Method for promoting immune cell proliferation

Abstract

To provide a method for promoting immune cell proliferation.SOLUTION: The method comprises the step for upregulating the expression of low-density lipoprotein receptor-related proteins or fragments thereof in immune cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a method for promoting immune cell proliferation. In particular, the present application relates to a method for increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the immune cell. [Background technology]

[0002] Chimeric antigen receptor T-cell immunotherapy (CAR-T) enhances the specific killing of tumors by genetically modifying T cells to express chimeric antigen receptors (CARs) and recognize antibodies against tumor cell surface antigens. CARs generally contain a single chain variable fragment (scFv) that specifically recognizes tumor associated antigens (TAAs), a hinge region, a transmembrane region, and an intracellular signaling region.

[0003] In recent years, CAR-T immunotherapy has shown clear efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma. Although efficacy has been shown, the proliferation capacity of immune cells expressing CAR is limited. Summary of the Invention

[0004] The present application provides genetically modified immune cells and methods for promoting immune cell proliferation. The method of the present application can increase the expression of low density lipoprotein receptor-related protein or a fragment thereof in the immune cells. The method of the present application can further promote the production of memory immune cells. The method of the present application can further inhibit immune cell differentiation. The method of the present application can further increase cytokine release by the immune cells. The method of the present application can further increase the tumor killing ability of the immune cells. Furthermore, the method of the present application can prevent tumor recurrence in a subject. The present application further provides a method of treating a tumor in a subject in need thereof. The present application further provides a composition comprising the genetically modified immune cells as described above, and the use of the genetically modified immune cells as described above and the composition in the preparation of a medicament. The present application further provides a method of preparing the genetically modified immune cells as described above.

[0005] On the other hand, the present application provides a method for promoting immune cell proliferation, comprising the step of increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the above-mentioned immune cells.

[0006] On the other hand, the present application provides a method for promoting production of memory immune cells, comprising the step of promoting differentiation of the aforementioned immune cells into memory immune cells by increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells.

[0007] On the other hand, the present application provides a method for inhibiting immune cell differentiation, comprising the step of inhibiting the differentiation of the aforementioned immune cells into differentiated immune cells by increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the aforementioned immune cells.

[0008] On the other hand, the present application provides a method for increasing cytokine release by immune cells, comprising the step of increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the aforementioned immune cells.

[0009] In one embodiment, the aforementioned cytokines are interleukins, interferons, In one embodiment, the aforementioned cytokines include IL-2, IL4, IL6, IL7, IL10, IL12, TNF-α, and / or IFNγ.

[0010] On the other hand, the present application provides a method for increasing the tumor-killing ability of immune cells, comprising the step of increasing the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in the above-mentioned immune cells.

[0011] Meanwhile, the present application provides a method for preventing the recurrence of a tumor in a subject, comprising administering to the tumor-susceptible subject immune cells having increased expression of low density lipoprotein receptor-related protein or a fragment thereof.

[0012] On the other hand, the present application provides a method for treating a tumor in a subject, which requires the step of administering to the subject immune cells having increased expression of low density lipoprotein receptor-related protein or a fragment thereof.

[0013] In some embodiments, the aforementioned tumor is selected from liver cancer, lung cancer, leukemia and mesothelioma.

[0014] In certain embodiments, the aforementioned methods include in vivo and in vitro methods.

[0015] In some embodiments, the immune cells include lymphocytes. In some embodiments, the immune cells include T cells. In some embodiments, the T cells include memory stem cell-like T cells (TSCM) and / or central memory T cells (TCM). In some embodiments, the TSCM are CCR7 + and / or CD62L + In one embodiment, the TSCM further comprises CD45RA +or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + One or more selected from the group It has the same characteristics.

[0016] In one embodiment, the aforementioned immune cells comprise genetically modified immune cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The genetically modified immune cells described above include genetically modified T cells.

[0017] In one embodiment, the aforementioned CAR contains an intracellular domain comprising a signaling domain and / or a costimulatory domain.

[0018] In one embodiment, the aforementioned signaling domain is a CD3ζ signaling domain. In one embodiment, the signaling domain comprises a portion selected from the group consisting of a CD3 delta signaling domain, a CD3 epsilon signaling domain, and a CD3 epsilon signaling domain. In one embodiment, the signaling domain comprises a portion selected from the group consisting of a CD3 delta signaling domain, a CD3 delta signaling domain, and a CD3 epsilon signaling domain. or an amino acid sequence having at least 80% homology thereto. In one embodiment, the nucleic acid molecule encoding the aforementioned signaling domain comprises a nucleic acid sequence set forth in SEQ ID NO:17, or a nucleic acid sequence having at least 80% homology thereto.

[0019] In one embodiment, the costimulatory domain comprises a portion selected from the group consisting of a CD27 costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. The costimulatory domain described above contains an amino acid sequence represented by any one of SEQ ID NO:14 and SEQ ID NO:16, or an amino acid sequence having at least 80% homology thereto. In some embodiments, the nucleic acid molecule encoding the costimulatory domain is a nucleic acid sequence represented by any one of SEQ ID NO:13 and SEQ ID NO:15, or a nucleic acid sequence having at least 80% identity thereto. The present invention contains a nucleic acid sequence having the following structure:

[0020] In one embodiment, the CAR comprises a hinge region. In one embodiment, the hinge region comprises a portion selected from the group of an IgG4 hinge region, an IgG1 hinge region, and a CD8 hinge region. In one embodiment, the hinge region comprises an amino acid sequence represented by SEQ ID NO: 31. In one embodiment, the nucleic acid molecule encoding the hinge region comprises a nucleotide sequence represented by SEQ ID NO: 32.

[0021] In one embodiment, the CAR comprises a transmembrane region. In one embodiment, the transmembrane region comprises a portion selected from the group of a CD8 transmembrane region, a CD28 transmembrane region, and a CD24 transmembrane region. In one embodiment, the transmembrane region comprises an amino acid sequence represented by SEQ ID NO: 33. In one embodiment, the nucleic acid molecule encoding the transmembrane region comprises a nucleotide sequence represented by SEQ ID NO: 34.

[0022] In some embodiments, the CAR comprises a targeting moiety. In some embodiments, the targeting moiety comprises an ScFv.

[0023] In one embodiment, the targeting moiety specifically binds and / or targets a tumor antigen. In one embodiment, the targeting moiety recognizes or recognizes a B-lymphocyte surface antigen. , TNF family members, HER family members and GPC family members. In one embodiment, the antibody specifically binds to and / or recognizes a target selected from the group consisting of: The aforementioned targeting moieties specifically bind to and / or recognize a target selected from the group of CD19, BCMA, HER2, Mesothelin and GPC3.

[0024] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 46. An antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes CD19, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 48 or an amino acid sequence having at least 80% identity thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 50 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 43 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 45 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 49 or an amino acid sequence having at least 80% identity thereto.

[0025] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 54. An antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 55 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 51 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 52 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 53 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 57 or an amino acid sequence having at least 80% identity thereto.

[0026] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 70. LCDR1 containing an amino acid sequence having at least 80% homology thereto, and an amino acid sequence having at least 80% homology thereto represented by SEQ ID NO:71. and an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region comprises an HCDR1 comprising an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the aforementioned heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 80% identity thereto.

[0027] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 62. and an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes mesothelin, the antibody or antigen-binding fragment comprising a light chain variable region comprising an LCDR1 containing an amino acid sequence represented by SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto, an LCDR2 containing an amino acid sequence represented by SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 59 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 60 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 61 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 65 or an amino acid sequence having at least 80% identity thereto.

[0028] In one embodiment, the targeting moiety is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 38. The antibody or antigen-binding fragment thereof specifically binds to and / or recognizes GPC3, comprising a light chain variable region including an LCDR1 containing an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, an LCDR2 containing an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region contains an amino acid sequence represented by SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 35 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 36 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto.

[0029] In one embodiment, the targeting moiety is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10. Any of the amino acid sequences shown below or amino acids having at least 80% homology thereto In one embodiment, the nucleic acid molecule encoding the targeting moiety described above comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence having at least 80% homology thereto.

[0030] In one embodiment, the method further comprises the steps of: + T lymph cells, CD8+ T lymphocytes, CD4 + By isolating T lymphocytes or regulatory T cells The method includes the step of obtaining a

[0031] In one embodiment, the method further comprises administering to the isolated PBMCs one or more or adding multiple T cell stimulatory factors. In one embodiment, the T cell stimulatory factors are selected from the group consisting of B lymphocyte surface antigen antibodies, TNF antibodies, intracellular polyesters, and antibiotics. In one embodiment, the T cell stimulatory factors are selected from the group consisting of CD3 antibodies, CD28 antibodies, 4-1BB antibodies, CD80 antibodies, CD86 antibodies, PHA, PMA, and ionomycin. .

[0032] In one embodiment, the T cell stimulatory factor includes a CD3 antibody at a concentration of 1 to 10,000 ng / mL. In one embodiment, the T cell stimulating factor comprises a CD28 antibody at a concentration of 1 to 10,000 ng / mL.

[0033] In one embodiment, the method further comprises administering to the isolated PBMCs one or more or the administration of multiple cytokine agents.

[0034] In certain embodiments, the cytokine agent comprises an interleukin.

[0035] In one embodiment, the interleukin comprises one or more interleukins selected from the group consisting of IL2, IL21, IL7 and IL15. In one embodiment, the IL2 concentration is 0.1 to 10,000 U / mL. In one embodiment, the interleukin contains IL21 at a concentration of 0.01 to 1000 ng / mL. The aforementioned interleukin contains IL7 at a concentration of 0.01 to 1000 ng / mL. In one embodiment, the aforementioned interleukin contains IL15 at a concentration of 0.01 to 1000 ng / mL.

[0036] In one embodiment, the low density lipoprotein receptor-related protein is one selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof is of human origin.

[0037] In one embodiment, the functional fragment comprises a fragment of the low density lipoprotein receptor-related protein or a truncated form thereof having the low density lipoprotein receptor-related protein activity. In one embodiment, the low density lipoprotein receptor-related protein is low density lipoprotein receptor-related protein 6 and truncated forms thereof, and and / or low density lipoprotein receptor-related protein 5 and truncated forms thereof. In one embodiment, the truncated forms of low density lipoprotein receptor-related protein 6 include the and / or The truncated form of the low density lipoprotein receptor-related protein 5 is In one embodiment, the antibody comprises an intracellular domain having a polypeptide receptor-associated protein 5. The truncated form of the low-density lipoprotein receptor-related protein 6 described above is The transmembrane domain of lipoprotein receptor-related protein 6 and the previously mentioned low density lipoproteins The low density lipoprotein receptor-related protein 5 includes the LDLR domain of the receptor-related protein 6 and / or the truncated form of the low density lipoprotein receptor-related protein 5 described above. The transmembrane domain containing protein 5 and the previously mentioned low density lipoprotein receptor-related protein In one embodiment, the low density lipoprotein receptor 5 domain is The human body-associated protein or a fragment thereof is represented by any of SEQ ID NOs: 22, 24, 26 and 28. or an amino acid sequence having at least 80% homology thereto. In one embodiment, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof is represented by any of SEQ ID NOs: 21, 23, 25, and 27. It contains a nucleic acid sequence or an amino acid sequence having at least 80% homology thereto.

[0038] On the other hand, the present application provides genetically modified immune cells that increase the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells by genetic modification.

[0039] In some embodiments, the genetically modified immune cells include lymphoid cells. In some embodiments, the genetically modified immune cells include genetically modified T cells. In one embodiment, the genetically modified immune cells include genetically modified memory stem cell-like T cells (TSCM) and / or genetically modified central memory T cells (TCM). In one embodiment, the TSCM are CCR7 + and / or CD62L + In one embodiment, the TSCM further comprises CD45RA + or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + The compound has one or more properties selected from the group consisting of:

[0040] In one embodiment, the aforementioned immune cells include genetically modified immune cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0041] In one embodiment, the CAR comprises an intracellular domain consisting of a signaling domain and / or a costimulatory domain. In one embodiment, the signaling domain comprises a CD3 zeta signaling domain, a CD3 delta signaling domain and a CD3 epsilon signaling domain. In one embodiment, the signaling domain comprises a portion selected from the group consisting of a signaling domain, a signaling domain, a polypeptide ... Thus, the nucleic acid molecule encoding the aforementioned signaling domain comprises the nucleic acid sequence depicted in SEQ ID NO:17 or a nucleic acid sequence having at least 80% homology thereto.

[0042] In one embodiment, the costimulatory domain comprises a portion selected from the group consisting of a CD27 costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. The costimulatory domain described above contains an amino acid sequence represented by any one of SEQ ID NO:14 and SEQ ID NO:16, or an amino acid sequence having at least 80% homology thereto. In some embodiments, the nucleic acid molecule encoding the costimulatory domain is a nucleic acid sequence represented by any one of SEQ ID NO:13 and SEQ ID NO:15, or a nucleic acid sequence having at least 80% identity thereto. The present invention contains a nucleic acid sequence having the following structure:

[0043] In one embodiment, the CAR comprises a hinge region. In one embodiment, the hinge region comprises a portion selected from the group of an IgG4 hinge region, an IgG1 hinge region, and a CD8 hinge region. In one embodiment, the hinge region comprises an amino acid sequence represented by SEQ ID NO: 31, or an amino acid sequence having at least 80% homology thereto. In one embodiment, the nucleic acid molecule encoding the hinge region comprises a nucleic acid sequence represented by SEQ ID NO: 32, or an amino acid sequence having at least 80% homology thereto.

[0044] In one embodiment, the CAR comprises a transmembrane region. In one embodiment, the transmembrane region comprises a portion selected from the group of CD8 transmembrane region, CD28 transmembrane region, and CD24 transmembrane region. In one embodiment, the transmembrane region comprises an amino acid sequence represented by SEQ ID NO: 33 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the nucleic acid molecule encoding the transmembrane region comprises a nucleic acid sequence represented by SEQ ID NO: 34 or a nucleic acid sequence having at least 80% homology thereto.

[0045] In some embodiments, the CAR comprises a targeting moiety. In some embodiments, the targeting moiety comprises an ScFv. In some embodiments, the targeting moiety specifically binds to and / or recognizes a tumor antigen. In some embodiments, the targeting moiety specifically binds to and / or recognizes a target selected from the group of B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members. In some embodiments, the targeting moiety specifically binds to and / or recognizes a target selected from the group of CD19, BCMA, HER2, Mesothelin, and GPC3.

[0046] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 46. An antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes CD19, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 48 or an amino acid sequence having at least 80% identity thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 50 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 43 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 45 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 49 or an amino acid sequence having at least 80% identity thereto.

[0047] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 54. An antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 55 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 51 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 52 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 53 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 57 or an amino acid sequence having at least 80% identity thereto.

[0048] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 70. LCDR1 containing an amino acid sequence having at least 80% homology thereto, and an amino acid sequence having at least 80% homology thereto represented by SEQ ID NO:71. and an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region comprises an HCDR1 comprising an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the aforementioned heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 80% identity thereto.

[0049] In one embodiment, the targeting moiety comprises the amino acid sequence represented by SEQ ID NO: 62. LCDR1 containing an amino acid sequence having at least 80% homology thereto, and an amino acid sequence having at least 80% homology thereto represented by SEQ ID NO:63. and an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 59 or an amino acid sequence having at least 80% homology thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 61 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the aforementioned heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 65, or an amino acid sequence having at least 80% identity thereto.

[0050] In one embodiment, the targeting moiety is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 38. The antibody or antigen-binding fragment thereof specifically binds to and / or recognizes GPC3, comprising a light chain variable region including an LCDR1 containing an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, an LCDR2 containing an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. In one embodiment, the light chain variable region contains an amino acid sequence represented by SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 35 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 36 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 37 or an amino acid sequence having at least 80% identity thereto. In certain embodiments, the aforementioned heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto.

[0051] In one embodiment, the targeting moiety is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10. Any of the amino acid sequences shown below or amino acids having at least 80% homology thereto In one embodiment, the nucleic acid molecule encoding the targeting moiety described above comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 1, 3, 5, 7 and 9, or a nucleic acid sequence having at least 80% homology thereto.

[0052] In one embodiment, the low density lipoprotein receptor-related protein is one selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof is of human origin.

[0053] In one embodiment, the functional fragment comprises a fragment of the low density lipoprotein receptor-related protein having the low density lipoprotein receptor-related protein activity, or a truncated form thereof. In one embodiment, the low density lipoprotein receptor-related protein is low density lipoprotein receptor-related protein 6, as well as truncated forms thereof, and and / or low density lipoprotein receptor-related protein 5 and truncated forms thereof. In one embodiment, the truncated forms of low density lipoprotein receptor-related protein 6 are The intracellular domain of the low density lipoprotein receptor-related protein 6 is included, and / or the truncated form of the low density lipoprotein receptor-related protein 5 is included, In one embodiment, the lipoprotein receptor-related protein 5 (LRP5) is a lipoprotein receptor-related protein 5 (LRP5) that is a lipoprotein receptor-related protein 5 (LRP5). The truncated form of the low-density lipoprotein receptor-related protein 6 described above is The transmembrane domain of protein receptor-related protein 6 and the previously described low density lipoprotein receptor and / or the truncated form of low density lipoprotein receptor-related protein 5 comprises the LDLR domain of low density lipoprotein receptor-related protein 6. In one embodiment, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence represented by any of SEQ ID NOs: 22, 24, 26, and 28. or an amino acid sequence having at least 80% homology thereto. In one embodiment, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof is a nucleic acid sequence represented by any one of SEQ ID NOs: 21, 23, 25 and 27. contains a nucleic acid sequence having at least 80% homology thereto.

[0054] On the other hand, the present application provides a composition comprising the aforementioned genetically modified immune cells.

[0055] In certain embodiments, the above-described compositions further comprise an optional pharma- ceutically acceptable carrier.

[0056] On the other hand, the present application relates to the use of the aforementioned in the preparation of a medicament for the treatment and / or prevention of tumors. Uses of the genetically modified cells and / or compositions described above are also provided.

[0057] In some embodiments, the aforementioned tumor is selected from liver cancer, lung cancer, leukemia and mesothelioma.

[0058] On the other hand, the present application provides a method for preparing the aforementioned composition, comprising the step of increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the aforementioned genetically modified immune cells.

[0059] In some embodiments, the method includes introducing into the genetically modified immune cell a vector that enhances expression of the low density lipoprotein receptor-related protein or a fragment thereof. In some embodiments, the vector is a retroviral vector. The vector is selected from the group consisting of a plasmid, a lentiviral vector and a transposon plasmid.

[0060] In one embodiment, the low density lipoprotein receptor-related protein or a fragment thereof is an amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26, and 28, or a fragment thereof. It contains an amino acid sequence having at least 80% homology thereto.

[0061] In some embodiments, the genetically modified immune cells include lymphoid cells. In some embodiments, the genetically modified immune cells include chimeric antigen receptor (CAR) In one embodiment, the method includes isolating the genetically modified immune cells and injecting a T cell medium onto the isolated genetically modified immune cells. The activation step includes providing a

[0062] In one embodiment, the aforementioned T cell medium contains one or more selected from the group consisting of DMEM medium, 1640 medium, MEM medium and X-VIVO medium.

[0063] In certain embodiments, the method further comprises administering to the genetically modified immune cells a T cell stimulatory factor.

[0064] Those skilled in the art will be able to easily grasp other aspects and advantages of the present disclosure from the following detailed description. In the following detailed description, only exemplary embodiments of the present disclosure are depicted and described. For those skilled in the art, the details of the present disclosure may be modified without departing from the spirit and scope of the present invention. Accordingly, in the present invention, the drawings and the description are merely examples and are not intended to limit the present invention. [Brief description of the drawings]

[0065] Specific features of the present invention are set forth in the appended claims. The features and advantages of the present invention will be more clearly understood with reference to the following detailed description of exemplary embodiments and drawings, which are generally described as follows:

[0066] FIG. 1 shows a schematic diagram of the construction of the above-mentioned CAR in the lentivirus according to the present invention.

[0067] FIG. 2 shows the genetic modification of the immune cells according to the present invention to promote memory immune cell production. The results are shown below.

[0068] FIG. 3 shows the results of suppressing immune cell differentiation by the genetically modified immune cells according to the present invention. show.

[0069] FIG. 4 shows the low density lipoprotein receptor-related protein or its analog in immune cells according to the present invention. 1 shows the results of the present invention showing that the antibody or a fragment thereof promotes immune cell proliferation.

[0070] 5A-5B show the results of increased cytokine release by genetically modified immune cells according to the present invention.

[0071] FIG. 6 shows the anti-tumor effect of the genetically modified immune cells according to the present invention.

[0072] FIG. 7 shows the effect of preventing tumor recurrence by the genetically modified immune cells according to the present invention. vinegar. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Hereinafter, the embodiments of the present invention will be described with reference to certain specific examples, but those skilled in the art can easily grasp other advantages and effects of the present invention from the contents disclosed in this specification.

[0074] In the present invention, the term immune cells generally refers to cells involved in or related to an immune response. The immune cells may include lymphocytes and various phagocytes. The immune cells may include natural and genetically modified immune cells. In the present invention, the genetically modified immune cells express a chimeric antigen receptor (CAR). Here, the lymphatic cells may include T lymphatic cells and B lymphatic cells. In the present invention, the immune cells may include T cells.

[0075] In the present invention, the term memory immune cells generally refers to immune cells with immune memory. The immune memory is said to be such that when a specific antigen is recognized and responded to, a rapid and effective immune response is exerted when the same antigen is encountered again. In the present invention, the memory immune cells may contain memory T cells. The memory T cells can be divided into memory stem cell-like T cells (TSCM) and central memory T cells (TCM).

[0076] In the present invention, the term "differentiated immune cells" generally refers to immune cells having a certain degree of differentiation. For example, the above-mentioned differentiated immune cells are T cells having a certain degree of differentiation. In the present invention, the differentiated immune cells can be obtained by differentiating the immune cells to a certain degree through culture. For example, the differentiated immune cells can be regulatory T cells (Treg ) may be included.

[0077] In the present invention, the term "regulatory T cells (Treg)" generally refers to a type of lymphocyte that negatively regulates the immune response of the body. Photographic factor Foxp3 + or CD127 - In the present invention, the regulatory T cells may be naturally occurring The naturally occurring CD4 + CD25 + Cells and induction Production is T R 1 Cells and T H It is three cells.

[0078] In the present invention, the term "subject susceptible to tumors" generally refers to a subject who is more likely to develop a tumor than a general subject. For example, the subject susceptible to tumors may be a subject who has a tumor but is cured, but is at risk of metastasis or recurrence. The subject susceptible to tumors may be a subject diagnosed with risk factors for developing tumors. For example, the risk factors may include some gene mutations (having deletions, additions or substitutions) that are proven to be correlated with tumors. In the present invention, the subject susceptible to tumors may be a subject who has been exposed to a carcinogenic environment for a long period of time. For example, the carcinogenic environment may include strong radiation and high concentrations of carcinogenic environmental substances.

[0079] In the present invention, the term genetic modification generally refers to a modification or alteration that occurs at the gene structure level. For example, the genetic modification may occur at the gene level, the transcription level and / or The genetic modification may be at the level of translation or at the level of expression. For example, the genetic modification may include any modification of genetic characteristics in an organism (such as its tissues, cells, DNA, mRNA, or a protein or a fragment thereof). The genetic modification may include being able to express a given protein or a fragment thereof in the organism. For example, the genetic modification may include providing the organism with a vector capable of expressing the given protein or a fragment thereof.

[0080] In the present invention, the term T cells, also referred to as T lymphocytes, refers to cells that play a major role in cell-mediated immunity. T cells are a subtype of white cell that plays a key role in the immune system. T cells are distinguished from other lymphoid cells, such as B cells and natural killer cells, by the T cell receptors present on their cell surface. In the present invention, the T cells may include memory stem cell-like T cells (TSCM) and central memory T cells (TCM).

[0081] In the present invention, the term "memory stem cell-like T cells (TSCM)" refers to cells that are in the early stage of differentiation of memory T cells, have stem cell characteristics, and have strong multipotency. In response to antigen stimulation, TSCM cells differentiate into central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEF).

[0082] In the present invention, the term "central memory T cells" (TCM) generally refers to long-term T cells that are generated after activating naive T cells with an antigen. Refers to T cells with memory. The biomarkers for the above TCM are CD62L + and CD45RO + With The central memory T cells described above return to the lymph nodes through lymphatic shielding, and at the same time, It is in an antigen-activated state.

[0083] In the present invention, the term T cell receptor, also commonly referred to as TCR, generally refers to the receptor that is a receptor for the anti-cancer agent by T cells. The T cell receptor is a molecular structure that specifically recognizes and binds to the original peptide-MHC molecule. The TCR may be present on the surface of a T cell in a complex with the CD3 molecule. TCRs are heterodimers anchored on the cell membrane, often composed of hypervariable α and β subunits, and rarely composed of γ and δ peptide chains, via a TCR domain. The constant region may be located proximal to the transmembrane region and connected to the intracellular terminus.

[0084] In the present invention, the term chimeric antigen receptor, also commonly referred to as CAR, generally refers to a chimeric antigen receptor that binds to an antigen. The term CAR refers to a fusion protein comprising an extracellular domain, which may be combined with at least one intracellular domain. In the present application, the above-mentioned CAR may comprise an intracellular domain comprising a signaling domain and / or a costimulatory domain. In the present invention, the CAR may comprise a group of polypeptides located on the same polypeptide chain (including, for example, a chimeric fusion protein), but may not be contiguous with each other, for example, located on different polypeptide chains. In the present invention, the signal involved in the induction is transmitted to the cytoplasm of the T cell via the CD3 and zeta chains. In the present invention, the intracellular domain may comprise one first level signaling domain (e.g., the main signal domain of CD3-zeta (zeta)). In one aspect, the cytoplasmic signal domain may comprise one or more costimulatory domains derived from at least one costimulatory molecule. For example, the costimulatory domain may be 4-1BB (i.e., CD137), CD27, ICOS and / or CD28. In the present invention, the CAR may comprise, for example, a chimeric fusion protein containing an optional leader sequence at the amino-terminus (N-ter), which optionally cleaves the antigen-binding domain (e.g., ScFv) upon cellular processing and localizes the CAR to the cell membrane.

[0085] In the present invention, the term signaling domain generally refers to a domain capable of transmitting a signal inside a cell. In the present invention, the intracellular signaling domain is capable of transmitting a signal into the cell. For example, the intracellular signaling domain is the intracellular signaling domain in the chimeric antigen receptor. In the present invention, the signaling domain is the intracellular signaling domain of CD3ζ, CD3δ and CD3ε signaling domain. The moiety is selected from the group:

[0086] In the present invention, the term costimulatory domain refers to a domain that crosses the cell membrane in a CAR, usually linking with an intracellular signaling domain and functioning to transmit a signal. In the present invention, the costimulatory domain is a group of CD27, CD28 and 4-1BB costimulatory domains. The present invention includes a portion selected from the group consisting of

[0087] In the present invention, the term hinge region generally refers to the linking region between the antigen-binding domain and the immune cell Fc receptor (FcR) binding domain. In the present invention, the hinge region may be a region between the heavy chain CH1 and CH2 functional domains in IgG1. In the present invention, the above-mentioned hinge region may be a single region between the scFv and the T cell membrane. The above-mentioned hinge region may be derived from IgG1 or IgG4, or from IgD or CD8. In the present invention, the hinge region may be derived from an IgG4 hinge region, an IgG1 hinge region, or a CD8 hinge region. The present invention includes a portion selected from the group consisting of:

[0088] In the present invention, the term "transmembrane domain" generally refers to CD3ζ, CD4, CD8, CD28, etc. The term "CAR" refers to a dimeric membrane protein that can anchor the CAR structure on the T cell membrane and link the extracellular antigen-binding domain and the intracellular signal domain. Different designs for the transmembrane domain can affect the expression of the introduced CAR gene. In the present invention, the transmembrane region comprises a portion selected from the group consisting of a CD8 transmembrane region, a CD28 transmembrane region, and a CD24 transmembrane region.

[0089] In the present invention, the term single chain antibody (ScFv) generally refers to an antibody in which the heavy chain variable region and the light chain variable region are linked by a linker. In the present invention, the linker may be a connecting peptide.

[0090] In the present invention, the term tumor antigen generally refers to an antigenic substance produced in or by a tumor cell that may be capable of eliciting an immune response in a host. For example, a tumor antigen constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. In some embodiments, the term tumor antigen can be a protein, polypeptide, peptide, or fragment thereof that is exclusively, preferentially, or differentially expressed on cancer cells and / or associated with cancer cells. The tumor antigen may be a tumor antigen (such as a tumor protein) that provides a preferential or specific target for cancer. For example, preferential expression may be conventional preferential expression over all other cells in the body, or preferential expression in a given area of ​​the body (e.g., in a given organ or tissue). In the present invention, the tumor antigen may be a B lymphocyte surface antigen, a TNF family member, a HER2 kinase, a HER2 kinase, or a HER2 kinase. It may be a member of the myelin family and a member of the GPC family.

[0091] In the present invention, the term B-lymphocyte surface antigen refers to an antigen that is usually produced by B-lymphocytes at different stages and is present on the surface of B-lymphocytes. For example, the B-lymphocyte surface antigen may include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39 and CD40. In the present invention, the B-lymphocyte surface antigen may include CD19.

[0092] In the present invention, the term CD19 may refer to the cluster of differentiation 19 protein, which is an antigenic determinant typically measurable on leukemic progenitor cells. Human CD19 has the UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 is available at GenBank accession no. The CD19 gene has the accession number NM_001178098. In the present invention, CD19 may further include proteins or functional fragments thereof that contain mutations, such as point mutations, fragments, insertions, deletions and splice variants of full-length wild-type CD19.

[0093] In the present invention, the term TNF family member generally refers to a member belonging to the TNF (Tumor Necrosis Factor) family. The TNF family members may include CD40LG (TNFSF5), CD70 (TNFSF7), EDA, FASLG (TNFSF6), LTA (TNFSF1), LTB (TNFSF3), TNFSF4 (OX40L), TNFSF8 (CD153), TNFSF9 (4-1BB), TNFSF10 (TRAIL), TNFSF11 (RANKL), TNFSF12 (TWEAK), TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF17 (BCMA) and TNFSF18. In the present invention, the above TNF family members may include BCMA and 4-1BB.

[0094] In the present invention, the term BCMA generally refers to B cell maturation antigen (BCMA, CD269). BCMA is a member of the tumor necrosis factor receptor (TNF) superfamily that can bind to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA is commonly found on the surface of plasma cells in multiple myeloma patients. Human BCMA has the GenBank accession number BAB60895.1.

[0095] The term CD137 protein, also called 4-1BB or TNFRS9, is usually used to describe a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS) and is an activation-induced It is a costimulatory molecule and an important regulator of immune responses. Studies to date have shown that CD137 agonists Monoclonal antibodies increase the expression of costimulatory molecules and significantly enhance cytolytic T lymphocyte responses in many models, exerting antitumor effects (see Vinay, Dass S., and Byoung S. Kwon. "4-1BB (CD137), an inducible costimulatory receptor, as a specific target for cancer therapy." BMB reports 47.3 (2014): 122). Human CD137 has the GenBank accession number NP_001552.2.

[0096] In the present invention, the term HER family member generally refers to a member belonging to the HER (human epidermal growth factor receptor) family. Family members may include EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). In the present invention, the HER family members may include HER2.

[0097] In the present invention, the term HER2 generally refers to human HER2, which is a member of the HER family. It refers to a protein. See, for example, Semba et al., PNAS(USA)82:6497-6501(1985) and Yamamoto et al., Nature 319:230-234(1986). Human HER2 may have the GenBank accession number XP_024306409.1.

[0098] In the present invention, the term GPC family member refers to phosphatidylinositol protein sugar chains (glypicans), which are generally identified as six types of glypicans, designated GPC1 to GPC6, in mammals. Abnormal expression of glypicans is observed in cancers including human hepatocellular carcinoma, ovarian cancer, mesothelioma, pancreatic cancer, glioma and breast cancer. In the present invention, the GPC family member may include GPC3.

[0099] In the present invention, the term GPC3 generally refers to an early marker of liver cancer and is encoded by phosphatidylinositol protein glycoprotein 3 (NCBI database gene ID: 2719). GPC3 is a protein that is highly expressed in hepatocellular carcinoma and can be measured in tissues from patients with early stage hepatocellular carcinoma. Human GPC3 has the GenBank accession number AAB87062.1 and is well known in the art. stomach.

[0100] In the present invention, the term Mesothelin (or MSLN) generally refers to a tumor differentiation antigen that is commonly present in mesothelial cells of the pleural, peritoneal and pericardial linings. It is highly expressed in carcinomas including malignant mesothelioma, pancreatic cancer, ovarian cancer and lung adenocarcinoma. Human Mesothelin may have the GenBank accession number AAH09272.1.

[0101] In the present invention, the term peripheral blood mononuclear cells generally refers to cells having a single nucleus in peripheral blood ( Peripheral blood mononuclear cells (PBMCs) are lymphocytes. In the present invention, the peripheral blood mononuclear cells are separated by utilizing Ficoll-hypaque (glucan-urografin) density gradient centrifugation due to the difference in specific gravity of each component in blood.

[0102] In the present invention, the term T cell activator generally refers to a substance that promotes the activation and proliferation of T cells. In the present invention, the T cell activator may include a B lymphocyte surface antigen antibody, a TNF antibody, an intracellular polyester, and / or an antibiotic. In the present invention, the T cell stimulator may be selected from the group of CD3 antibody, CD28 antibody, 4-1BB antibody, CD80 antibody, CD86 antibody, PHA, PMA, and ionomycin.

[0103] In the present invention, the term B-lymphocyte surface antigen antibody generally refers to an antibody that specifically binds to a B-lymphocyte surface antigen. In the present invention, the B-lymphocyte surface antigen antibody may include CD3 antibody, CD28 antibody, CD80 antibody, and CD86 antibody.

[0104] In the present invention, the term TNF antibody generally refers to an antibody that specifically binds to a TNF family member. In the present invention, the TNF antibody may include the 4-1BB antibody.

[0105] In the present invention, the term intracellular polyester refers to a natural polymeric biomaterial that is usually synthesized by microorganisms and exists as an inclusion body within a cell. Intracellular polyesters have excellent biological properties. In the present invention, intracellular polyesters may include polyhydroxyalkanoates (PHAs).

[0106] In the present invention, the term antibiotics generally refers to anti-pathogens or other active metabolic products produced by microorganisms or plants or animals that inhibit the growth and useful functions of other living cells. In the present invention, the antibiotics include β-lactams, aminoglycosides, amphotericin, and cyclosporine. For example, the antibiotic may include ionomycin, cyclosporine, macrolides, polypeptides, nitroimidazoles, and tetracyclines.

[0107] In the present invention, the term PHA generally refers to intracellular polysaccharides synthesized by many types of bacteria. PHA is a type of polyhydroxyalkanoate that exists as discontinuous inclusion bodies in the cytoplasm of living organisms. It has the same physical and chemical properties as synthetic plastics. The materials may have the following properties: biodegradability, biocompatibility, optical activity, piezoelectric effect, gas barrier properties, etc.

[0108] In the present invention, the term PMA generally refers to phorbol-12-myristate-13-acetate.

[0109] In the present invention, the term CD3 antibody generally refers to an antibody or an antigen-binding fragment thereof that specifically binds to CD3. CD3 is an important differentiation antigen in the T cell membrane that transmits T cell activation signals. The CD3 antibody may be huOKT3g1 or HuM291.

[0110] In the present invention, the term CD28 antibody generally refers to an antibody or antigen-binding fragment thereof that specifically binds to CD28. Human CD28 is located at 2q33 and contains similar exons and introns to CTLA4, which is a member of the B7 family whose ligands both include B7-1 (CD80) and B7-2 (CD86).

[0111] In the present invention, the term cytokine generally refers to a small protein molecule with a wide range of biological activities synthesized and secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (e.g., endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokines may have a wide variety of functions, such as regulating innate and adaptive immunity, hematopoiesis, cell proliferation, APSC pluripotent cells, and damaged tissue repair. In the present invention, the cytokines may include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemotactic factors, and growth factors. For example, the cytokine may be an interleukin.

[0112] In the present invention, the term interleukin is generally used to refer to T and / or B lymphocytes. and / or secreted proteins capable of promoting the development and differentiation of hematopoietic cells; or Interleukins refer to signaling molecules. Interleukins are synthesized by accessory CD4 T lymphocytes, as well as monocytes, macrophages and endothelial cells. In the present invention, the term interleukin refers to a compound that essentially retains a biological activity corresponding to a wild-type interleukin (e.g., a biological activity corresponding to a wild-type interleukin having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even at least 100% of the biological activity). The interleukin may comprise a full length interleukin or a fragment (e.g., truncated) or variant thereof. The interleukin as used herein may be from any mammalian species. In one embodiment, the interleukin is from a species selected from human, horse, cow, mouse, pig, rabbit, cat, dog, rat, goat, sheep and non-human primates. In one embodiment, the interleukin is a mutant. For example, in the present invention, the cytokine may comprise one or more selected from the group of IL2, IL21, IL7 and IL15.

[0113] In the present invention, the term IL2 generally refers to T cell growth factor, TCGF. It is produced by the immune system and, in an autocrine and paracrine manner, activates T cells, promotes cytokine production, stimulates NK cell proliferation, induces LAK cell production, promotes B cell proliferation and antibody secretion, and inhibits macrophages. It acts to trigger phage activation.

[0114] In the present invention, the term IL21 refers to a molecule that is homologous to the spatial structure of IL-2, IL-4, and IL-15. , normally promotes the proliferation and differentiation of bone marrow NK cells, and stimulates B cell proliferation in conjunction with anti-CD40 antibodies and T cell proliferation in conjunction with anti-CD3 antibodies.

[0115] In the present invention, the term IL7 refers to a glycoprotein secreted by bone marrow stromal cells, the gene for which is usually located on chromosome 8. The target cells for IL7 are in particular human and mouse bone marrow It is a lymphoid cell that can promote the proliferation of B cell precursors, thymocytes, and peripheral mature T cells derived from the IL-7. At high concentrations, IL-7 can enhance the cytotoxic activity of macrophages and induce cytokine secretion from monocytes.

[0116] In the present invention, the term IL15 is generally used to refer to activated monocytes, epidermal cells and It is produced by many types of cells, including fibroblasts and lymphocytes, and has many similarities to IL2. IL15 is a member of the IL2 family and can control the activation and proliferation of T and NK cells and induce B cell proliferation by altering the expression of Bcl-1 family members (e.g., Bcl-2 and Bcl-XL).

[0117] In the present invention, the term low density lipoprotein receptor-related protein (LRP) generally refers to an endogenous protein containing 839 amino acids (with the 21 amino acid signal peptide removed). It is embedded in the outer phospholipid layer of LDL (Low Density Lipoprotein) particles, belongs to endocytic receptors that can mediate endocytosis of cholesterol-rich LDL, and is a member of the low density lipoprotein receptor (LDLR) gene family. The LRP is most prominently expressed in bronchial epithelial cells, adrenal gland and cortical tissue. In the present invention, the low density lipoprotein receptor-related protein may include one or more types selected from the group of low density lipoprotein receptor-related proteins 1 to 12 or truncated forms thereof.

[0118] In the present invention, the terms low density lipoprotein receptor-related protein 6 (LRP-6) and low density protein receptor-related protein 5 (LRP-5) generally refer to a single subgroup in the low density lipoprotein receptor (LDLR) family. Human LRP-6 has UniProt accession number O75581. Human LRP-5 has UniProt accession number O75197.

[0119] In the present invention, the term truncated protein generally refers to a shortened protein. The truncated forms are obtained by removing the N- or C-terminal portions of the protein by proteolysis or structural gene engineering, or by premature termination of translation by the introduction of a stop codon in the structural gene by a nonsense mutation.

[0120] In the present invention, the term intracellular region generally refers to a domain in which a protein is located in a cell membrane. In the present invention, the intracellular region may refer to a domain in the cell membrane of the low density lipoprotein receptor-related protein. In the present invention, the intracellular region may include a sequence of 24 to 243 of SEQ ID NO. 24 or a sequence having at least 80% homology thereto. Alternatively, it may have an amino acid sequence of amino acids 24 to 231 of SEQ ID NO. 28 or an amino acid sequence having at least 80% homology thereto.

[0121] In the present invention, the term transmembrane domain generally refers to a domain in a protein that spans a cell membrane. In the present invention, the transmembrane domain may be the domain in which the low density lipoprotein receptor-related protein spans a cell membrane. The transmembrane domain is composed of 23 hydrophobic amino acids. The domain provides an anchor function for LDLR to bind to a cell membrane. In the present invention, the transmembrane domain may comprise the sequence of 1 to 23 of SEQ NO. 24 or an amino acid sequence having at least 80% homology thereto, or the sequence of 1 to 23 of SEQ NO. 28 or an amino acid sequence having at least 80% homology thereto.

[0122] In the present invention, the term LDLR domain generally refers to a domain adjacent to the N segment of the low density lipoprotein receptor-related protein other than the transmembrane region. In the present invention, the LDLR domain is an amino acid sequence of positions 5 to 119 of SEQ ID NO. 22 or an amino acid sequence having at least 80% homology thereto, Alternatively, it may have the sequence from base 4 to base 119 of SEQ ID NO. 26 or an amino acid sequence having at least 80% homology thereto.

[0123] In the present invention, the term ribosome hopping site, also called internal ribosome entry site (IRES), refers to a segment of nucleotide sequence that is usually located in the center of an mRNA sequence and is used for translation initiation. The IRES site enables initiation of cap-independent translation. The IRES is usually located in the 5'UTR. In the present invention, the ribosome hopping site is a sequence of 1 to 578 of SEQ ID NO.29. The eye may have an array of eyes.

[0124] In the present invention, the term 2A sequence generally refers to a segment of protease-independent Generally, it refers to a self-cleaving amino acid sequence. The 2A sequence is involved in the transcription synthesis of two types of proteins. In the present invention, the 2A sequence may comprise the sequence from base 1 to base 54 of SEQ ID NO.75.

[0125] In the present invention, the term pharma- ceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or non-ionic surfactants, and the like.

[0126] In the present invention, the term retroviral vector generally refers to a vector that delivers a complementary strand of DNA to an infected cell. This refers to an RNA virus that reverse transcribes and synthesizes a single strand of DNA, uses this single strand of DNA as a template to synthesize a second strand of DNA, and integrates it into the cellular genome DNA. The above retrovirus vector uses the enzymes of the host cell to self-transcribe and replicate the RNA synthesis protein, packaging the virus, releasing it from the cell, and making it an infectious virus. The above retrovirus has high transfer efficiency and can improve the transfection rate of genes.

[0127] In the present invention, the term lentiviral vector refers to a vector that is capable of infecting HIV-1 (human immunodeficiency virus type I) Lentivirus is a gene therapy vector that has been developed based on the lentivirus. Both lentivirus vectors have the ability to infect dividing and non-dividing cells. They can effectively infect almost all mammalian cells, including neuronal cells, hepatocytes, etc., and have a high infection efficiency. Lentivirus achieves sustained expression by integrating foreign genes into the host chromosomes.

[0128] In the present invention, the term transposon plasmid refers to an autosomal recessive gene that is normally found in chromosomal DNA. The transposon-based plasmid is a basic unit capable of self-replicating and transferring. The transposon-based plasmid "jumps" from one location in the genome to another through a series of processes including cleavage and reintegration.

[0129] In the present invention, the term "tumor" refers to a neoplasm that is generated by clonal abnormal proliferation of a certain cell in a local tissue in a living body, usually under the action of various carcinogenic factors, as the cell loses normal control of proliferation at the genetic level, and such a neoplasm is also called a neoplasm because it has many space-occupying blocks. In the present invention, the tumor may include solid tumors and non-solid tumors. In the present invention, the tumor may include liver cancer, lung cancer, leukemia, and mesothelioma.

[0130] In the present invention, the term "significant increase" generally refers to an increase in the level (e.g., protein expression level, cell number), P<0.05, P<0.04, P<0.03, P<0.02, P<0.01, P<0.005, or P<0.001. For example, the above-mentioned "significant increase" may be 5% or more, 10% or more, or the like. , an increase of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

[0131] In the present invention, the term essentially unincreased typically means that the levels (e.g. protein expression level, cell number) remain the same or are increased by 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less compared to before.

[0132] In the present invention, the term about generally refers to a range of 0.5%-10% more or less than a given numerical value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% more or less than a given numerical value.

[0133] Low density lipoprotein receptor-related protein or fragment thereof In the present invention, the low density lipoprotein receptor-related protein is one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. May include a variety.

[0134] In the present invention, the low density lipoprotein receptor-related protein or a fragment thereof may be derived from a mammal, for example, from a human, a macaque, a rat, or a mouse.

[0135] In the present invention, the functional fragment may include a fragment of the low-density lipoprotein receptor-related protein having the low-density lipoprotein receptor-related protein activity or a truncated form thereof. For example, the low-density lipoprotein receptor-related protein may include low-density lipoprotein receptor-related protein 6 and truncated forms thereof, and / or low-density lipoprotein receptor-related protein 5 and truncated forms thereof.

[0136] In the present invention, the truncated form of the low density lipoprotein receptor-related protein 6 is It may comprise the intracellular domain of low-density lipoprotein receptor-related protein 6, and / or the truncated form of the low-density lipoprotein receptor-related protein 5 may comprise the intracellular domain of the low-density lipoprotein receptor-related protein 5. Also, for example, the truncated form of the low-density lipoprotein receptor-related protein 6 may comprise the transmembrane domain of the low-density lipoprotein receptor-related protein 6 and the LDLR domain of the low-density lipoprotein receptor-related protein 6, and / or the truncated form of the low-density lipoprotein receptor-related protein 5 may comprise the transmembrane domain of the low-density lipoprotein receptor-related protein 5 and the LDLR domain of the low-density lipoprotein receptor-related protein 5.

[0137] In the present invention, the low density lipoprotein receptor-related protein or a fragment thereof is an amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26 and 28 or at least one sequence similar thereto. Both contain amino acid sequences with 80% homology.

[0138] In the present invention, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof contains a nucleic acid sequence represented by any one of SEQ ID NOs: 21, 23, 25 and 27, or a nucleic acid sequence having at least 80% homology thereto.

[0139] Genetically modified immune cells The present invention provides genetically modified immune cells that enhance the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in said immune cells.

[0140] In the present invention, the genetically modified immune cells may comprise lymphoid cells. In the present invention, the genetically modified immune cells comprise genetically modified T cells. In the present invention, the genetically modified immune cells are genetically modified memory stem cell-like T cells (TSCM) and / or genetically modified central memory T cells (TCM) In the present invention, the TSCM is CCR7 + and / or CD62L + In the present invention, the TSCM further contains CD45RA + or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + The present invention includes one or more characteristics selected from the group consisting of:

[0141] In the present invention, the immune cells may comprise genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR).

[0142] In the present invention, the CAR may comprise an intracellular domain, the intracellular domain comprising: It may comprise a signalling domain and / or a costimulatory domain.

[0143] For example, the signaling domain may comprise a portion selected from the group consisting of a CD3 zeta signaling domain, a CD3 delta signaling domain, and a CD3 epsilon signaling domain. For example, the signaling domain may have the amino acid sequence represented by SEQ ID NO: 18 or a sequence similar thereto. The signal transduction domain may contain an amino acid sequence having at least 80% homology thereto. The nucleic acid molecule encoding the gene may comprise the nucleic acid sequence depicted in SEQ ID NO:17 or a nucleic acid sequence having at least 80% homology thereto.

[0144] For example, the costimulatory domain may comprise a portion selected from the group consisting of a CD27 costimulatory domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. For example, the costimulatory domain may comprise an amino acid sequence set forth in any of SEQ ID NO:14 and SEQ ID NO:16, or an amino acid sequence having at least 80% identity thereto, and the nucleic acid molecule encoding the costimulatory domain may comprise a nucleic acid sequence set forth in any of SEQ ID NO:13 and SEQ ID NO:15, or a nucleic acid sequence having at least 80% identity thereto.

[0145] In the present invention, the CAR may comprise a hinge region. For example, the hinge region may comprise a portion selected from the group of IgG4 hinge region, IgG1 hinge region and CD8 hinge region. For example, the hinge region may comprise an amino acid sequence represented by SEQ ID NO: 31 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the hinge region may comprise a nucleic acid sequence represented by SEQ ID NO: 32 or a nucleic acid sequence having at least 80% homology thereto.

[0146] In the present invention, the CAR may comprise a transmembrane region. For example, the transmembrane region may comprise a portion selected from the group of CD8 transmembrane region, CD28 transmembrane region and CD24 transmembrane region. For example, the transmembrane region may comprise an amino acid sequence represented by SEQ ID NO: 33 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the transmembrane region may comprise a nucleic acid sequence represented by SEQ ID NO: 34 or a nucleic acid sequence having at least 80% homology thereto.

[0147] In the present invention, the CAR may comprise a targeting moiety. The targeting moiety may comprise an antibody or an antigen-binding fragment. The antigen-binding fragment may be selected from the group of Fab, Fab', F(ab)2, F(ab')2, Fv and ScFv fragments. For example, the targeting moiety may be an ScFv.

[0148] In the present invention, the targeting moiety specifically binds and / or recognizes a tumor antigen. For example, the targeting moiety can specifically bind and / or recognize a target selected from the group consisting of a B-lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member. is capable of specifically binding to and / or recognizing a target selected from the group of CD19, BCMA, HER2, Mesothelin and GPC3.

[0149] In the present invention, the targeting moiety is an antibody that specifically binds and / or recognizes CD19. The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 46 or an amino acid sequence having at least 80% identity thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 48 or an amino acid sequence having at least 80% identity thereto. The light chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 50 or an amino acid sequence having at least 80% identity thereto. In the present invention, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 43 or an amino acid sequence having at least 80% identity thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 45 or an amino acid sequence having at least 80% identity thereto. The heavy chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 49 or an amino acid sequence having at least 80% identity thereto.

[0150] In the present invention, the targeting moiety is an amino acid sequence represented by SEQ ID NO: 54 or LCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 55 or an amino acid sequence having at least 80% homology thereto. and an LCDR2 having an amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto. and an LCDR3 comprising an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto. In the present invention, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 51 or an amino acid sequence having at least 80% homology thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 53 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.

[0151] In the present invention, the targeting moiety is an amino acid sequence represented by SEQ ID NO: 70 or LCDR1 containing an amino acid sequence having at least 80% homology thereto, and an amino acid sequence represented by SEQ ID NO: 71 or an amino acid sequence having at least 80% homology thereto. LCDR2 having an amino acid sequence represented by SEQ ID NO: 72 or at least 80% homology thereto. and an LCDR3 containing an amino acid sequence having at least 80% homology thereto. The light chain variable region may contain an amino acid sequence represented by SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto. In the present invention, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region containing an HCDR1 containing an amino acid sequence represented by SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto, an HCDR2 containing an amino acid sequence represented by SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 containing an amino acid sequence represented by SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may contain an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.

[0152] In the present invention, the targeting moiety is an amino acid sequence represented by SEQ ID NO: 62 or LCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto. and an LCDR2 having an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% identity thereto. and an LCDR3 comprising an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 59 or an amino acid sequence having at least 80% homology thereto, an HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 61 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.

[0153] In the present invention, the targeting moiety is an amino acid sequence represented by SEQ ID NO: 38 or LCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto. and an LCDR2 having an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80% identity thereto. and an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, the antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an HCDR1 containing an amino acid sequence represented by SEQ ID NO: 35 or an amino acid sequence having at least 80% homology thereto, an HCDR2 containing an amino acid sequence represented by SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 37. The light chain variable region may comprise an HCDR1 containing an amino acid sequence represented by SEQ ID NO: 35 or an amino acid sequence having at least 80% homology thereto, an HCDR2 containing an amino acid sequence represented by SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 37. and an HCDR3 containing an amino acid sequence as set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto. The heavy chain variable region may contain an amino acid sequence as set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto.

[0154] In the present invention, the targeting moiety is any of SEQ ID NOs: 2, 4, 6, 8 and 10. or an amino acid sequence having at least 80% homology thereto. In the present invention, the nucleic acid molecule encoding the targeting moiety may be a nucleic acid sequence represented by any of SEQ ID NOs: 1, 3, 5, 7 and 9, or a nucleic acid sequence having at least 80% homology thereto. The nucleic acid sequence may include

[0155] In the present invention, the enthalpy of an amino acid position of a CDR is calculated according to Kabat.

[0156] The proteins, polypeptides, amino acid sequences and / or nucleic acid sequences according to the invention are At least, a mutant or homologue having the same or similar function as the above-mentioned protein or polypeptide, and / or a mutant or homologue having the same or similar function as the above-mentioned protein or polypeptide. It should also be understood that the scope includes nucleic acid sequences encoding variants or homologs with similar functions.

[0157] In the present invention, the mutant is the protein and / or the polypeptide (e.g. For example, an antibody or a fragment thereof that specifically binds to the GPC3 protein, or a low-density lipoprotein receptor-related protein or a fragment thereof that has one or more amino acid sequences that are related to the low-density lipoprotein receptor-related protein or a fragment thereof, The functional variant may be a protein or polypeptide in which at least one amino acid has been substituted, deleted or added. For example, the functional variant may comprise a protein or polypeptide that already contains an amino acid change by substitution, deletion and / or insertion of at least one, for example 1-30, 1-20 or 1-10, for example 1, 2, 3, 4 or 5 amino acids. The functional variant may essentially retain the biological properties of the protein or polypeptide before the change (for example, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (for example, antigen binding ability or biological function of the low density lipoprotein receptor-related protein or fragment thereof) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0158] In the present invention, the homologues are the proteins and / or polypeptides (e.g. For example, an antibody or a fragment thereof that specifically binds to the GPC3 protein, or a low-density lipoprotein receptor-related protein or a fragment thereof that has at least about 85% ( For example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% The nucleic acid may be a protein or polypeptide having sequence homology thereto (about 98%, about 99% or more).

[0159] In the present invention, the above-mentioned homology generally refers to identity, similarity, etc. between two or more sequences. "Percent sequence identity" refers to the degree of similarity or correlation between two aligned sequences. By comparing over a comparison window, identical nucleobases (e.g. The number of matching positions is calculated by counting the number of positions where the same amino acid residue (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present, and the number of matching positions is divided by the total number of positions in the comparison window (i.e., the size of the window), and the result is multiplied by 100 to obtain the sequence homology percentage. Alignment to determine the sequence homology percentage can be achieved by various means well known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including the full-length sequence coverage being compared and any algorithms required to achieve maximum alignment in the sequence region of interest. The above homology can also be measured using the FASTA and BLAST algorithms. The FASTA algorithm is described in W. R. Pearson and D. J. Lipman, "An Improved Tool for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and D. J. Lipman and W. R. Pearson, "Fast and Sensitive Searches for Protein Identity," Science, 227:1435-1441, 1989. The BLAST algorithm is described in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990.

[0160] In the present invention, the CAR comprises from the N-terminus to the C-terminus the targeting moiety, the hinge region, The polypeptide may sequentially comprise said domain, said transmembrane domain, said costimulatory domain and said signalling domain.

[0161] In the present invention, the polynucleotide molecule encoding the CAR is The polypeptide may comprise, in order, a nucleotide sequence encoding the targeting moiety, a nucleotide sequence encoding the hinge region, a nucleotide sequence encoding the transmembrane region, a nucleotide sequence encoding the transmembrane region, a nucleotide sequence encoding the costimulatory domain, and a nucleotide sequence encoding the signaling domain.

[0162] In the present invention, the polynucleotide molecule encoding the CAR further comprises a leader sequence. For example, the nucleic acid molecule encoding the leader sequence may comprise SEQ ID NO: 30 or a nucleic acid sequence having at least 80% homology thereto. The nucleic acid molecule encoding the leader sequence may be located 5' to the nucleotide sequence encoding the targeting moiety.

[0163] In the present invention, the genetically modified immune cell is capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof. For example, the genetically modified immune cell may have a vector capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof. The vector may include a nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof. For example, the polynucleotide molecule may include a nucleotide molecule represented by any of SEQ ID NOs: 21, 23, 25 and 27. The nucleic acid sequence may contain a nucleic acid sequence having at least 80% homology thereto. For example, the vector may be a retroviral vector, a lentiviral vector and / or It may be selected from transposon plasmids.

[0164] In the present invention, the genetically modified immune cells contain the chimeric antigen receptor (CAR). For example, the genetically modified immune cells may comprise a vector capable of expressing the chimeric antigen receptor (CAR). The vector may also include, for example, a nucleotide molecule encoding a cellular antigen receptor (CAR). The vectors can be retroviral, lentiviral and / or transposons. The plasmid may be selected from the group of plasmids.

[0165] In the present invention, the vector capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) capable of expressing the chimeric antigen receptor (CAR) are The vectors that can be used may be the same vector or different vectors, and the one or more vectors may be the low density lipoprotein receptor-related protein or and the chimeric antigen receptor (CAR), The immune cells thus obtained may then have both the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR).

[0166] For example, the vector capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof and the vector capable of expressing the chimeric antigen receptor (CAR) The vector may be an identical vector, in which the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof and the nucleotide molecule encoding the chimeric antigen receptor (CAR) are contained in the same expression cassette. For example, the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof may be located on the It may be located at the 3' end of the nucleotide molecule.

[0167] In the present invention, the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof may be directly or indirectly linked to the nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the indirect link may be performed by linking sequences. The 5' end of the linking sequence may be linked to the 3' end of the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof, and the 3' end of the linking sequence may be linked to the 5' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR). In the present invention, the linking sequence may contain a nucleic acid sequence represented by any of SEQ ID NOs: 19 and 29 or a nucleic acid sequence having at least 80% homology thereto.

[0168] In the present invention, the low density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cell may be two independent proteins, i.e., the two are not linked to each other at all, The low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) obtained by expression of the genetically modified immune cells may form any form of di(polymer) or protein complex. However, the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) may be linked to each other. For example, the low density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor are not completely cleaved by the translation of these two proteins, and the proteins are not completely cleaved by the translation of these two proteins. In some cases, amalgamations were formed.

[0169] Preparation method The present invention provides a method for preparing the genetically modified immune cells, comprising the step of increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cells.

[0170] The present invention further provides a method of preparing the composition, comprising increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cell.

[0171] In the present invention, the above method further comprises the steps of: + T lymphocytes, CD8 + T lymphocytes, CD4 + The method may include a step of obtaining by isolating T lymphocytes or regulatory T cells.

[0172] In the present invention, the method may comprise a step of isolating and activating the genetically modified immune cells, comprising administering a T cell medium to the isolated genetically modified immune cells.

[0173] In the present invention, said T cell medium may be selected from one or more of the following group: DMEM medium, 1640 medium, MEM medium and X-VIVO medium.

[0174] In the present invention, the above method further comprises the step of: Alternatively, the method may include adding a variety of T cell stimulating factors.

[0175] For example, the T cell stimulatory factor may be a B lymphocyte surface antigen antibody, a TNF antibody, an intracellular polypeptide, For example, the T cell stimulator may be selected from the group consisting of CD3 antibody, CD28 antibody, 4-1BB antibody, CD80 antibody, CD86 antibody, PHA, PMA, and ionomycin.

[0176] In the present invention, the T cell stimulating factor may include a CD3 antibody, which may have a concentration of 1 to 10,000 ng / mL. For example, the CD3 antibody may have a concentration of 1 to 9,000 ng / mL, 1 to 5,000 ng / mL, 1 to 4,000 ng / mL, 1 to 3,000 ng / mL, 1 to 1,000 ng / mL, 1 to 500 ng / mL, 1 to 400 ng / mL, 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, 1 to 10 ng / mL, or 1 to 5 ng / mL. In the present invention, the T cell stimulating factor may include a CD28 antibody, which may have a concentration of 1 to 10,000 ng / mL. For example, the concentration of the CD28 antibody may be 1 to 9000 ng / mL, 1 to 5000 ng / mL, 1 to 4000 ng / mL, 1 to 3000 ng / mL, 1 to 1000 ng / mL, 1 to 500 ng / mL, 1 to 400 ng / mL, 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, 1 to 10 ng / mL or 1 to 5 ng / mL.

[0177] In the present invention, the method further comprises administering one or more subpopulations to the isolated PBMCs. In the present invention, the cytokine agent may include an interleukin. For example, the interleukin may include one or more interleukins selected from the group consisting of IL2, IL21, IL7, and IL15.

[0178] In the present invention, the interleukin includes IL2, which may have a concentration of 0.1 to 10,000 U / mL. For example, the IL2 may have a concentration of 0.1 to 8000 U / mL, 0.1 to 6000 U / mL, 0.1 to 4000 U / mL, 0.1 to 2000 U / mL, 5 to 2000 U / mL, 5 to 1900 U / mL, 5 to 1800 U / mL, 5 to 1700 U / mL, 5 to 1600 U / mL, 5 to 1500 U / mL, 5 to 1400 U / mL, 5 to 1300 U / mL, 5 to 1200 U / mL, 5 to 1100 U / mL, 5 to 1000 U / mL, L, 5-900 U / mL, 5-800 U / mL, 5-700 U / mL, 5-600 U / mL, 5-500 U / mL, 5-400 U / mL, 5-300 U / mL, 5-200 U / mL, 5-500 U / mL, 5-400 U / mL, 5-300 U / mL, 5-200 U / mL, 5-100 U / mL, 5-50 U / mL, 5-40 U / mL, 5-30 U / mL, 5-20 U / mL, 5-100 U / mL, 5-50 U / mL, 5-40 U / mL, 5-30 U / mL, 5-20 U / mL or 5-10 U / mL.

[0179] In the present invention, the interleukin is IL21, which may have a concentration of 0.01 to 1000 ng / mL. For example, the concentration of the IL21 may be 0.01 to 800 ng / mL, 0.01 to 600 ng / mL, 0.01 to 400 ng / mL, 0.01 to 200 ng / mL, 0.01 to 100 ng / mL, 0.1 to 100 ng / mL, 0.1 to 90 ng / mL, 0.1 to 80 ng / mL, 0.1 to 70 ng / mL, 0.1 to 60 ng / mL, 0.1 to 50 ng / mL, 0.1 to 40 ng / mL, 0.1 to 30 ng / mL, 0.1 to 20 ng / mL, 0.1 to 10 ng / mL, or 0.1 to 5 ng / mL.

[0180] In the present invention, the interleukin may contain IL7, which may have a concentration of 0.01 to 1000 ng / mL. For example, the IL7 may have a concentration of 0.01 to 800 ng / mL, 0.01 to 600 ng / mL, 0.01 to 400 ng / mL, 0.01 to 200 ng / mL, 0.01 to 100 ng / mL, 0.1 to 100 ng / mL, 0.1 to 90 ng / mL, 0.1 to 80 ng / mL, 0.1 to 70 ng / mL, 0.1 to 60 ng / mL, 0.1 to 50 ng / mL, 0.1 to 40 ng / mL, 0.1 to 30 ng / mL, 0.1 to 20 ng / mL, 0.1 to 10 ng / mL, or 0.1 to 5 ng / mL.

[0181] In the present invention, the interleukin is IL15, which may have a concentration of 0.01 to 1000 ng / mL. For example, the concentration of the IL15 may be 0.01 to 800 ng / mL, 0.01 to 600 ng / mL, 0.01 to 400 ng / mL, 0.01 to 200 ng / mL, 0.01 to 100 ng / mL, 0.1 to 100 ng / mL, 0.1 to 90 ng / mL, 0.1 to 80 ng / mL, 0.1 to 70 ng / mL, 0.1 to 60 ng / mL, 0.1 to 50 ng / mL, 0.1 to 40 ng / mL, 0.1 to 30 ng / mL, 0.1 to 20 ng / mL, 0.1 to 10 ng / mL, or 0.1 to 5 ng / mL.

[0182] Promotes immune cell proliferation, promotes memory immune cell production, inhibits immune cell differentiation, enhances cytokine release, and increases tumor killing ability On the other hand, the present invention provides a method for promoting immune cell proliferation, comprising the step of increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells.

[0183] On the other hand, the present invention provides a method for promoting production of memory immune cells, comprising a step of promoting differentiation of the immune cells into memory immune cells by increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0184] On the other hand, the present invention provides a method for inhibiting immune cell differentiation, comprising a step of inhibiting differentiation of the immune cell into a differentiated immune cell by increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cell.

[0185] On the other hand, the present invention provides a method for enhancing cytokine release by immune cells, comprising the step of increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0186] In the present invention, the cytokines are interleukins, interferons and / or In the present invention, the cytokine may include IL-2, IL4, or tumor necrosis factor. , IL6, IL7, IL10, IL21, TNF-α and / or IFNγ.

[0187] On the other hand, the present invention provides a method for enhancing the tumor-killing ability of an immune cell, comprising the step of increasing the expression level of a low-density lipoprotein receptor-related protein or a fragment thereof in the immune cell.

[0188] On the other hand, the present invention provides a method for preventing tumor recurrence in a subject, comprising administering to a tumor-susceptible subject immune cells having increased expression of low density lipoprotein receptor-related protein or a fragment thereof.

[0189] Meanwhile, the present invention provides a method of treating a tumor in a subject in need thereof, comprising the step of administering to said subject immune cells having increased expression of low density lipoprotein receptor-related protein or a fragment thereof.

[0190] In the present invention, the tumor may be selected from liver cancer, lung cancer, leukemia and mesothelioma.In the present invention, the method may include an in vivo method and an in vitro method.

[0191] In the present invention, the expression level is increased by increasing the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells to a level significantly higher than that in control immune cells (for example, 5% or more, 10% or more, 20% or more, 30% or more). or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more), wherein said control immune cell is a corresponding immune cell in which the expression level of said low density lipoprotein receptor-related protein or fragment thereof is not essentially enhanced.

[0192] In the present invention, the promotion of memory immune cell production means increasing the number of memory immune cells produced by the immune cells significantly (e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more) than the number of memory immune cells produced by the control immune cells. or more, 80% or more, 90% or more, 100% or more) wherein the control immune cell is a corresponding immune cell in which the expression level of the low density lipoprotein receptor-related protein or a fragment thereof is not essentially enhanced.

[0193] In the present invention, the above-mentioned inhibition of immune cell differentiation refers to the inhibition of the number of differentiated immune cells produced by the immune cells by a significant amount (e.g., 5% or more, 10% or more, 20% or more, 30% or more) compared to the number of memory immune cells produced by the control immune cells. or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more), wherein the control immune cell is a corresponding immune cell in which the expression of low density lipoprotein receptor-related protein or a fragment thereof is not essentially elevated.

[0194] In the present invention, enhancing the release of cytokines by the above-mentioned immune cells may mean increasing the number of cytokines released by the immune cells significantly (e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more) compared to the number of cytokines released by control immune cells, provided that the control immune cells are corresponding immune cells in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is not essentially increased.

[0195] In the present invention, increasing the tumor-killing ability of the aforementioned immune cells may mean improving the tumor-killing ability of the immune cells by a greater extent (e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more) than the tumor-killing ability of a control immune cell, provided that the control immune cell is a corresponding immune cell in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is not essentially increased.

[0196] In the present invention, the above-mentioned prevention of tumor recurrence in a subject means that the ability of the immune cells to prevent tumor recurrence in a subject is significantly greater (e.g., 5% or more, 10% or less) than the ability of control immune cells to prevent tumor recurrence in a subject. or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more), wherein the control immune cell is a corresponding immune cell in which the expression level of the low density lipoprotein receptor-related protein or fragment thereof is not essentially enhanced.

[0197] In the present invention, treating a tumor in a subject in need thereof may mean increasing the tumor therapeutic effect of the immune cells by more significantly (e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more) than the tumor therapeutic effect of a control immune cell, provided that: The control immune cells are those that express low-density lipoprotein receptor-related protein or The corresponding immune cells do not essentially express the fragments at elevated levels.

[0198] For example, the corresponding immune cells may include lymphocytes. In the present invention, the corresponding immune cells may include T cells. In the present invention, the T cells may include memory stem cell-like T cells (TSCM) and / or central memory T cells (TCM). In the present invention, the TSCM may include CCR7 + and / or CD62L + In the present invention, the TSCM may further include CD45RA + or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8+ The present invention may have one or more properties selected from the group consisting of In the present invention, the corresponding immune cells are those that express a chimeric antigen receptor (CAR). In the present invention, the corresponding immune cells may comprise genetically modified T cells.

[0199] In the present invention, the content of the low-density lipoprotein receptor-related protein or a fragment thereof in the corresponding immune cells is merely the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in a normal individual, or the expression level is not essentially elevated compared to the content of the low-density lipoprotein receptor-related protein or a fragment thereof in a normal individual. For example, the expression level not essentially elevated refers to at most 4.5%, at most 4%, at most 3%, at most 2%, at most 1%, at most 0.5%, at most 0.3%, at most 0.1%, at most 0.01%, or less. The corresponding immune cells may not contain a vector carrying a nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof, and / or The corresponding immune cells may not contain a nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof.

[0200] In the present invention, the method for measuring the expression level of the low density lipoprotein receptor-related protein or a fragment thereof, and / or the cytokine may include quantitative PCR, western blot and immunohistochemistry.

[0201] In the present invention, the memory immune cells and / or the differentiated immune cells are measured. Methods may include flow cytometry, immunofluorescence, magnetic bead separation, ELISA, ELISPOT and quantitative PCR.

[0202] In the present invention, the efficacy of tumor treatment can be evaluated using an index selected from the group consisting of tumor volume, overall survival (OS), complete remission period (DOR), stable disease period, disease-free survival (DFS), progression-free survival (PFS), disease control rate (DCR), objective response rate (ORR) and / or clinical usefulness.

[0203] In the present invention, the preventive effect against tumor recurrence in a subject can be evaluated using an indicator selected from the group consisting of expression levels of tumor markers (e.g., tumor-associated antigens, tumor-specific genes, and tumor suppressor genes), histological detection results (e.g., epithelial hyperplasia, degree of polyp disappearance), imaging findings (e.g., mammography test results), and / or expression levels of serum markers (e.g., serum free DNA, methylated DNA).

[0204] Compositions and uses thereof The present invention provides a composition comprising the genetically modified immune cells.

[0205] In the present invention, the composition may further optionally contain a pharma- ceutically acceptable carrier, which may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a non-ionic surfactant.

[0206] In the present invention, the composition is administered orally, intravenously, intramuscularly, in situ at the tumor site, by inhalation, rectally, intravaginally, transdermally or by subcutaneous storage. It can be prepared for.

[0207] The pharmaceutical compositions are used to inhibit tumor growth, for example, the pharmaceutical compositions of the present invention can inhibit or slow the development or progression of a disease, reduce the size of a tumor (and thus essentially eliminate the tumor), and / or reduce and / or stabilize a disease state.

[0208] The compositions of the invention may comprise a therapeutically effective amount of the antibody or antigen-binding fragment thereof, the therapeutically effective amount being sufficient to treat a subject at risk of developing a disease (e.g., cancer) and / or any condition thereof. The dose is for preventing and / or treating (at least partially treating) a comorbidity.

[0209] The present invention also relates to the use of said gene in the preparation of a medicament for treating and / or preventing tumors. Uses of the modified cells and / or compositions described above are provided.

[0210] In the present invention, the tumor may be selected from liver cancer, lung cancer, leukemia and mesothelioma.

[0211] The following examples are provided to illustrate the subject matter and the mode of operation of the methods and systems of the present invention without being bound by any theory, and are not intended to limit the scope of the present invention.

[0212] Working Example Example 1 Construction of lentiviral vector The above CAR-T is a method to artificially synthesize fragments containing the CAR structure, for example, targeting GPC3, CD19, Mesothelin (MSLN), HER2, and BCMA, and deliver them to a lentivirus vector (LV100A, System The vectors were constructed in Biosciences (Hepatitis C Biosciences, Inc.) and lentiviruses were generated by transfection (as shown in FIG. 1 ) as described herein to obtain GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-TL6, GPC3-CD28, GPC3-CD28-L6, GPC3-CD28-TL6, GPC3-41BB-L5, GPC3-41BB-TL5, BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-TL6, CD19-41BB, CD19-41BB-L6, CD19-41BB-TL6, MSLN-41BB, MSLN-41BB-L6, MSLN-41BB-TL6, HER2-41BB, HER2-41BB-L6 and HER2-41BB-TL6 lentiviruses, respectively.

[0213] GPC3-41BB is constructed by sequentially splicing a leader sequence, GPC3 ScFv, a CD8 hinge region and transmembrane region, 41BB, and CD3zeta from the 5' end to the 3' end, and its nucleotide sequence is as shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0214] GPC3-41BB-L6 is composed of a leader sequence, GPC3 ScFv, CD8 hinge and transmembrane domain, 41BB, CD3zeta, 2A, and L6 spliced ​​in sequence from the 5' to 3' end. The nucleotide sequences of the respective nucleic acids are as shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.21. It is.

[0215] GPC3-41BB-TL6 contains a leader sequence, GPC3 ScFv, a CD8 hinge region and a transmembrane region, and 41BB The nucleotide sequences of the 5'-3' spliced ​​TL6, CD3zeta, 2A and TL6 are shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.23, respectively. It is.

[0216] GPC3-41BB-L5 contains a leader sequence, GPC3 ScFv, a CD8 hinge region and a transmembrane region, 41BB, It is constructed by sequentially splicing CD3zeta, 2A, and L5 from the 5' to the 3' end. The nucleotide sequences of the nucleic acids are as shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.25, respectively. It is.

[0217] GPC3-41BB-TL5 contains a leader sequence, GPC3 ScFv, a CD8 hinge region and a transmembrane region, and 41BB The nucleotide sequences of the TL5, CD3zeta, 2A, and TL5 are shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.27, respectively. It is.

[0218] GPC3-CD28 is constructed by sequentially splicing a leader sequence, GPC3 ScFv, CD8 hinge and transmembrane domain, CD28, and CD3zeta from the 5' to 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.17, respectively.

[0219] GPC3-CD28-L6 is composed of a leader sequence, GPC3 ScFv, CD8 hinge and transmembrane domain, CD28, CD3zeta, 2A, and L6 spliced ​​sequentially from the 5' to 3' end. The nucleotide sequences of the nucleic acids are as shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.21, respectively. It is.

[0220] GPC3-CD28-TL6 is constructed by sequentially splicing a leader sequence, GPC3ScFv, a CD8 hinge region and transmembrane region, CD28, CD3zeta, 2A, and TL6 from the 5' end to the 3' end, and its nucleotide sequence is as shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.23, respectively. It is.

[0221] BCMA-41BB is constructed by sequentially splicing a leader sequence, BCMAScFv, a CD8 hinge and transmembrane region, 41BB, and CD3zeta from the 5' to 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0222] BCMA-41BB-L6 is composed of a leader sequence, BCMAScFv, CD8 hinge and transmembrane domain, 41BB, CD3zeta, 2A, and L6 spliced ​​sequentially from the 5' to 3' end. The nucleotide sequences of the respective nucleic acids are as shown in SEQ ID NO.30, SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.21. It is.

[0223] BCMA-41BB-TL6 contains a leader sequence, BCMAScFv, CD8 hinge and transmembrane domains, 41BB The nucleotide sequences of the 5'-3' spliced ​​TL6, CD3zeta, 2A and TL6 are shown in SEQ ID NO.30, SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.23, respectively. It is.

[0224] CD19-41BB is constructed by sequentially splicing a leader sequence, CD19ScFv, a CD8 hinge and transmembrane region, 41BB, and CD3zeta from the 5' to 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0225] CD19-41BB-L6 is constructed by sequentially splicing a leader sequence, CD19ScFv, a CD8 hinge and transmembrane region, 41BB, CD3zeta, 2A, and L6 from the 5' to 3' end, and its nucleotide sequence is as shown in SEQ ID NO.30, SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.21, respectively.

[0226] CD19-41BB-TL6 is constructed by sequentially splicing a leader sequence, CD19ScFv, a CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 from the 5' end to the 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.23, respectively. It is.

[0227] MSLN-41BB is constructed by sequentially splicing a leader sequence, MSLNScFv, a CD8 hinge and transmembrane region, 41BB, and CD3zeta from the 5' to 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0228] MSLN-41BB-L6 is composed of a leader sequence, MSLNScFv, CD8 hinge and transmembrane domain, 41BB, CD3zeta, 2A, and L6 spliced ​​sequentially from the 5' to 3' end. The nucleotide sequences of the respective nucleic acids are as shown in SEQ ID NO.30, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.21. It is.

[0229] MSLN-41BB-TL6 contains a leader sequence, MSLNScFv, a CD8 hinge region and a transmembrane region, and 41BB The nucleotide sequences of the 5'-3' spliced ​​TL6, CD3zeta, 2A and TL6 are shown in SEQ ID NO.30, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.23, respectively. It is.

[0230] HER2-41BB is constructed by sequentially splicing a leader sequence, HER2ScFv, a CD8 hinge region and transmembrane region, 41BB, and CD3zeta from the 5' end to the 3' end, and its nucleotide sequence is shown in SEQ ID NO.30, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17, respectively.

[0231] HER2-41BB-L6 is constructed by sequentially splicing a leader sequence, HER2ScFv, a CD8 hinge and transmembrane region, 41BB, CD3zeta, 2A, and L6 from the 5' end to the 3' end, and its nucleotide sequence is as shown in SEQ ID NO.30, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.21, respectively.

[0232] HER2-41BB-TL6 contains a leader sequence, HER2 ScFv, CD8 hinge and transmembrane domains, 41BB The nucleotide sequences of the 5'-3' spliced ​​TL6, CD3zeta, 2A and TL6 are shown in SEQ ID NO.30, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.23, respectively. It is.

[0233] Example 2 Lentivirus-infected T cells Ficoll separation solution was used to separate 1×10 7 Larger peripheral blood mononuclear lymphocytes (PBMCs) were isolated and diluted with anti-human CD3 and anti-human CD4 antibodies at a final concentration of 1 μg / ml in PBS. The human CD28 antibody was diluted, and the diluted antibody mixture was added to the cell culture dish, and The cells were evenly spread on a cell culture dish and incubated at room temperature for 2 hours. After 2 hours, the antibody mixture was diluted with PBS. The combined solution was washed once, and the isolated PBMCs were then added to a final concentration of 1 × 10 6The T cells were activated by resuspending them in lymphocyte culture medium containing Xvivo15 medium, 5% FBS, and 200 U / ml IL2T or Xvivo15 medium, 5% FBS, 20 ng / ml IL21, and 10 ng / ml IL7 to a concentration of 1 cell / ml, adding them to a culture dish containing the antibody mixture, and culturing at 37°C, 5% CO2 for 24 hours.

[0234] A certain amount of T cell culture fluid was taken, mixed evenly in synperonic F108 with a final concentration of 1mg / ml, heated to 37℃ in a water bath, and mixed with non-infectious reagents. Then, the cell culture dishes required for the experiment were prepared. First, 1mg / ml anti-human CD3 antibody and 0.5mg / ml anti-human CD28 antibody were taken, diluted with PBS buffer at a volume ratio of 1:1000 and mixed evenly, then diluted with retronectin (1mg / ml) reagent at a volume ratio of 1:40 and mixed evenly, then spread evenly on the cell culture dish and incubated at room temperature for 2 hours. After 2 hours, the cell culture dish was washed with PBS and the preparation was completed.

[0235] The activated T cells were diluted with the prepared infection reagent at an MOI of 3. The prepared lentiviruses were added to the culture dish and mixed uniformly. Then, the culture dish was spread evenly and lentivirus infection was performed to obtain GPC3-41BB, GPC3-41BB-L6, and GPC3-41BB-L6. T cells expressing CD4+, GPC3-41BB-TL6, GPC3-CD28, GPC3-CD28-L6, GPC3-CD28-TL6, GPC3-41BB-L5, GPC3-41BB-TL5, BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-TL6, CD19-41BB, CD19-41BB-L6, CD19-41BB-TL6, MSLN-41BB, MSLN-41BB-L6, MSLN-41BB-TL6, HER2-41BB, HER2-41BB-L6 and HER2-41BB-TL6 were obtained. The cell density after infection was 1 × 10 6 The cell density was monitored to maintain 10–100 cells / ml. After 14 days, the cell number increased 10–100 fold.

[0236] Example 3 Promotion of memory T cell formation by LRP6, truncated LRP6, LRP5 or truncated LRP5 In a cell culture incubator at 37°C and 5% CO2, T cells expressing GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-TL6, GPC3-CD28, GPC3-CD28-L6, GPC3-CD28-TL6, GPC3-41BB-L5, GPC3-41BB-TL5, BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-TL6, CD19-41BB, CD19-41BB-L6, CD19-41BB-TL6, MSLN-41BB, MSLN-41BB-L6, MSLN-41BB-TL6, HER2-41BB, HER2-41BB-L6 and HER2-41BB-TL6 obtained in vitro were cultured for a total of 9 or 13 days.

[0237] The expression of CD3, CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was measured using BD flow cytometry, and the protein expression results are shown in Figure 2.

[0238] These results indicate that the proportion of memory stem cell-like T cells (TSCM) and central memory T cells (TCM) in the total number of T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6 is higher than that of T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6. In T cells expressing GPC3-CD28-L6 or GPC3-CD28-TL6, the proportions of TSCM and TCM in the total number of cells were significantly higher than in the control group. In addition, in T cells expressing GPC3-41BB-L5 or GPC3-41BB-TL5, the proportions of TSCM and TCM in the total number of cells were significantly higher than those in the control group; in T cells expressing BCMA-41BB-L6 or BCMA-41BB-TL6, the proportions of TSCM and TCM in the total number of cells were significantly higher than those in the control group; in T cells expressing CD19-41BB-L6 or CD19-41BB-TL6, the proportions of TSCM and TCM in the total number of cells were significantly higher than those in the control group; in T cells expressing MSLN-41BB-L6 or MSLN-41BB-TL6, the proportions of TSCM and TCM in the total number of cells were significantly higher than those in the control group; and in T cells expressing HER2-41BB-L6 or HER2-41BB-TL6, the proportions of TSCM and TCM in the total number of cells were also significantly higher than those in the control group.

[0239] Example 4 Differentiation of LRP6 or its truncated form suppressor Treg cells The T cells expressing GPC3-41BB and GPC3-41BB-L6 obtained in Example 2 were cultured in vitro in a cell culture incubator at 37° C. and 5% CO for a total of 9 or 12 days. BD flow cytometry revealed that CD3, CD4, CD25, CD127, and FoxP3 proteins were expressed in T cells. The expression of the protein was measured, and the results are shown in Figure 3. The results showed that GPC3-41BB-L6-expressing T cells were associated with increased CD4 + The proportion of regulatory T cells (Treg) among T cells was shown to be significantly lower than that of T cells expressing GPC3-41BB in the control group.

[0240] Example 5 Specific CAR-T for tumor antigen induction by LRP6, LRP6 truncated form, LRP5 or LRP5 truncated form Promoting cell proliferation T cells expressing GPC3-41BB, GPC3-41BB-L6, or GPC3-41BB-TL6 obtained in Example 2 were co-cultured with irradiated Huh7 or HepG2 cells (purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China)) in a 1:1 cell ratio in Xvivo15 medium, and the Huh7 or HepG2 cells were supplementally irradiated again on the 5th day, and stimulated three times each time by counting with trypan blue staining. T cells expressing GPC3-41BB, GPC3-41BB-L5, or GPC3-41BB-TL5 were cultured and cocultured with irradiated Huh7 or T cells expressing HER2-41BB, HER2-41BB-L6, or HER2-41BB-TL6 were co-cultured with irradiated SKOV3 cells (purchased from the Chinese Academy of Sciences (Shanghai, China) cell bank) in a 1:1 cell ratio in Xvivo15 medium with Huh7 or HepG2 cells that were given a supplementary irradiation again on the 5th day, and stimulated three times each time by counting by trypan blue staining. T cells expressing HER2-41BB, HER2-41BB-L6, or HER2-41BB-TL6 were co-cultured with irradiated SKOV3 cells (purchased from the Chinese Academy of Sciences (Shanghai, China) cell bank) in a 1:1 cell ratio in Xvivo15 medium with irradiated SKOV3 cells that were given a supplementary irradiation again on the 5th day, and stimulated three times each time by counting by trypan blue staining. The cell proliferation patterns of each group were as shown in Figure 4. These results showed that the proliferation fold of T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6 was significantly higher than that of the control group (T cells expressing GPC3-41BB), the proliferation fold of T cells expressing GPC3-41BB-L5 or GPC3-41BB-TL5 was significantly higher than that of the control group, and the proliferation fold of T cells expressing HER2-41BB-L6 or HER2-41BB-TL6 was also significantly higher than that of the control group (T cells expressing HER2-41BB).

[0241] Example 6 CAR-T cell in vivo proliferation and cytokine release by LRP6 or its truncated forms Promoting NSG mice (purchased from Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were inoculated with Huh7 cells (1 × 10 7 14 days later, the tumor volume of the mice was approximately 100 mm 3 was measured as At this time, the mice were divided into 4 groups, each of which had 6 mice, namely, the T cell group, the GPC3-41BB group, the GPC3-41BB-L6 group, and the GPC3-41BB-TL6 group. Then, cells were injected into each tail vein (i.e., one group each of unmodified T cells, T cells expressing GPC3-41BB, T cells expressing GPC3-41BB-L6, and T cells expressing GPC3-41BB-TL6, at a dose of 3 × 10 6 Each mouse was injected with 100 μl of IgG (100 μg / mouse), and on the 8th day, 50 μl of blood was taken from the tail of the mouse. The expression of human CD8 and CD4 proteins was measured for each group by BD flow cytometry. The results are shown in Figure 5A. The results in Figure 5A show that the proportion of cells expressing CD8 and CD4 proteins in the total number of cells in the GPC3-41BB-L6 or GPC3-41BB-TL6 group was significantly higher than that of the other control groups.

[0242] NSG mice (purchased from Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were inoculated with Huh7 cells (1 × 10 7 14 days later, the tumor volume of the mice was approximately 100 mm 3 was measured as At this time, the mice were divided into five groups, each of which had six mice, and each group was divided into a T cell group, a GPC3-41BB group, and a The T cell groups were then injected with T cells (i.e., unmodified T cells, one group at a time) via the tail vein, with a dose of 2 × 10 6 cells / animal), and GPC3-41BB-expressing T cells (at a dose of 8 × 10 5 (1 / animal), GPC3-41BB-L6 GPC3-41BB-L6 expressing T cells (at a dose of 3 × 10 5 pieces / fish, 8×10 5 Pieces / animal or 2×10 6 On day 8, 50 μl of blood was collected from the tail of the mice. The expression of human IL-2, IL4, IL6, IL10, TNF-α and IFNγ cytokines was measured for each group by using a ELISA. The results are shown in Figure 5B. The results in Figure 5B show that the expression of IFNγ cytokines in the GPC3-41BB-L6 group was significantly higher than that in the other control groups.

[0243] Example 7 Enhancement of CAR-T cell antitumor effect by LRP6 or its truncated form NSG mice (purchased from Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were inoculated with Huh7 cells (at a dose of 1 × 10 7 After 14 days, the tumor volume of the mice was approximately 100 mm 3 and The mice were divided into 4 groups, each containing 8 mice, which were the T cell group, the GPC3-41BB group, and the GPC3-41BB-L6 group. T cells (i.e., unmodified T cells were injected into each group via the tail vein, with a dose of 8 × 10 5 cells / animal), and GPC3-41BB-expressing T cells (at a dose of 8 × 10 5 cells / animal), and GPC3-41BB-L6-expressing T cells (at a dose of 3 × 10 5 Pieces / animal or 8×10 5 The mice were injected with 100 μg / animal.

[0244] Tumor volume size was measured and mouse deaths were recorded every Monday and Thursday. The results are shown in Figure 6. From these results, the GPC3-41BB-L6 group The antitumor effect and mouse survival rate were both approximately three times higher than in the control group. I understand.

[0245] Example 8: LRP6 or its truncated form promotes CAR-T cells in vivo for long-term survival and prevention of tumor recurrence NSG mice (purchased from Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were inoculated with Huh7 cells (at a dose of 1 × 10 7 After 14 days, the tumor volume of the mice was approximately 100 mm 3 and The mice were divided into three groups of nine mice each: the T cell group, the GPC3-41BB group, and the GPC3-41BB-L6 group. T cells (at a dose of 2 × 10 6 cells / animal), and GPC3-41BB-expressing T cells (at a dose of 2 × 10 6 The number of mice expressing GPC3-41BB-L6 was 100 / animal, and the number of mice expressing GPC3-41BB-L6 was 100 / animal. T cells (at a dose of 2 × 10 6 The mice were injected with 100 μg / animal.

[0246] Eighty-five days after the tumor had completely disappeared, NSG mice were reinjected with Huh7 cells (at a dose of 3 × 10 7 On day 99, 50 μl of blood was collected from the tail of the mice and human CD8, CD4 and CD3 protein expression was measured for each group by BD flow cytometry, and on day 120, bone marrow was removed from the mice and human CD8, CD4 and CD3 protein expression was measured for each group by BD flow cytometry.

[0247] The results were as shown in Figure 7. As a result, for the GPC3-41BB-L6 group, The percentage of cells expressing CD8 or CD4 protein in the total number of cells in peripheral blood and the percentage of cells expressing CD3 protein in the total number of cells in bone marrow were both far higher than those in the GPC3-41BB group, and mice in the GPC3-41BB-L6 group were unable to form tumors even after being reinoculated with tumor cells, indicating that LRP6 or its truncated form can promote the long-term survival of CAR-T cells in vivo and has the advantage of preventing tumor recurrence.

[0248] The above detailed description is given by way of interpretation or example, but is not intended to limit the scope of the appended claims. It is obvious to those skilled in the art that there are various modifications to the embodiments described hereinabove, and they fall within the scope of the appended claims and the corresponding embodiments.

Claims

1. Increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in immune cells. A method for promoting immune cell proliferation.

2. The method includes a step of promoting differentiation of immune cells into memory immune cells by increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the immune cells. A method for promoting memory immune cell production.

3. Inhibiting differentiation of the immune cells into differentiated immune cells by increasing the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells. A method for inhibiting immune cell differentiation.

4. The differentiated immune cells include regulatory T cells (Tregs). The method of claim 3.

5. Increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the immune cell. A method for increasing cytokine release by immune cells.

6. The cytokines may be interleukins, interferons and / or tumor necrosis factors. Including children, The method of claim 5.

7. The cytokines are IL-2, IL4, IL6, IL7, IL10, IL21, TNF-α and / or IFNγ. Including, The method according to any one of claims 5 to 6.

8. Increasing the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the immune cell. A method for increasing the tumor-killing ability of immune cells.

9. Administering to a subject susceptible to a tumor an immune cell that has increased expression of a low density lipoprotein receptor-related protein or a fragment thereof. A method for preventing tumor recurrence in a subject.

10. Administering to a subject immune cells having increased expression of low density lipoprotein receptor-related protein or a fragment thereof; A method of treating a tumor in a subject in need thereof.

11. The tumor is selected from liver cancer, lung cancer, leukemia and mesothelioma; The method according to any one of claims 8 to 10.

12. The method is an in vitro or ex vivo method. The method according to any one of claims 8 to 11.

13. The immune cells include lymphocytes. The method according to any one of claims 1 to 12.

14. The immune cells include T cells. The method according to any one of claims 1 to 13.

15. The T cells include memory stem cell-like T cells (TSCM) and / or central memory T cells (TCM); The method of claim 14.

16. The TSCM is CCR7 + and / or CD62L + That is, The method of claim 15.

17. The TSCM further comprises CD45RA + or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + One or more characteristics selected from the group having The method according to any one of claims 15 to 16.

18. The immune cells are engineered to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Containing genetically modified immune cells, The method according to any one of claims 1 to 17.

19. The genetically modified immune cells include genetically modified T cells. The method of claim 18.

20. The CAR contains an intracellular region comprising a signaling domain and / or a costimulatory domain.

20. The method of claim 19.

21. The signaling domain comprises a portion selected from the group consisting of a CD3 zeta signaling domain, a CD3 delta signaling domain, and a CD3 epsilon signaling domain; The method of claim 20.

22. The signaling domain may have the amino acid sequence represented by SEQ ID NO: 18 or at least one amino acid sequence similar thereto. Contains an amino acid sequence with at least 80% homology The method according to any one of claims 20 to 21.

23. The nucleic acid molecule encoding the signaling domain comprises a nucleic acid sequence represented by SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.

23. The method according to any one of claims 20 to 22.

24. The costimulatory domain includes a CD27 costimulatory domain, a CD28 costimulatory domain and a 4-1BB costimulatory domain. containing a portion selected from the group of domains, The method according to any one of claims 20 to 23.

25. The costimulatory domain comprises an amino acid sequence represented by any one of SEQ ID NO: 14 and SEQ ID NO: 16, or an amino acid sequence having at least 80% homology thereto; The method according to any one of claims 20 to 24.

26. The nucleic acid molecule encoding the costimulatory domain comprises a nucleic acid sequence represented by any one of SEQ ID NO:13 and SEQ ID NO:15, or a nucleic acid sequence having at least 80% homology thereto. R, The method according to any one of claims 20 to 25.

27. The CAR comprises a hinge region. The method according to any one of claims 18 to 26.

28. The hinge region is selected from the group consisting of IgG4 hinge region, IgG1 hinge region and CD8 hinge region. including a portion selected from 28. The method of claim 27.

29. The hinge region comprises an amino acid sequence represented by SEQ ID NO: 31 or an amino acid sequence having at least 80% homology thereto; The method according to any one of claims 27 to 28.

30. The nucleic acid molecule encoding the hinge region may have the nucleic acid sequence represented by SEQ ID NO: 32 or Contains a nucleic acid sequence having at least 80% homology thereto, 30. The method according to any one of claims 27 to 29.

31. The CAR comprises a transmembrane domain. The method according to any one of claims 18 to 30.

32. The transmembrane domain comprises a portion selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain and / or a CD24 transmembrane domain; The method of claim 31.

33. The transmembrane domain comprises an amino acid sequence represented by SEQ ID NO: 33 or an amino acid sequence having at least 80% homology thereto; 33. The method according to any one of claims 31 to 32.

34. The nucleic acid molecule encoding the transmembrane domain may have the nucleic acid sequence represented by SEQ ID NO: 34 or Contains a nucleic acid sequence having at least 80% homology thereto, 34. The method according to any one of claims 31 to 33.

35. The CAR comprises a targeting moiety. The method according to any one of claims 18 to 34.

36. The targeting moiety comprises an ScFv. The method of claim 35.

37. The targeting moiety specifically binds and / or recognizes a tumor antigen; The method according to any one of claims 35 to 36.

38. The targeting moiety may be a B lymphocyte surface antigen, a TNF family member, a HER family member, and GPC family members, and / or recognize, 38. The method according to any one of claims 35 to 37.

39. the targeting moiety specifically binds to and / or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin and GPC3; 39. The method according to any one of claims 35 to 38.

40. the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes CD19, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 46 or an amino acid sequence having at least 80% identity thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 48 or an amino acid sequence having at least 80% identity thereto; 40. The method according to any one of claims 35 to 39.

41. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 50 or an amino acid sequence having at least 80% identity thereto. The method of claim 40.

42. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 43 or its equivalent. HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 45 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

42. The method according to any one of claims 40 to 41.

43. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 49 or an amino acid sequence having at least 80% identity thereto.

43. The method of claim 42.

44. the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA, comprising a light chain variable region comprising: an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 54 or an amino acid sequence having at least 80% identity thereto; an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 55 or an amino acid sequence having at least 80% identity thereto; and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto; 40. The method according to any one of claims 35 to 39.

45. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having at least 80% identity thereto. The method of claim 44.

46. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 51 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 53 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

46. ​​The method according to any one of claims 44 to 45.

47. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 57 or an amino acid sequence having at least 80% identity thereto. The method of claim 46.

48. The targeting moiety comprises an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto, and an amino acid sequence represented by SEQ ID NO:

71. or an amino acid sequence having at least 80% homology thereto; and An antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes HER2, comprising a light chain variable region comprising an LCDR3 containing an amino acid sequence represented by ID NO: 72 or an amino acid sequence having at least 80% homology thereto.

40. The method according to any one of claims 35 to 39.

49. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 74 or an amino acid sequence having at least 80% identity thereto. The method of claim 48.

50. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 67 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 69 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

50. The method according to any one of claims 48 to 49.

51. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% identity thereto. The method of claim 50.

52. The targeting portion specifically binds to Mesothelin, comprising a light chain variable region including an LCDR1 having an amino acid sequence represented by SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto, an LCDR2 having an amino acid sequence represented by SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 having an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. and / or an antibody or antigen-binding fragment thereof that recognizes 40. The method according to any one of claims 35 to 39.

53. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 66 or an amino acid sequence having at least 80% identity thereto.

53. The method of claim 52.

54. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 59 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 61 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

54. The method according to any one of claims 52 to 53.

55. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 65 or an amino acid sequence having at least 80% identity thereto.

55. The method of claim 54.

56. The targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto.

40. The method according to any one of claims 35 to 39.

57. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 42 or an amino acid sequence having at least 80% identity thereto. The method of claim 56.

58. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 35 or its equivalent. HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 37 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

58. The method of any one of claims 56 to 57.

59. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto.

59. The method of claim 58.

60. The targeting moiety may be an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, and 10. or an amino acid sequence having at least 80% homology thereto, 60. The method of any one of claims 35 to 59.

61. The nucleic acid molecule encoding the targeting moiety comprises a nucleic acid sequence represented by any of SEQ ID NOs: 1, 3, 5, 7 and 9, or a nucleic acid sequence having at least 80% homology thereto; The method according to any one of claims 35 to 60.

62. The method further comprises the steps of: + T lymphocytes, CD8 + T lymphocytes, CD4 + The method includes a step of obtaining T lymphocytes or regulatory T cells by isolating the T lymphocytes or regulatory T cells. The method of any one of claims 1 to 61.

63. The method may further comprise adding one or more T cell stimulatory factors to the isolated PBMCs.

63. The method of claim 62.

64. The T cell stimulatory factors include B lymphocyte surface antigen antibodies, TNF antibodies, intracellular polyesters, and and antibiotics, 64. The method of claim 63.

65. The T cell stimulatory factor is selected from the group consisting of CD3 antibody, CD28 antibody, 4-1BB antibody, CD80 antibody, CD86 antibody, PHA, PMA and ionomycin; 65. The method of any one of claims 63 to 64.

66. The T cell stimulator comprises a CD3 antibody at a concentration of 1 to 10,000 ng / mL.

66. The method according to any one of claims 63 to 65.

67. The T cell stimulator comprises a CD28 antibody at a concentration of 1 to 10,000 ng / mL.

67. The method of any one of claims 63 to 66.

68. The method further comprises administering to the isolated PBMC one or more cytokine agents. Including 68. The method of any one of claims 63 to 67.

69. The cytokine agent comprises an interleukin.

69. The method of claim 68.

70. The interleukin is one or more selected from the group consisting of IL2, IL21, IL7 and IL15. Contains types of 70. The method of claim 69.

71. The interleukin contains IL2 at a concentration of 0.1 to 10000 U / mL.

71. The method of any one of claims 69 to 70.

72. The interleukin contains IL21 at a concentration of 0.01 to 1000 ng / mL.

72. The method of any one of claims 69 to 71.

73. The interleukin contains IL7 at a concentration of 0.01 to 1000 ng / mL.

73. The method of any one of claims 69 to 72.

74. The interleukin contains IL15 at a concentration of 0.01 to 1000 ng / mL.

74. The method of any one of claims 69 to 73.

75. The low density lipoprotein receptor-related protein or a fragment thereof is one or more proteins selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. Containing the following:

75. The method of any one of claims 1 to 74.

76. The low density lipoprotein receptor-related protein or a fragment thereof is of human origin.

76. The method of any one of claims 1 to 75.

77. The functional fragment comprises a fragment of the low density lipoprotein receptor-related protein or a truncated form thereof that has the low density lipoprotein receptor-related protein activity.

77. The method of any one of claims 75 to 76.

78. The low density lipoprotein receptor-related protein includes low density lipoprotein receptor-related protein 6 and truncations thereof, and / or low density lipoprotein receptor-related protein 5 and truncations thereof; 78. The method of any one of claims 1 to 77.

79. The truncated form of the low density lipoprotein receptor-related protein 6 is and / or the truncated form of said low density lipoprotein receptor-related protein 5 comprises the intracellular domain of said low density lipoprotein receptor-related protein 6. comprising an intracellular region having quality 5; 79. The method of claim 78.

80. The truncated form of the low density lipoprotein receptor-related protein 6 is Transmembrane domain of low density lipoprotein receptor-associated protein 6 and the low density lipoprotein receptor-associated protein and / or the truncated form of low density lipoprotein receptor-related protein 5 comprises the transmembrane region of low density lipoprotein receptor-related protein 5 and the LDLR domain of low density lipoprotein receptor-related protein 5.

80. The method of any one of claims 78 to 79.

81. The low density lipoprotein receptor-related protein or a fragment thereof is represented by SEQ ID NO: 22, 4, 26 and 28, or an amino acid sequence having at least 80% homology thereto; 81. The method of any one of claims 1 to 80.

82. The nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof has a nucleic acid sequence represented by any one of SEQ ID NOs: 21, 23, 25 and 27 or at least one thereof. Contains a nucleic acid sequence having 80% homology with 82. The method according to any one of claims 1 to 81.

83. Genetically modifying the immune system to increase the expression of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells; Genetically modified immune cells.

84. The genetically modified immune cells include lymphoid cells. The immune cell of claim 83.

85. The genetically modified immune cells include genetically modified T cells. An immune cell according to any one of claims 83 to 84.

86. The genetically modified immune cells are genetically modified memory stem cell-like T cells (TSCM). and / or genetically modified central memory T cells (TCM), An immune cell according to any one of claims 83 to 85.

87. The TSCM is CCR7+ and / or CD62L+. The immune cell of claim 86.

88. The TSCM further comprises CD45RA + or CD45RA - , CD45RO + or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + The compound has one or more properties selected from the group consisting of do, 88. An immune cell according to any one of claims 86 to 87.

89. The genetically modified immune cells may be chimeric antigen receptors (CARs) or T cell receptors (TCRs). ) An immune cell according to any one of claims 83 to 88.

90. The CAR contains an intracellular domain consisting of a signaling domain and / or a costimulatory domain. The immune cell of claim 89.

91. The signaling domain comprises a portion selected from the group consisting of a CD3 zeta signaling domain, a CD3 delta signaling domain, and a CD3 epsilon signaling domain; The immune cell of claim 90.

92. The signaling domain comprises an amino acid sequence represented by SEQ ID NO:

18. An immune cell according to any one of claims 90 to 91.

93. The nucleic acid molecule encoding the signaling domain comprises a nucleic acid sequence represented by SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto. An immune cell according to any one of claims 90 to 92.

94. The costimulatory domain includes a CD27 costimulatory domain, a CD28 costimulatory domain and a 4-1BB costimulatory domain. containing a portion selected from the group of domains, An immune cell according to any one of claims 90 to 93.

95. The costimulatory domain comprises an amino acid sequence represented by any one of SEQ ID NO: 14 and SEQ ID NO: 16, or an amino acid sequence having at least 80% homology thereto; An immune cell according to any one of claims 90 to 94.

96. The nucleic acid molecule encoding the costimulatory domain comprises a nucleic acid sequence represented by any one of SEQ ID NO:13 and SEQ ID NO:15, or a nucleic acid sequence having at least 80% homology thereto. R, An immune cell according to any one of claims 90 to 95.

97. The CAR comprises a hinge region. An immune cell according to any one of claims 89 to 96.

98. The hinge region is selected from the group consisting of IgG4 hinge region, IgG1 hinge region and CD8 hinge region. including a portion selected from The immune cell of claim 97.

99. The hinge region comprises an amino acid sequence represented by SEQ ID NO: 31 or an amino acid sequence having at least 80% homology thereto; An immune cell according to any one of claims 97 to 98.

100. The nucleic acid molecule encoding the hinge region may have the nucleic acid sequence represented by SEQ ID NO: 32 or Contains a nucleic acid sequence having at least 80% homology thereto, 100. An immune cell according to any one of claims 97 to 99.

101. The CAR comprises a transmembrane domain. An immune cell according to any one of claims 98 to 100.

102. The transmembrane domain comprises a portion selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain and / or a CD24 transmembrane domain; The immune cell of claim 101.

103. The transmembrane domain comprises an amino acid sequence represented by SEQ ID NO: 33 or an amino acid sequence having at least 80% homology thereto; An immune cell according to any one of claims 101 to 102.

104. The nucleic acid molecule encoding the transmembrane domain may have the nucleic acid sequence represented by SEQ ID NO: 34 or Contains a nucleic acid sequence having at least 80% homology thereto, An immune cell according to any one of claims 101 to 103.

105. The CAR comprises a targeting moiety. An immune cell according to any one of claims 89 to 104.

106. The targeting moiety comprises an ScFv. The immune cell of claim 105.

107. The targeting moiety specifically binds and / or recognizes a tumor antigen; An immune cell according to any one of claims 105 to 106.

108. The targeting moiety may be a B lymphocyte surface antigen, a TNF family member, a HER family member, and GPC family members, and / or recognize, An immune cell according to any one of claims 105 to 107.

109. the targeting moiety specifically binds to and / or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin and GPC3; An immune cell according to any one of claims 105 to 108.

110. the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes CD19, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 46 or an amino acid sequence having at least 80% identity thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 47 or an amino acid sequence having at least 80% identity thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 48 or an amino acid sequence having at least 80% identity thereto; An immune cell according to any one of claims 105 to 109.

111. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 50 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 110.

112. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 43 or its equivalent. HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 45 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure: An immune cell according to any one of claims 110 to 111.

113. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 49 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 112.

114. the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA, comprising a light chain variable region comprising: an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 54 or an amino acid sequence having at least 80% identity thereto; an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 55 or an amino acid sequence having at least 80% identity thereto; and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having at least 80% identity thereto; An immune cell according to any one of claims 105 to 109.

115. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 114.

116. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 51 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 53 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure: An immune cell according to any one of claims 114 to 115.

117. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 57 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 116.

118. The targeting moiety comprises an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto, and an amino acid sequence represented by SEQ ID NO:

71. an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes HER2, comprising a light chain variable region comprising an LCDR2 containing an amino acid sequence represented by SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 containing an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto; An immune cell according to any one of claims 105 to 109.

119. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 74 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 118.

120. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 67 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 69 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure:

120. An immune cell according to any one of claims 118 to 119.

121. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 120.

122. The targeting moiety comprises an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto, and an amino acid sequence represented by SEQ ID NO:

63. and an LCDR2 comprising an LCDR3 having an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto, and a light chain variable region comprising an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto, and / or an antibody or antigen-binding fragment thereof that recognizes An immune cell according to any one of claims 105 to 109.

123. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 66 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 122.

124. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 59 or HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 61 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure: An immune cell according to any one of claims 122 to 123.

125. The heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 65 or at least 8 amino acids identical thereto. Contains an amino acid sequence with 0% homology The immune cell of claim 124.

126. The targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, comprising a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto, an LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto, and an LCDR3 comprising an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. An immune cell according to any one of claims 105 to 109.

127. The light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 42 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 126.

128. The antibody or antigen-binding fragment thereof has the amino acid sequence represented by SEQ ID NO: 35 or its equivalent. HCDR1 containing an amino acid sequence having at least 80% homology thereto, and SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto. and an amino acid sequence represented by SEQ ID NO: 37 or at least 80% homologous thereto. and a heavy chain variable region comprising an HCDR3 having an amino acid sequence having the following structure: An immune cell according to any one of claims 126 to 127.

129. The heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 41 or an amino acid sequence having at least 80% identity thereto. The immune cell of claim 128.

130. The targeting moiety may be an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, and 10. or an amino acid sequence having at least 80% homology thereto, 130. An immune cell according to any one of claims 105 to 129.

131. The nucleic acid molecule encoding the targeting moiety comprises a nucleic acid sequence represented by any of SEQ ID NOs: 1, 3, 5, 7 and 9, or a nucleic acid sequence having at least 80% homology thereto; An immune cell according to any one of claims 105 to 130.

132. The low density lipoprotein receptor-related protein or a fragment thereof includes one or more proteins selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof; An immune cell according to any one of claims 83 to 131.

133. The low density lipoprotein receptor-related protein or a fragment thereof is of human origin. An immune cell according to any one of claims 83 to 132.

134. The functional fragment comprises a fragment of the low density lipoprotein receptor-related protein or a truncated form thereof that has the low density lipoprotein receptor-related protein activity. An immune cell according to any one of claims 132 to 133.

135. The low density lipoprotein receptor-related protein includes low density lipoprotein receptor-related protein 6 and truncations thereof, and / or low density lipoprotein receptor-related protein 5 and truncations thereof; An immune cell according to any one of claims 83 to 134.

136. The truncated form of the low density lipoprotein receptor-related protein 6 is and / or the truncated form of said low density lipoprotein receptor-related protein 5 comprises the intracellular domain of said low density lipoprotein receptor-related protein 6. containing the intracellular region of protein 5, The immune cell of claim 135.

137. The truncated form of the low density lipoprotein receptor-related protein 6 is The transmembrane domain of the low density lipoprotein receptor-associated protein 6 and the low density lipoprotein receptor-associated protein and / or said truncated form of low density lipoprotein receptor-related protein 5 comprises the transmembrane region of low density lipoprotein receptor-related protein 5 and the LDLR domain of low density lipoprotein receptor-related protein 5; An immune cell according to any one of claims 135 to 136.

138. The low density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26 and 28, or an amino acid sequence having at least 80% homology thereto. An immune cell according to any one of claims 83 to 137.

139. The nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof has a nucleic acid sequence represented by any one of SEQ ID NOs: 21, 23, 25 and 27 or at least one thereof. Contains a nucleic acid sequence having 80% homology with An immune cell according to any one of claims 83 to 138.

140. The genetically modified immune cell according to any one of claims 83 to 139. composition.

141. and optionally further comprising a pharma- ceutically acceptable carrier. The composition of claim 140.

142. A genetically modified immune cell according to any one of claims 83 to 139 and / or a genetically modified immune cell according to any one of claims 140 to 141 in the preparation of a medicinal product for the treatment and / or prevention of tumors. Uses of the above-mentioned compositions.

143. The tumor is selected from liver cancer, lung cancer, leukemia and mesothelioma; 143. The use of claim 142.

144. Preparation of drugs used to promote immune cell proliferation, increase memory immune cell production, inhibit immune cell differentiation, increase cytokine release by immune cells, and / or increase tumor killing ability by immune cells The present invention relates to a method for producing a low-density lipoprotein receptor-related protein or a fragment thereof, and a method for producing the same.

145. The low density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26 and 28, or an amino acid sequence having at least 80% homology thereto.

145. The use of claim 144.

146. Increasing the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cells. A method for preparing a genetically modified immune cell according to any one of claims 83 to 139.

147. Introducing into the genetically modified immune cells a vector that enhances the expression of the low density lipoprotein receptor-related protein or a fragment thereof. The method of claim 146.

148. The vector is selected from the group consisting of retroviral vectors, lentiviral vectors and transposon plasmids; The method of claim 147.

149. The low density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26 and 28, or an amino acid sequence having at least 80% homology thereto.

149. The method of any one of claims 146 to 148.

150. The genetically modified immune cells include lymphoid cells.

150. The method of any one of claims 146 to 149.

151. The genetically modified immune cells express a chimeric antigen receptor (CAR).

151. The method of any one of claims 146 to 150.

152. and isolating and activating the genetically modified immune cells, the activation comprising administering a T cell medium to the isolated genetically modified immune cells. The method of claim 151.

153. The T cell medium is one selected from the group consisting of DMEM medium, 1640 medium, MEM medium and X-VIVO medium. Containing one or more types, The method of claim 152.

154. further comprising administering to the genetically modified immune cells a T cell stimulatory factor.

154. The method of any one of claims 151 to 153.

Images