) and Comparative Example 3B (abbreviated as CE3B) TIFF2025059072000011.tif38165

[0107] Functional virus titer is 1×10 6 TU / mL or more, T cells could be successfully transduced and a lower amount of lentivirus could be used to transduce CAR-T cells, while a lower amount of lentivirus used for transduction also tended to correlate with better growth of T cells for CAR-T generation. Therefore, according to the results in Table 11, Example 3B (with CD28 hinge domain) met the above criteria, which provides additional benefits for the preparation of CAR-T cells. It was also found that the functional virus titer of Example 3B was comparable to that of Comparative Example 3B.

[0108] <Example 3C: Preparation of CAR-T cells expressing anti-BCMA-NKp30> The preparation method of the CAR-T cells containing anti-BCMA-NKp30 of Example 3C was the same as the preparation method of Examples 1-2C, except that the lentivirus of Example 3B was used to transduce T cells, and the MOI of lentiviral transduction was 3. On the other hand, the CAR-T cells of Comparative Example 3C were prepared by transduction with the lentivirus of Comparative Example 3B, and the MOI of lentiviral transduction was 3. The structural schematic diagram of the CAR-T cells containing anti-BCMA-NKp30 of Example 3C and the CAR-T cells of Comparative Example 3C is shown in FIG. 15.

[0109] <Test Example 14: Cell proliferation rate of CAR-T cells containing anti-BCMA-NKp30> The method of Test Example 14 was the same as that of Test Example 2, except that the cell proliferation rate of CAR-T cells containing anti-BCMA-NKp30 of Example 3C was detected, and the results are shown in Table 12.

[0110] Table 12: Fold cell proliferation of CAR-T cells of Example 3C (abbreviated as E3C) and Comparative Example 3C (abbreviated as CE3C) TIFF2025059072000012.tif38165

[0111] If the cell proliferation rate was more than 5 times, it was beneficial for the subsequent T cell functional test. Therefore, according to the results in Table 12, for the CAR-T cells of Example 3C, the cell proliferation rate met the above criteria and was significantly higher than that of the CAR-T cells of Comparative Example 3C.

[0112] <Test Example 15: Results of CAR-T cell CAR-positive expression in Example 3C and Comparative Example 3C showing transduction efficiency of lentiviruses in Example 3B and Comparative Example 3B> The method of Test Example 15 was the same as that of Test Example 3, except that Biotinylated BCMA Protein, His, Avitag (His&AVI Tag, purchased from Sino Biological, Cat: 10620-H40H-B) was used to target anti-BCMA receptor, and 500-fold diluted streptavidin-conjugated PE (purchased from Invitrogen, 12-4317-87) was used to detect the expression of anti-BCMA receptor. The results of Example 3C and Comparative Example 3C analyzed by flow cytometry are shown in Figure 16A, Figure 16B, and Figure 16C, respectively. For Example 3B or Comparative Example 3B, the lentiviral transduction efficiency was determined based on the percentage of cells that expressed anti-BCMA receptor, as listed in Table 13.

[0113] Table 13: Lentiviral transduction efficiency of Example 3B (abbreviated as E3B) and Comparative Example 3B (abbreviated as CE3B) TIFF2025059072000013.tif38165

[0114] If the transduction efficiency during the preparation of CAR-T cells was higher than 25%, it was beneficial for the subsequent functional testing of the expanded CAR-T cells. Therefore, according to the results in Table 13 and Figure 16A, the lentivirus of Example 3B showed a transduction efficiency that met the above criteria.

[0115] <Test Example 16: T cell subpopulation analysis of CAR-T cells of Example 3C> The method of Test Example 16 was the same as that of Test Example 4, except that the analysis was performed on the 7th day after the preparation of CAR-T cells containing anti-BCMA-NKp30 of Example 3C. The percentages of Tscm in the CAR-T cells of Example 3C and Comparative Example 3C and the T cells of the non-transduced group are shown in Table 14, and the distribution of the subpopulations of T cells of Example 3C, Comparative Example 3C and the non-transduced group analyzed by flow cytometry are shown in Figures 17A, 17B and 17C, respectively. Here, T cells that simultaneously express CD45RA and CCR7 were considered as Tscm.

[0116] Table 14: Percentage of Tscm in Example 3C (abbreviated as E3C) and Comparative Example 3C (abbreviated as CE3C), and percentage of Tscm in T cells of the non-transduced group (abbreviated as UTD) TIFF2025059072000014.tif47165

[0117] When the proportion of Tscm in CAR-T cells is higher than 40%, it can potentially reduce the cytokine release syndrome and neurotoxicity caused by CAR-T cell therapy. According to the results of Table 14 and Figures 17A, 17B and 17C, Example 3C (having the hinge domain of CD28) has a high proportion of Tscm, which is higher than that in the conventional single-chain CAR-T cells of Comparative Example 3C and is equivalent to that in the activated T cells of the non-transduced group. Therefore, it is expected that the CAR-T cells of Example 3C can potentially reduce the cytokine release syndrome and neurotoxicity caused by such treatment.

[0118] <Test Example 17: Cytotoxicity assay analyzed by flow cytometry> To test the antitumor activity of CAR-T cells containing anti-BCMA-NKp30, in Test Example 17, the H929 myeloma cell line (purchased from BCRC, 60386; hereinafter referred to as H929 cells) was cultured in a 50-well plate using CellLTRace TM The cells were labeled with Violet, cultured in RPMI-1640 medium (purchased from Gibco), and used in a cytotoxicity assay analyzed by flow cytometry. In Test Example 17, the effector cells were the CAR-T cells of Example 3C and Comparative Example 3C, and non-transduced T cells were used as the non-transduced group (control group). Specifically, the cell density was 1×10 4Target cells were seeded at 100 / mL in a 96-well microplate, and then effector cells were added to the microplate at E / T ratios of 0.1:1, 0.3:1, and 1:1. Co-culture was performed for 24 hours at 37°C under 5% CO2. Experiments were performed in triplicate. After co-culture, the 96-well microplate was centrifuged at 300×g for 5 minutes to pellet the remaining cells, and the supernatant was removed. The cell pellet in each well was washed with 200 μL of PBS containing 2% fetal calf serum (FCS) and then centrifuged again. The supernatant was then removed from the washed cell pellet. The cells were resuspended and harvested by applying 300 μL of flow buffer (PBS containing 2% FCS). To perform absolute quantification of the remaining cancer cells per treatment, the cell suspension was stained with counting microbeads (CountBright TM 15μL of 100% PBS (Purchased from Invitrogen) was added and analyzed by flow cytometry. The cytotoxicity results for each group were calculated using the following formula:

[0119] Cytotoxicity (%) = (number of target cells in the target cell-only group - number of target cells in the co-culture group) / number of target cells in the target-only group × 100%

[0120] As shown in FIG. 18, when the E / T ratio was 0.1:1, the cytotoxicity of Example 3C was 73.54%, which was equivalent to the cytotoxicity of Comparative Example 3C of 74.78%, while when the E / T ratio was 0.3:1 and 1:1, the cytotoxicity of Example 3C was approximately 80% to 85%, both of which were better than the cytotoxicity of Comparative Example 3C at the corresponding E / T ratios.

[0121] <Test Example 18: Real-time cytotoxicity assay for analysis of Example 3C and Comparative Example 3C on H929 cancer cells> A human BCMA fragment was cloned from H929 cell line (purchased from BCRC, 60386). A cDNA fragment encoding a truncated BCMA (shown as SEQ ID NO:97) was amplified by PCR. Here, the truncated BCMA cDNA fragment (amino acids 1-98) refers to a cDNA fragment containing the extracellular and transmembrane domains but lacking the intracellular and cytoplasmic signaling domains. The truncated BCMA cDNA fragment was ligated into pLAS5w.Ppuro vector (purchased from Academia Sinica RNAi Core Facility, product number: C6-8-39) using the restriction enzyme sites HpaI and NheI, and the resulting product was then introduced into SKOV3 cells (human ovarian cancer cell line, purchased from ATCC, HTB-77) by applying Polyjet transfection reagent (purchased from SignaGen Laboratories, SL100688). The deleted BCMA transfected SKOV3 cells were cultured in McCoy's 5A medium (purchased from Gibco) containing 2 μg / mL puromycin and 10% FBS to select SKOV3 cell lines that were puromycin resistant and simultaneously expressed BCMA. Finally, the expression of BCMA was reconfirmed by flow cytometry analysis. The SKOV3 cell lines expressing BCMA were used as target cells for real-time cytotoxicity assays performed using the xCELLigence system.

[0122] The method of Test Example 18 was the same as that of Test Example 6, except that the effector cells were the CAR-T cells of Example 3C or Comparative Example 3C. The target cells were the SKOV3 cells expressing BCMA, and the cell density was 1.2 × 10 4 The target cells were seeded at 100 / mL in a 96-well microplate (E-plate, Agilent Technologies, 300600910) and left overnight to ensure that the target cells adhered. To ensure sufficient cancer cell growth, back impedance measurements were performed for a total of 22 hours. At this time, effector cells were added to the microplate and co-cultured at E / T ratios of 0.3:1 and 0.1:1.

[0123] The experimental results at E / T ratios of 0.3:1 and 0.1:1 are shown in Figures 19A and 19B, respectively, and show that after 36 hours of co-culture and 58 hours after the start of the experiment, the CAR-T cells of Example 3C killed 100% of the BCMA-expressing SKOV3 cells at both E / T ratios. The normalized cell index curves of Example 3C look almost identical to those of the total lysis group, indicating that the cytotoxicity of the CAR-T cells of Example 3C is slightly stronger than that of the single chain CAR-T cells of Comparative Example 3C.

[0124] In summary, the lentivirus of Example 3B (with the hinge domain of CD28) had moderate to high transduction efficiency, and the CAR-T cells of Example 3C proliferated well, showed good cytotoxicity, and had a high proportion of Tscm, potentially leading to reduced cytokine release syndrome and neurotoxicity caused by such treatment. Therefore, the hinge domain of CD28 was a suitable hinge domain to fit the anti-BCMA receptor.

[0125] Although many of the characteristics and advantages of the present invention have been described above, together with details of the construction and characteristics of the invention, the disclosure is by way of example only, and changes may be made in details, particularly in the shape, size and arrangement of parts, within the scope of the principles of the invention to the maximum extent indicated by the broad and general meaning of the terms used in the appended claims.

Claims

1. 1. An isolated nucleic acid molecule having a nucleic acid sequence, the nucleic acid sequence being (a) an extracellular antigen-binding domain having a heavy chain variable region; (b) a hinge domain; and (c) the NKp30 transmembrane domain; and (d) an isolated nucleic acid molecule encoding the NKp30 cytoplasmic domain.

2. The isolated nucleic acid molecule of claim 1, wherein the extracellular antigen-binding domain comprises a heavy chain variable region of an anti-mesothelin antibody, a heavy chain variable region of an anti-CD22 antibody, a heavy chain variable region of an anti-CD19 antibody, a heavy chain variable region of an anti-BCMA antibody, a heavy chain variable region of an anti-CD123 antibody, a heavy chain variable region of an anti-GPRC5D antibody or a heavy chain variable region of an anti-TSHR antibody.

3. The hinge domain is ４ S) ３ , CD8 hinge domain, CD28 hinge domain, IgG4 hinge domain, EAAAKGGGGS, (EAAAK) ３ , the hinge domain of GST, the hinge domain of EAAAK-GS, the hinge domain of IgD, the IgG4-CH3 domain, and (AP) ６ The isolated nucleic acid molecule of claim 1 selected from the group consisting of:

4. The isolated nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding an adaptor.

5. 2. The isolated nucleic acid molecule of claim 1, wherein the hinge domain and the NKp30 transmembrane domain are connected by the NKp30 stalk domain.

6. The extracellular antigen-binding domain comprises a heavy chain variable region of an anti-mesothelin antibody, and the hinge domain comprises ４ S) ３ 2. The isolated nucleic acid molecule of claim 1, which is

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

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

9. 9. The method of claim 8, wherein the isolated nucleic acid molecule is introduced into the nucleated cell by lentiviral transduction.

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

11. The isolated cell of claim 10, comprising a T cell, a natural killer cell, a natural killer T cell or an adipose-derived stem cell.

12. 11. A pharmaceutical composition for the treatment or alleviation of cancer, comprising an effective amount of the isolated cells of claim 10.

13. 13. A pharmaceutical composition for treating or alleviating an autoimmune disease, comprising an effective amount of the isolated cells of claim 10, wherein the autoimmune disease comprises systemic lupus erythematosus, idiopathic inflammatory myopathy, systemic sclerosis or multiple sclerosis.

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