Method for promoting the proliferation of immune cells

Abstract

The present application discloses a method for promoting the proliferation of immune cells. The method comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

Description

Technical Field

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

[0002] Chimeric antigen receptor T-cell immunotherapy (CAR-T) is a method of genetically engineering T cells to express a chimeric antigen receptor (CAR) that recognizes the antibody of the antigen on the surface of tumor cells, thereby enhancing the specific killing of tumors by T cells. CAR generally comprises a single-chain variable domain (scFv) that specifically recognizes a tumor-associated antigen (TAA), a hinge region, a transmembrane region, and an intracellular signaling region.

[0003] In recent years, CAR-T immunotherapy has shown significant efficacy in the treatment of acute leukemia and non-Hodgkin lymphoma, but the proliferative capacity of immune cells expressing CAR is extremely limited. Summary of the Invention

[0004] The present application provides a genetically modified immune cell, and a method for promoting the proliferation of immune cells. The method provided by the present application can up-regulate the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells. The method provided by the present application can also promote the generation of memory immune cells. The method provided by the present application can also inhibit the differentiation of immune cells. The method provided by the present application can also enhance the release of cytokines by immune cells. The method provided by the present application can also enhance the tumor killing ability of immune cells. In addition, the method provided by the present application can be used to prevent tumor recurrence in a subject. The present application also provides a method for treating tumors in a subject in need thereof. The present application also provides a composition comprising the genetically modified immune cell, and the use of the genetically modified immune cell and the composition in the preparation of a drug. The present application also provides a method for preparing the genetically modified immune cell.

[0005] On the one hand, the present application provides a method for promoting the proliferation of immune cells, which comprises the following steps: up-regulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0006] On the one hand, the present application provides a method for promoting the generation of memory immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells, thereby promoting the differentiation of the immune cells into memory immune cells.

[0007] On the one hand, the present application provides a method for inhibiting the differentiation of immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells, thereby inhibiting the differentiation of the immune cells into differentiated immune cells.

[0008] On the one hand, the present application provides a method for enhancing the release of cytokines by immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0009] In certain embodiments, the cytokines include interleukins, interferons, and / or tumor necrosis factors. In certain embodiments, the cytokines include IL-2, IL4, IL6, IL7, IL10, IL12, TNF-α, and / or IFNγ.

[0010] On the one hand, the present application provides a method for enhancing the tumor-killing ability of immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0011] On the one hand, the present application provides a method for preventing tumor recurrence in a subject, the method comprising: administering immune cells to a subject susceptible to tumors, wherein the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells is upregulated.

[0012] On the one hand, the present application provides a method for treating tumors in a subject in need thereof, which comprises the following steps: administering immune cells to the subject, wherein the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells is upregulated.

[0013] In certain embodiments, the tumors are selected from liver cancer, lung cancer, leukemia, and mesothelioma.

[0014] In certain embodiments, the method includes in vivo methods and in vitro methods.

[0015] In certain embodiments, the immune cells include lymphocytes. In certain embodiments, the immune cells include T cells. In certain embodiments, the T cells include memory stem cell-like T cells (TSCM) and / or central memory T cells (TCM). In certain embodiments, the TSCM is CCR7 + and / or CD62L +。In certain embodiments, the TSCM further has one or more properties selected from the group consisting of: CD45RA + or CD45RA - 、CD45RO + or CD45RO - 、CD27 + 、CD28 + 、CD127 + 、CD122 + 、CD3 + 、CD4 + and CD8 + 。

[0016] In certain embodiments, the immune cells comprise genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain embodiments, the genetically modified immune cells include genetically modified T cells.

[0017] In certain embodiments, the CAR comprises an intracellular domain, and the intracellular domain includes a signaling domain and / or a co-stimulatory domain.

[0018] In certain embodiments, the signaling domain comprises a portion selected from the group consisting of: the signaling domain of CD3ζ, the signaling domain of CD3δ, and the signaling domain of CD3ε. In certain embodiments, the signaling domain comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the nucleic acid molecule encoding the signaling domain comprises the nucleic acid sequence shown in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.

[0019] In certain embodiments, the co-stimulatory domain comprises a portion selected from the group consisting of: the co-stimulatory domain of CD27, the co-stimulatory domain of CD28, and the co-stimulatory domain of 4-1BB. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence shown in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 14 and SEQ ID NO: 16. In certain embodiments, the nucleic acid molecule encoding the co-stimulatory domain comprises the nucleic acid sequence shown in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 13 and SEQ ID NO: 15.

[0020] In certain embodiments, the CAR comprises a hinge region. In certain embodiments, the hinge region comprises a portion selected from the group consisting of the hinge region of IgG4, the hinge region of IgG1, and the hinge region of CD8. In certain embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the nucleic acid molecule encoding the hinge region comprises the nucleotide sequence shown in SEQ ID NO: 32.

[0021] In certain embodiments, the CAR comprises a transmembrane region. In certain embodiments, the transmembrane region comprises a portion selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD24. In certain embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 33. In certain embodiments, the nucleic acid molecule encoding the transmembrane region comprises the nucleotide sequence shown in SEQ ID NO: 34.

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

[0023] In certain embodiments, the targeting moiety specifically binds and / or recognizes a tumor antigen. In certain embodiments, the targeting moiety specifically binds and / or recognizes a target selected from the group consisting of B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members. In certain embodiments, the targeting moiety specifically binds and / or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.

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

[0025] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA, and the antibody or the antigen-binding fragment thereof comprises a light-chain variable region, and the light-chain variable region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 80% homology thereto; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region, and the heavy-chain variable region comprises HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence having at least 80% homology thereto; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.

[0026] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes HER2, and the antibody or the antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 80% homology thereto; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.

[0027] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes Mesothelin. The antibody or antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.

[0028] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, and the antibody or the antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.

[0029] In certain embodiments, the targeting moiety comprises an amino acid sequence shown in any one of the following: SEQ ID NO: 2, 4, 6, 8, and 10 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the nucleic acid molecule encoding the targeting moiety comprises a nucleic acid sequence shown in any one of the following: SEQ ID NO: 1, 3, 5, 7, and 9 or an amino acid sequence having at least 80% homology thereto.

[0030] In certain embodiments, the method further comprises the following steps: isolating and obtaining peripheral blood mononuclear cells PBMC, CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes or regulatory T cells.

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

[0032] In certain embodiments, the T cell stimulating factor comprises CD3 antibody, and the concentration of the CD3 antibody is 1 - 10,000 ng / mL. In certain embodiments, the T cell stimulating factor comprises CD28 antibody, and the concentration of the CD28 antibody is 1 - 10,000 ng / mL.

[0033] In certain embodiments, the method further comprises: adding one or more cytokine agents to the isolated PBMC.

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

[0035] In certain embodiments, the interleukin comprises one or more selected from the group consisting of: IL2, IL21, IL7, and IL15. In certain embodiments, the interleukin comprises IL2, and the concentration of the IL2 is 0.1 - 10,000 U / mL. In certain embodiments, the interleukin comprises IL21, and the concentration of the IL21 is 0.01 - 1,000 ng / mL. In certain embodiments, the interleukin comprises IL7, and the concentration of the IL7 is 0.01 - 1,000 ng / mL. In certain embodiments, the interleukin comprises IL15, and the concentration of the IL15 is 0.01 - 1,000 ng / mL.

[0036] In certain embodiments, the low density lipoprotein receptor-related protein comprises one or more selected from the group consisting of: low density lipoprotein receptor-related proteins 1 - 12 and functional fragments thereof. In certain embodiments, the low density lipoprotein receptor-related protein or its fragment is derived from a human.

[0037] In certain embodiments, the functional fragment comprises a fragment or truncation of the low-density lipoprotein receptor-related protein having the activity of the low-density lipoprotein receptor-related protein. In certain embodiments, the low-density lipoprotein receptor-related protein comprises low-density lipoprotein receptor-related protein 6 and its truncations, and / or low-density lipoprotein receptor-related protein 5 and its truncations. In certain embodiments, the truncation of the low-density lipoprotein receptor-related protein 6 comprises the intracellular region of the low-density lipoprotein receptor-related protein 6; and / or, the truncation of the low-density lipoprotein receptor-related protein 5 comprises the intracellular region of the low-density lipoprotein receptor-related protein 5. In certain embodiments, the truncation of the low-density lipoprotein receptor-related protein 6 comprises the transmembrane region of the low-density lipoprotein receptor-related protein 6 and the LDLR region of the low-density lipoprotein receptor-related protein 6; and / or, the truncation of the low-density lipoprotein receptor-related protein 5 comprises the transmembrane region of the low-density lipoprotein receptor-related protein 5 and the LDLR region of the low-density lipoprotein receptor-related protein 5. In certain embodiments, the low-density lipoprotein receptor-related protein or its fragment comprises an amino acid sequence shown in any one of the following: SEQ ID NO: 22, 24, 26, and 28 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or its fragment comprises a nucleic acid sequence shown in any one of the following: SEQ ID NO: 21, 23, 25, and 27 or an amino acid sequence having at least 80% homology thereto.

[0038] On the other hand, the present application provides a genetically modified immune cell, wherein the genetic modification upregulates the expression level of the low-density lipoprotein receptor-related protein or its fragment in the immune cell.

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

[0040] In certain embodiments, the immune cells comprise genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0041] In certain embodiments, the CAR comprises an intracellular domain, and the intracellular domain includes a signaling domain and / or a co-stimulatory domain. In certain embodiments, the signaling domain comprises a portion selected from the group consisting of the signaling domain of CD3ζ, the signaling domain of CD3δ, and the signaling domain of CD3ε. In certain embodiments, the signaling domain comprises the amino acid sequence shown in SEQ ID NO: 18. In certain embodiments, the nucleic acid molecule encoding the signaling domain comprises the nucleic acid sequence shown in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.

[0042] In certain embodiments, the co-stimulatory domain comprises a portion selected from the group consisting of the co-stimulatory domain of CD27, the co-stimulatory domain of CD28, and the co-stimulatory domain of 4-1BB. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence shown in any one of the following: SEQ ID NO: 14 and SEQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the nucleic acid molecule encoding the co-stimulatory domain comprises the nucleic acid sequence shown in any one of the following: SEQ ID NO: 13 and SEQ ID NO: 15 or a nucleic acid sequence having at least 80% homology thereto.

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

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

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

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

[0047] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA. The antibody or antigen-binding fragment thereof comprises a light-chain variable region, and the light-chain variable region comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 80% homology thereto; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region, and the heavy-chain variable region comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.

[0048] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes HER2. The antibody or antigen-binding fragment thereof comprises a light chain variable region, which comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 80% homology thereto; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, which comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.

[0049] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes Mesothelin, and the antibody or the antigen-binding fragment thereof comprises a light chain variable region that comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that comprises HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80% homology thereto; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.

[0050] In certain embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, and the antibody or antigen-binding fragment thereof comprises a light chain variable region, which comprises LCDR1-LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, which comprises HCDR1-HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 80% homology thereto; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% homology thereto. In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.

[0051] In certain embodiments, the targeting moiety comprises the amino acid sequence shown in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 2, 4, 6, 8, and 10. In certain embodiments, the nucleic acid molecule encoding the targeting moiety comprises the nucleic acid sequence shown in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 1, 3, 5, 7, and 9.

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

[0053] In certain embodiments, the functional fragment comprises a fragment or truncation of the low-density lipoprotein receptor-related protein having the activity of the low-density lipoprotein receptor-related protein. In certain embodiments, the low-density lipoprotein receptor-related protein comprises low-density lipoprotein receptor-related protein 6 and its truncations, and / or low-density lipoprotein receptor-related protein 5 and its truncations. In certain embodiments, the truncation of the low-density lipoprotein receptor-related protein 6 comprises the intracellular region of the low-density lipoprotein receptor-related protein 6; and / or, the truncation of the low-density lipoprotein receptor-related protein 5 comprises the intracellular region of the low-density lipoprotein receptor-related protein 5. In certain embodiments, the truncation of the low-density lipoprotein receptor-related protein 6 comprises the transmembrane region of the low-density lipoprotein receptor-related protein 6 and the LDLR region of the low-density lipoprotein receptor-related protein 6; and / or, the truncation of the low-density lipoprotein receptor-related protein 5 comprises the transmembrane region of the low-density lipoprotein receptor-related protein 5 and the LDLR region of the low-density lipoprotein receptor-related protein 5. In certain embodiments, the low-density lipoprotein receptor-related protein or its fragment comprises the amino acid sequence shown in any of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 22, 24, 26, and 28. In certain embodiments, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or its fragment comprises the nucleic acid sequence shown in any of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 21, 23, 25, and 27.

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

[0055] In certain embodiments, the composition further comprises an optionally pharmaceutically acceptable carrier.

[0056] On the other hand, the present application provides the use of the genetically modified cells described above and / or the composition in the preparation of a drug for treating and / or preventing tumors.

[0057] In certain embodiments, the 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 composition, the method comprising the following steps: upregulating the expression level of the low-density lipoprotein receptor-related protein or its fragment in the genetically modified immune cells.

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

[0060] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence shown in any of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 22, 24, 26, and 28.

[0061] In certain embodiments, the genetically modified immune cell comprises a lymphocyte. In certain embodiments, the genetically modified immune cell expresses a chimeric antigen receptor (CAR). In certain embodiments, the method comprises the step of isolating and activating the genetically modified immune cell, wherein the activation comprises administering a T cell medium to the isolated genetically modified immune cell.

[0062] In certain embodiments, the T cell medium is selected from one or more of the following groups: DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.

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

[0064] Those skilled in the art can easily insight into other aspects and advantages of the present disclosure from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present disclosure enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0066] Figure 1 Shows a schematic diagram of the structure of the CAR in the lentivirus described in the present application.

[0067] Figure 2 Shows the results of the genetically modified immune cell described in the present application promoting the generation of memory immune cells.

[0068] Figure 3 Shown are the results of the inhibition of immune cell differentiation by the genetically modified immune cells described in the present application.

[0069] Figure 4 Shown are the results of the promotion of the proliferation of the immune cells described in the present application by low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells described in the present application.

[0070] Figure 5 Shown in A-5B are the results of the enhancement of cytokine release by the genetically modified immune cells described in the present application.

[0071] Figure 6 Shown is the anti-tumor effect of the genetically modified immune cells described in the present application.

[0072] Figure 7 Shown is the effect of the genetically modified immune cells described in the present application in preventing tumor recurrence. Detailed implementation manners

[0073] The following specific embodiments illustrate the implementation manners of the invention of the present application. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0074] In the present application, the term "immune cell" generally refers to cells participating in or related to immune responses. The immune cells may include lymphocytes and various phagocytes. The immune cells may also include natural and genetically modified immune cells. In the present application, the genetically modified immune cells may express a chimeric antigen receptor (CAR). Among them, the lymphocytes may include T lymphocytes and B lymphocytes. In the present application, the immune cells may include T cells.

[0075] In the present application, the term "memory immune cell" generally refers to an immune cell with immune memory. The immune memory may refer to the ability to produce a specific recognition and response to a certain antigen and then, when encountering the same antigen again, be able to produce a rapid and strong immune response. In the present application, the memory immune cells may include memory T cells. The memory T cells may be divided into memory stem cell-like T cells (TSCM) and central memory T cells (TCM).

[0076] In the present application, the term "differentiated immune cell" generally refers to an immune cell with a certain degree of differentiation. For example, the differentiated immune cell may be a T cell with a certain degree of differentiation. In the present application, the differentiated immune cells may be obtained by culturing the immune cells to a certain degree of differentiation. For example, the differentiated immune cells may include regulatory T cells (Treg).

[0077] In the present application, the term "regulatory T cell" (Treg) generally refers to a group of lymphocytes that negatively regulate the body's immune response. The molecular marker of the regulatory T cell can be a transcription factor Foxp3 + or CD127 - . In the present application, the regulatory T cells can be classified into two categories: naturally occurring and induced. Among them, the naturally occurring ones are CD4 + CD25 + cells, and the induced ones are T R 1 cells and T H 3 cells.

[0078] In the present application, the term "subject susceptible to tumor" generally refers to a subject with an increased probability of developing a tumor compared to a normal subject. For example, the subject susceptible to tumor can be a subject who has had a tumor cured but is at risk of metastasis and recurrence. The subject susceptible to tumor can also be a subject who has been diagnosed with risk factors for developing a tumor. For example, the risk factors can include some gene mutations (including deletions, insertions or substitutions) proven to be related to tumors. In the present application, the subject susceptible to tumor can also be a subject who has been exposed to a carcinogenic environment for a long time. For example, the carcinogenic environment can include strong radiation and high concentrations of carcinogens

[0079] In the present application, the term "genetic modification" generally refers to changes or modifications occurring at the genetic structure level. For example, the genetic modification can be a modification at the gene level, transcription level and / or translation level. For another example, the genetic modification can include any change in the genetic characteristics of an organism (including its tissues, cells, DNA, mRNA or proteins and their fragments, etc.). The genetic modification can include making the organism express a specific protein or its fragment. For example, the genetic modification can include making the organism contain a vector capable of expressing the specific protein or its fragment.

[0080] In the present application, the term "T cell", also known as T lymphocyte, is a subtype of white blood cells and plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes such as B cells and natural killer cells by the T cell receptors present on their cell surface. In the present application, the T cells can include memory stem cell-like T cells (TSCM) and central memory T cells (TCM).

[0081] In the present application, the term "memory stem cell-like T cells" (T memory stem cells, TSCM) generally refers to cells that are in the early differentiation stage of memory T cells, have stem cell characteristics, and have strong multi-directional differentiation potential. After responding to antigen stimulation, TSCM cells can differentiate into central memory T cells (central memory T cells, TCM), effector memory T cells (effector memory T cells, TEM), and effector T cells (effector T cells, TEF).

[0082] In the present application, the term "central memory T cells" (central memory T cells, TCM) generally refers to T cells with long-term memory generated after naive T cells are activated by antigens. The biomarkers of the TCM may include CD62L + and CD45RO + . The central memory T cells can return to the lymph nodes through the lymphatic barrier and are in a state of being activated by antigens at the same time.

[0083] In the present application, the term "T cell receptor" is usually also referred to as "TCR", and generally refers to the molecular structure by which T cells specifically recognize and bind antigen peptide-MHC molecules. The T cell receptor can exist on the surface of T cells in the form of a complex with the CD3 molecule. The TCR can be a heterodimer fixed on the cell membrane, and most are composed of a highly variable α subunit and a β subunit connected by a disulfide bond; a small number are composed of γ and δ peptide chains. The TCR can include a variable region and a constant region, where the constant region can be close to the cell membrane, connecting the transmembrane region and the intracellular terminus, and the variable region is responsible for recognizing the polypeptide / MHC complex.

[0084] In the present application, the term "chimeric antigen receptor" is usually also referred to as "CAR", and generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. In the present application, the CAR may comprise an intracellular domain, and the intracellular domain includes a signal transduction domain and / or a co-stimulatory domain. In the present application, a set of polypeptides of the CAR may be located in the same polypeptide chain (e.g., comprising a chimeric fusion protein), or may not be contiguous with each other, e.g., may be located in different polypeptide chains. In the present application, the signal involved in induction can be transduced into the cytoplasm of T cells via CD3 and the ζ chain. In the present application, the intracellular domain may comprise a primary signal transduction binding domain (e.g., the major signal domain of CD3-zeta (ζ)). In one aspect, the cytoplasmic signal domain may further comprise one or more co-stimulatory domains derived from at least one co-stimulatory molecule. For example, the co-stimulatory domain may be 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In the present application, the CAR may comprise a chimeric fusion protein, e.g., containing an optional leader sequence at the amino terminus (N-ter). Among them, the leader sequence optionally cleaves the antigen-binding domain (e.g., ScFv) during cell processing and localizes the CAR to the cell membrane.

[0085] In the present application, the term "signal transduction domain" generally refers to a domain located inside the cell that can transduce signals. In the present application, the intracellular signal transduction domain can transduce signals into the cell. For example, the intracellular signal transduction domain is the intracellular signal transduction domain of the chimeric antigen receptor. In the present application, the signal transduction domain may comprise a part selected from the group consisting of the signal transduction domains of CD3ζ, CD3δ, and CD3ε.

[0086] In the present application, the term "co-stimulatory domain" generally refers to a domain in the CAR that crosses the cell membrane and is connected to the intracellular signal transduction domain, playing a role in transmitting signals. In the present application, the co-stimulatory domain may comprise a part selected from the group consisting of the co-stimulatory domains of CD27, CD28, and 4-1BB.

[0087] In the present application, the term "hinge region" generally refers to the connecting region between the antigen-binding region and the immune cell Fc receptor (FcR) binding region. For example, the hinge region may be the region between the CHl and CH2 functional regions of the heavy chain in an immunoglobulin. In the present application, the hinge region may be a region located between the scFv and the T cell membrane. The hinge region may be derived from IgG1 or IgG4, and may also be derived from IgD or CD8. In the present application, the hinge region may comprise a part selected from the group consisting of the hinge region of IgG4, the hinge region of IgG1, and the hinge region of CD8.

[0088] In the present application, the term "transmembrane region" generally refers to a transmembrane region that connects an extracellular antigen-binding domain and an intracellular signaling domain, which is generally composed of dimeric membrane proteins, mainly including CD3ζ, CD4, CD8, CD28, etc., and can anchor the CAR structure to the T cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene. In the present application, the transmembrane region may comprise a portion selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD24.

[0089] In the present application, the term "single-chain antibody" (ScFv) generally refers to an antibody formed by connecting the heavy-chain variable region and the light-chain variable region through a linker. In the present application, the linker may be a linking peptide.

[0090] In the present application, the term "tumor antigen" generally refers to an antigenic substance present within or produced by tumor cells, which may have the ability to trigger an immune response in the host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that forms part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" may also refer to a biomolecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is specifically or preferentially or differentially expressed on cancer cells and / or is associated with cancer cells, thereby providing a cancer-preferred or -specific target. For example, preferential expression may be conventional preferential expression compared to any other cells in an organism, or preferential expression within a specific region of an organism (e.g., within a specific organ or tissue). In the present application, the tumor antigen may include B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members.

[0091] In the present application, the term "B lymphocyte surface antigen" generally refers to an antigen located on the surface of B lymphocytes produced at different stages. For example, the B lymphocyte surface antigen may include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, and CD40. In the present application, the B lymphocyte surface antigen may include CD19.

[0092] In the present application, the term "CD19" generally refers to the cluster of differentiation 19 protein, which is an antigenic determinant detectable on pre-leukemic cells. The accession number of human CD19 in UniProt / Swiss-Prot is P15391, and the nucleotide sequence encoding human CD19 has an accession number of NM_001178098 in GenBank. In the present application, CD19 may also include a protein containing a mutation or a functional fragment thereof, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19.

[0093] In the present application, the term "TNF family member" generally refers to a member belonging to the TNF (Tumor Necrosis Factor) family. 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 application, the TNF family members may include BCMA and 4-1BB.

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

[0095] The term "CD137 protein", also known as 4-1BB or TNFRS9, generally refers to a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), which is an activation-induced co-stimulatory molecule and an important regulator of the immune response. Studies have shown that CD137 agonist monoclonal antibodies increase the expression of co-stimulatory molecules in many models and significantly enhance the cytolytic T lymphocyte response, playing an anti-tumor role (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). The GenBank accession number of human CD137 is NP_001552.2.

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

[0097] In the present application, the term "HER2" generally refers to the human HER2 protein, which belongs to the HER family members. For example, see Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al., Nature 319: 230-234 (1986). The GenBank accession number of human HER2 can include XP_024306409.1.

[0098] In the present application, the term "GPC family member" generally refers to glypican. Six glypicans have been identified in mammals, which are respectively called GPC1 to GPC6. There are abnormal expressions of glypicans in cancers, including human hepatocellular carcinoma, ovarian cancer, mesothelioma, pancreatic cancer, glioma, and breast cancer. In the present application, the GPC family member can include GPC3.

[0099] In the present application, the term "GPC3" generally refers to the protein encoded by glypican 3 (Gene ID in NCBI database: 2719), which is an early marker of liver cancer. GPC3 is highly expressed in hepatocellular carcinoma and is detected in the tissues of patients with early-stage hepatocellular carcinoma. The GenBank accession number of human GPC3 can be AAB87062.1.

[0100] In the present application, the term "Mesothelin" (mesothelin, or abbreviated as MSLN) generally refers to a tumor differentiation antigen, which is usually present on mesothelial cells lining the pleura, peritoneum, and pericardium. It is highly expressed in cancers, including malignant mesothelioma, pancreatic cancer, ovarian cancer, and lung adenocarcinoma. The GenBank accession number of human Mesothelin can be AAH09272.1.

[0101] In the present application, the term "peripheral blood mononuclear cell" generally refers to cells with a single nucleus in peripheral blood (Peripheral blood mononuclear cell, PBMC). The peripheral blood mononuclear cells may include lymphocytes and monocytes. In the present application, the peripheral blood mononuclear cells can be isolated using the Ficoll-hypaque density gradient centrifugation method and separated according to the specific gravity differences of the components in the blood.

[0102] In the present application, the term "T cell activator" generally refers to a substance that promotes the activation and proliferation of T cells. In the present application, the T cell activator may include B lymphocyte surface antigen antibodies, TNF antibodies, intracellular polyesters, and / or antibiotics. In the present application, the T cell stimulatory factor is selected from the following group: CD3 antibody, CD28 antibody, 4-1BB antibody, CD80 antibody, CD86 antibody, PHA, PMA, and ionomycin.

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

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

[0105] In the present application, the term "intracellular polyester" generally refers to a natural polymer biomaterial synthesized by microorganisms and present in the form of inclusion bodies within cells. Intracellular polyesters have good biological properties. In the present application, intracellular polyesters may include polyhydroxyalkanoates (PHA).

[0106] In the present application, the term "antibiotic" generally refers to a metabolite produced by microorganisms or animals and plants that has anti-pathogen or other activities and can interfere with the development and function of other living cells and play a role. In the present application, the antibiotics may include β-lactams, aminoglycosides, amide alcohols, macrolides, polypeptides, nitroimidazoles, and tetracyclines. For example, the antibiotics may include ionomycin.

[0107] In the present application, the term "PHA" generally refers to polyhydroxyalkanoate, which belongs to intracellular polyesters synthesized by various bacteria and can exist in the form of discontinuous inclusion bodies in the cytoplasm within an organism. PHA may have physical and chemical properties similar to synthetic plastics, as well as biodegradability, biocompatibility, optical activity, piezoelectricity, gas barrier properties, etc.

[0108] In the present application, the term "PMA" generally refers to Phorbol-12-myristate-13-acetate.

[0109] In the present application, 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 on the T cell membrane and can transmit signals for T cell activation. The CD3 antibody can be huOKT3g1 or HuM291.

[0110] In the present application, the term "CD28 antibody" generally refers to an antibody or an antigen-binding fragment thereof that specifically binds to CD28. Human CD28 is located at 2q33, has similar exons and introns to CTLA4, and the ligands of both are the B7 family, including B7-1 (CD80) and B7-2 (CD86).

[0111] In the present application, the term "cytokine" generally refers to a class of small molecular proteins with a wide range of biological activities synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokine can have multiple functions such as regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and repair of damaged tissues. In the present application, the cytokine can include interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. For example, the cytokine can be an interleukin.

[0112] In the present application, the term "interleukin" generally refers to a secreted protein or signaling molecule that can promote the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes, as well as by monocytes, macrophages, and endothelial cells. In the present application, the term "interleukin" can include full-length interleukins or fragments (such as truncated forms) or variants thereof that substantially retain the biological activity of the corresponding wild-type interleukin (e.g., 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 of the corresponding wild-type interleukin). The interleukins used herein can be from any mammalian species. In certain embodiments, the interleukin is from a species selected from humans, horses, cows, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In certain embodiments, the interleukin can be a mutant form. For example, in the present application, the cytokine includes one or more selected from the group consisting of IL2, IL21, IL7, and IL15.

[0113] In the present application, the term "IL2" generally refers to the T cell growth factor, TCGF. It is produced by T cells and exerts its effects in an autocrine and paracrine manner. It can activate T cells, promote the production of cytokines; stimulate the proliferation of NK cells and induce the production of LAK cells; promote the proliferation of B cells and the secretion of antibodies, and can also activate macrophages.

[0114] In the present application, the term "IL21" is homologous in spatial structure to IL-2, IL-4, and IL-15. It generally can promote the proliferation and differentiation of bone marrow NK cells, synergistically stimulate the proliferation of B cells with anti-CD40 antibody, and synergistically stimulate the proliferation of T cells with anti-CD3 antibody.

[0115] In the present application, the term "IL7" generally refers to a glycoprotein secreted by bone marrow stromal cells, and its gene is located on chromosome 8. The target cells of IL7 are lymphocytes, especially it can promote the growth of B progenitor cells, thymocytes and peripheral mature T cells derived from human or mouse bone marrow. At higher concentrations, IL-7 can also enhance the cytotoxic activity of macrophages and induce monocytes to secrete cytokines.

[0116] In the present application, the term "IL15" is generally produced by various cells such as activated monocytes-macrophages, epidermal cells and fibroblasts, and has many similarities with IL2. IL15 belongs to the IL2 family members and regulates the activation and proliferation of T and NK cells by changing the expression of Bcl-1 family members (such as Bcl-2 and Bcl-XL), and can also induce the proliferation of B cells.

[0117] In the present application, the term "low density lipoprotein receptor-related protein" (LRP) generally refers to an integral protein containing 839 amino acids (after removing a 21-amino acid signal peptide). Embedded in the outer phospholipid layer of LDL (Low density lipoprotein) particles, it belongs to an endocytic receptor and can mediate the endocytosis of cholesterol-rich LDL. It is a member of the low density lipoprotein receptor (LDLR) gene family. The LRP is most significantly expressed in bronchial epithelial cells and adrenal and cortical tissues. In the present application, the low density lipoprotein receptor-related protein may include one or more selected from the group consisting of low density lipoprotein receptor-related protein 1-12 or truncated forms.

[0118] In the present application, the terms "low density lipoprotein receptor-related protein 6" (LRP-6) and "low density lipoprotein receptor-related protein 5" (LRP-5) generally refer to a unique subgroup within the low density lipoprotein receptor (LDLR) family. The accession number of human LRP-6 in UniProt is O75581. The accession number of human LRP-5 in UniProt is O75197.

[0119] In the present application, the term "truncated protein" generally refers to a truncated protein. The truncated protein can be obtained by proteolysis or by manipulating the structural gene to eliminate the N- or C-terminal portion of the protein. Alternatively, the truncated protein can be obtained by a nonsense mutation that results in the presence of a stop codon in the structural gene, thereby prematurely terminating translation.

[0120] In the present application, the term "intracellular region" generally refers to the domain of a protein that is located within the cell membrane. In the present application, the intracellular domain may refer to the domain of the low density lipoprotein receptor-related protein within the cell membrane. In the present application, the intracellular region may include the sequence from position 24 to position 243 in SEQ NO.24 or an amino acid sequence having at least 80% homology thereto, or include the sequence from position 24 to position 231 in SEQ NO.28 or an amino acid sequence having at least 80% homology thereto.

[0121] In the present application, the term "transmembrane region" generally refers to the domain of a protein that spans the cell membrane. In the present application, the transmembrane region may refer to the domain of the low density lipoprotein receptor-related protein that spans the cell membrane. The transmembrane region may be composed of 23 hydrophobic amino acids. This domain mainly provides an anchoring function for the binding of LDLR to the cell membrane. In the present application, the transmembrane region may include the sequence from position 1 to position 23 in SEQ NO.24 or an amino acid sequence having at least 80% homology thereto, or include the sequence from position 1 to position 23 in SEQ NO.28 or an amino acid sequence having at least 80% homology thereto.

[0122] In the present application, the term "LDLR region" generally refers to the domain of the low density lipoprotein receptor-related protein that is close to the N-terminus outside the transmembrane region. This domain may have the function of enhancing the Wnt signal. In the present application, the LDLR region may include the sequence from position 5 to position 119 in SEQ NO.22 or an amino acid sequence having at least 80% homology thereto, or the sequence from position 4 to position 119 in SEQ NO 26 or an amino acid sequence having at least 80% homology thereto.

[0123] In the present application, the term "ribosome skipping site" is also called an Internal Ribosome Entry Site (IRES), and generally refers to a nucleotide sequence located in the middle of the mRNA sequence and reserved for translation initiation. The ribosome skipping site can allow cap-independent translation initiation. IRES is usually located in the 5' UTR. In the present application, the ribosome skipping site may include the sequence from position 1 to position 578 in SEQ NO.29.

[0124] In the present application, the term "2A sequence" generally refers to a protease-independent self-cleaving amino acid sequence. The 2A sequence can help transcribe two proteins. In the present application, the 2A sequence may include the sequence from position 1 to position 54 in SEQ NO 75.

[0125] In the present application, the term "pharmaceutically acceptable carrier" may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants, etc.

[0126] In the present application, the term "retroviral vector" generally refers to an RNA virus that can reverse transcribe to produce a complementary DNA strand in an infected cell, and use this single-stranded DNA as a template to synthesize the second DNA strand, which is then incorporated into the cellular genomic DNA. The retroviral vector can utilize the enzymes of the host cell to transcribe and replicate RNA to synthesize proteins by itself, and then package the virus and release it from the cell to become an infectious virus. The transduction efficiency of the retrovirus is high, and it can effectively improve the gene transfection rate.

[0127] In the present application, the term "lentiviral vector" refers to a gene therapy vector developed based on HIV-1 (human immunodeficiency virus type I). The lentiviral vector can infect both dividing and non-dividing cells. It can effectively infect almost all mammalian cells, including neuronal cells and hepatocytes, with high infection efficiency. Lentivirus can effectively integrate foreign genes into the host chromosome, thereby achieving persistent expression.

[0128] In the present application, the term "transposon plasmid" generally refers to the basic unit that exists on chromosomal DNA and can replicate and transpose autonomously. The transposon plasmid can "jump" from one position to another in the genome through a series of processes such as cleavage and reintegration.

[0129] In the present application, the term "tumor" generally refers to a neogrowth formed by a single cell in a local tissue losing normal regulation of its growth at the gene level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation. Since such neogrowths often present as mass-like protrusions occupying space, they are also called neoplasms. In the present application, the tumor may include solid tumors and non-solid tumors. In the present application, the tumor may include liver cancer, lung cancer, leukemia, and mesothelioma.

[0130] In the present application, the term "significantly increased" generally refers to an increase in level (such as protein expression level, cell number), where 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 significantly increased may refer to an increase of 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.

[0131] In the present application, the term "basically not increased" generally refers to the level (such as protein expression level, cell number) remaining the same as the original, or increasing 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.

[0132] In the present application, the term "about" generally refers to a variation within the range of 0.5% - 10% above or below the specified value. For example, it may vary within the range of 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% above or below the specified value.

[0133] Low-density lipoprotein receptor-related protein or a fragment thereof

[0134] In the present application, the low-density lipoprotein receptor-related protein may include one or more selected from the following group: low-density lipoprotein receptor-related protein 1-12 and its functional fragments.

[0135] In the present application, the low-density lipoprotein receptor-related protein or its fragment may be derived from a mammal. For example, it may be derived from a human, macaque, rat, or mouse.

[0136] In the present application, the functional fragment may comprise a fragment or truncation of the low density lipoprotein receptor-related protein having the activity of the low density lipoprotein receptor-related protein. For example, the low density lipoprotein receptor-related protein may comprise low density lipoprotein receptor-related protein 6 and its truncations, and / or low density lipoprotein receptor-related protein 5 and its truncations.

[0137] In the present application, the truncation of the low density lipoprotein receptor-related protein 6 may comprise the intracellular region of the low density lipoprotein receptor-related protein 6; and / or, the truncation of the low density lipoprotein receptor-related protein 5 may comprise the intracellular region of the low density lipoprotein receptor-related protein 5. Again, for example, the truncation of the low density lipoprotein receptor-related protein 6 may comprise the transmembrane region of the low density lipoprotein receptor-related protein 6 and the LDLR region of the low density lipoprotein receptor-related protein 6; and / or, the truncation of the low density lipoprotein receptor-related protein 5 may comprise the transmembrane region of the low density lipoprotein receptor-related protein 5 and the LDLR region of the low density lipoprotein receptor-related protein 5.

[0138] In the present application, the low density lipoprotein receptor-related protein or its fragment comprises an amino acid sequence shown in any of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 22, 24, 26 and 28.

[0139] In the present application, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or its fragment comprises a nucleic acid sequence shown in any of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 21, 23, 25 and 27.

[0140] Genetically modified immune cells

[0141] The present application provides a genetically modified immune cell, wherein the expression level of the low density lipoprotein receptor-related protein or its fragment in the immune cell is up-regulated.

[0142] In the present application, the genetically modified immune cell may comprise lymphocytes. In the present application, the genetically modified immune cell comprises genetically modified T cells. In the present application, the genetically modified immune cell comprises genetically modified memory stem cell-like T cells (TSCM) and / or genetically modified central memory T cells (TCM). In the present application, the TSCM comprises CCR7 + and / or CD62L + . In the present application, the TSCM further comprises one or more properties selected from the group consisting of: CD45RA + or CD45RA - , CD45RO+ or CD45RO - , CD27 + , CD28 + , CD127 + , CD122 + , CD3 + , CD4 + and CD8 + 。

[0143] In the present application, the immune cells may include genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR).

[0144] In the present application, the CAR may include an intracellular domain, and the intracellular domain may include a signal transduction domain and / or a co-stimulatory domain.

[0145] For example, the signal transduction domain may include a portion selected from the group consisting of the signal transduction domain of CD3ζ, the signal transduction domain of CD3δ, and the signal transduction domain of CD3ε. For example, the signal transduction domain may include the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the signal transduction domain may include the nucleic acid sequence shown in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.

[0146] For example, the co-stimulatory domain may include a portion selected from the group consisting of the co-stimulatory domain of CD27, the co-stimulatory domain of CD28, and the co-stimulatory domain of 4-1BB. For example, the co-stimulatory domain may include the amino acid sequence shown in any one of the following: SEQ ID NO: 14 and SEQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the co-stimulatory domain may include the nucleic acid sequence shown in any one of the following: SEQ ID NO: 13 and SEQ ID NO: 15 or a nucleic acid sequence having at least 80% homology thereto.

[0147] In the present application, the CAR may include a hinge region. For example, the hinge region may include a portion selected from the group consisting of the hinge region of IgG4, the hinge region of IgG1, and the hinge region of CD8. For example, the hinge region may include the amino acid sequence shown in 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 include the nucleic acid sequence shown in SEQ ID NO: 32 or a nucleic acid sequence having at least 80% homology thereto.

[0148] In the present application, the CAR may comprise a transmembrane region. For example, the transmembrane region may comprise a portion selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD24. For example, the transmembrane region may comprise the amino acid sequence shown in 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 the nucleic acid sequence shown in SEQ ID NO: 34 or a nucleic acid sequence having at least 80% homology thereto.

[0149] In the present application, the CAR may comprise a targeting moiety. In the present application, the targeting moiety may include an antibody or an antigen-binding fragment. The antigen-binding fragment may be selected from the group consisting of Fab, Fab’, F(ab)2, F(ab’)2, Fv, and ScFv fragments. For example, the targeting moiety may be ScFv.

[0150] In the present application, the targeting moiety may specifically bind to and / or recognize a tumor antigen. For example, the targeting moiety may specifically bind to and / or recognize a target selected from the group consisting of B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members. For another example, the targeting moiety may specifically bind to and / or recognize a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.

[0151] In the present application, the targeting moiety may be an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes CD19. The antibody or the antigen-binding fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise LCDR1-LCDR3, wherein LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto; LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80% homology thereto; LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 48 or an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 50 or an amino acid sequence having at least 80% homology thereto. In the present application, the antibody or the antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-HCDR3, wherein HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology thereto; HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 80% homology thereto.

[0152] In the present application, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes BCMA. The antibody or the antigen-binding fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise LCDR1-LCDR3, wherein LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 80% homology thereto; LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto. In the present application, the antibody or the antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-HCDR3, wherein HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence having at least 80% homology thereto; HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.

[0153] In the present application, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes HER2. The antibody or the antigen-binding fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise LCDR1-LCDR3, wherein LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 71 or an amino acid sequence having at least 80% homology thereto; LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto. In the present application, the antibody or the antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-HCDR3, wherein HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 67 or an amino acid sequence having at least 80% homology thereto; HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.

[0154] In the present application, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes Mesothelin. The antibody or the antigen-binding fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise LCDR1-LCDR3, wherein LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 63 or an amino acid sequence having at least 80% homology thereto; LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto. The antibody or the antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-HCDR3, wherein HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80% homology thereto; HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.

[0155] In the present application, the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3. The antibody or the antigen-binding fragment thereof may comprise a light chain variable region, and the light chain variable region may comprise LCDR1-LCDR3, wherein LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology thereto; LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto. The light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto. In the present application, the antibody or the antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-HCDR3, wherein HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 80% homology thereto; HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 80% homology thereto. The heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.

[0156] In the present application, the targeting moiety may comprise the amino acid sequence shown in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 2, 4, 6, 8, and 10. In the present application, the nucleic acid molecule encoding the targeting moiety may comprise the nucleic acid sequence shown in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 1, 3, 5, 7, and 9.

[0157] In the present application, the starting and ending positions of the amino acid sites of the CDR are calculated in the Kabat manner.

[0158] In the present application, the proteins, polypeptides, amino acid sequences, and / or nucleic acid sequences involved should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the said protein or polypeptide. And / or nucleic acid sequences encoding variants or homologs having the same or similar functions as the said protein or polypeptide.

[0159] In the present application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to the GPC3 protein, or the low-density lipoprotein receptor-related protein or a fragment thereof). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by substitution, deletion and / or insertion of at least 1, such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4 or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (such as substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (such as antigen-binding ability, or the biological function of the low-density lipoprotein receptor-related protein or a fragment thereof) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0160] In the present application, the homolog may be a protein or polypeptide having at least about 85% (such as having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to the GPC3 protein, or the low-density lipoprotein receptor-related protein or a fragment thereof).

[0161] In the present application, the homology generally refers to the similarity, analogy or correlation between two or more sequences. The "percent sequence homology" can be calculated by the following method: comparing two sequences to be aligned in a comparison window, determining the number of positions where the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exist in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to generate the percent sequence homology. The alignment for determining the percent sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment within the full-length sequences being compared or within the target sequence region. The homology can also be determined by the following methods: FASTA and BLAST. The description of the FASTA algorithm can be found in "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and "Rapid and Sensitive Protein Similarity Searches" by D.J. Lipman and W.R. Pearson, Science, 227: 1435-1441, 1989. The description of the BLAST algorithm can be found in "Basic Local Alignment Search Tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, Journal of Molecular Biology, 215: 403-410, 1990.

[0162] In the present application, the CAR may sequentially include the targeting portion, the hinge region, the transmembrane region, the co-stimulatory domain and the signaling domain from the N-terminus to the C-terminus.

[0163] In the present application, the polynucleotide molecule encoding the CAR may sequentially include the nucleotide sequence encoding the targeting portion, the nucleotide sequence encoding the hinge region, the nucleotide sequence encoding the transmembrane region, the nucleotide sequence encoding the transmembrane region, the nucleotide sequence encoding the co-stimulatory domain and the nucleotide sequence encoding the signaling domain from the 5'-end to the 3'-end.

[0164] In the present application, the polynucleotide molecule encoding the CAR may further comprise a leader sequence. For example, the nucleic acid molecule encoding the leader sequence may comprise the nucleic acid sequence shown in 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 at the 5' end of the nucleotide sequence encoding the targeting moiety.

[0165] In the present application, the genetically modified immune cell may express the low density lipoprotein receptor related protein or a fragment thereof. For example, the genetically modified immune cell may comprise a vector capable of expressing the low density lipoprotein receptor related protein or a fragment thereof. The vector may contain a nucleotide molecule encoding the low density lipoprotein receptor related protein or a fragment thereof. For example, the polynucleotide molecule may comprise the nucleic acid sequence shown in any one of the following: SEQ ID NO: 21, 23, 25, and 27 or a nucleic acid sequence having at least 80% homology thereto. Again, for example, the vector may be selected from the group consisting of: retroviral vectors, lentiviral vectors, and / or transposon plasmids.

[0166] In the present application, the genetically modified immune cell may express the chimeric antigen receptor (CAR). For example, the genetically modified immune cell may comprise a vector capable of expressing the chimeric antigen receptor (CAR). The vector may contain a nucleotide molecule encoding the chimeric antigen receptor (CAR). Again, for example, the vector may be selected from the group consisting of: retroviral vectors, lentiviral vectors, and / or transposon plasmids.

[0167] In the present application, 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) may be the same vector or different vectors, as long as the one or more vectors can express the low density lipoprotein receptor related protein or a fragment thereof and the chimeric antigen receptor (CAR), so that the genetically modified immune cell simultaneously has the low density lipoprotein receptor related protein or a fragment thereof and the chimeric antigen receptor (CAR).

[0168] 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) may be the same vector. In this vector, 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) may be located in the same expression frame. For example, the nucleotide molecule encoding the low density lipoprotein receptor related protein or a fragment thereof may be located at the 3' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).

[0169] In the present application, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment can be directly or indirectly linked to the nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the indirect linkage can be achieved through a linker sequence. The 5' end of the linker sequence can be linked to the 3' end of the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment, and the 3' end of the linker sequence can be linked to the 5' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR). In the present application, the linker sequence can comprise a nucleic acid sequence shown in any one of the following: SEQ ID NO: 19 and 29 or a nucleic acid sequence having at least 80% homology thereto.

[0170] In the present application, the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cell can be two independent proteins. That is, there is no interconnection between the two to form any form of dimer or protein complex. However, the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cell can also be interconnected. For example, in some cases, the two proteins formed by translation are not completely cleaved, and the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor form a complex.

[0171] Preparation method

[0172] The present application provides a method for preparing the genetically modified immune cell, the method comprising the following steps: upregulating the expression level of the low-density lipoprotein receptor-related protein or its fragment in the genetically modified immune cell.

[0173] The present application also provides a method for preparing the composition, the method comprising the following steps: upregulating the expression level of the low-density lipoprotein receptor-related protein or its fragment in the genetically modified immune cell.

[0174] In the present application, the method may further comprise the following steps: isolating peripheral blood mononuclear cells PBMC, CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes or regulatory T cells.

[0175] In the present application, the method may comprise the step of isolating and activating the genetically modified immune cell, wherein the activation comprises administering a T cell culture medium to the isolated genetically modified immune cell.

[0176] In the present application, the T cell culture medium can be selected from one or more of the following groups: DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.

[0177] In the present application, the method may further include the following step: adding one or more T cell stimulating factors to the isolated peripheral blood mononuclear cells PBMC.

[0178] For example, the T cell stimulating factors can be selected from the following groups: B lymphocyte surface antigen antibodies, TNF antibodies, intracellular polyesters, and antibiotics. Again, for example, the T cell stimulating factors can be selected from the following groups: CD3 antibody, CD28 antibody, 4-1BB antibody, CD80 antibody, CD86 antibody, PHA, PMA, and ionomycin.

[0179] In the present application, the T cell stimulating factor can include a CD3 antibody, and the concentration of the CD3 antibody can be 1-10000 ng / mL. For example, the concentration of the CD3 antibody can be 1-9000 ng / mL, 1-5000 ng / mL, 1-4000 ng / mL, 1-3000 ng / mL, 1-1000 ng / mL, 1-500 ng / mL, 1-400 ng / mL, 1-300 ng / mL, 1-200 ng / mL, 1-100 ng / mL, 1-50 ng / mL, 1-40 ng / mL, 1-30 ng / mL, 1-20 ng / mL, 1-10 ng / mL, or 1-5 ng / mL. In the present application, the T cell stimulating factor can include a CD28 antibody, and the concentration of the CD28 antibody can be 1-10000 ng / mL. For example, the concentration of the CD28 antibody can be 1-9000 ng / mL, 1-5000 ng / mL, 1-4000 ng / mL, 1-3000 ng / mL, 1-1000 ng / mL, 1-500 ng / mL, 1-400 ng / mL, 1-300 ng / mL, 1-200 ng / mL, 1-100 ng / mL, 1-50 ng / mL, 1-40 ng / mL, 1-30 ng / mL, 1-20 ng / mL, 1-10 ng / mL, or 1-5 ng / mL.

[0180] In the present application, the method may further include: adding one or more cytokine agents to the isolated PBMC. In the present application, the cytokine agent can include interleukin. For example, the interleukin can include one or more selected from the following group: IL2, IL21, IL7, and IL15.

[0181] In the present application, the interleukin may include IL2, and the concentration of the IL2 may be 0.1 - 10000 U / mL. For example, the concentration of the IL2 may be 0.1 - 8000 U / mL, 0.1 - 6000 U / mL, 0.1 - 4000 U / mL, 0.1 - 2000 U / mL, 5 - 2000 U / mL, 5 - 1900 U / mL, 5 - 1800 U / mL, 5 - 1700 U / mL, 5 - 1600 U / mL, 5 - 1500 U / mL, 5 - 1400 U / mL, 5 - 1300 U / mL, 5 - 1200 U / mL, 5 - 1100 U / mL, 5 - 1000 U / mL, 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 or 5 - 10 U / mL.

[0182] In the present application, the interleukin may include IL21, and the concentration of the IL21 may be 0.01 - 1000 ng / mL. For example, the concentration of the IL21 may be 0.01 - 800 ng / mL, 0.01 - 600 ng / mL, 0.01 - 400 ng / mL, 0.01 - 200 ng / mL, 0.01 - 100 ng / mL, 0.1 - 100 ng / mL, 0.1 - 90 ng / mL, 0.1 - 80 ng / mL, 0.1 - 70 ng / mL, 0.1 - 60 ng / mL, 0.1 - 50 ng / mL, 0.1 - 40 ng / mL, 0.1 - 30 ng / mL, 0.1 - 20 ng / mL, 0.1 - 10 ng / mL or 0.1 - 5 ng / mL.

[0183] In the present application, the interleukin may include IL7, and the concentration of the IL7 may be 0.01 - 1000 ng / mL. For example, the concentration of the IL7 may be 0.01 - 800 ng / mL, 0.01 - 600 ng / mL, 0.01 - 400 ng / mL, 0.01 - 200 ng / mL, 0.01 - 100 ng / mL, 0.1 - 100 ng / mL, 0.1 - 90 ng / mL, 0.1 - 80 ng / mL, 0.1 - 70 ng / mL, 0.1 - 60 ng / mL, 0.1 - 50 ng / mL, 0.1 - 40 ng / mL, 0.1 - 30 ng / mL, 0.1 - 20 ng / mL, 0.1 - 10 ng / mL or 0.1 - 5 ng / mL.

[0184] In the present application, the interleukin may include IL15, and the concentration of the IL15 may be 0.01 - 1000 ng / mL. For example, the concentration of the IL15 may be 0.01 - 800 ng / mL, 0.01 - 600 ng / mL, 0.01 - 400 ng / mL, 0.01 - 200 ng / mL, 0.01 - 100 ng / mL, 0.1 - 100 ng / mL, 0.1 - 90 ng / mL, 0.1 - 80 ng / mL, 0.1 - 70 ng / mL, 0.1 - 60 ng / mL, 0.1 - 50 ng / mL, 0.1 - 40 ng / mL, 0.1 - 30 ng / mL, 0.1 - 20 ng / mL, 0.1 - 10 ng / mL, or 0.1 - 5 ng / mL.

[0185] Promote the proliferation of immune cells, promote the generation of memory immune cells, inhibit the differentiation of immune cells, enhance the release of cells Factors and enhance the killing ability against tumors

[0186] On the one hand, the present application provides a method for promoting the proliferation of immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0187] On the one hand, the present application provides a method for promoting the generation of memory immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in immune cells, so as to promote the differentiation of the immune cells into memory immune cells.

[0188] On the one hand, the present application provides a method for inhibiting the differentiation of immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells, so as to inhibit the differentiation of the immune cells into differentiated immune cells.

[0189] On the one hand, the present application provides a method for enhancing the release of cytokines by immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0190] In the present application, the cytokine may include interleukin, interferon, and / or tumor necrosis factor. In the present application, the cytokine may include IL-2, IL4, IL6, IL7, IL10, IL21, TNF-α, and / or IFNγ.

[0191] On the one hand, the present application provides a method for enhancing the tumor-killing ability of immune cells, which comprises the following steps: upregulating the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells.

[0192] On the one hand, the present application provides a method for preventing tumor recurrence in a subject, the method comprising: administering immune cells to a subject susceptible to tumors, wherein the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells is up-regulated.

[0193] On the one hand, the present application provides a method for treating tumors in a subject in need thereof, which comprises the following steps: administering immune cells to the subject, wherein the expression level of low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells is up-regulated.

[0194] In the present application, the tumor may be selected from liver cancer, lung cancer, leukemia, and mesothelioma. In the present application, the method may include in vivo methods and in vitro methods.

[0195] In the present application, the up-regulation of the expression level may be such that the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in the immune cells is significantly up-regulated compared to the expression level of the control immune cells (e.g., up-regulated by 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), wherein the control immune cells are the corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is substantially not up-regulated.

[0196] In the present application, the promotion of the generation of memory immune cells may be such that the number of memory immune cells generated by the immune cells is significantly up-regulated compared to the number of memory immune cells generated by the control immune cells (e.g., up-regulated by 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), wherein the control immune cells are the corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is substantially not up-regulated.

[0197] In the present application, the inhibition of immune cell differentiation may be such that the number of differentiated immune cells generated by the immune cells is significantly down-regulated compared to the number of memory immune cells generated by the control immune cells (e.g., down-regulated by 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), wherein the control immune cells are the corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is substantially not up-regulated.

[0198] In the present application, the enhancement of cytokine release by immune cells can be a significant upregulation (e.g., by 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) in the amount of cytokines released by the immune cells compared to the amount of cytokines released by control immune cells, where the control immune cells are the corresponding immune cells in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is substantially not upregulated.

[0199] In the present application, the enhancement of the tumor-killing ability of immune cells can be a significant increase (e.g., by 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) in the tumor-killing ability of the immune cells compared to the tumor-killing ability of control immune cells, where the control immune cells are the corresponding immune cells in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is substantially not upregulated.

[0200] In the present application, the prevention of tumor recurrence in a subject can be a significant increase (e.g., by 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) in the ability of the immune cells to prevent tumor recurrence in the subject compared to the ability of control immune cells to prevent tumor recurrence in the subject, where the control immune cells are the corresponding immune cells in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is substantially not upregulated.

[0201] In the present application, the treatment of tumors in a subject in need thereof can be a significant increase (e.g., by 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) in the therapeutic effect of the immune cells on tumors compared to the therapeutic effect of control immune cells on tumors, where the control immune cells are the corresponding immune cells in which the expression level of low-density lipoprotein receptor-related protein or a fragment thereof is substantially not upregulated.

[0202] For example, the corresponding immune cells can include lymphocytes. In the present application, the corresponding immune cells can include T cells. In the present application, the T cells can include memory stem cell-like T cells (TSCM) and / or central memory T cells (TCM). In the present application, the TSCM can be CCR7+ and / or CD62L + In the present application, the TSCM may also have one or more properties selected from the group consisting of: CD45RA + or CD45RA - 、CD45RO + or CD45RO - 、CD27 + 、CD28 + 、CD127 + 、CD122 + 、CD3 + 、CD4 + and CD8 + In the present application, the corresponding immune cells may include genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR). In the present application, the corresponding immune cells may include genetically modified T cells.

[0203] In the present application, the content of the low-density lipoprotein receptor-related protein or its fragment in the corresponding immune cells may be only the expression level of the low-density lipoprotein receptor-related protein or its fragment in a normal organism, or an expression level that is not substantially upregulated compared to the content of the low-density lipoprotein receptor-related protein or its fragment in a normal organism. For example, the "not substantially upregulated" may mean upregulated by 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. In the present application, the corresponding immune cells may not contain a vector comprising a nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment, and / or, the corresponding immune cells may not comprise a nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment.

[0204] In the present application, the methods for detecting the expression level of the low-density lipoprotein receptor-related protein or its fragment, and / or the cytokine may include quantitative PCR, western blot, and immunohistochemistry.

[0205] In the present application, the methods for detecting the memory immune cells and / or the differentiated immune cells may include flow cytometry, immunofluorescence, magnetic bead separation, ELISA, ELISPOT, and quantitative PCR.

[0206] In the present application, the therapeutic effect of a tumor can be evaluated using an index selected from the group consisting of: tumor volume, overall survival (OS), duration of overall response (DOR), duration of stable disease, disease-free survival time (DFS), progression-free survival time (PFS), disease control rate (DCR), objective response rate (ORR), and / or clinical benefit response ratio.

[0207] In the present application, the effect of preventing tumor recurrence in a subject can be evaluated using an index selected from the group consisting of: the expression level of a tumor marker (e.g., tumor-associated antigen, tumor-specific gene, and tumor suppressor gene), histological test results (e.g., epithelial hyperplasia, degree of polyp disappearance), imaging test results (e.g., mammography test results), and / or the expression level of a serum marker (e.g., serum-free DNA, methylated DNA).

[0208] Composition and its uses

[0209] The present application provides a composition comprising the genetically modified immune cells described above.

[0210] In the present application, the composition may further comprise an optionally pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes, and / or nonionic surfactants, etc.

[0211] In the present application, the composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot.

[0212] The composition can be used to inhibit tumor growth. For example, the composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (even substantially eliminate the tumor), and / or can alleviate and / or stabilize the disease state.

[0213] The composition described in the present application may comprise a therapeutically effective amount of the antibody or its antigen-binding fragment. The therapeutically effective amount is the dose required to prevent and / or treat (at least partially treat) a disorder or disorder (e.g., cancer) and / or any of its complications in a subject suffering from or at risk of developing the disorder.

[0214] The present application provides the use of the genetically modified cell and / or the composition described above in the preparation of a drug, wherein the drug is used for treating and / or preventing tumors.

[0215] In the present application, the tumor may be selected from liver cancer, lung cancer, leukemia, and mesothelioma.

[0216] Without being limited by any theory, the embodiments described below are only for explaining the working modes of the devices, methods and systems of the present application, and are not used to limit the scope of the invention of the present application.

[0217] Embodiment

[0218] Embodiment 1 Construction of lentiviral vectors

[0219] Taking CAR-T targeting GPC3, CD19, Mesothelin (MSLN), HER2, BCMA as examples, fragments containing the CAR structure were artificially synthesized and constructed into lentiviral vectors (LV100A, System Biosciences), and then lentiviruses were transfected in accordance with the methods described in their instructions (as Figure 1 shown), and lentiviruses of 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 were obtained respectively.

[0220] GPC3-41BB was constructed by splicing the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.15 and SEQ ID NO.17 respectively.

[0221] GPC3-41BB-L6 was constructed by splicing the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.21 respectively.

[0222] GPC3-41BB-TL6 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.

[0223] GPC3-41BB-L5 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L5 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.25 respectively.

[0224] GPC3-41BB-TL5 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL5 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.

[0225] GPC3-CD28 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.17 respectively.

[0226] GPC3-CD28-L6 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are 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.

[0227] GPC3-CD28-TL6 is constructed by splicing a leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are 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.

[0228] BCMA-41BB is constructed by splicing a leader sequence, BCMAScFv, CD8 hinge region and transmembrane region, 41BB, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17 respectively.

[0229] BCMA-41BB-L6 is constructed by splicing a leader sequence, BCMA ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.5, SEQID NO.11, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.21 respectively.

[0230] BCMA-41BB-TL6 is constructed by splicing a leader sequence, BCMA ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.

[0231] CD19-41BB is constructed by splicing a leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.3, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17 respectively.

[0232] CD19-41BB-L6 is constructed by splicing a leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are 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.

[0233] CD19-41BB-TL6 is constructed by splicing a leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are 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.

[0234] MSLN-41BB is constructed by splicing a leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17 respectively.

[0235] MSLN-41BB-L6 is constructed by splicing a leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.21 respectively.

[0236] MSLN-41BB-TL6 is constructed by splicing a leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.

[0237] HER2-41BB is constructed by splicing a leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, and CD3zeta in sequence from the 5'-end to the 3'-end. Their nucleotide sequences are shown in SEQ ID NO.30, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.17 respectively.

[0238] HER2-41BB-L6 is constructed by splicing a leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.21 respectively.

[0239] HER2-41BB-TL6 is constructed by splicing a leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and TL6 in sequence from the 5'-end to the 3'-end. Their nucleotide sequences 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.

[0240] Example 2 Lentivirus Infection of T Cells

[0241] Fresh human peripheral blood was separated by ficoll separation solution to obtain peripheral blood mononuclear lymphocytes (PBMCs) greater than 1×10 7 . Anti-human CD3 and anti-human CD28 antibodies were diluted with PBS to a final concentration of 1 μg / ml. Then, the diluted antibody mixture was added to the cell culture dish and evenly spread in the cell culture dish, and incubated at room temperature for 2 hours. After 2 hours, the antibody mixture was washed once with PBS. Subsequently, the separated PBMCs were resuspended with lymphocyte culture medium containing Xvivo15 medium, 5% FBS, 200 U / ml IL2T or Xvivo15 medium, 5% FBS, 20 ng / ml IL21, 10 ng / ml IL7 to a final concentration of 1×10 6 cells / ml, and added to the culture dish containing the antibody mixture and cultured at 37°C, 5% CO 2 for 24 hours to activate T cells.

[0242] Take a certain amount of T cell culture medium, add synperonic F108 with a final concentration of 1 mg / ml and mix well. Heat it to 37°C in a water bath to prepare the uninfected reagent. Subsequently, prepare the cell culture dishes required for the experiment. First, dilute 1 mg / ml anti-human CD3 antibody and 0.5 mg / ml anti-human CD28 antibody with PBS buffer at a volume ratio of 1:1000 and mix well. Then, dilute with retronectin (1 mg / ml) reagent at a volume ratio of 1:40 and mix well. Then spread it evenly on the cell culture dishes and incubate at room temperature for 2 hours. After 2 hours, wash with PBS, and the cell culture dishes are ready.

[0243] Dilute the activated T cells with the prepared infection reagent, and add each lentivirus prepared in Example 1 at a ratio of MOI = 3 and mix well. Subsequently, spread it evenly in the cell culture dishes for lentivirus infection, so as to obtain 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 respectively. Monitor the cell density after infection to maintain the density of these infected cells at 1×10 6 cells / ml. After 14 days, the cell number can be amplified 10 - 100 times.

[0244] Example 3 LRP6, truncated LRP6, LRP5 or truncated LRP5 promotes the formation of memory T cells

[0245] At 37°C, 5% CO 2The 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 Example 2 of in vitro culture were co-cultured for 9 days or 13 days in a cell incubator.

[0246] The expression levels of CD3, CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, CD28 proteins in T cells were detected by BD flow cytometry. Among them, the measured protein expression results are as Figure 2 shown.

[0247] The results showed that in T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6, the proportions of memory stem cell-like T cells (TSCM) and central memory T cells (TCM) in the total cells were significantly higher than those in the control group; in T cells expressing GPC3-CD28-L6 or GPC3-CD28-TL6, the percentages of TSCM and TCM in the total cells were significantly higher than those in the control group; in T cells expressing GPC3-41BB-L5 or GPC3-41BB-TL5, the percentages of TSCM and TCM in the total cells were significantly higher than those in the control group; in T cells expressing BCMA-41BB-L6 or BCMA-41BB-TL6, the percentages of TSCM and TCM in the total cells were significantly higher than those in the control group; in T cells expressing CD19-41BB-L6 or CD19-41BB-TL6, the percentages of TSCM and TCM in the total cells were significantly higher than those in the control group; in T cells expressing MSLN-41BB-L6 or MSLN-41BB-TL6, the percentages of TSCM and TCM in the total cells were significantly higher than those in the control group; in T cells expressing HER2-41BB-L6 or HER2-41BB-TL6, the percentages of TSCM and TCM in the total cells were also significantly higher than those in the control group.

[0248] Example 4 Inhibition of Treg cell differentiation by LRP6 or its truncated form

[0249] At 37°C, 5% CO 2In vitro culture the T cells expressing GPC3-41BB and GPC3-41BB-L6 obtained in Example 2 in a cell incubator for 9 days or 12 days. Use BD flow cytometry to detect the expression of CD3, CD4, CD25, CD127, and FoxP3 proteins in the T cells, and the measured protein expression results are as Figure 3 shown. The results show that in the T cells expressing GPC3-41BB-L6, the percentage of regulatory T cells (Tregs) in CD4+ T cells is significantly lower than that in the control group of T cells expressing GPC3-41BB.

[0250] Example 5 LRP6, truncated LRP6, LRP5 or truncated LRP5 promotes the expansion of specific CAR-T cells induced by tumor antigens

[0251] Co-culture the T cells expressing GPC3-41BB, GPC3-41BB-L6 or GPC3-41BB-TL6 obtained in Example 2 with irradiated Huh7 or HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) at a cell number ratio of 1:1 in Xvivo15 medium, and replenish irradiated Huh7 or HepG2 every 5 days for stimulation. Stimulate 3 times, and perform counting by trypan blue staining each time; co-culture the T cells expressing GPC3-41BB, GPC3-41BB-L5 or GPC3-41BB-TL5 with irradiated Huh7 or HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) at a cell number ratio of 1:1 in Xvivo15 medium, and replenish irradiated Huh7 or HepG2 every 5 days for stimulation. Stimulate 3 times, and perform counting by trypan blue staining each time; co-culture the T cells expressing HER2-41BB, HER2-41BB-L6 or HER2-41BB-TL6 with irradiated SKOV3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) at a cell number ratio of 1:1 in Xvivo15 medium, and replenish irradiated SKOV3 every 5 days for stimulation. Stimulate 3 times, and perform counting by trypan blue staining each time; the cell proliferation of each group is as Figure 4 shown. The results show that the amplification multiples of the T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6 are much higher than those of the control group (T cells expressing GPC3-41BB), the amplification multiples of the T cells expressing GPC3-41BB-L5 or GPC3-41BB-TL5 are much higher than those of the control group, and the amplification multiples of the T cells expressing HER2-41BB-L6 or HER2-41BB-TL6 are also much higher than those of the control group (T cells expressing HER2-41BB).

[0252] Example 6 LRP6 or its truncated form promotes the in vivo proliferation of CAR-T cells and the release of cytokines

[0253] Subcutaneously inoculate Huh7 cells (1×10 7 cells / mouse) into NSG mice (purchased from Beijing Biocytogen Co., Ltd.). After 14 days, the tumor volume of the mice was measured to be approximately 100 mm 3 . At this time, the mice were divided into 4 groups, namely the T cell group, the GPC3-41BB group, the GPC3-41BB-L6 group, and the GPC3-41BB-TL6 group, with 6 mice in each group. Then, cells were injected into them via the tail vein (i.e., the unmodified T cells, the T cells expressing GPC3-41BB, the T cells expressing GPC3-41BB-L6, and the T cells expressing GPC3-41BB-TL6 were injected into each group respectively, with a dose of 3×10 6 cells / mouse). On the 8th day, 50 μl of blood was taken from the tails of the mice. The expression of human CD8 and CD4 proteins in each group was detected by BD flow cytometry. The results are shown in Figure 5 A. Figure 5 The results in A show that the proportion of cells expressing CD8 and CD4 proteins in the GPC3-41BB-L6 or GPC3-41BB-TL6 group was much higher than that in other control groups.

[0254] Subcutaneously inoculate Huh7 cells (1×10 7 cells / mouse) into NSG mice (purchased from Beijing Biocytogen Co., Ltd.). After 14 days, the tumor volume of the mice was measured to be approximately 100 mm 3 . At this time, the mice were divided into 5 groups, namely the T cell group, the GPC3-41BB group, and the GPC3-41BB-L6 group, with 6 mice in each group. Then, T cells were injected into the T cell group via the tail vein (i.e., unmodified T cells were injected, with a dose of 2×10 6 cells / mouse), the T cells expressing GPC3-41BB were injected into the GPC3-41BB group (with a dose of 8×10 5 cells / mouse), and the T cells expressing GPC3-41BB-L6 were injected into the GPC3-41BB-L6 group (with doses of 3×10 5 cells / mouse, 8×10 5 cells / mouse, or 2×10 6 cells / mouse). On the 8th day, 50 μl of blood was taken from the tails of the mice. The expression of human IL-2, IL4, IL6, IL10, TNF-α, and IFNγ cytokines in each group was detected by BD flow cytometry. The results are shown in Figure 5 B. Figure 5 The results in B show that the expression of IFNγ cytokine in the GPC3-41BB-L6 group was significantly higher than that in other control groups.

[0255] Example 7 Enhancement of the anti-tumor effect of CAR-T cells by LRP6 or its truncated form

[0256] NSG mice (purchased from Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with Huh7 cells (at a dose of 1×10 7 cells / mouse). After 14 days, the tumor volume of the mice was measured to be approximately 100 mm 3 . At this time, the mice were divided into 4 groups, namely the T cell group, the GPC3-41BB group, and the GPC3-41BB-L6 group, with 8 mice in each group. Then, T cells were injected into the T cell group via the tail vein (i.e., each injection of unmodified T cells, at a dose of 8×10 5 cells / mouse), GPC3-41BB-expressing T cells were injected into the GPC3-41BB group (at a dose of 8×10 5 cells / mouse), and GPC3-41BB-L6-expressing T cells were injected into the GPC3-41BB-L6 group (at a dose of 3×10 5 cells / mouse or 8×10 5 cells / mouse).

[0257] The tumor volume was measured on Mondays and Thursdays every week, and the death of the mice was recorded. The results are as Figure 6 shown. The results showed that both the tumor suppression effect and the survival rate of the mice in the GPC3-41BB-L6 group were significantly higher than those of the other control groups, approximately 3 times that of the control group GPC3-41BB group.

[0258] Example 8 LRP6 or its truncated form promotes the long-term survival of CAR-T cells in vivo and prevents tumor recurrence

[0259] NSG mice (purchased from Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with Huh7 cells (at a dose of 1×10 7 cells / mouse). After 14 days, the tumor volume of the mice was measured to be approximately 100 mm 3 . At this time, the mice were divided into 3 groups, namely the T cell group, the GPC3-41BB group, and the GPC3-41BB-L6 group, with 9 mice in each group. Then, T cells were injected into the T cell group via the tail vein (at a dose of 2×10 6 cells / mouse), GPC3-41BB-expressing T cells were injected into the GPC3-41BB group (at a dose of 2×10 6 cells / mouse), and GPC3-41BB-L6-expressing T cells were injected into the GPC3-41BB-L6 group (at a dose of 2×10 6 cells / mouse).

[0260] 85 days after the tumors completely disappeared, Huh7 cells (at a dose of 3×10 7On the 99th day, 50 μl of blood was collected from the tails of the mice, and the expression of human CD8, CD4, and CD3 proteins in each group was detected by BD flow cytometry. On the 120th day, the bone marrow of the mice was collected, and the expression of human CD8, CD4, and CD3 proteins in each group was detected by BD flow cytometry.

[0261] The results are as Figure 7 shown. The results showed that the proportions of cells expressing CD8 or CD4 proteins in the peripheral blood and the proportion of cells expressing CD3 protein in the bone marrow in the GPC3-41BB-L6 group were much higher than those in the GPC3-41BB group. Moreover, even when the mice in the GPC3-41BB-L6 group were re-inoculated with tumor cells, tumors could not form. This indicates that LRP6 or its truncated form promotes the long-term survival of CAR-T cells in vivo and has the characteristic of preventing tumor recurrence.

[0262] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed herein are obvious to those of ordinary skill in the art and are within the scope of the appended claims and their equivalents.

Claims

1. A genetically modified immune cell, wherein the genetic modification upregulates the expression level of low-density lipoprotein receptor-related protein or a functional fragment thereof in the immune cell, and the low-density lipoprotein receptor-related protein or a functional fragment thereof is selected from: low-density lipoprotein receptor-related protein 5 or a functional fragment thereof, or low-density lipoprotein receptor-related protein 6 or a functional fragment thereof, and the functional fragment contains the intracellular domain of the low-density lipoprotein receptor-related protein. The immune cell is a T cell, and the immune cell further expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

2. The immune cell according to claim 1, wherein the amino acid sequence of the low-density lipoprotein receptor-related protein 6 or a functional fragment thereof is shown in SEQ ID NO: 24, and the amino acid sequence of the low-density lipoprotein receptor-related protein 5 or a functional fragment thereof is shown in SEQ ID NO:

28.

3. The immune cell according to claim 1, wherein the amino acid sequence of the low-density lipoprotein receptor-related protein 6 or a functional fragment thereof is as shown in SEQ ID NO: 22 or SEQ ID NO: 24, and the amino acid sequence of the low-density lipoprotein receptor-related protein 5 or a functional fragment thereof is as shown in SEQ ID NO: 26 or 28.

4. The immune cell according to claim 1, wherein the CAR contains an intracellular domain, and the intracellular domain contains a signal transduction domain and / or a co-stimulatory domain.

5. The immune cell according to claim 4, wherein the signal transduction domain contains a part selected from the group consisting of: the signal transduction domain of CD3ζ, the signal transduction domain of CD3δ, and the signal transduction domain of CD3ε.

6. The immune cell according to claim 5, wherein the amino acid sequence of the signal transduction domain is as shown in SEQ ID NO:

18.

7. The immune cell according to claim 5, wherein the nucleic acid sequence of the nucleic acid molecule encoding the signal transduction domain is as shown in SEQ ID NO:

17.

8. The immune cell according to claim 4, wherein the co-stimulatory domain is selected from the group consisting of: the co-stimulatory domain of CD27, the co-stimulatory domain of CD28, and the co-stimulatory domain of 4-1BB.

9. The immune cell according to claim 8, wherein the amino acid sequence of the co-stimulatory domain is as shown in SEQ ID NO: 14 or SEQ ID NO:

16.

10. The immune cell according to claim 8, wherein the nucleic acid sequence of the nucleic acid molecule encoding the co-stimulatory domain is as shown in SEQ ID NO: 13 or SEQ ID NO:

15.

11. The immune cell according to claim 4, wherein the CAR contains a hinge region.

12. The immune cell according to claim 11, wherein the hinge region is selected from the group consisting of: the hinge region of IgG4, the hinge region of IgG1, and the hinge region of CD8.

13. The immune cell according to claim 11, wherein the amino acid sequence of the hinge region is as shown in SEQ ID NO:

31.

14. The immune cell according to claim 11, wherein the nucleic acid sequence of the nucleic acid molecule encoding the hinge region is as shown in SEQ ID NO:

32.

15. The immune cell according to claim 4, wherein the CAR comprises a transmembrane region.

16. The immune cell according to claim 15, wherein the transmembrane region is selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD28, and / or the transmembrane region of CD24.

17. The immune cell according to claim 15, wherein the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

33.

18. The immune cell according to claim 15, wherein the nucleic acid sequence of the nucleic acid molecule encoding the transmembrane region is as shown in SEQ ID NO:

34.

19. The immune cell according to claim 4, wherein the CAR comprises a targeting moiety.

20. The immune cell according to claim 19, wherein the targeting moiety comprises a ScFv.

21. The immune cell according to claim 19, wherein the targeting moiety specifically binds and / or recognizes a tumor antigen.

22. The immune cell according to claim 19, wherein the targeting moiety specifically binds and / or recognizes a target selected from the group consisting of B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members.

23. The immune cell according to claim 19, wherein the targeting moiety specifically binds and / or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.

24. The immune cell according to claim 19, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds and / or recognizes CD19, the antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1-LCDR3, the heavy chain variable region comprising HCDR1-HCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 46; the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 47; the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 48; the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 43; the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 44; and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

45.

25. The immune cell according to claim 24, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 50, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

49.

26. The immune cell according to claim 19, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds and / or recognizes BCMA, the antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-LCDR3, the heavy chain variable region comprises HCDR1-HCDR3, wherein the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:54; the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:55; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:56; the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:51; the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:52; and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

53.

27. The immune cell according to claim 26, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:58, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

57.

28. The immune cell according to claim 19, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds and / or recognizes HER2, the antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-LCDR3, the heavy chain variable region comprises HCDR1-HCDR3, wherein the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:70; the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:71; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:72; the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:67; the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:68; and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

69.

29. The immune cell according to claim 28, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:74, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

73.

30. The immune cell according to claim 19, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes Mesothelin, the antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-LCDR3, the heavy chain variable region comprises HCDR1-HCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:62; the amino acid sequence of LCDR2 is as shown in SEQ ID NO:63; the amino acid sequence of LCDR3 is as shown in SEQ ID NO:64; the amino acid sequence of HCDR1 is as shown in SEQ ID NO:59; the amino acid sequence of HCDR2 is as shown in SEQ ID NO:60; and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

61.

31. The immune cell according to claim 30, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:66, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

65.

32. The immune cell according to claim 19, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, the antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-LCDR3, the heavy chain variable region comprises HCDR1-HCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:38; the amino acid sequence of LCDR2 is as shown in SEQ ID NO:39; the amino acid sequence of LCDR3 is as shown in SEQ ID NO:40; the amino acid sequence of HCDR1 is as shown in SEQ ID NO:35; the amino acid sequence of HCDR2 is as shown in SEQ ID NO:36; and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

37.

33. The immune cell according to claim 32, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:42, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

41.

34. The immune cell according to claim 19, wherein the amino acid sequence of the targeting moiety is as shown in SEQ ID NO:2, 4, 6, 8 or 10.

35. The immune cell according to claim 19, wherein the nucleic acid sequence of the nucleic acid molecule encoding the targeting moiety is as shown in SEQ ID NO:1, 3, 5, 7 or 9.

36. A composition comprising the genetically modified immune cell according to any one of claims 1-35.

37. The composition according to claim 36, which further comprises an optionally pharmaceutically acceptable carrier. Use of the genetically modified immune cell according to any one of claims 1-35 and / or the composition according to any one of claims 36-37 in the preparation of a medicament, wherein the medicament is for treating a tumor selected from liver cancer, lung cancer, leukemia and mesothelioma.

40. A method for preparing the genetically modified immune cell according to any one of claims 1-35, the method comprising the step of upregulating the expression level of low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cell.

41. The method according to claim 40, which comprises the following steps: introducing into the genetically modified immune cell a vector that upregulates the expression level of the low density lipoprotein receptor-related protein or a fragment thereof.

42. The method according to claim 40, wherein the vector is selected from the following group: retroviral vector, lentiviral vector and transposon plasmid.

43. The method according to any one of claims 40-42, wherein the low density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence shown in any one of the following: SEQ ID NO: 22, 24, 26 and 28.

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