WO2019184886A1 - 促进免疫细胞增殖的方法 - Google Patents

促进免疫细胞增殖的方法 Download PDF

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WO2019184886A1
WO2019184886A1 PCT/CN2019/079576 CN2019079576W WO2019184886A1 WO 2019184886 A1 WO2019184886 A1 WO 2019184886A1 CN 2019079576 W CN2019079576 W CN 2019079576W WO 2019184886 A1 WO2019184886 A1 WO 2019184886A1
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acid sequence
amino acid
seq
homology
immune cell
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English (en)
French (fr)
Inventor
凌有国
郭昊
马海立
李慧姣
王华菁
应碧
杨焕凤
何晓文
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Shanghai OriginCell Medical Technology Co Ltd
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Shanghai OriginCell Medical Technology Co Ltd
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Priority to CN201980022452.0A priority Critical patent/CN111918964B/zh
Priority to US17/040,865 priority patent/US12005080B2/en
Priority to JP2020550874A priority patent/JP2021518149A/ja
Priority to EP19776983.9A priority patent/EP3778876A4/en
Publication of WO2019184886A1 publication Critical patent/WO2019184886A1/zh
Anticipated expiration legal-status Critical
Priority to US18/652,232 priority patent/US20240400989A1/en
Priority to JP2024193020A priority patent/JP2025023986A/ja
Ceased legal-status Critical Current

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    • C12N5/0634—Cells from the blood or the immune system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/53—Liver
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07K2319/00—Fusion polypeptide
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present application relates to a method of promoting proliferation of immune cells.
  • the method of the present application can up-regulate the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • Chimeric Antigen Receptor T-Cell Immunotherapy is a method of genetically modifying T cells to express Chimeric antigen receptor (CAR) and recognize tumors.
  • CAR generally comprises a single chain variable fragment (scFv), a hinge region, a transmembrane region, and an intracellular signaling region that specifically recognize a Tumor associated antigen (TAA).
  • scFv single chain variable fragment
  • TAA Tumor associated antigen
  • CAR-T immunotherapy has been shown to be effective in the treatment of acute leukemia and non-Hodgkin's lymphoma, but the proliferative capacity of CAR-expressing immune cells is extremely limited.
  • the present application provides a genetically modified immune cell, and a method of promoting proliferation of immune cells.
  • the method provided herein can upregulate the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • the methods provided herein can also promote memory immune cell production.
  • the methods provided herein can also inhibit immune cell differentiation.
  • the methods provided herein can also enhance the release of cytokines by immune cells.
  • the method provided by the present application can also enhance the ability of immune cells to kill tumors.
  • the methods provided herein can be used to prevent tumor recurrence in a subject.
  • the present application also provides a method of treating a tumor in a subject in need thereof.
  • the application also provides compositions comprising the genetically modified immune cells, and the genetically modified immune cells and the use of the compositions in the preparation of a medicament.
  • the present application also provides methods of making the genetically modified immune cells.
  • the application provides a method of promoting proliferation of immune cells comprising the step of upregulating expression of a low density lipoprotein receptor associated protein or fragment thereof in said immune cell.
  • the application provides a method for promoting the production of memory immune cells, comprising the steps of: upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in an immune cell, thereby promoting differentiation of the immune cell For memory immune cells.
  • the application provides a method for inhibiting differentiation of immune cells, comprising the steps of: upregulating expression of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell, thereby inhibiting differentiation of the immune cell For differentiated immune cells.
  • the present application provides a method of enhancing release of a cytokine by an immune cell, comprising the step of upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • the cytokine comprises an interleukin, an interferon, and/or a tumor necrosis factor. In certain embodiments, the cytokine comprises IL-2, IL4, IL6, IL7, IL10, IL12, TNF-[alpha], and/or IFN[gamma].
  • the application provides a method of enhancing the ability of an immune cell to kill a tumor, comprising the step of upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • the application provides a method of preventing tumor recurrence in a subject, the method comprising: administering an immune cell to a subject susceptible to the tumor, wherein the low density lipoprotein receptor-related protein in the immune cell The expression level of the fragment or its fragment is up-regulated.
  • the application provides a method of treating a tumor in a subject in need thereof, comprising the steps of administering to the subject an immune cell, wherein the immune cell is associated with a low density lipoprotein receptor The expression level of the protein or its fragment is up-regulated.
  • the tumor is selected from the group consisting of liver cancer, lung cancer, leukemia, and mesothelioma.
  • the methods include in vivo methods and in vitro methods.
  • the immune cell comprises a lymphocyte. In certain embodiments, the immune cell comprises a T cell. In certain embodiments, the T cell comprises a memory stem cell-like T cell (TSCM) and/or a central memory T cell (TCM). In certain embodiments, the TSCM is CCR7 + and/or CD62L + . In certain embodiments, the TSCM further selected from the group having one or more properties: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD3 + , CD4 + and CD8 + .
  • TSCM memory stem cell-like T cell
  • TCM central memory T cell
  • the TSCM is CCR7 + and/or CD62L + . In certain embodiments, the TSCM further selected from the group having one or more properties: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +
  • the immune cell comprises a genetically modified immune cell and the genetically modified immune cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain embodiments, the genetically modified immune cell comprises a genetically modified T cell.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the CAR comprises an intracellular domain comprising a signaling domain and/or a costimulatory domain.
  • the signaling domain comprises a portion selected from the group consisting of: a signaling domain of CD3 ⁇ , a signaling domain of CD3 ⁇ , and a signaling domain of CD3 ⁇ .
  • the signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the signaling domain comprises the nucleic acid sequence set forth in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.
  • the costimulatory domain comprises a portion selected from the group consisting of a costimulatory domain of CD27, a costimulatory domain of CD28, and a costimulatory domain of 4-1BB.
  • the costimulatory domain comprises an amino acid sequence set forth 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.
  • the nucleic acid molecule encoding the costimulatory domain comprises a nucleic acid sequence set forth 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.
  • the CAR comprises a hinge region.
  • the hinge region comprises a portion selected from the group consisting of a hinge region of IgG4, a hinge region of IgGl, and a hinge region of CD8.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO:31.
  • the nucleic acid molecule encoding the hinge region comprises the nucleotide sequence set forth in SEQ ID NO:32.
  • the CAR comprises a transmembrane region.
  • the transmembrane region comprises a portion selected from the group consisting of a transmembrane region of CD8, a transmembrane region of CD28, and a transmembrane region of CD24.
  • the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:33.
  • the nucleic acid molecule encoding the transmembrane region comprises the nucleotide sequence set forth in SEQ ID NO:34.
  • the CAR comprises a targeting moiety.
  • the targeting moiety comprises a ScFv.
  • the targeting moiety specifically binds to and/or recognizes a tumor antigen. In certain embodiments, the targeting moiety specifically binds to and/or recognizes a target selected from the group consisting of a B lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member. In certain embodiments, the targeting moiety specifically binds to and/or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes CD 19, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain
  • the variable region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47 or An amino acid sequence having at least 80% homology thereto; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:43 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 45 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 49 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes BCMA, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 51 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 53 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes HER2, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:67 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 69 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes Mesothelin, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 62 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:59 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 61 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes GPC3, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 35 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 37 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, and 10 or an amino acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the targeting moiety comprises a nucleic acid sequence set forth in any one of: SEQ ID NOs: 1, 3, 5, 7, and 9 or at least 80% homologous thereto Amino acid sequence.
  • the method further comprises the step of isolating peripheral blood mononuclear cells PBMC, CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes, or regulatory T cells.
  • the method further comprises adding one or more T cell stimulating factors to the isolated PBMC.
  • the T cell stimulating factor is selected from the group consisting of a B lymphocyte surface antigen antibody, a TNF antibody, an intracellular polyester, and an antibiotic.
  • the T cell stimulating factor is selected from the group consisting of a CD3 antibody, a CD28 antibody, a 4-1BB antibody, a CD80 antibody, a CD86 antibody, PHA, PMA, and ionomycin.
  • the T cell stimulating factor comprises a CD3 antibody, and the concentration of the CD3 antibody is 1-1000 ng/mL. In certain embodiments, the T cell stimulating factor comprises a CD28 antibody, and the concentration of the CD28 antibody is 1-1000 ng/mL.
  • the method further comprises adding one or more cytokine agents to the isolated PBMC.
  • the cytokine agent comprises an interleukin.
  • the interleukin comprises one or more selected from the group consisting of IL2, IL21, IL7, and IL15.
  • the interleukin comprises IL2 and the concentration of the IL2 is between 0.1 and 10000 U/mL.
  • the interleukin comprises IL21 and the concentration of the IL21 is from 0.01 to 1000 ng/mL.
  • the interleukin comprises IL7 and the concentration of the IL7 is from 0.01 to 1000 ng/mL.
  • the interleukin comprises IL15 and the concentration of the IL15 is from 0.01 to 1000 ng/mL.
  • the low density lipoprotein receptor associated protein comprises one or more selected from the group consisting of low density lipoprotein receptor associated proteins 1-12 and functional fragments thereof. In certain embodiments, the low density lipoprotein receptor associated protein or fragment thereof is derived from a human.
  • the functional fragment comprises a fragment or truncation of the low density lipoprotein receptor associated protein having the low density lipoprotein receptor associated protein activity.
  • the low density lipoprotein receptor associated protein comprises low density lipoprotein receptor associated protein 6 and truncation thereof, and/or low density lipoprotein receptor associated protein 5 and truncation thereof .
  • the truncation of the low density lipoprotein receptor associated protein 6 comprises an intracellular region of the low density lipoprotein receptor associated protein 6; and/or the low density lipoprotein is The truncated body of the body-associated protein 5 comprises the intracellular region of the low density lipoprotein receptor associated protein 5.
  • the truncated form of low density lipoprotein receptor associated protein 6 comprises a transmembrane region of said low density lipoprotein receptor associated protein 6 and said low density lipoprotein receptor associated protein 6 LDLR region; and/or the truncated body of low density lipoprotein receptor-associated protein 5 comprises a transmembrane region of said low density lipoprotein receptor associated protein 5 and said low density lipoprotein receptor associated protein 5 LDLR area.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in any one of the following: SEQ ID NOs: 22, 24, 26, and 28 or at least 80% identical thereto A source amino acid sequence.
  • the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or fragment thereof comprises the nucleic acid sequence set forth in any one of the following: SEQ ID NO: 21, 23, 25, and 27 or An amino acid sequence of at least 80% homology.
  • the application provides a genetically modified immune cell, wherein the genetic modification is such that the expression level of a low density lipoprotein receptor-related protein or fragment thereof in the immune cell is up-regulated.
  • the genetically modified immune cell comprises a lymphocyte. In certain embodiments, the genetically modified immune cell comprises a genetically modified T cell. 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 selected from the group having one or more properties: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD3 + , CD4 + and CD8 + .
  • CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 +, CD127 +, CD122 +, CD3 + , CD4 + and CD8 + CD45RA + or CD45RA -, CD45RO +, CD27 +, CD28 + , CD127 +, CD122 +
  • the immune cell comprises a genetically modified immune cell and the genetically modified immune cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the CAR comprises an intracellular domain comprising a signaling domain and/or a costimulatory domain.
  • the signaling domain comprises a portion selected from the group consisting of: a signaling domain of CD3 ⁇ , a signaling domain of CD3 ⁇ , and a signaling domain of CD3 ⁇ .
  • the signaling domain comprises the amino acid sequence set forth in SEQ ID NO:18.
  • the nucleic acid molecule encoding the signaling domain comprises the nucleic acid sequence set forth in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.
  • the costimulatory domain comprises a portion selected from the group consisting of a costimulatory domain of CD27, a costimulatory domain of CD28, and a costimulatory domain of 4-1BB.
  • the costimulatory domain comprises the amino acid sequence set forth in any one of SEQ ID NO: 14 and SEQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the costimulatory domain comprises a nucleic acid sequence set forth in any one of: SEQ ID NO: 13 and SEQ ID NO: 15 or at least 80% homologous thereto Nucleic acid sequence.
  • the CAR comprises a hinge region.
  • the hinge region comprises a portion selected from the group consisting of a hinge region of IgG4, a hinge region of IgGl, and a hinge region of CD8.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the hinge region comprises the nucleic acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 80% homology thereto.
  • the CAR comprises a transmembrane region.
  • the transmembrane region comprises a portion selected from the group consisting of a transmembrane region of CD8, a transmembrane region of CD28, and a transmembrane region of CD24.
  • the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the transmembrane region comprises the nucleic acid sequence set forth in SEQ ID NO: 34 or a nucleic acid sequence having at least 80% homology thereto.
  • the CAR comprises a targeting moiety.
  • the targeting moiety comprises a ScFv.
  • the targeting moiety specifically binds to and/or recognizes a tumor antigen.
  • the targeting moiety specifically binds to and/or recognizes a target selected from the group consisting of a B lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member.
  • the targeting moiety specifically binds to and/or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes CD 19, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain
  • the variable region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47 or An amino acid sequence having at least 80% homology thereto; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:43 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 45 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 49 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes BCMA, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 51 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 53 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes HER2, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:67 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 69 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes Mesothelin, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 63 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:59 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 61 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes GPC3, the antibody or antigen-binding fragment thereof comprising a light chain variable region, the light chain variable The region comprises LCDR1-LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39 or An amino acid sequence having at least 80% homology; the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 35 or An amino acid sequence having at least 80% homology thereto; the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; the HCDR3 comprising SEQ ID NO: 37 Amino acid sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety comprises an amino acid sequence set forth in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NOs: 2, 4, 6, 8, and 10.
  • the nucleic acid molecule encoding the targeting moiety comprises a nucleic acid sequence set forth in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the low density lipoprotein receptor associated protein comprises one or more selected from the group consisting of low density lipoprotein receptor associated proteins 1-12 and functional fragments thereof. In certain embodiments, the low density lipoprotein receptor associated protein or fragment thereof is derived from a human.
  • the functional fragment comprises a fragment or truncation of the low density lipoprotein receptor associated protein having the low density lipoprotein receptor associated protein activity.
  • the low density lipoprotein receptor associated protein comprises low density lipoprotein receptor associated protein 6 and truncation thereof, and/or low density lipoprotein receptor associated protein 5 and truncation thereof .
  • the truncation of the low density lipoprotein receptor associated protein 6 comprises an intracellular region of the low density lipoprotein receptor associated protein 6; and/or the low density lipoprotein is The truncated body of the body-associated protein 5 comprises the intracellular region of the low density lipoprotein receptor associated protein 5.
  • the truncated form of low density lipoprotein receptor associated protein 6 comprises a transmembrane region of said low density lipoprotein receptor associated protein 6 and said low density lipoprotein receptor associated protein 6 LDLR region; and/or the truncated body of low density lipoprotein receptor-associated protein 5 comprises a transmembrane region of said low density lipoprotein receptor associated protein 5 and said low density lipoprotein receptor associated protein 5 LDLR area.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises an amino acid sequence set forth in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO:22, 24, 26 and 28.
  • the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or fragment thereof comprises a nucleic acid sequence set forth in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 21, 23, 25 and 27.
  • the application provides a composition comprising the genetically modified immune cell.
  • composition further comprises an optionally pharmaceutically acceptable carrier.
  • the application provides the use of the genetically modified cell and/or the composition for the preparation of a medicament, wherein the medicament is for treating and/or preventing a tumor.
  • the tumor is selected from the group consisting of liver cancer, lung cancer, leukemia, and mesothelioma.
  • the present application provides a method of preparing the composition, the method comprising the steps of: expressing a low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cell Up.
  • 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 associated protein or fragment thereof.
  • the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, and a transposon plasmid.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises an amino acid sequence set forth in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO:22, 24, 26 and 28.
  • 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 activating comprises administering a T cell culture medium to the isolated genetically modified immune cell.
  • CAR chimeric antigen receptor
  • the T cell culture medium is selected from one or more of the group consisting of DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.
  • the method further comprises administering a T cell stimulating factor to the genetically modified immune cell.
  • Figure 1 is a schematic view showing the structure of the CAR in the lentivirus described in the present application.
  • Figure 2 shows the results of genetically modified immune cells described herein for promoting the production of memory immune cells.
  • Figure 3 shows the results of inhibition of immune cell differentiation by genetically modified immune cells described herein.
  • Figure 4 shows the results of the low density lipoprotein receptor-related protein or fragment thereof in the immune cells described herein promoting the proliferation of immune cells as described herein.
  • Figures 5A-5B show the results of enhanced cytokine release by genetically modified immune cells described herein.
  • Figure 6 shows the anti-tumor effect of the genetically modified immune cells described herein.
  • Figure 7 shows the effect of genetically modified immune cells described herein to prevent tumor recurrence.
  • the term "immune cell” generally refers to a cell involved in or associated with an immune response.
  • the immune cells can include lymphocytes and various phagocytic cells.
  • the immune cells can also comprise natural and genetically modified immune cells.
  • the genetically modified immune cell can express a chimeric antigen receptor (CAR).
  • the lymphocytes may comprise T lymphocytes and B lymphocytes.
  • the immune cells may comprise T cells.
  • the term "memory-type immune cells” generally refers to immune cells having immunological memory.
  • the immunological memory can refer to a specific recognition and response to an antigen, and the same antigen can again produce a rapid and strong immune response.
  • the memory type immune cells may include memory type T cells.
  • the memory type T cells can be classified into memory stem cell-like T cells (TSCM) and central memory T cells (TCM).
  • the term "differentiated immune cells” generally refers to immune cells having a certain degree of differentiation.
  • the differentiated immune cells may be T cells having a certain degree of differentiation.
  • the differentiated immune cells can be obtained by culturing the immune cells to differentiate them to a certain extent.
  • the differentiated immune cells can comprise regulatory T cells (Tregs).
  • the term "regulatory T cell” generally refers to a population of lymphocytes that have a negative regulatory immune response.
  • the molecular marker of the regulatory T cell may be the transcription factor Foxp3 + or CD127 ⁇ .
  • the regulatory T cells can be classified into two types, natural presence and induction. Wherein the naturally occurring CD4 + CD25 + cells to induce the production of T R 1 cells and T H 3 cells.
  • the term "subject susceptible to a tumor” generally refers to a subject having an increased probability of having a tumor compared to a normal subject.
  • the subject susceptible to a tumor may be a subject who has been cured of a tumor but has a risk of metastasis, recurrence.
  • the subject susceptible to tumors may also be a subject who has been diagnosed with the risk factors for having a tumor.
  • the risk factors may include genetic mutations (including deletions, additions, or substitutions) that are associated with a tumor.
  • the subject susceptible to tumors may also be a subject who is exposed to a carcinogenic environment for a prolonged period of time.
  • the carcinogenic environment may include strong radiation, high concentration of carcinogens
  • the term "genetic modification” generally refers to alterations or modifications that occur at the genetic structure level.
  • the genetic modification can be a modification of a gene level, a transcription level, and/or a translation level.
  • the genetic modification can include any alteration in genetic characteristics of an organism, including its tissues, cells, DNA, mRNA or proteins, and fragments thereof.
  • the genetic modification can include causing the organism to express a particular protein or fragment thereof.
  • the genetic modification can include causing the organism to comprise a vector capable of expressing the particular protein or fragment thereof.
  • T cell also known as T lymphocyte
  • T lymphocyte is a subtype of leukocytes that 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 T cell receptors present on the cell surface.
  • the T cells may include memory stem cell-like T cells (TSCM) and central memory T cells (TCM).
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • TEM effector memory T cells
  • TEF effector T cells
  • central memory T cells generally refers to long-term memory T cells produced by antigenic activation of naive T cells.
  • the biomarker of the TCM can comprise CD62L + and CD45RO + .
  • the central memory T cells are capable of returning to the lymph nodes through lymphatic shielding while being in a state activated by the antigen.
  • T cell receptor is also commonly 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 may be present on the surface of the T cell as a complex with the CD3 molecule.
  • the TCR may be a heterodimer immobilized on a cell membrane, most of which consist of a highly variable alpha subunit and a beta subunit joined by a disulfide bond; a few consist of a gamma and delta peptide chain.
  • the TCR can comprise a variable region and a constant region, wherein the constant region can be adjacent to a cell membrane, ligating a transmembrane region and an intracellular end, and the variable region is responsible for recognizing the polypeptide/MHC complex.
  • chimeric antigen receptor is also commonly 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.
  • the CAR may comprise an intracellular domain comprising a signaling domain and/or a costimulatory domain.
  • a group of polypeptides of a CAR may be located in the same polypeptide chain (eg, comprising a chimeric fusion protein) or may be non-contiguous with each other, eg, may be located in a different polypeptide chain.
  • the signal involved in induction can be transduced into the cytoplasm of T cells via CD3 and ⁇ chain.
  • an intracellular domain may comprise a primary signaling binding domain (eg, the major signaling domain of CD3-zeta ( ⁇ )).
  • the cytoplasmic signal domain can further comprise one or more costimulatory domains derived from at least one costimulatory molecule.
  • the costimulatory domain can be 4-1BB (ie, CD137), CD27, ICOS, and/or CD28.
  • a CAR may comprise a chimeric fusion protein, for example, comprising an optional leader sequence at the amino terminus (N-ter). Wherein the leader sequence optionally cleaves an antigen binding domain (eg, ScFv) and localizes the CAR to the cell membrane during cell processing.
  • the term "signaling domain” generally refers to a domain located within a cell capable of transducing a signal.
  • the intracellular signaling domain can signal a signal into a cell.
  • the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor.
  • the signaling domain may comprise a portion selected from the group consisting of CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ signaling domains.
  • costimulatory domain generally refers to a domain in a CAR that crosses a cell membrane, which is linked to an intracellular signal transduction domain and functions to transmit a signal.
  • the costimulatory domain may comprise a portion selected from the group consisting of CD27, CD28 and 4-1BB co-stimulatory domains.
  • the term "hinge region” generally refers to a junction region between an antigen binding region and an immune cell Fc receptor (FcR) binding region.
  • the hinge region can be a region between the heavy chain CH1 and CH2 functional regions in an immunoglobulin.
  • the hinge region may be a region between the scFv and the T cell membrane.
  • the hinge region may be derived from IgGl or IgG4, and may also be derived from IgD or CD8.
  • the hinge region may comprise a portion selected from the group consisting of a hinge region of IgG4, a hinge region of IgG1, and a hinge region of CD8.
  • the term "transmembrane region” generally refers to a transmembrane region joining an extracellular antigen binding domain and an intracellular signal domain, generally consisting of a dimeric membrane protein, mainly including CD3, CD4, CD8, CD28, etc.
  • the CAR structure is anchored to the T cell membrane.
  • Different designs of the transmembrane region can affect the expression of the introduced CAR gene.
  • the transmembrane region may comprise a portion selected from the group consisting of a transmembrane region of CD8, a transmembrane region of CD28, and a transmembrane region of CD24.
  • single-chain antibody generally refers to an antibody obtained by linking the heavy chain variable region and the light chain variable region via a linker.
  • the linker may be a linker peptide.
  • tumor antigen generally refers to an antigenic substance produced within or by a tumor cell that may have the ability to trigger an immune response in the host.
  • the tumor antigen can 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.
  • tumor antigen may also refer to a biomolecule (eg, protein, carbohydrate, glycoprotein, etc.) that is specifically or preferentially or differentially expressed on cancer cells and/or associated with cancer cells to provide cancer priority. Or a specific target.
  • preferential expression can be a conventional preferential expression, or a preferential expression within a particular region of an organism (eg, within a particular organ or tissue) as compared to any other cell in the organism.
  • the tumor antigen may include a B lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member.
  • B lymphocyte surface antigen generally refers to an antigen located on the surface of B lymphocytes produced by B lymphocytes at different stages.
  • the B lymphocyte surface antigen may include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, and CD40.
  • the B lymphocyte surface antigen may include CD19.
  • CD19 generally refers to a group 19 protein that is an antigenic determinant detectable on leukemia precursor cells.
  • the accession number of human CD19 in UniProt/Swiss-Prot is P15391, and the nucleotide sequence encoding human CD19 is in GenBank under accession number NM_001178098.
  • CD19 may also include a protein comprising a mutation or a functional fragment thereof, such as point mutations, fragments, insertions, deletions, and splice variants of full length wild type CD19.
  • the term "TNF family member” generally refers to a member belonging to the TNF (Tumor Necrosis Factor) family.
  • TNF family 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.
  • the TNF family members may include BCMA and 4-1BB.
  • BCMA generally refers to B cell maturation Antigen (BCMA, CD269).
  • BCMA is a member of the tumor necrosis factor receptor (TNF) superfamily and binds to B cell activating factor (BAFF) and proliferation inducing ligand (APRIL).
  • TNF tumor necrosis factor receptor
  • APRIL proliferation inducing ligand
  • BCMA is common on the surface of plasma cells in patients with multiple myeloma.
  • the accession number of BCMA in GenBank is BAB60895.1.
  • 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 important regulator of activation-induced costimulatory molecules that are immune responses.
  • TNFRS9 tumor necrosis factor receptor superfamily
  • HER family member generally refers to a member belonging to the family of HER (human epidermal growth factor receptor).
  • Members of the HER family may include EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4).
  • the HER family member may comprise HER2.
  • HER2 generally refers to the human HER2 protein, which belongs to the HER family members. See, for example, Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al., Nature 319: 230-234 (1986).
  • GenBank accession number of human HER2 may include XP_024306409.1.
  • the term "member of the GPC family” generally refers to glypican, which is identified in mammals as having six glypicans, referred to as GPC1 to GPC6, respectively.
  • Abnormal expression of glypicans in cancer including human hepatocellular carcinoma, ovarian cancer, mesothelioma, pancreatic cancer, glioma, and breast cancer.
  • the GPC family members may include GPC3.
  • GPC3 generally refers to a protein encoded by Glypican 3 (NCBI database gene ID: 2719), which is an early marker of liver cancer. GPC3 is highly expressed in hepatocellular carcinoma and is detected in tissues of patients with early hepatocellular carcinoma.
  • GenBank accession number of human GPC3 can be AAB87062.1.
  • MSLN mesothelin, or abbreviated MSLN
  • MSLN tumor differentiation antigen
  • peripheral blood mononuclear cells generally refers to a peripheral blood mononuclear cell (PBMC) in peripheral blood.
  • the peripheral blood mononuclear cells may include lymphocytes and monocytes.
  • the separation of the peripheral blood mononuclear cells can be performed using Ficoll-hypaque density gradient centrifugation according to the difference in specific gravity of each component in the blood.
  • the term "T cell activating factor” generally refers to a substance that promotes T cell activation and proliferation.
  • the T cell activating factor may include a B lymphocyte surface antigen antibody, a TNF antibody, an intracellular polyester, and/or an antibiotic.
  • the T cell stimulating factor may be selected from the group consisting of a CD3 antibody, a CD28 antibody, a 4-1BB antibody, a CD80 antibody, a CD86 antibody, PHA, PMA, and ionomycin.
  • B lymphocyte surface antigen antibody generally refers to an antibody that specifically binds to a B lymphocyte surface antigen.
  • the B lymphocyte surface antigen antibody may include a CD3 antibody, a CD28 antibody, a CD80 antibody, and a CD86 antibody.
  • the term "TNF antibody” generally refers to an antibody that specifically binds to a member of the TNF family.
  • the TNF antibody may comprise a 4-1BB antibody.
  • Intracellular polyester generally refers to a natural polymeric biomaterial synthesized by microorganisms in the form of inclusion bodies in cells. Intracellular polyester has good biological properties.
  • the intracellular polyester may include a polyhydroxyalkanoate (PHA).
  • the term "antibiotic” generally refers to a metabolic product produced by a microorganism or an animal or plant that has an anti-pathogenic or other activity that interferes with the developmental functions and functions of other living cells.
  • the antibiotic may include ⁇ -lactams, aminoglycosides, amide alcohols, macrolides, polypeptides, nitroimidazoles, and tetracyclines.
  • the antibiotic can include ionomycin.
  • PHA generally refers to polyhydroxyalkanoates which are intracellular polyesters synthesized by a variety of bacteria and which may exist in vivo as discrete inclusion bodies within the cytoplasm.
  • PHA can have physical, chemical properties similar to synthetic plastics, as well as biodegradability, biocompatibility, optical activity, piezoelectricity, gas barrier properties, and the like.
  • PMA generally refers to Phorbol-12-myristate-13-acetate.
  • CD3 antibody generally refers to an antibody or antigen-binding fragment thereof that specifically binds to CD3.
  • CD3 can be an important differentiation antigen on the T cell membrane and can transmit signals of T cell activation.
  • the CD3 antibody can be huOKT3g1 or HuM291.
  • CD28 antibody generally refers to an antibody or antigen-binding fragment thereof that specifically binds to CD28.
  • Human CD28 is located at 2q33 and has similar exons and introns to CTLA4.
  • the ligands for both are the B7 family, including B7-1 (CD80) and B7-2 (CD86).
  • the term "cytokine” generally refers to immune cells (eg, mononuclear, macrophage, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts).
  • the cytokine may have various functions such as regulation of innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and repair of damaged tissues.
  • the cytokine may include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, and growth factors.
  • the cytokine can be an interleukin.
  • interleukin generally refers to a secreted protein or signal molecule capable of promoting 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.
  • the term "interleukin” may include full-length interleukins or fragments (eg, truncated forms) or variants thereof that substantially retain the biological activity of the corresponding wild-type interleukin (eg, biologically active) 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.
  • the interleukin is from a species selected from the group consisting of human, horse, cow, mouse, pig, rabbit, cat, dog, rat, goat, sheep, and non-human primate.
  • the interleukin can be in a mutated form.
  • the cytokine comprises one or more selected from the group consisting of IL2, IL21, IL7 and IL15.
  • IL2 generally refers to 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, induce the production of LAK cells, promote the proliferation of B cells and secrete antibodies, and activate giants. Phagocytes.
  • IL21 is homologous to the spatial structure of IL-2, IL-4, IL-15, and generally promotes proliferation and differentiation of bone marrow NK cells, and synergizes with anti-CD40 antibody to stimulate proliferation of B cells.
  • CD3 antibodies synergistically stimulate the proliferation of T cells.
  • IL7 generally refers to a glycoprotein secreted by bone marrow stromal cells whose gene is located on chromosome 8.
  • the targeting cells of IL7 are lymphocytes, especially those capable of promoting B progenitor cells derived from human or mouse bone marrow, thymocytes, and peripheral mature T cells. At higher concentrations, IL-7 also enhances the cytotoxic activity of macrophages and induces the secretion of cytokines by monocytes.
  • IL15 generally refers to a variety of cells produced by activated mononuclear-macrophages, epidermal cells, and fibroblasts, and has many similarities to IL2.
  • IL15 belongs to the IL2 family and regulates the activation and proliferation of T and NK cells by altering the expression of Bcl-1 family members (such as Bcl-2 and Bcl-XL), and also induces B cell proliferation.
  • Bcl-1 family members such as Bcl-2 and Bcl-XL
  • LRP Low density lipoprotein receptor-related protein
  • Embedded in the outer phospholipid layer of LDL (Low density lipoprotein) particles is an endocytic receptor that mediates the endocytosis of cholesterol-rich LDL. It is a low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • the LRP is most prominently expressed in bronchial epithelial cells and adrenal and cortical tissues.
  • the low density lipoprotein receptor-related protein may comprise one or more selected from the group consisting of low density lipoprotein receptor associated proteins 1-12 or truncations.
  • low-density lipoprotein receptor-related protein 6 LRP-6
  • LRP-5 low-density protein receptor-associated protein 5"
  • LDLR low-density lipoprotein receptor
  • Truncated protein generally refers to a truncated protein.
  • the truncated body can be obtained by protein hydrolysis or by manipulation of a structural gene to eliminate the N- or C-terminal portion of the protein.
  • the truncated body can be obtained by nonsense mutation resulting in the presence of a stop codon in the structural gene to terminate the translation early.
  • the term "intracellular region” generally refers to a domain in which a protein is located within a cell membrane.
  • the intracellular domain may refer to a domain within the cell membrane of the low density lipoprotein receptor associated protein.
  • the intracellular region may comprise a sequence from position 24 to position 243 of SEQ NO. 24 or an amino acid sequence having at least 80% homology thereto, or comprising the first of SEQ NO. The sequence from position 24 to position 231 or an amino acid sequence having at least 80% homology thereto.
  • the term "transmembrane region” generally refers to a domain in a protein that spans a cell membrane.
  • the transmembrane region may refer to a domain in which the low density lipoprotein receptor-related protein spans the cell membrane.
  • the transmembrane region can be composed of 23 hydrophobic amino acids. This domain provides anchoring function primarily for the binding of LDLR on the cell membrane.
  • the transmembrane region may comprise a sequence from position 1 to position 23 in SEQ NO. 24 or an amino acid sequence having at least 80% homology thereto, or a fragment comprising SEQ NO. The sequence from position 1 to position 23 or an amino acid sequence having at least 80% homology thereto.
  • the term "LDLR region” generally refers to a domain of the low density lipoprotein receptor-related protein that is adjacent to the N segment outside the transmembrane region. This domain may have the function of enhancing the Wnt signal.
  • the LDLR region may comprise amino acid sequences from positions 5 to 119 of SEQ NO. 22 or having at least 80% homology thereto, or positions 4 to 119 of SEQ NO. A sequence of positions or an amino acid sequence having at least 80% homology thereto.
  • ribosomal hopping site also known as the internal ribosome entry site (IRES)
  • IRES internal ribosome entry site
  • the ribosome hopping site may allow translation to be initiated in a hat-independent manner.
  • the IRES is usually located in the 5' UTR.
  • the ribosome skip site may comprise the sequence of positions 1 to 578 of SEQ NO.
  • the term "2A sequence” generally refers to a self-cleaving amino acid sequence that is not protease dependent.
  • the 2A sequence can facilitate transcription to produce two proteins.
  • the 2A sequence may include the sequence from position 1 to position 54 of SEQ NO.
  • 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.
  • the term "retroviral vector” generally refers to an RNA virus which can be reverse transcribed in a infected cell to produce a DNA complementary strand and synthesize a second DNA strand using the DNA single strand as a template, and Incorporation into cellular genomic DNA.
  • the retroviral vector can self-transcribe and replicate the RNA synthesis protein by using the enzyme of the host cell, and then package the virus and release it from the cell to become an infectious virus.
  • the retrovirus has high transduction efficiency and can effectively increase the transfection rate of the gene.
  • lentiviral vector refers to a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency type I virus).
  • HIV-1 human immunodeficiency type I virus
  • the lentiviral vector can have an ability to infect both dividing cells and non-dividing cells. It can effectively infect almost all mammalian cells including neuronal cells, hepatocytes, and the like, and has high infection efficiency. Lentiviruses can efficiently integrate foreign genes into the host chromosome to achieve persistent expression.
  • transposon plasmid generally refers to the basic unit of autonomous replication and displacement present on chromosomal DNA.
  • the transposon plasmid can be "jumped" from one location of the genome to another by a series of processes such as cleavage, re-integration, and the like.
  • the term "tumor” generally refers to a new organism in which a certain cell of a local tissue loses its normal regulation at the genetic level under the action of various carcinogenic factors, resulting in clonal abnormal proliferation. (neogrowth), because this new organism is mostly a occupying block, also known as neoplasm.
  • the tumor may comprise a solid tumor and a non-solid tumor.
  • the tumor may include liver cancer, lung cancer, leukemia, and mesothelioma.
  • the term "significantly increased” generally refers to an increase in levels (eg, protein expression, cell number), P ⁇ 0.05, P ⁇ 0.04, P ⁇ 0.03, P ⁇ 0.02, P ⁇ 0.01, P ⁇ 0.005 or P. ⁇ 0.001.
  • the significant increase may mean 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.
  • the term "substantially not increased” generally means that the level (eg, protein expression amount, cell number) is consistent with the original, or increases by 3% or less, increases by 2% or less, and increases by 1%. Or less, an increase of 0.5% or less, an increase of 0.4% or less, an increase of 0.3% or less, an increase of 0.2% or less, an increase of 0.1% or less.
  • the term "about” generally means a range of 0.5% to 10% above or below a specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5% above or below a specified value, Variations within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • Low-density lipoprotein receptor-related protein or fragment thereof Low-density lipoprotein receptor-related protein or fragment thereof
  • the low density lipoprotein receptor-related protein may comprise one or more selected from the group consisting of low density lipoprotein receptor associated proteins 1-12 and functional fragments thereof.
  • the low density lipoprotein receptor associated protein or fragment thereof may be derived from a mammal.
  • it can be derived from humans, macaques, rats, and mice.
  • 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.
  • the low density lipoprotein receptor-related protein may comprise a low density lipoprotein receptor associated protein 6 and a truncated body thereof, and/or a low density lipoprotein receptor associated protein 5 and a truncated body thereof.
  • the truncation of the low density lipoprotein receptor-related protein 6 may comprise an intracellular region of the low density lipoprotein receptor associated protein 6; and/or the low density lipoprotein receptor
  • the truncated body of the related protein 5 may comprise the intracellular region of the low density lipoprotein receptor associated protein 5.
  • the truncation of the low density lipoprotein receptor-related protein 6 may comprise a transmembrane region of the low density lipoprotein receptor associated protein 6 and an LDLR region of the low density lipoprotein receptor associated protein 6.
  • the truncated body of the low density lipoprotein receptor-related protein 5 may comprise a transmembrane region of the low density lipoprotein receptor associated protein 5 and the low density lipoprotein receptor associated protein 5 LDLR area.
  • the low-density lipoprotein receptor-related protein or a fragment thereof 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: 22, 24, 26 and 28.
  • the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof 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: 21, 23, 25 and 27.
  • the present application provides a genetically modified immune cell wherein the expression level of a low density lipoprotein receptor-related protein or a fragment thereof is up-regulated in the immune cell.
  • the genetically modified immune cells may comprise lymphocytes.
  • the genetically modified immune cell comprises a genetically modified T cell.
  • the genetically modified immune cell comprises a genetically modified memory stem cell-like T cell (TSCM) and/or a genetically modified central memory T cell (TCM).
  • the TSCM is comprised as CCR7 + and/or CD62L + .
  • 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 + .
  • the immune cells may comprise genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR may comprise an intracellular domain, which may comprise a signaling domain and/or a costimulatory domain.
  • the signaling domain may comprise a portion selected from the group consisting of a signaling domain of CD3[zeta], a signaling domain of CD3[delta], and a signaling domain of CD3[epsilon].
  • the signaling domain may comprise an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto
  • the nucleic acid molecule encoding the signaling domain may comprise SEQ ID NO: 17.
  • the costimulatory domain can comprise a portion selected from the group consisting of a costimulatory domain of CD27, a costimulatory domain of CD28, and a costimulatory domain of 4-1BB.
  • the costimulatory domain may comprise an amino acid sequence set forth in any one of SEQ ID NO: 14 and SEQ ID NO: 16 or an amino acid sequence having at least 80% homology thereto, encoding the costimulatory
  • the nucleic acid molecule of the domain may comprise the nucleic acid sequence set forth in any one of SEQ ID NO: 13 and SEQ ID NO: 15 or a nucleic acid sequence having at least 80% homology thereto.
  • the CAR may comprise a hinge region.
  • the hinge region may comprise a portion selected from the group consisting of a hinge region of IgG4, a hinge region of IgG1, and a hinge region of CD8.
  • the hinge region may comprise an amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence having at least 80% homology thereto
  • the nucleic acid molecule encoding the hinge region may comprise the nucleic acid set forth in SEQ ID NO: A sequence or a nucleic acid sequence having at least 80% homology thereto.
  • the CAR may comprise a transmembrane region.
  • the transmembrane region can comprise a portion selected from the group consisting of a transmembrane region of CD8, a transmembrane region of CD28, and a transmembrane region of CD24.
  • the transmembrane region may comprise an amino acid sequence set forth 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 sequence set forth in SEQ ID NO:34.
  • the CAR may comprise a targeting moiety.
  • the targeting moiety can comprise an antibody or 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.
  • the targeting moiety can be a ScFv.
  • the targeting moiety can specifically bind to and/or recognize a tumor antigen.
  • the targeting moiety can specifically bind to and/or recognize a target selected from the group consisting of a B lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member.
  • the targeting moiety can specifically bind to and/or recognize a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.
  • the targeting moiety may be an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes CD19.
  • the antibody or antigen-binding fragment thereof may comprise a light chain variable region, which may comprise LCDR1-LCDR3, wherein the LCDR1 may comprise or have at least 80 of the amino acid sequence set forth in SEQ ID NO:46 % homologous amino acid sequence; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 47 or an amino acid sequence having at least 80% homology thereto; the LCDR3 may comprise the amino acid set forth in SEQ ID NO:48 A sequence or an amino acid sequence having at least 80% homology thereto.
  • the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise an HCDR1-HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:43 Or an amino acid sequence having at least 80% homology thereto; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; the HCDR3 may comprise SEQ ID NO: The amino acid sequence shown at 45 or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 49 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes BCMA, and the antibody or antigen-binding fragment thereof may comprise a light chain variable region, the light chain variable region LCDR1-LCDR3 may be included, wherein the LCDR1 may comprise an amino acid sequence set forth in SEQ ID NO: 54 or an amino acid sequence having at least 80% homology thereto; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55 Or an amino acid sequence having at least 80% homology thereto; the LCDR3 may comprise the amino acid sequence set forth 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 set forth in SEQ ID NO: 58 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, which may comprise an HCDR1-HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:51 Or an amino acid sequence having at least 80% homology thereto; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 52 or an amino acid sequence having at least 80% homology thereto; the HCDR3 may comprise SEQ ID NO: The amino acid sequence shown at 53 or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes HER2, and the antibody or antigen-binding fragment thereof may comprise a light chain variable region
  • the light chain variable region LCDR1-LCDR3 may be included, wherein the LCDR1 may comprise an amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence having at least 80% homology thereto; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:71 Or an amino acid sequence having at least 80% homology thereto; the LCDR3 may comprise the amino acid sequence of 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 set forth in SEQ ID NO: 74 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise an HCDR1-HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:67 Or an amino acid sequence having at least 80% homology thereto; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 68 or an amino acid sequence having at least 80% homology thereto; the HCDR3 may comprise SEQ ID NO: The amino acid sequence shown at 69 or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 73 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes Mesothelin, and the antibody or antigen-binding fragment thereof may comprise a light chain variable region, the light chain variable region LCDR1-LCDR3 may be included, wherein the LCDR1 may comprise an amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence having at least 80% homology thereto; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:63 Or an amino acid sequence having at least 80% homology thereto; the LCDR3 may comprise the amino acid sequence set forth 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 set forth in SEQ ID NO: 66 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, which may comprise an HCDR1-HCDR3, wherein the HCDR1 may comprise or have at least 80 of the amino acid sequence set forth in SEQ ID NO:59 % homologous amino acid sequence; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence having at least 80% homology thereto; the HCDR3 may comprise the amino acid set forth in SEQ ID NO: 61 A sequence or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 65 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and/or recognizes GPC3, and the antibody or antigen-binding fragment thereof may comprise a light chain variable region, the light chain variable region LCDR1-LCDR3 may be included, wherein the LCDR1 may comprise an amino acid sequence set forth in SEQ ID NO: 38 or an amino acid sequence having at least 80% homology thereto; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:39 Or an amino acid sequence having at least 80% homology thereto; the LCDR3 may comprise the amino acid sequence set forth 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 set forth in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, and the heavy chain variable region may comprise an HCDR1-HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:35 Or an amino acid sequence having at least 80% homology thereto; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology thereto; the HCDR3 may comprise SEQ ID NO: The amino acid sequence shown at 37 or an amino acid sequence having at least 80% homology thereto.
  • the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto.
  • the targeting moiety may comprise an amino acid sequence set forth in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NOs: 2, 4, 6, 8, and 10.
  • the nucleic acid molecule encoding the targeting moiety may comprise a nucleic acid sequence as set forth 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.
  • the enthalpy of the amino acid position of the CDR is calculated in the form of Kabat.
  • a protein, polypeptide, amino acid sequence and/or nucleic acid sequence referred to in the present application is also understood to include at least the following range: a variant or homologue having the same or similar function as the protein or polypeptide. And/or a nucleic acid sequence encoding a variant or homolog of the same or similar function as the protein or polypeptide.
  • the variant may be, in the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to a GPC3 protein, or the low density lipoprotein receptor-related protein or A protein or polypeptide in which the amino acid sequence of a fragment has been substituted, deleted or added with one or more amino acids.
  • the functional variant may comprise having been substituted by at least 1, for example 1-30, 1-20 or 1-10, for example 1 , 2, 3, 4 or 5 amino acids.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion or addition).
  • the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity, or, The biological function of a low density lipoprotein receptor-related protein or a fragment thereof).
  • the substitution can be a conservative substitution.
  • the homologue may be, together with the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to a GPC3 protein, or the low density lipoprotein receptor-related protein or
  • the amino acid sequence thereof has at least about 85% (eg, 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 more of a sequence homologous protein or polypeptide.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology” can be calculated by comparing two sequences to be aligned in a comparison window to determine the presence of the same nucleic acid base in both sequences (eg, A, T, C, G, I) Or the same amino acid residues (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met)
  • the number of positions is obtained to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to generate a sequence homology percentage.
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • Megalign DNASTAR
  • One of skill in the art can determine suitable parameters for aligning the sequences, including any algorithms needed to achieve maximum alignment within the full length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad.
  • the CAR may comprise the targeting moiety, the hinge region, the transmembrane region, the costimulatory domain, and the signaling domain in sequence from N-terminus to C-terminus.
  • the polynucleotide molecule encoding the CAR may comprise, in sequence from the 5' end to the 3' end, a nucleotide sequence encoding the targeting portion, a nucleotide sequence encoding the hinge region, and a coding house.
  • a polynucleotide molecule encoding the CAR may further comprise a leader sequence.
  • a nucleic acid molecule encoding the leader sequence may comprise a nucleic acid sequence set forth in SEQ ID NO: 30 or a nucleic acid sequence having at least 80% homology thereto.
  • the nucleic acid molecule encoding the leader sequence can be located at the 5' end of the nucleotide sequence encoding the targeting moiety.
  • the genetically modified immune cell can express the low density lipoprotein receptor associated protein or a fragment thereof.
  • the genetically modified immune cell can comprise a vector capable of expressing the low density lipoprotein receptor associated protein or a fragment thereof.
  • a nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof may be included in the vector.
  • the polynucleotide molecule may comprise a nucleic acid sequence set forth 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.
  • the vector can be selected from the group consisting of a retroviral vector, a lentiviral vector, and/or a transposon plasmid.
  • the genetically modified immune cell can express the chimeric antigen receptor (CAR).
  • the genetically modified immune cell can comprise a vector capable of expressing the chimeric antigen receptor (CAR).
  • a nucleotide molecule encoding the chimeric antigen receptor (CAR) can be included in the vector.
  • the vector can be selected from the group consisting of a retroviral vector, a lentiviral vector, and/or a transposon plasmid.
  • the vector capable of expressing the low density lipoprotein receptor-related protein or a fragment thereof may be the same vector as the vector capable of expressing the chimeric antigen receptor (CAR), or may be 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), thereby allowing the genetically modified immune cells to have both The low density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) may be used.
  • 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.
  • 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 An expression box.
  • the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof can be located at the 3' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).
  • the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof may be directly or together with the nucleotide molecule encoding the chimeric antigen receptor (CAR).
  • the indirect connections can be connected by a connection sequence.
  • the 5' end of the ligation sequence may be ligated to the 3' end of the nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof, and the 3' end of the ligation sequence may be The 5' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR) is ligated.
  • the linker sequence may comprise the nucleic acid sequence set forth in any one of the following: SEQ ID NO: 19 and 29 or a nucleic acid sequence having at least 80% homology thereto.
  • the low density lipoprotein receptor-related protein or fragment thereof and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cell may be two independent proteins. That is, the two do not have any inter-connected relationship to form any form of di(poly)mer or protein complex.
  • the low density lipoprotein receptor-related protein or fragment thereof expressed by the genetically modified immune cell and the chimeric antigen receptor (CAR) may also be linked to each other.
  • the two proteins formed by translation are not cleaved completely, and the low density lipoprotein receptor-related protein or fragment thereof and the chimeric antigen receptor form a complex.
  • the present application provides a method of preparing the genetically modified immune cell, the method comprising the steps of: expressing a low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cell Up.
  • the present application also provides a method of preparing the composition, the method comprising the step of upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the genetically modified immune cell.
  • the method may further comprise the step of isolating peripheral blood mononuclear cells PBMC, CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes or regulatory T cells.
  • the method can include the step of isolating and activating the genetically modified immune cells, wherein the activating comprises administering a T cell culture medium to the isolated genetically modified immune cells.
  • the T cell culture medium may be selected from one or more of the group consisting of DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.
  • the method may further comprise the step of adding one or more T cell stimulating factors to the isolated peripheral blood mononuclear cell PBMC.
  • the T cell stimulating factor can be selected from the group consisting of a B lymphocyte surface antigen antibody, a TNF antibody, an intracellular polyester, and an antibiotic.
  • the T cell stimulating factor can be selected from the group consisting of a CD3 antibody, a CD28 antibody, a 4-1BB antibody, a CD80 antibody, a CD86 antibody, PHA, PMA, and ionomycin.
  • the T cell stimulating factor may include a CD3 antibody, and the concentration of the CD3 antibody may be 1-1000 ng/mL.
  • the concentration of the CD3 antibody may 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.
  • the T cell stimulating factor may include a CD28 antibody, and the concentration of the CD28 antibody may be 1-1000 ng/mL.
  • the concentration of the CD28 antibody may 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.
  • the method may further comprise adding one or more cytokine agents to the isolated PBMC.
  • the cytokine agent may include an interleukin.
  • the interleukin can comprise one or more selected from the group consisting of IL2, IL21, IL7 and IL15.
  • the interleukin may include IL2, and the concentration of the IL2 may be 0.1-10000 U/mL.
  • 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.
  • the interleukin may include IL21, and the concentration of the IL21 may be 0.01-1000 ng/mL.
  • 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.
  • the interleukin may include IL7, and the concentration of the IL7 may be 0.01-1000 ng/mL.
  • 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.
  • the interleukin may include IL15, and the concentration of the IL15 may be 0.01-1000 ng/mL.
  • 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.
  • the application provides a method of promoting proliferation of immune cells comprising the step of upregulating expression of a low density lipoprotein receptor associated protein or fragment thereof in said immune cell.
  • the application provides a method for promoting the production of memory immune cells, comprising the steps of: upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in an immune cell, thereby promoting differentiation of the immune cell For memory immune cells.
  • the application provides a method for inhibiting differentiation of immune cells, comprising the steps of: upregulating expression of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell, thereby inhibiting differentiation of the immune cell For differentiated immune cells.
  • the application provides a method of enhancing the release of a cytokine by an immune cell, comprising the step of upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • the cytokine may comprise an interleukin, an interferon and/or a tumor necrosis factor.
  • the cytokine may comprise IL-2, IL4, IL6, IL7, IL10, IL21, TNF- ⁇ and/or IFN ⁇ .
  • the application provides a method of enhancing the ability of an immune cell to kill a tumor, comprising the step of upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the immune cell.
  • the application provides a method of preventing tumor recurrence in a subject, the method comprising: administering an immune cell to a subject susceptible to the tumor, wherein the low density lipoprotein receptor-related protein in the immune cell The expression level of the fragment or its fragment is up-regulated.
  • the application provides a method of treating a tumor in a subject in need thereof, comprising the steps of administering to the subject an immune cell, wherein the immune cell is associated with a low density lipoprotein receptor The expression level of the protein or its fragment is up-regulated.
  • the tumor may be selected from the group consisting of liver cancer, lung cancer, leukemia, and mesothelioma.
  • the methods can include in vivo methods and in vitro methods.
  • the expression level is up-regulated such that the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in the immune cell is significantly up-regulated compared to the expression level of the control immune cell (for example, up-regulated) 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 cell is a corresponding immune cell in which the expression level of the low density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the control immune cell for example, up-regulated 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
  • the promoting memory immune cell production may be such that the number of memory immune cells produced by the immune cells is significantly up-regulated compared to the number of memory immune cells produced by the control immune cells (eg, up 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 More, 90% or more, 100% or more), wherein the control immune cells are corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the control immune cells are corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the inhibiting immune cell differentiation may be such that the number of differentiated immune cells produced by the immune cells is significantly down-regulated compared to the number of memory immune cells produced by the control immune cells (eg, down 5% or more). 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 corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the enhanced immune cell release cytokine may be such that the amount of cytokine released by the immune cell is significantly up-regulated compared to the amount of cytokine released by the control immune cell (eg, up 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 corresponding immune cells in which the expression level of the low density lipoprotein receptor-related protein or a fragment thereof is not substantially upregulated.
  • the control immune cells are corresponding immune cells in which the expression level of the low density lipoprotein receptor-related protein or a fragment thereof is not substantially upregulated.
  • the enhancing immune cell's ability to kill tumors may be such that the killing ability of the immune cells against the tumor is significantly improved compared to the killing ability of the control immune cells against the tumor (for example, 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 corresponding immune cells in which the expression level of the low density lipoprotein receptor-related protein or a fragment thereof is not substantially upregulated.
  • the prevention of tumor recurrence in a subject may be such that the ability of the immune cell to prevent tumor recurrence in a subject is significantly improved compared to the ability of a control immune cell to prevent tumor recurrence in a subject (eg, Increased 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 corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the tumor in the subject in need of treatment may be such that the therapeutic effect of the immune cell on the tumor is significantly improved compared to the therapeutic effect of the control immune cell on the tumor (for example, 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), wherein the control immune cells are corresponding immune cells in which the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof is not substantially up-regulated.
  • the corresponding immune cells can comprise lymphocytes.
  • the corresponding immune cells may comprise T cells.
  • the T cell may comprise a memory stem cell-like T cell (TSCM) and/or a central memory T cell (TCM).
  • the TSCM may be CCR7 + and/or CD62L + .
  • 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 + .
  • the corresponding immune cells may comprise genetically modified immune cells, and the genetically modified immune cells express a chimeric antigen receptor (CAR).
  • the corresponding immune cells may comprise genetically modified T cells.
  • the content of the low-density lipoprotein receptor-related protein or a fragment thereof in the corresponding immune cell may be only the expression level of the low-density lipoprotein receptor-related protein or a fragment thereof in a normal organism.
  • substantially unregulated can mean up to 4.5%, up to 4%, up to 3%, up to 2%, up to 1%, up to 0.5%, up to 0.3%, up to 0.1%, up to 0.01% or less.
  • the corresponding immune cell may not contain a vector comprising a nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof, and/or the corresponding immune cell may not comprise A nucleotide molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof.
  • methods for detecting the expression amount of the low density lipoprotein receptor-related protein or a fragment thereof, and/or the cytokine may include quantitative PCR, western blot, and immunohistochemistry.
  • 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.
  • the evaluation of the therapeutic effect of the tumor may use an indicator selected from the group consisting of tumor volume, overall survival (OS), total remission (DOR), stable duration of disease, disease-free survival time (DFS), no Progressive survival time (PFS), disease control rate (DCR), objective response rate (ORR), and/or clinical benefit response ratio.
  • OS overall survival
  • DOR total remission
  • DFS disease-free survival time
  • PFS no Progressive survival time
  • DCR disease control rate
  • ORR objective response rate
  • the evaluation of the effect of preventing tumor recurrence in a subject may use an indicator selected from the group consisting of expression levels of tumor markers (eg, tumor-associated antigens, tumor-specific genes, and tumor suppressor genes), histological detection Results (eg, epithelial hyperplasia, degree of disappearance of polyps), imaging findings (eg, mammography test results), and/or expression levels of serum markers (eg, serum free DNA, methylated DNA).
  • tumor markers eg, tumor-associated antigens, tumor-specific genes, and tumor suppressor genes
  • histological detection Results eg, epithelial hyperplasia, degree of disappearance of polyps
  • imaging findings eg, mammography test results
  • serum markers eg, serum free DNA, methylated DNA
  • the application provides a composition comprising the genetically modified immune cells.
  • the composition may also comprise an optionally pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or nonionic surface active agents. Agents, etc.
  • 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.
  • the drug is administered or administered through a subcutaneous reservoir.
  • compositions of the present application can inhibit or delay the progression or progression of the disease, can reduce tumor size (even substantially eliminate tumors), and/or can alleviate and/or stabilize disease states.
  • compositions described herein can comprise a therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • the therapeutically effective amount is a dose that is capable of preventing and/or treating (at least partially treating) a condition or disorder (eg, cancer) and/or any of its complications in a subject having or at risk of developing.
  • the application provides the use of the genetically modified cell and/or the composition for the preparation of a medicament for the treatment and/or prevention of a tumor.
  • the tumor may be selected from the group consisting of liver cancer, lung cancer, leukemia, and mesothelioma.
  • the CAR-T artificially synthesizes a fragment containing a CAR structure by targeting GPC3, CD19, Mesothelin (MSLN), HER2, BCMA, and constructs into a lentiviral vector (LV100A, System Biosciences), and then according to the description thereof.
  • Lentivirus was obtained by transfection (as shown in Figure 1), and GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-TL6, GPC3-CD28, GPC3-CD28-L6, GPC3-CD28-TL6, GPC3- were obtained, respectively.
  • GPC3-41BB was constructed by splicing the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta from 5' end to 3' end.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO.
  • GPC3-41BB-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and L6. Its nucleotide sequence is 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. .
  • GPC3-41BB-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL6. Its nucleotide sequence is 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. .
  • GPC3-41BB-L5 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A, and L5.
  • the nucleotide sequences thereof 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. .
  • GPC3-41BB-TL5 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL5.
  • the nucleotide sequences thereof 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. .
  • GPC3-CD28 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28 and CD3zeta.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO.
  • GPC3-CD28-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28, CD3zeta, 2A and L6. Its nucleotide sequence is 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. .
  • GPC3-CD28-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, GPC3 ScFv, CD8 hinge region and transmembrane region, CD28, CD3zeta, 2A and TL6. Its 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. .
  • BCMA-41BB was constructed by splicing the leader sequence, BCMA ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta from 5' end to 3' end.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO.
  • BCMA-41BB-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, BCMA ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and L6. Its nucleotide sequence is 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. .
  • BCMA-41BB-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, BCMA ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL6.
  • the nucleotide sequences thereof 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. .
  • CD19-41BB was constructed by splicing the leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta from 5' end to 3' end.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO.
  • CD19-41BB-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and L6. Its nucleotide sequence is shown in SEQ ID NO. 30, SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19 and SEQ ID NO. .
  • CD19-41BB-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, CD19 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL6. Its nucleotide sequence is shown in SEQ ID NO. 30, SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19 and SEQ ID NO. .
  • MSLN-41BB was constructed by splicing the leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta from 5' end to 3' end.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO.
  • MSLN-41BB-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and L6. Its nucleotide sequence is 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. .
  • MSLN-41BB-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, MSLN ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL6.
  • the nucleotide sequences thereof 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. .
  • HER2-41BB was constructed by splicing the leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta from 5' end to 3' end.
  • the nucleotide sequences thereof are shown in SEQ ID NO. 30, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO.
  • HER2-41BB-L6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and L6. Its nucleotide sequence is 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. .
  • HER2-41BB-TL6 was constructed by splicing from the 5' end to the 3' end of the leader sequence, HER2 ScFv, CD8 hinge region and transmembrane region, 41BB, CD3zeta, 2A and TL6. Its nucleotide sequence is 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. .
  • PBMC peripheral blood mononuclear lymphocytes
  • the isolated PBMCs were resuspended in a lymphocyte culture medium containing Xvivo15 medium, 5% FBS, 200 U/ml IL2 T or Xvivo15 medium, 5% FBS, 20 ng/ml IL21, 10 ng/ml IL7.
  • the final concentration was 1 ⁇ 10 6 cells/ml, and the cells were cultured at 37 ° C, 5% CO 2 for 24 hours in a Petri dish in which the antibody mixture was placed to activate T cells.
  • T cell culture solution was taken, mixed with synperonic F108 at a final concentration of 1 mg/ml, and heated to 37 ° C in a water bath to prepare an uninfected reagent. Subsequently, prepare the cell culture dish required for the experiment.
  • 1 mg/ml anti-human CD3 antibody and 0.5 mg/ml anti-human CD28 antibody were first diluted and mixed with PBS buffer at a volume ratio of 1:1000, and then with a dose ratio of 1:40 with retronectin (1 mg/ml) reagent. Dilute and mix, then spread evenly into cell culture dishes and incubate for 2 hours at room temperature. After 2 hours, washing was carried out with PBS, and the cell culture dish was ready to be completed.
  • 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, T cells of MSLN-41BB-L6, MSLN-41BB-TL6, HER2-41BB, HER2-41BB-L6 and HER2-41BB-TL6.
  • the cell density was monitored after infection, and the density of these infected cells was maintained at 1 ⁇ 10 6 cells/ml. After 14 days, the number of cells can be amplified 10-100 times.
  • Example 3 LRP6, LRP6 truncation, LRP5 or LRP5 truncation promotes memory T cell formation
  • GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-TL6, GPC3-CD28, GPC3-CD28-L6, GPC3-, respectively, obtained in Example 2 was cultured in vitro at 37 ° C in a 5% CO 2 cell culture incubator.
  • 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- T cells of 41BB, MSLN-41BB-L6, MSLN-41BB-TL6, HER2-41BB, HER2-41BB-L6 and HER2-41BB-TL6 were co-cultured for 9 days or 13 days.
  • CD3, CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometry. Among them, the measured protein expression results are shown in Figure 2.
  • T cells expressing GPC3-41BB-L6 or GPC3-41BB-TL6 the ratio of memory stem cell-like T cells (TSCM) and central memory T cells (TCM) was significantly higher than that of the control group;
  • T cells expressing GPC3-CD28-L6 or GPC3-CD28-TL6 the percentage of total cells in TSCM and TCM was significantly higher than that in the control group;
  • T cells expressing GPC3-41BB-L5 or GPC3-41BB-TL5 The percentage of total cells in TSCM and TCM was significantly higher than that in the control group.
  • the percentage of total cells in TSCM and TCM was significantly higher than that in the control group; Among the T cells of 41BB-L6 or CD19-41BB-TL6, TSCM and TCM accounted for a significantly higher percentage of total cells than the control group; in T cells expressing MSLN-41BB-L6 or MSLN-41BB-TL6, TSCM and TCM accounted for The percentage of total cells was significantly higher than that of the control group. In the T cells expressing HER2-41BB-L6 or HER2-41BB-TL6, the percentage of total cells in TSCM and TCM was also significantly higher than that in the control group.
  • Example 4 LRP6 or its truncation inhibits differentiation of Treg cells
  • T cells expressing GPC3-41BB and GPC3-41BB-L6, respectively, obtained in Example 2 were cultured in vitro at 37 ° C in a 5% CO 2 cell culture incubator for 9 days or 12 days.
  • the expression of CD3, CD4, CD25, CD127 and FoxP3 protein in T cells was detected by BD flow, and the measured protein expression results are shown in Fig. 3.
  • Example 5 LRP6, LRP6 truncation, LRP5 or LRP5 truncation promotes tumor antigen-induced specific CAR-T cell expansion
  • T cells expressing GPC3-41BB, GPC3-41BB-L6 or GPC3-41BB-TL6 obtained in Example 2 were compared with irradiated Huh7 or HepG2 cells (purchased by the Chinese Academy of Sciences cell bank) by a ratio of 1:1 cells.
  • Co-culture with Xvivo15 medium re-supplemented with irradiated Huh7 or HepG2 every 5 days, stimulated 3 times, each time counted with trypan blue staining; will express GPC3-41BB, GPC3-41BB-L5 or GPC3 -41BB-TL5 T cells and irradiated Huh7 or HepG2 cells (purchased by the Chinese Academy of Sciences cell bank) were co-cultured with Xvivo15 medium at a ratio of 1:1 cells, and irradiated with Huh7 or re-irradiated every 5 days.
  • HepG2 was stimulated, stimulated 3 times, each time counted with trypan blue staining; T cells expressing HER2-41BB, HER2-41BB-L6 or HER2-41BB-TL6 and irradiated SKOV3 cells (Chinese Academy of Sciences cell bank purchased The cells were co-cultured with Xvivo15 medium at a ratio of 1:1 cells, stimulated with SKOV3 supplemented every 5 days, stimulated 3 times, each time counted with trypan blue staining; cell proliferation of each group As shown in Figure 4.
  • Example 6 LRP6 or its truncated body promotes CAR-T cell proliferation and cytokine release in vivo
  • Huh7 cells (1 ⁇ 10 7 cells/cell) were subcutaneously inoculated into NSG mice (purchased by Biotech), and after 14 days, the tumor volume of the mice was measured to be about 100 mm 3 .
  • the mice were divided into 4 groups: T cell group, GPC3-41BB group, GPC3-41BB-L6 group and GPC3-41BB-TL6 group, with 6 rats in each group.
  • cells were injected into the tail vein separately (ie, each group was injected with unmodified T cells, T cells expressing GPC3-41BB, T cells expressing GPC3-41BB-L6, and T cells expressing GPC3-41BB-TL6, respectively.
  • the dose was 3 ⁇ 10 6 /head), and 50 ⁇ l of blood was taken from the tail of the mouse on the 8th day.
  • the expression of human CD8 and CD4 proteins in each group was detected by BD flow.
  • the result is shown in Figure 5A.
  • the results in Fig. 5A indicate that the percentage of cells expressing CD8 and CD4 proteins in the GPC3-41BB-L6 or GPC3-41BB-TL6 group was much higher than that of the other control groups.
  • Huh7 cells (1 ⁇ 10 7 cells/cell) were subcutaneously inoculated into NSG mice (purchased by Biotech), and after 14 days, the tumor volume of the mice was measured to be about 100 mm 3 . At this time, the mice were divided into 5 groups: T cell group, GPC3-41 BB group and GPC3-41 BB-L6 group, with 6 rats in each group.
  • T cells were injected into the T cell group through the tail vein (ie, each injection of unmodified T cells at a dose of 2 ⁇ 10 6 cells/only), and GPC3-41BB-expressing T cells were injected into the GPC3-41BB group (dose was 8 ⁇ 10 5 /only), GPC3-41BB-L6 group was injected with T cells expressing GPC3-41BB-L6 (dose was 3 ⁇ 10 5 /8, 8 ⁇ 10 5 /only or 2 ⁇ 10 6 ) /only), 50 ⁇ l of blood was taken from the tail of the mouse on the 8th day.
  • Example 7 LRP6 or its truncation enhances anti-tumor effect of CAR-T cells
  • Huh7 cells (a dose of 1 ⁇ 10 7 cells/cell) were subcutaneously inoculated into NSG mice (available from Biotech), and after 14 days, the tumor volume of the mice was measured to be about 100 mm 3 . At this time, the mice were divided into 4 groups: T cell group, GPC3-41BB group and GPC3-41BB-L6 group, with 8 rats in each group.
  • T cells were injected into the T cell group through the tail vein (ie, each injection of unmodified T cells at a dose of 8 ⁇ 10 5 cells/only), and GPC3-41BB-expressing T cells were injected into the GPC3-41BB group (dose was 8 ⁇ 10 5 / each), GPC3-41BB-L6 group was injected with T cells expressing GPC3-41BB-L6 (dose was 3 ⁇ 10 5 / 8 or 8 ⁇ 10 5 / only).
  • Example 8 LRP6 or its truncated body promotes long-term survival of CAR-T cells in vivo and prevents tumor recurrence
  • Huh7 cells (a dose of 1 ⁇ 10 7 cells/cell) were subcutaneously inoculated into NSG mice (available from Biotech), and after 14 days, the tumor volume of the mice was measured to be about 100 mm 3 .
  • the mice were divided into three groups, T cell group, GPC3-41BB group and GPC3-41BB-L6 group, with 9 rats in each group.
  • T cells were injected into the T cell group through the tail vein (dose was 2 ⁇ 10 6 cells/only), and GPC3-41BB-expressing T cells (dose was 2 ⁇ 10 6 cells/only) were injected into the GPC3-41BB group.
  • the GPC3-41BB-L6 group was injected with T cells expressing GPC3-41BB-L6 (dose was 2 ⁇ 10 6 /head).
  • mice were subcutaneously inoculated with Huh7 cells (dose of 3 ⁇ 10 7 /head).
  • Huh7 cells dose of 3 ⁇ 10 7 /head.
  • 50 ⁇ l of blood was taken from the tail of the mice, and the expression of human CD8, CD4 and CD3 proteins in each group was detected by BD flow.
  • the mouse bone marrow was taken, and the human CD8 in each group was detected by BD flow. Expression of CD4 and CD3 proteins.

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Abstract

本申请公开了一种促进免疫细胞增殖的方法。所述方法包括以下步骤：使该免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。

Description

[根据细则37.2由ISA制定的发明名称]　促进免疫细胞增殖的方法 技术领域
本申请涉及一种促进免疫细胞增殖的方法。特别地，本申请的方法可以使该免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
背景技术
嵌合抗原受体T细胞免疫疗法(CAR-T，Chimeric Antigen Receptor T-Cell Immunotherapy)，是通过基因工程手段修饰T细胞，使其表达嵌合抗原受体(Chimeric antigen receptor，CAR)并识别肿瘤细胞表面抗原的抗体，以此增强T细胞对肿瘤的特异性杀伤。CAR一般包含特异性识别肿瘤相关抗原(Tumor associated antigen，TAA)的单链可变区结构域(Single chain variable fragment，scFv)、铰链区、跨膜区和胞内信号传导区。
近年来，CAR-T免疫疗法在急性白血病和非霍奇金淋巴瘤的治疗上有显著的疗效，但表达CAR的免疫细胞的增殖能力极为有限。
发明内容
本申请提供了一种经遗传修饰的免疫细胞，以及一种促进免疫细胞增殖的方法。本申请提供的方法可以使该免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。本申请提供的方法还可以促进记忆性免疫细胞产生。本申请提供的方法还可以抑制免疫细胞分化。本申请提供的方法还可以增强免疫细胞释放细胞因子。本申请提供的方法还可以增强免疫细胞对肿瘤杀伤能力。此外，本申请提供的方法能够用于预防受试者中肿瘤复发。本申请还提供了一种治疗有需要的受试者中的肿瘤的方法。本申请还提供了包含所述经遗传修饰的免疫细胞的组合物，以及所述经遗传修饰的免疫细胞和所述组合物在制备药物中的用途。本申请还提供了制备所述经遗传修改的免疫细胞的方法。
一方面，本申请提供了一种促进免疫细胞增殖的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种促进记忆性免疫细胞产生的方法，其包含以下步骤：使免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而促进所述免疫细胞分化为记忆性免疫细胞。
一方面，本申请提供了一种抑制免疫细胞分化的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而抑制所述免疫细胞分化为分化 型免疫细胞。
一方面，本申请提供了一种增强免疫细胞释放细胞因子的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在某些实施方式中，所述细胞因子包含白介素、干扰素和/或肿瘤坏死因子。在某些实施方式中，所述细胞因子包含IL-2、IL4、IL6、IL7、IL10、IL12、TNF-α和/或IFNγ。
一方面，本申请提供了一种增强免疫细胞对肿瘤杀伤能力的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种预防受试者中肿瘤复发的方法，所述方法包含：向易患肿瘤的受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种治疗有需要的受试者中的肿瘤的方法，其包含以下步骤：向所述受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在某些实施方式中，所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
在某些实施方式中，所述方法包括体内方法和体外方法。
在某些实施方式中，所述免疫细胞包含淋巴细胞。在某些实施方式中，所述免疫细胞包含T细胞。在某些实施方式中，所述T细胞包含记忆性干细胞样T细胞(TSCM)和/或中央记忆性T细胞(TCM)。在某些实施方式中，所述TSCM为CCR7 +和/或CD62L +。在某些实施方式中，所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。
在某些实施方式中，所述免疫细胞包含经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。在某些实施方式中，所述经遗传修饰的免疫细胞包括经遗传修饰的T细胞。
在某些实施方式中，所述CAR包含细胞内结构域，所述细胞内结构域包括信号传导结构域和/或共刺激结构域。
在某些实施方式中，所述信号传导结构域包含选自下组的部分：CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。在某些实施方式中，所述信号传导结构域包含SEQ ID NO：18所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，编码所述信号传导结构域的核酸分子包含SEQ ID NO：17所示的核酸序列或与其具备至少80％同源性的核酸序列。
在某些实施方式中，所述共刺激结构域包含选自下组的部分：CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。在某些实施方式中，所述共刺激结构域包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：14和SEQ ID NO：16。在某些实施方式中，编码所述共刺激结构域的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：13和SEQ ID NO：15。
在某些实施方式中，所述CAR包含铰链区。在某些实施方式中，所述铰链区包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。在某些实施方式中，所述铰链区包含SEQ ID NO：31所示的氨基酸序列。在某些实施方式中，编码所述铰链区的核酸分子包含SEQ ID NO：32所示的核苷酸序列。
在某些实施方式中，所述CAR包含跨膜区。在某些实施方式中，所述跨膜区包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。在某些实施方式中，所述跨膜区包含SEQ ID NO：33所示的氨基酸序列。在某些实施方式中，编码所述跨膜区的核酸分子包含SEQ ID NO：34所示的核苷酸序列。
在某些实施方式中，所述CAR包含靶向部分。在某些实施方式中，所述靶向部分包括ScFv。
在某些实施方式中，所述靶向部分特异性结合和/或识别肿瘤抗原。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
在某些实施方式中，所述靶向部分为特异性结合和/或识别CD 19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：46所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：47所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：48所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：50所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：43所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：44所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：45所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中， 所述重链可变区包含SEQ ID NO：49所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：54所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：55所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：56所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：58所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：51所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：52所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：53所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：57所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：70所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：71所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：72所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：74所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：67所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：68所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：69所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：73所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别Mesothelin的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3， 其中所述LCDR1包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：64所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：66所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：59所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：60所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：61所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：65所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别GPC3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：38所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：39所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：40所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：42所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：35所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：36所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：37所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：41所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分包含下述任一项所示的氨基酸序列：SEQ ID NO：2、4、6、8和10或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，编码所述靶向部分的核酸分子包含下述任一项所示的核酸序列：SEQ ID NO：1、3、5、7和9或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述方法还包括以下步骤：分离获得外周血单核细胞PBMC、CD3 +T淋巴细胞、CD8 +T淋巴细胞、CD4 +T淋巴细胞或调节T细胞。
在某些实施方式中，所述方法还包括：向经分离的所述PBMC加入一种或多种T细胞刺激因子。在某些实施方式中，所述T细胞刺激因子选自以下组：B淋巴细胞表面抗原抗体、TNF抗体、细胞内聚酯和抗生素。在某些实施方式中，所述T细胞刺激因子选自以下组：CD3抗体、CD28抗体、4-1BB抗体、CD80抗体、CD86抗体、PHA、PMA和离子霉素。
在某些实施方式中，所述T细胞刺激因子包括CD3抗体，且所述CD3抗体的浓度为1-10000ng/mL。在某些实施方式中，所述T细胞刺激因子包括CD28抗体，且所述CD28抗体的浓度为1-10000ng/mL。
在某些实施方式中，所述方法还包括：向经分离的所述PBMC加入一种或多种细胞因子剂。
在某些实施方式中，所述细胞因子剂包括白细胞介素。
在某些实施方式中，所述白细胞介素包括选自下组的一种或多种：IL2、IL21、IL7和IL15。在某些实施方式中，所述白细胞介素包括IL2，且所述IL2的浓度为0.1-10000U/mL。在某些实施方式中，所述白细胞介素包括IL21，且所述IL21的浓度为0.01-1000ng/mL。在某些实施方式中，所述白细胞介素包括IL7，且所述IL7的浓度为0.01-1000ng/mL。在某些实施方式中，所述白细胞介素包括IL15，且所述IL15的浓度为0.01-1000ng/mL。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白包括选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段来源于人。
在某些实施方式中，所述功能性片段包含具备所述低密度脂蛋白受体相关蛋白活性的所述低密度脂蛋白受体相关蛋白的片段或截短体。在某些实施方式中，所述低密度脂蛋白受体相关蛋白包含低密度脂蛋白受体相关蛋白6和其截短体，和/或低密度脂蛋白受体相关蛋白5和其截短体。在某些实施方式中，所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的胞内区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的胞内区。在某些实施方式中，所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的跨膜区和所述低密度脂蛋白受体相关蛋白6的LDLR区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的跨膜区和所述低密度脂蛋白受体相关蛋白5的LDLR区。在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列：SEQ ID NO：22、24、26和28或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子包含下述任一项所示的核酸序列： SEQ ID NO：21、23、25和27或与其具备至少80％同源性的氨基酸序列。
另一方面，本申请提供了一种经遗传修饰的免疫细胞，其中所述遗传修饰使得该免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在某些实施方式中，所述经遗传修饰的免疫细胞包含淋巴细胞。在某些实施方式中，所述经遗传修饰的免疫细胞包括经遗传修饰的T细胞。在某些实施方式中，所述经遗传修饰的免疫细胞包含经遗传修饰的记忆性干细胞样T细胞(TSCM)和/或经遗传修饰的中央记忆性T细胞(TCM)。在某些实施方式中，所述TSCM为CCR7 +和/或CD62L +。在某些实施方式中，所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。
在某些实施方式中，所述免疫细胞包含经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。
在某些实施方式中，所述CAR包含细胞内结构域，所述细胞内结构域包括信号传导结构域和/或共刺激结构域。在某些实施方式中，所述信号传导结构域包含选自下组的部分：CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。在某些实施方式中，所述信号传导结构域包含SEQ ID NO：18所示的氨基酸序列。在某些实施方式中，编码所述信号传导结构域的核酸分子包含SEQ ID NO：17所示的核酸序列或与其具备至少80％同源性的核酸序列。
在某些实施方式中，所述共刺激结构域包含选自下组的部分：CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。在某些实施方式中，所述共刺激结构域包含下述任一项所示的氨基酸序列：SEQ ID NO：14和SEQ ID NO：16或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，编码所述共刺激结构域的核酸分子包含下述任一项所示的核酸序列：SEQ ID NO：13和SEQ ID NO：15或与其具备至少80％同源性的核酸序列。
在某些实施方式中，所述CAR包含铰链区。在某些实施方式中，所述铰链区包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。在某些实施方式中，所述铰链区包含SEQ ID NO：31所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，编码所述铰链区的核酸分子包含SEQ ID NO：32所示的核酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述CAR包含跨膜区。在某些实施方式中，所述跨膜区包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。在某些实施方式中，所述跨膜区包含SEQ ID NO：33所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些 实施方式中，编码所述跨膜区的核酸分子包含SEQ ID NO：34所示的核酸序列或与其具备至少80％同源性的核酸序列。
在某些实施方式中，所述CAR包含靶向部分。在某些实施方式中，所述靶向部分包括ScFv。在某些实施方式中，所述靶向部分特异性结合和/或识别肿瘤抗原。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
在某些实施方式中，所述靶向部分为特异性结合和/或识别CD 19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：46所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：47所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：48所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：50所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：43所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：44所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：45所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：49所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：54所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：55所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：56所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：58所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：51所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：52所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含 SEQ ID NO：53所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：57所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：70所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：71所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：72所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：74所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：67所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：68所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：69所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：73所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别Mesothelin的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：63所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：64所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：66所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：59所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：60所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：61所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：65所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分为特异性结合和/或识别GPC3的抗体或其抗原结合片 段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：38所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：39所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：40所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述轻链可变区包含SEQ ID NO：42所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：35所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：36所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：37所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在某些实施方式中，所述重链可变区包含SEQ ID NO：41所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在某些实施方式中，所述靶向部分包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：2、4、6、8和10。在某些实施方式中，编码所述靶向部分的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：1、3、5、7和9。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白包括选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段来源于人。
在某些实施方式中，所述功能性片段包含具备所述低密度脂蛋白受体相关蛋白活性的所述低密度脂蛋白受体相关蛋白的片段或截短体。在某些实施方式中，所述低密度脂蛋白受体相关蛋白包含低密度脂蛋白受体相关蛋白6和其截短体，和/或低密度脂蛋白受体相关蛋白5和其截短体。在某些实施方式中，所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的胞内区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的胞内区。在某些实施方式中，所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的跨膜区和所述低密度脂蛋白受体相关蛋白6的LDLR区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的跨膜区和所述低密度脂蛋白受体相关蛋白5的LDLR区。在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。在某些实施方式中， 编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：21、23、25和27。
另一方面，本申请提供了一种组合物，其包含所述的经遗传修饰的免疫细胞。
在某些实施方式中，所述组合物还包含任选地药学上可接受的载体。
另一方面，本申请提供了一种所述经遗传修饰的细胞和/或所述的组合物在制备药物中的用途，其中所述药物用于治疗和/或预防肿瘤。
在某些实施方式中，所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
另一方面，本申请提供了一种制备所述组合物的方法，所述方法包括以下的步骤：使所述经遗传修饰的免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在某些实施方式中，所述的方法包括以下步骤：向所述经遗传修饰的免疫细胞中引入使得所述低密度脂蛋白受体相关蛋白或其片段的表达量上调的载体。在某些实施方式中，所述载体选自以下组：逆转录病毒载体、慢病毒载体和转座子质粒。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
在某些实施方式中，所述经遗传修饰的免疫细胞包含淋巴细胞。在某些实施方式中，所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。在某些实施方式中，所述方法包括分离并激活所述经遗传修饰的免疫细胞的步骤，其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基。
在某些实施方式中，所述T细胞培养基选自以下组中的一种或多种：DMEM培养基、1640培养基、MEM培养基和X-VIVO培养基。
在某些实施方式中，所述的方法还包括向所述经遗传修饰的免疫细胞施用T细胞刺激因子。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书 如下：
图1显示的是本申请所述慢病毒中所述CAR的结构示意图。
图2显示的是本申请所述经遗传修饰的免疫细胞促进记忆性免疫细胞产生的结果。
图3显示的是本申请所述经遗传修饰的免疫细胞抑制免疫细胞分化的结果。
图4显示的是本申请所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段促进本申请所述免疫细胞增殖的结果。
图5A-5B显示的是本申请所述经遗传修饰的免疫细胞增强细胞因子释放的结果。
图6显示的是本申请所述经遗传修饰的免疫细胞抗肿瘤的效果。
图7显示的是本申请所述经遗传修饰的免疫细胞预防肿瘤复发的效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“免疫细胞”通常是指参与免疫应答或与免疫应答相关的细胞。所述免疫细胞可以包括淋巴细胞和各种吞噬细胞。所述免疫细胞也可以包含天然的和经遗传修饰的免疫细胞。在本申请中，所述经遗传修饰的免疫细胞可以表达嵌合抗原受体(CAR)。其中，所述淋巴细胞可以包含T淋巴细胞和B淋巴细胞。在本申请中，所述免疫细胞可以包括T细胞。
在本申请中，术语“记忆型免疫细胞”通常是指具备免疫记忆的免疫细胞。所述免疫记忆可以指对某一抗原产生特异性识别及应答后，再次遇到同一抗原能够产生快速和强烈的免疫应答。在本申请中，所述记忆型免疫细胞可以包括记忆型T细胞。所述记忆型T细胞可以分为记忆性干细胞样T细胞(TSCM)和中央记忆性T细胞(TCM)。
在本申请中，术语“分化型免疫细胞”通常是指具有一定分化程度的免疫细胞。例如，所述分化型免疫细胞可以为具备一定分化程度的T细胞。在本申请中，所述分化型免疫细胞可以通过培养所述免疫细胞使其分化至一定程度而获得。例如，所述分化型免疫细胞可以包含调节性T细胞(Treg)。
在本申请中，术语“调节性T细胞”(regulatory T cell，Treg)通常是指一群具有负调节机体免疫反应的淋巴细胞。所述调节性T细胞的分子标记可以为种转录因子Foxp3 +或CD127 -。在本申请中，所述调节性T细胞可被分为自然存在和诱导产生两类。其中自然存在为CD4 +CD25 +细胞，诱导产生为T R1细胞和T H3细胞。
在本申请中，术语“易患有肿瘤的受试者”通常是指与普通受试者相比，患有肿瘤的概率 增大的受试者。例如，所述易患有肿瘤的受试者可以为曾经患有肿瘤被治愈，但是存在转移、复发的风险的受试者。所述易患有肿瘤的受试者也可以为已经被诊断出存在患有肿瘤的危险因素的受试者。例如，所述危险因素可以包括一些证明与肿瘤相关的基因突变(包括缺失、增加或替换)。在本申请中，所述易患有肿瘤的受试者也可以为长时间接触致癌环境的受试者。例如，所述致癌环境可以包括强辐射、高浓度的致癌物质
在本申请中，术语“遗传修饰”通常是指遗传结构层面上发生的改动或修饰。例如，所述遗传修饰可以为基因水平、转录水平和/或翻译水平的修饰。又例如，所述遗传修饰可以包括生物体(包括其组织、细胞、DNA、mRNA或蛋白质及其片段等)上任何遗传特性的改变。所述的遗传修饰可以包括使所述生物体表达特定的蛋白质或其片段。例如，所述遗传修饰可以包括使所述生物体包含能够表达所述特定的蛋白质或其片段的载体。
在本申请中，术语“T细胞”又称T淋巴细胞，是白细胞的一种亚型，其在细胞介导的免疫中发挥中心作用。T细胞可以通过细胞表面存在的T细胞受体与其他淋巴细胞如B细胞和自然杀伤细胞区分开来。在本申请中，所述T细胞可以包括记忆性干细胞样T细胞(TSCM)和中央记忆性T细胞(TCM)。
在本申请中，术语“记忆性干细胞样T细胞”(T memory stem cells，TSCM)通常是指通常是指处于记忆性T细胞早期分化阶段，具有干细胞特征，具有较强的多向分化潜能的细胞。TSCM细胞在应答抗原刺激后，能够分化为中央记忆性T细胞(central memory T cells，TCM)、效应记忆性T细胞(effector memory T cells，TEM)和效应性T细胞(effector T cells，TEF)。
在本申请中，术语“中央记忆性T细胞”(central memory T cells，TCM)通常是指是幼稚T细胞(Naive T Cell)经过抗原激活后，产生的具有长期记忆性的T细胞。所述TCM的生物标记可以包含CD62L +和CD45RO +。所述中央记忆性T细胞能够通过淋巴屏蔽，回归淋巴结，同时处于被抗原激活的状态。
在本申请中，术语“T细胞受体”通常又称“TCR”，通常是指T细胞特异性识别和结合抗原肽-MHC分子的分子结构。所述T细胞受体可以与CD3分子呈复合物形式存在于T细胞表面。所述TCR可以是一个固定在细胞膜上的异源二聚体，多数由高度易变的α亚基和β亚基通过二硫键连接组成；少数由γ和δ肽链组成。所述TCR可以包含可变区和恒定区，其中所述恒定区可以靠近细胞膜，连接着跨膜区和胞内的末端，而所述可变区负责识别多肽/MHC复合体。
在本申请中，术语“嵌合抗原受体”通常又称“CAR”，通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。本申请中，所述CAR可包含细胞内结构域，所述 细胞内结构域包括信号传导结构域和/或共刺激结构域。在本申请中，CAR的一组多肽可位于相同的多肽链中(例如，包含嵌合融合蛋白)，也可以彼此不连续，例如，可位于不同的多肽链中。在本申请中，参与诱导的信号可以经CD3和ζ链转导入T细胞的细胞质。在本申请中，细胞内结构域可包含一个一级信号传导结合域(例如，CD3-zeta(ζ)的主要信号结构域)。在一个方面，细胞质信号结构域还可包含一种或多种衍生自至少一种共刺激分子的共刺激结构域。例如，所述共刺激结构域可为4-1BB(即CD137)，CD27，ICOS和/或CD28。在本申请中，CAR可包含嵌合融合蛋白，例如，在氨基末端(N-ter)上含有任选的前导序列。其中，所述前导序列任选地在细胞加工过程中将抗原结合结构域(例如，ScFv)切割并将CAR定位于细胞膜。
在本申请中，术语“信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。例如，所述胞内信号传导结构域是所述嵌合抗原受体的胞内信号传导结构域。在本申请中，所述信号传导结构域可以包含选自下组的部分：CD3ζ、CD3δ和CD3ε的信号传导结构域。
在本申请中，术语“共刺激结构域”通常是指CAR中穿过细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。在本申请中，所述共刺激结构域可以包含选自下组的部分：CD27、CD28和4-1BB的共刺激结构域。
在本申请中，术语“铰链区”通常是指抗原结合区和免疫细胞Fc受体(FcR)结合区之间的连接区。例如，所述铰链区可以为免疫球蛋白中重链CH1和CH2功能区之间的区域。在本申请中，所述铰链区可以为位于scFv和T细胞膜之间的一段区域。所述铰链区可以来源于IgG1或IgG4，还可以来源于IgD或CD8。在本申请中，铰链区可以包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。
在本申请中，术语“跨膜区”通常是指跨膜区连接胞外抗原结合域和胞内信号域，一般由二聚体膜蛋白组成，主要包括CD3ζ、CD4、CD8、CD28等，能将CAR结构锚定于T细胞膜上。跨膜区不同的设计能影响导入的CAR基因的表达。在本申请中，所述跨膜区可以包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。
在本申请中，术语“单链抗体”(ScFv)通常是指由所述重链可变区和所述轻链可变区通过连接子连接而成的抗体。在本申请中，所述连接子可以为连接肽。
在本申请中，术语“肿瘤抗原”通常是指在肿瘤细胞内或由肿瘤细胞产生的抗原物质，其可能具有在宿主中触发免疫应答的能力。例如，肿瘤抗原可以是构成肿瘤细胞的一部分并且能够诱导肿瘤特异性细胞毒性T淋巴细胞的蛋白质，多肽，肽或其片段。在一些实施方案中， 术语“肿瘤抗原”还可以指在癌细胞上专门或优先或差异表达和/或与癌细胞相关的生物分子(例如，蛋白质，碳水化合物，糖蛋白等)从而提供癌症优先或特异性的靶标。例如，与生物体中的任何其他细胞相比，优先表达可以为常规的优先表达，或者在生物体的特定区域内的优先表达(例如在特定器官或组织内)。在本申请中，所述肿瘤抗原可以包括B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。
在本申请中，术语“B淋巴细胞表面抗原”通常是指B淋巴细胞在不同阶段产生的位于B淋巴细胞表面的抗原。例如，所述B淋巴细胞表面抗原可以包括CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD37、CD38、CD39和CD40。在本申请中，B淋巴细胞表面抗原可以包括CD19。
在本申请中，术语“CD19”通常是指是指分化群19蛋白，其是可在白血病前体细胞上检测的抗原决定簇。人CD19在UniProt/Swiss-Prot的登录号为P15391，编码人CD19的核苷酸序列在GenBank的登录号为NM_001178098。在本申请中，CD19还可以包括包含突变的蛋白质或其功能性片段，例如全长野生型CD19的点突变、片段、插入、缺失和剪接变体。
在本申请中，术语“TNF家族成员”通常是指属于TNF(肿瘤坏死因子Tumor Necrosis Factor)家族的成员。TNF家族成员可以包括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)和TNFSF18。在本申请中，所述TNF家族成员可以包括BCMA和4-1BB。
在本申请中，术语“BCMA”通常是指B细胞成熟抗原(B Cell Maturation Antigen，BCMA，CD269)。BCMA是肿瘤坏死因子受体(TNF)超家族的成员，可结合B细胞激活因子(BAFF)和增殖诱导配体(APRIL)。BCMA常见于多发性骨髓瘤患者的浆细胞表面。人BCMA在GenBank的登录号为BAB60895.1。
术语“CD137蛋白”，又名4-1BB或TNFRS9，通常是指肿瘤坏死因子受体超家族(TNFRS)的一种跨膜蛋白质，是激活诱导的共刺激分子是免疫应答的重要调节剂。研究表明CD137激动型单克隆抗体在许多模型中增加共刺激分子表达，并且显著增强细胞溶解性T淋巴细胞应答，发挥抗肿瘤作用(参见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)。人CD137在GenBank的登录号为NP_001552.2。
在本申请中，术语“HER家族成员”通常是指属于HER(人表皮生长因子human epidermal  growth factor receptor)家族的成员。HER家族成员可以包括EGFR(ErbB-1)、HER2/c-neu(ErbB-2)、Her3(ErbB-3)以及Her4(ErbB-4)。在本申请中，所述HER家族成员可以包括HER2。
在本申请中，术语“HER2”通常是指人类HER2蛋白，其属于HER家族成员。例如，参见Semba et al.，PNAS(USA)82：6497-6501(1985)和Yamamoto et al.，Nature 319：230-234(1986)。人HER2的GenBank登录号可以包括XP_024306409.1。
在本申请中，术语“GPC家族成员”通常是指磷脂酰肌醇蛋白聚糖(glypican)，在哺乳动物中鉴定存在6种glypican，分别称为GPC1至GPC6。癌症中glypicans存在异常表达，所述癌症包括人肝细胞癌、卵巢癌、间皮瘤、胰腺癌、神经胶质瘤和乳腺癌。在本申请中，所述GPC家族成员可以包括GPC3。
在本申请中，术语“GPC3”通常是指由磷脂酰肌醇蛋白聚糖3(NCBI数据库基因ID：2719)编码的蛋白质，其是肝癌的早期标志物。GPC3在肝细胞癌中高度表达，并在早期肝细胞癌患者组织中检测到。人GPC3的GenBank登录号可以为AAB87062.1。
在本申请中，术语“Mesothelin”(间皮素，或缩写为MSLN)通常是指一种肿瘤分化抗原，通常存在于胸膜、腹膜和心包膜衬里的间皮细胞上。其在癌症中高度表达，所述癌症包括恶性间皮瘤、胰腺癌、卵巢癌和肺腺癌。人Mesothelin的GenBank登录号可以为AAH09272.1。
在本申请中，术语“外周血单核细胞”通常是指外周血中具有单个核的细胞(Peripheral blood mononuclear cell，PBMC)。所述外周血单核细胞可以包括淋巴细胞和单核细胞。在本申请中，分离所述外周血单核细胞可使用Ficoll-hypaque(葡聚糖-泛影葡胺)密度梯度离心法，根据血液中各成分的比重差异进行分离。
在本申请中，术语“T细胞激活因子”通常是指促进T细胞活化、增殖的物质。在本申请中，所述T细胞激活因子可以包括B淋巴细胞表面抗原抗体、TNF抗体、细胞内聚酯和/或抗生素。在本申请中，所述T细胞刺激因子可选自以下组：CD3抗体、CD28抗体、4-1BB抗体、CD80抗体、CD86抗体、PHA、PMA和离子霉素。
在本申请中，术语“B淋巴细胞表面抗原抗体”通常是指与B淋巴细胞表面抗原特异性结合的抗体。在本申请中，B淋巴细胞表面抗原抗体可以包括CD3抗体、CD28抗体、CD80抗体和CD86抗体。
在本申请中，术语“TNF抗体”通常是指与TNF家族成员特异性结合的抗体。在本申请中，所述TNF抗体可以包括4-1BB抗体。
在本申请中，术语“细胞内聚酯”通常是指微生物合成的、在细胞内以包涵体形式存在的 天然高分子生物材料。细胞内聚酯具有良好的生物学性能。在本申请中，细胞内聚酯可以包括聚羟基脂肪酸酯(PHA)。
在本申请中，术语“抗生素”通常是指由微生物或动植物所产生的具有抗病原体或其它活性的代谢产物，其能干扰其他生活细胞发育功能及发挥作用。在本申请中，所述抗生素可以包括β-内酰胺类、氨基糖苷类、酰胺醇类、大内环酯类、多肽类、硝基咪唑类和四环素类。例如，所述抗生素可以包括离子霉素。
在本申请中，术语“PHA”通常是指聚羟基脂肪酸酯，其属于由多种细菌合成的细胞内聚酯，在生物体内可以以细胞质内不连续的包涵体的形式而存在。PHA可以具有类似于合成塑料的物理、化学特性，以及生物可降解性、生物相容性、光学活性、压电性、气体相隔性等。
在本申请中，术语“PMA”通常是指丙二醇甲醚醋酸酯(Phorbol-12-myristate-13-acetate)。
在本申请中，术语“CD3抗体”通常是指特异性结合CD3的抗体或其抗原结合片段。CD3可以为T细胞膜上重要的分化抗原，可以传递T细胞活化的信号。所述CD3抗体可以为huOKT3g1或HuM291。
在本申请中，术语“CD28抗体”通常是指特异性结合CD28的抗体或其抗原结合片段。人CD28定位于2q33，与CTLA4有相似的外显子和内含子，两者的配体是B7家族，包括B7-1(CD80)和B7-2(CD86)。
在本申请中，术语“细胞因子”通常是指由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。所述细胞因子可具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。在本申请中，所述细胞因子可包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子和生长因子。例如，所述细胞因子可以为白细胞介素。
在本申请中，术语“白细胞介素”通常是指能够促进T和/或B淋巴细胞和/或造血细胞的发育和分化的分泌蛋白或信号分子。白介素可以由辅助CD4T淋巴细胞，以及通过单核细胞，巨噬细胞和内皮细胞合成。在本申请中，术语“白细胞介素”可以包括全长白介素或片段(例如截短形式)或其变体，其基本上保持相应野生型白细胞介素的生物学活性(例如，具有生物活性至少80％，至少90％，至少95％，至少98％，至少99％，或甚至至少100％的相应野生型白细胞介素的生物活性)。本文所用的白细胞介素可以来自任何哺乳动物物种。在某些实施方式中，白细胞介素来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。在某些实施方式中，白细胞介素可以是突变形式。例如，在本申请 中，所述细胞因子包括选自下组的一种或多种：IL2、IL21、IL7和IL15。
在本申请中，术语“IL2”通常是指T细胞生长因子，TCGF。其由T细胞产生，以自分泌和旁分泌的方式发挥效益，能够活化T细胞，促进细胞因子的产生；刺激NK细胞增殖，诱导LAK细胞产生；促进B细胞增殖和分泌抗体，还能激活巨噬细胞。
在本申请中，术语“IL21”与IL-2、IL-4、IL-15空间结构同源，通常可以促进骨髓NK细胞的增殖与分化，与抗CD40抗体协同刺激B细胞的增殖，与抗CD3抗体协同刺激T细胞的增殖。
在本申请中，术语“IL7”通常是指由骨髓基质细胞分泌的糖蛋白，其基因位于第8号染色体。IL7的靶向细胞为淋巴细胞，尤其是能够促进来自人或小鼠骨髓的B祖细胞、胸腺细胞及外周成熟的T细胞的生长。在较高浓度时，IL-7还能增强巨噬细胞的细胞毒活性，诱导单核细胞分泌细胞因子。
在本申请中，术语“IL15”通常是指由活化的单核-巨噬细胞、表皮细胞和成纤维细胞等多种细胞产生，与IL2有很多相似之处。IL15属于IL2家族成员，通过改变Bcl-1家族成员(如Bcl-2和Bcl-XL)的表达来调节T和NK细胞的激活和增殖，也可诱导B细胞增殖。
在本申请中，术语“低密度脂蛋白受体相关蛋白”(Low density lipoprotein receptor-related protein，LRP)通常是指一种包含839个氨基酸(除去21个氨基酸信号肽后)的镶嵌蛋白。嵌入在LDL(Low density lipoprotein)颗粒的外部磷脂层中属于一种内吞性受体，可以介导富含胆固醇的LDL的内吞作用，是低密度脂蛋白受体(Low density lipoprotein receptor，LDLR)基因家族中的一员。所述LRP在支气管上皮细胞和肾上腺和皮质组织中表达最显著。在本申请中，所述低密度脂蛋白受体相关蛋白可以包括选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12或截短体。
在本申请中，术语“低密度脂蛋白受体相关蛋白6”(LRP-6)和“低密度蛋白受体相关蛋白5”(LRP-5)通常是指低密度脂蛋白受体(LDLR)家族中独特的亚群。人LRP-6在UniProt中的登录号为O75581。人LRP-5在UniProt中的登录号为O75197。
在本申请中，术语“截短体”(Truncated protein)通常是指截短的蛋白质。所述截短体可以通过蛋白质水解或操作结构基因消除蛋白质的N-或C-末端部分而得到。或者，所述截短体可以通过无义突变而导致结构基因中存在终止密码子而提前终止翻译而得到。
在本申请中，术语“胞内区”通常是指蛋白质位于细胞膜内的结构域。在本申请中，所述胞内域可以指所述低密度脂蛋白受体相关蛋白细胞膜内的结构域。在本申请中，所述胞内区可包括SEQ NO.24中的第24位至第243位的序列或与其具备至少80％同源性的氨基酸序列， 或者，包括SEQ NO.28中的第24位至第231位的序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，术语“跨膜区”通常是指蛋白质中横跨细胞膜的结构域。在本申请中，所述跨膜区可以指所述低密度脂蛋白受体相关蛋白横跨细胞膜的结构域。所述跨膜区可以由23个疏水性氨基酸所组成。该结构域主要为LDLR在细胞膜上的结合提供锚定的功能。在本申请中，所述跨膜区可以包括SEQ NO.24中的第1位至第23位的序列或与其具备至少80％同源性的氨基酸序列，或者，包括SEQ NO.28中的第1位至第23位的序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，术语“LDLR区”通常是指所述低密度脂蛋白受体相关蛋白在跨膜区以外靠近N段的结构域。该结构域该结构域可以具有增强Wnt信号的功能。在本申请中，所述LDLR区可包括SEQ NO.22中的第5位至第119位或与其具备至少80％同源性的氨基酸序列，或SEQ NO.26中的第4位至第119位的序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，术语“核糖体跳跃位点”又叫内部核糖体进入位点(Internal ribosome entry site，IRES)，通常是指位于mRNA序列中部为转译起始而备用的一段核苷酸序列。所述核糖体跳跃位点可以允许以不依赖于帽子的方式起始翻译。IRES通常位于5’UTR中。在本申请中，所述核糖体跳跃位点可包括SEQ NO.29中的第1位至第578位序列。
在本申请中，术语“2A序列”通常是指一段不依赖于蛋白酶的自剪切氨基酸序列。所述2A序列可以有助于转录产生两种蛋白。在本申请中，所述2A序列可以包括SEQ NO.75中的第1位至第54位的序列。
在本申请中，术语“药学上可接受的载体”可包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，术语“逆转录病毒载体”通常是指一种RNA病毒，其可在受染细胞内逆转录产生DNA互补链，并以此DNA单链作为模板合成第二条DNA链，并掺入细胞基因组DNA中。所述逆转录病毒载体可利用宿主细胞的酶自行转录与复制RNA合成蛋白，再包装病毒，从胞内释放，成为感染性病毒。所述逆转录病毒的转导效率高，能够有效提高基因的转染率。
在本申请中，术语“慢病毒载体”是指以HIV-1(人类免疫缺陷I型病毒)为基础发展起来的基因治疗载体。所述慢病毒载体可以对分裂细胞和非分裂细胞均具有感染能力。其可以有效地感染包括神经元细胞、肝细胞等几乎所有的哺乳动物细胞，感染效率高。慢病毒可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达。
在本申请中，术语“转座子质粒”通常是指存在于染色体DNA上可自主复制和位移的基本单位。所述转座子质粒可以通过切割、重新整合等一系列过程从基因组的一个位置“跳跃”到另一个位置。
在本申请中，术语“肿瘤”通常是指机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的新生物(neogrowth)，因为这种新生物多呈占位性块状突起，也称赘生物(neoplasm)。在本申请中，所述肿瘤可以包括实体瘤和非实体瘤。在本申请中，所述肿瘤可以包括肝癌、肺癌、白血病和间皮瘤。
在本申请中，术语“显著增加”通常是指水平(例如蛋白表达量、细胞数量)增加，P＜0.05、P＜0.04、P＜0.03、P＜0.02、P＜0.01、P＜0.005或P＜0.001。又例如，所述显著增加可以指增加了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多。
在本申请中，术语“基本上未增加”通常是指水平(例如蛋白表达量、细胞数量)与原来一致，或增加了3％或更少、增加了2％或更少、增加了1％或更少、增加了0.5％或更少、增加了0.4％或更少、增加了0.3％或更少、增加了0.2％或更少、增加了0.1％或更少。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
低密度脂蛋白受体相关蛋白或其片段
在本申请中，所述低密度脂蛋白受体相关蛋白可包括选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在本申请中，所述低密度脂蛋白受体相关蛋白或其片段可以来源于哺乳动物。例如，可以来源于人、猕猴、大鼠和小鼠。
在本申请中，所述功能性片段可包含具备所述低密度脂蛋白受体相关蛋白活性的所述低密度脂蛋白受体相关蛋白的片段或截短体。例如，所述低密度脂蛋白受体相关蛋白可以包含低密度脂蛋白受体相关蛋白6和其截短体，和/或，低密度脂蛋白受体相关蛋白5和其截短体。
在本申请中，所述低密度脂蛋白受体相关蛋白6的截短体可以包含所述低密度脂蛋白受体相关蛋白6的胞内区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体可以包含所述低密度脂蛋白受体相关蛋白5的胞内区。又例如，所述低密度脂蛋白受体相关蛋白6的截短体可以包含所述低密度脂蛋白受体相关蛋白6的跨膜区和所述低密度脂蛋白受体相关蛋白6的LDLR区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体可以包含所述低密度脂蛋白受 体相关蛋白5的跨膜区和所述低密度脂蛋白受体相关蛋白5的LDLR区。
在本申请中，所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
在本申请中，编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：21、23、25和27。
经遗传修饰的免疫细胞
本申请提供了一种经遗传修饰的免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在本申请中，所述经遗传修饰的免疫细胞可以包含淋巴细胞。在本申请中，所述经遗传修饰的免疫细胞包含包括经遗传修饰的T细胞。在本申请中，所述经遗传修饰的免疫细胞包含包含经遗传修饰的记忆性干细胞样T细胞(TSCM)和/或经遗传修饰的中央记忆性T细胞(TCM)。在本申请中，所述TSCM包含为CCR7 +和/或CD62L +。在本申请中，所述TSCM还包含具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。
在本申请中，所述免疫细胞可以包含经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。
在本申请中，所述CAR可以包含细胞内结构域，所述细胞内结构域可以包括信号传导结构域和/或共刺激结构域。
例如，所述信号传导结构域可以包含选自下组的部分：CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。例如，所述信号传导结构域可以包含SEQ ID NO：18所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列，编码所述信号传导结构域的核酸分子可以包含SEQ ID NO：17所示的核酸序列或与其具备至少80％同源性的核酸序列。
例如，所述共刺激结构域可以包含选自下组的部分：CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。例如，所述共刺激结构域可以包含下述任一项所示的氨基酸序列：SEQ ID NO：14和SEQ ID NO：16或与其具备至少80％同源性的氨基酸序列，编码所述共刺激结构域的核酸分子可以包含下述任一项所示的核酸序列：SEQ ID NO：13和SEQ ID NO：15或与其具备至少80％同源性的核酸序列。
在本申请中，所述CAR可以包含铰链区。例如，所述铰链区可以包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。例如，所述铰链区可以包含SEQ ID NO：31 所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列，编码所述铰链区的核酸分子可以包含SEQ ID NO：32所示的核酸序列或与其具备至少80％同源性的核酸序列。
在本申请中，所述CAR可以包含跨膜区。例如，所述跨膜区可以包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。例如，所述跨膜区可以包含SEQ ID NO：33所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列，编码所述跨膜区的核酸分子可以包含SEQ ID NO：34所示的核酸序列或与其具备至少80％同源性的核酸序列。
在本申请中，所述CAR可以包含靶向部分。在本申请中，所述靶向部分可以包括抗体或抗原结合片段。所述抗原结合片段可以选自下组：Fab、Fab’、F(ab)2、F(ab’)2、Fv和ScFv片段。例如，所述靶向部分可以为ScFv。
在本申请中，所述靶向部分可以特异性结合和/或识别肿瘤抗原。例如，所述靶向部分可以特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。又例如，所述靶向部分可以特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
在本申请中，所述靶向部分可以为特异性结合和/或识别CD19的抗体或其抗原结合片段。所述抗体或其抗原结合片段可以包含轻链可变区，所述轻链可变区可以包含LCDR1-LCDR3，其中所述LCDR1可以包含SEQ ID NO：46所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2可以包含SEQ ID NO：47所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3可以包含SEQ ID NO：48所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述轻链可变区可以包含SEQ ID NO：50所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在本申请中，所述抗体或其抗原结合片段可以包含重链可变区，所述重链可变区可以包含HCDR1-HCDR3，其中所述HCDR1可以包含SEQ ID NO：43所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2可以包含SEQ ID NO：44所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3可以包含SEQ ID NO：45所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述重链可变区可以包含SEQ ID NO：49所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，所述靶向部分为特异性结合和/或识别BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段可以包含轻链可变区，所述轻链可变区可以包含LCDR1-LCDR3，其中所述LCDR1可以包含SEQ ID NO：54所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2可以包含SEQ ID NO：55所示的氨基酸序列或与其具备至少80％ 同源性的氨基酸序列；所述LCDR3可以包含SEQ ID NO：56所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述轻链可变区可以包含SEQ ID NO：58所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在本申请中，所述抗体或其抗原结合片段可以包含重链可变区，所述重链可变区可以包含HCDR1-HCDR3，其中所述HCDR1可以包含SEQ ID NO：51所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2可以包含SEQ ID NO：52所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3可以包含SEQ ID NO：53所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述重链可变区可以包含SEQ ID NO：57所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，所述靶向部分为特异性结合和/或识别HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段可以包含轻链可变区，所述轻链可变区可以包含LCDR1-LCDR3，其中所述LCDR1可以包含SEQ ID NO：70所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2可以包含SEQ ID NO：71所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3可以包含SEQ ID NO：72所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述轻链可变区可以包含SEQ ID NO：74所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在本申请中，所述抗体或其抗原结合片段可以包含重链可变区，所述重链可变区可以包含HCDR1-HCDR3，其中所述HCDR1可以包含SEQ ID NO：67所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2可以包含SEQ ID NO：68所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3可以包含SEQ ID NO：69所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述重链可变区可以包含SEQ ID NO：73所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，所述靶向部分为特异性结合和/或识别Mesothelin的抗体或其抗原结合片段，所述抗体或其抗原结合片段可以包含轻链可变区，所述轻链可变区可以包含LCDR1-LCDR3，其中所述LCDR1可以包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2可以包含SEQ ID NO：63所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3可以包含SEQ ID NO：64所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述轻链可变区可以包含SEQ ID NO：66所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述抗体或其抗原结合片段可以包含重链可变区，所述重链可变区可以包含HCDR1-HCDR3，其中所述HCDR1可以包含SEQ ID NO：59所示 的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2可以包含SEQ ID NO：60所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3可以包含SEQ ID NO：61所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述重链可变区可以包含SEQ ID NO：65所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，所述靶向部分为特异性结合和/或识别GPC3的抗体或其抗原结合片段，所述抗体或其抗原结合片段可以包含轻链可变区，所述轻链可变区可以包含LCDR1-LCDR3，其中所述LCDR1可以包含SEQ ID NO：38所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2可以包含SEQ ID NO：39所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3可以包含SEQ ID NO：40所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述轻链可变区可以包含SEQ ID NO：42所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。在本申请中，所述抗体或其抗原结合片段可以包含重链可变区，所述重链可变区可以包含HCDR1-HCDR3，其中所述HCDR1可以包含SEQ ID NO：35所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2可以包含SEQ ID NO：36所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3可以包含SEQ ID NO：37所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。所述重链可变区可以包含SEQ ID NO：41所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
在本申请中，所述靶向部分可以包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：2、4、6、8和10。在本申请中，编码所述靶向部分的核酸分子可以包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：1、3、5、7和9。
本申请中，CDR的氨基酸位点的起讫以Kabat的方式计算。
在本申请中涉及的蛋白质、多肽、氨基酸序列和/或核酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。和/或编码与该所述蛋白质或多肽具备相同或类似功能的变体或同源物的核酸序列。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，特异性结合GPC3蛋白的抗体或其片段，或者，所述低密度脂蛋白受体相关蛋白或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改 变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力，或者，所述低密度脂蛋白受体相关蛋白或其片段的生物学功能)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合GPC3蛋白的抗体或其片段，或者，所述低密度脂蛋白受体相关蛋白或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，所述CAR可以从N端到C端依次包含所述靶向部分、所述铰链区、所述跨膜区、所述共刺激结构域和所述信号传导结构域。
在本申请中，编码所述CAR的多核苷酸分子可以从5’端到3’端依次包含编码所述靶向部分的核苷酸序列、编码所述铰链区的核苷酸序列、编码所述跨膜区的核苷酸序列、编码所述跨膜区的核苷酸序列、编码所述共刺激结构域的核苷酸序列和编码所述信号传导结构域的核苷酸序列。
在本申请中，编码所述CAR的多核苷酸分子还可以包含前导序列。例如，编码所述前导 序列的核酸分子可以包含SEQ ID NO：30所示的核酸序列或与其具备至少80％同源性的核酸序列。编码所述前导序列的核酸分子可以位于编码所述靶向部分的核苷酸序列的5’端。
在本申请中，所述经遗传修饰的免疫细胞可以表达所述低密度脂蛋白受体相关蛋白或其片段。例如，所述经遗传修饰的免疫细胞可以包含能够表达所述低密度脂蛋白受体相关蛋白或其片段的载体。所述载体中可包含编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子。例如，所述多核苷酸分子可以包含下述任一项所示的核酸序列：SEQ ID NO：21、23、25和27或与其具备至少80％同源性的核酸序列。又例如，所述载体可以选自下组：逆转录病毒载体、慢病毒载体和/或转座子质粒。
在本申请中，所述经遗传修饰的免疫细胞可以表达所述嵌合抗原受体(CAR)。例如，所述经遗传修饰的免疫细胞可以包含能够表达所述所述嵌合抗原受体(CAR)的载体。所述载体中可包含编码所述所述嵌合抗原受体(CAR)的核苷酸分子。又例如，所述载体可以选自下组：逆转录病毒载体、慢病毒载体和/或转座子质粒。
在本申请中，所述能够表达所述低密度脂蛋白受体相关蛋白或其片段的载体与所述能够表达所述嵌合抗原受体(CAR)的载体可以为同一个载体，也可以为不同的载体，只要这一个或多个载体可以表达所述低密度脂蛋白受体相关蛋白或其片段和所述嵌合抗原受体(CAR)，从而使所述经遗传修饰的免疫细胞同时具备所述低密度脂蛋白受体相关蛋白或其片段和所述嵌合抗原受体(CAR)即可。
例如，所述能够表达所述低密度脂蛋白受体相关蛋白或其片段的载体与所述能够表达所述嵌合抗原受体(CAR)的载体可以为同一个载体。在该载体中，所述编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子和所述编码所述所述嵌合抗原受体(CAR)的核苷酸分子可以位于同一个表达框中。例如，所述编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子可以位于所述编码所述所述嵌合抗原受体(CAR)的核苷酸分子的3’端。
在本申请中，所述编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子可以与所述编码所述所述嵌合抗原受体(CAR)的核苷酸分子直接或间接连接。例如，所述的间接连接可以通过连接序列进行连接。所述连接序列的5’端可以与所述编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子的3’端连接，且所述连接序列的3’端可以与所述编码所述所述嵌合抗原受体(CAR)的核苷酸分子的5’端连接。在本申请中，所述连接序列可以包含下述任一项所示的核酸序列：SEQ ID NO：19和29或与其具备至少80％同源性的核酸序列。
在本申请中，所述经遗传修饰的免疫细胞所表达得到的所述低密度脂蛋白受体相关蛋白或其片段和所述嵌合抗原受体(CAR)可以是两个独立的蛋白。即，两者没有任何的相互连 接关系而形成任何形式的二(多)聚体或蛋白质复合物。然而，所述经遗传修饰的免疫细胞所表达得到的所述低密度脂蛋白受体相关蛋白或其片段和所述嵌合抗原受体(CAR)也可以是相互连接的。例如，在某些情况下，翻译形成的这两种蛋白没有被切割完全，所述低密度脂蛋白受体相关蛋白或其片段和所述嵌合抗原受体形成了复合体。
制备方法
本申请提供了一种制备所述经遗传修饰的免疫细胞的方法，所述方法包括以下的步骤：使所述经遗传修饰的免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
本申请还提供了一种制备所述组合物的方法，所述方法包括以下的步骤：使所述经遗传修饰的免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在本申请中，所述方法可以还包括以下步骤：分离获得外周血单核细胞PBMC、CD3 +T淋巴细胞、CD8 +T淋巴细胞、CD4 +T淋巴细胞或调节T细胞。
在本申请中，所述方法可以包括分离并激活所述经遗传修饰的免疫细胞的步骤，其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基。
在本申请中，所述T细胞培养基可以选自以下组中的一种或多种：DMEM培养基、1640培养基、MEM培养基和X-VIVO培养基。
在本申请中，所述方法还可以包括以下的步骤：向经分离的所述外周血单核细胞PBMC加入一种或多种T细胞刺激因子。
例如，所述T细胞刺激因子可以选自以下组：B淋巴细胞表面抗原抗体、TNF抗体、细胞内聚酯和抗生素。又例如，所述T细胞刺激因子可以选自以下组：CD3抗体、CD28抗体、4-1BB抗体、CD80抗体、CD86抗体、PHA、PMA和离子霉素。
在本申请中，所述T细胞刺激因子可以包括CD3抗体，且所述CD3抗体的浓度可以为1-10000ng/mL。例如，所述CD3抗体的浓度可以为1-9000ng/mL、1-5000ng/mL、1-4000ng/mL、1-3000ng/mL、1-1000ng/mL、1-500ng/mL、1-400ng/mL、1-300ng/mL、1-200ng/mL、1-100ng/mL、1-50ng/mL、1-40ng/mL、1-30ng/mL、1-20ng/mL、1-10ng/mL或1-5ng/mL。在本申请中，所述T细胞刺激因子可以包括CD28抗体，且所述CD28抗体的浓度可以为1-10000ng/mL。例如，所述CD28抗体的浓度可以为1-9000ng/mL、1-5000ng/mL、1-4000ng/mL、1-3000ng/mL、1-1000ng/mL、1-500ng/mL、1-400ng/mL、1-300ng/mL、1-200ng/mL、1-100ng/mL、1-50ng/mL、1-40ng/mL、1-30ng/mL、1-20ng/mL、1-10ng/mL或1-5ng/mL。
在本申请中，所述方法还可以包括：向经分离的所述PBMC加入一种或多种细胞因子剂。在本申请中，所述细胞因子剂可以包括白细胞介素。例如，所述白细胞介素可以包括选自下 组的一种或多种：IL2、IL21、IL7和IL15。
在本申请中，所述白细胞介素可以包括IL2，且所述IL2的浓度可以为0.1-10000U/mL。例如，所述IL2的浓度可以为0.1-8000U/mL、0.1-6000U/mL、0.1-4000U/mL、0.1-2000U/mL、5-2000U/mL、5-1900U/mL、5-1800U/mL、5-1700U/mL、5-1600U/mL、5-1500U/mL、5-1400U/mL、5-1300U/mL、5-1200U/mL、5-1100U/mL、5-1000U/mL、5-900U/mL、5-800U/mL、5-700U/mL、5-600U/mL、5-500U/mL、5-400U/mL、5-300U/mL、5-200U/mL、5-500U/mL、5-400U/mL、5-300U/mL、5-200U/mL、5-100U/mL、5-50U/mL、5-40U/mL、5-30U/mL、5-20U/mL或5-10U/mL。
在本申请中，所述白细胞介素可以包括IL21，且所述IL21的浓度可以为0.01-1000ng/mL。例如，所述IL21的浓度可以为0.01-800ng/mL、0.01-600ng/mL、0.01-400ng/mL、0.01-200ng/mL、0.01-100ng/mL、0.1-100ng/mL、0.1-90ng/mL、0.1-80ng/mL、0.1-70ng/mL、0.1-60ng/mL、0.1-50ng/mL、0.1-40ng/mL、0.1-30ng/mL、0.1-20ng/mL、0.1-10ng/mL或0.1-5ng/mL。
在本申请中，所述白细胞介素可以包括IL7，且所述IL7的浓度可以为0.01-1000ng/mL。例如，所述IL7的浓度可以为0.01-800ng/mL、0.01-600ng/mL、0.01-400ng/mL、0.01-200ng/mL、0.01-100ng/mL、0.1-100ng/mL、0.1-90ng/mL、0.1-80ng/mL、0.1-70ng/mL、0.1-60ng/mL、0.1-50ng/mL、0.1-40ng/mL、0.1-30ng/mL、0.1-20ng/mL、0.1-10ng/mL或0.1-5ng/mL。
在本申请中，所述白细胞介素可以包括IL15，且所述IL15的浓度可以为0.01-1000ng/mL。例如，所述IL15的浓度可以为0.01-800ng/mL、0.01-600ng/mL、0.01-400ng/mL、0.01-200ng/mL、0.01-100ng/mL、0.1-100ng/mL、0.1-90ng/mL、0.1-80ng/mL、0.1-70ng/mL、0.1-60ng/mL、0.1-50ng/mL、0.1-40ng/mL、0.1-30ng/mL、0.1-20ng/mL、0.1-10ng/mL或0.1-5ng/mL。
促进免疫细胞增殖、促进记忆性免疫细胞产生、抑制免疫细胞分化、增强释放细胞因子 和增强对肿瘤的杀伤能力
一方面，本申请提供了一种促进免疫细胞增殖的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种促进记忆性免疫细胞产生的方法，其包含以下步骤：使免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而促进所述免疫细胞分化为记忆性免疫细胞。
一方面，本申请提供了一种抑制免疫细胞分化的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而抑制所述免疫细胞分化为分化型免疫细胞。
一方面，本申请提供了一种增强免疫细胞释放细胞因子的方法，其包含以下步骤：使所 述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在本申请中，所述细胞因子可以包含白介素、干扰素和/或肿瘤坏死因子。在本申请中，所述细胞因子可以包含IL-2、IL4、IL6、IL7、IL10、IL21、TNF-α和/或IFNγ。
一方面，本申请提供了一种增强免疫细胞对肿瘤杀伤能力的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种预防受试者中肿瘤复发的方法，所述方法包含：向易患肿瘤的受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
一方面，本申请提供了一种治疗有需要的受试者中的肿瘤的方法，其包含以下步骤：向所述受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
在本申请中，所述肿瘤可以选自肝癌、肺癌、白血病和间皮瘤。在本申请中，所述方法可以包括体内方法和体外方法。
在本申请中，所述表达量上调可为使所述免疫细胞中所述低密度脂蛋白受体相关蛋白或其片段的表达量与对照免疫细胞的表达量相比显著上调(例如，上调了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中所述低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述促进记忆性免疫细胞产生可以为使所述免疫细胞产生记忆性免疫细胞的数量与对照免疫细胞所产生记忆性免疫细胞的数量相比显著上调(例如，上调了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述抑制免疫细胞分化可以为使所述免疫细胞产生分化型免疫细胞的数量与对照免疫细胞所产生记忆性免疫细胞的数量相比显著下调(例如，下调了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述增强免疫细胞释放细胞因子可以为使所述免疫细胞释放细胞因子的数量与对照免疫细胞所释放的细胞因子的数量相比显著上调(例如，上调了5％或更多、10％或 更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述增强免疫细胞对肿瘤杀伤能力可以为使所述免疫细胞对肿瘤的杀伤能力与对照免疫细胞对肿瘤的杀伤能力相比显著提高(例如，提高了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述预防受试者中肿瘤复发可以为使所述免疫细胞针对预防受试者中肿瘤复发能力与对照免疫细胞针对预防受试者中肿瘤复发能力相比显著提高(例如，提高了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
在本申请中，所述治疗有需要的受试者中的肿瘤可以为使所述免疫细胞对肿瘤的治疗效果与对照免疫细胞对肿瘤的治疗效果相比显著提高(例如，提高了5％或更多、10％或更多、20％或更多、30％或更多、40％或更多、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、100％或更多)，其中所述对照免疫细胞为其中低密度脂蛋白受体相关蛋白或其片段的表达量基本上未上调的相应免疫细胞。
例如，所述相应的免疫细胞可以包含淋巴细胞。在本申请中，所述相应的免疫细胞可以包含T细胞。在本申请中，所述T细胞可以包含记忆性干细胞样T细胞(TSCM)和/或中央记忆性T细胞(TCM)。在本申请中，所述TSCM可以为CCR7 +和/或CD62L +。在本申请中，所述TSCM还可以具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。在本申请中，所述相应的免疫细胞可以包含经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。在本申请中，所述相应的免疫细胞可以包括经遗传修饰的T细胞。
在本申请中，所述相应的免疫细胞中所述低密度脂蛋白受体相关蛋白或其片段的含量可以仅仅为正常机体中所述低密度脂蛋白受体相关蛋白或其片段的表达量，或者为与正常机体中所述低密度脂蛋白受体相关蛋白或其片段的含量相比基本上未上调的表达量。例如，所述基本上未上调可以指上调至多4.5％、至多4％、至多3％、至多2％、至多1％、至多0.5％、至多0.3％、至多0.1％、至多0.01％或更少。在本申请中，所述相应的免疫细胞可以不含有包 含编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子的载体，和/或，所述相应的免疫细胞可以不包含编码所述低密度脂蛋白受体相关蛋白或其片段的核苷酸分子。
在本申请中，检测所述低密度脂蛋白受体相关蛋白或其片段，和/或所述细胞因子的表达量的方法可以包括定量PCR、western blot和免疫组化法。
在本申请中，检测所述记忆性免疫细胞和/或所述分化型免疫细胞的方法可以包括流式细胞术、免疫荧光法、磁珠分离法、ELISA、ELISPOT和定量PCR。
在本申请中，评价肿瘤的治疗效果可以使用选自下组的指标：肿瘤体积、总生存期(OS)、总缓解期(DOR)、病情稳定持续时间、无病生存时间(DFS)、无进展生存时间(PFS)、疾病控制率(DCR)、客观缓解率(ORR)和/或临床受益反应比率。
在本申请中，评价预防受试者中肿瘤复发的效果可以使用选自下组的指标：肿瘤标志物(例如，肿瘤相关抗原、肿瘤特异性基因和抑癌基因)的表达水平、组织学检测结果(例如上皮型增生、息肉消失的程度)、影像学检测结果(例如钼靶检测结果)和/或血清标志物(例如血清游离DNA、甲基化DNA)的表达水平。
组合物及其用途
本申请提供了一种组合物，其包含所述的经遗传修饰的免疫细胞。
在本申请中，所述组合物还可以包含任选地药学上可接受的载体。所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，所述组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
所述组合物可以用于抑制肿瘤生长。例如，本申请的组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的组合物可以包含治疗有效量的所述抗体或其抗原结合片段。所述治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的病症或病症(例如癌症)和/或其任何并发症而所需的剂量。
本申请提供了一种所述经遗传修饰的细胞和/或所述的组合物在制备药物中的用途，其中所述药物用于治疗和/或预防肿瘤。
在本申请中，所述肿瘤可以选自肝癌、肺癌、白血病和间皮瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的装置、方法和系统的工作方式，而不用于限制本申请发明的范围。
实施例
实施例1慢病毒载体的构建
所述CAR-T以靶向GPC3、CD19、Mesothelin(MSLN)、HER2、BCMA为例，人工合成包含CAR结构的片段，并构建到慢病毒载体(LV100A，SystemBiosciences公司)，随后依照其说明书记载的方式转染获得慢病毒(如图1所示)，分别得到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和HER2-41BB-TL6慢病毒。
GPC3-41BB由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.15和SEQ ID NO.17所示。
GPC3-41BB-L6由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
GPC3-41BB-TL6由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
GPC3-41BB-L5由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L5从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.25所示。
GPC3-41BB-TL5由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL5从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.27所示。
GPC3-CD28由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、CD28、CD3zeta从5’端至3’端依次拼接构建。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.13和SEQ ID NO.17所示。
GPC3-CD28-L6由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、CD28、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
GPC3-CD28-TL6由前导序列、GPC3 ScFv、CD8铰链区和跨膜区、CD28、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
BCMA-41BB由前导序列、BCMA ScFv、CD8铰链区和跨膜区、41BB、CD3zeta从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.5、SEQ ID NO.11、SEQ ID NO.15和SEQ ID NO.17所示。
BCMA-41BB-L6由前导序列、BCMA ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.5、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
BCMA-41BB-TL6由前导序列、BCMA ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.5、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
CD19-41BB由前导序列、CD19 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.3、SEQ ID NO.11、SEQ ID NO.15和SEQ ID NO.17所示。
CD19-41BB-L6由前导序列、CD19 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.3、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
CD19-41BB-TL6由前导序列、CD19 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.3、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
MSLN-41BB由前导序列、MSLN ScFv、CD8铰链区和跨膜区、41BB、CD3zeta从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.7、SEQ ID NO.11、SEQ ID NO.15和SEQ ID NO.17所示。
MSLN-41BB-L6由前导序列、MSLN ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.7、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
MSLN-41BB-TL6由前导序列、MSLN ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID  NO.7、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
HER2-41BB由前导序列、HER2 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.15和SEQ ID NO.17所示。
HER2-41BB-L6由前导序列、HER2 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、L6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.21所示。
HER2-41BB-TL6由前导序列、HER2 ScFv、CD8铰链区和跨膜区、41BB、CD3zeta、2A、TL6从5’端至3’端依次拼接构建而成。其核苷酸序列分别如SEQ ID NO.30、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19和SEQ ID NO.23所示。
实施例2慢病毒感染T细胞
通过ficoll分离液分离新鲜人外周血，获得大于1×10 7的外周血单核淋巴细胞(PBMC)。用PBS稀释抗人CD3及抗人CD28抗体，使其终浓度达到1μg/ml。然后，向细胞培养皿中加入稀释后的抗体混合液，然后将其均匀铺至细胞培养皿中，于室温孵育2小时。2小时后，用PBS洗涤一次抗体混合液。随后，将分离的PBMC用含有Xvivo15培养基、5％FBS、200U/ml IL2 T或Xvivo15培养基、5％FBS、20ng/ml IL21、10ng/ml IL7的淋巴细胞培养液进行重悬，使其终浓度达到1×10 6个细胞/ml，加入放置抗体混合液的培养皿中于37℃、5％CO 2培养24小时，以激活T细胞。
取一定量的T细胞培养液，加入终浓度为1mg/ml的synperonic F108混匀，于水浴锅加热至37℃，配制未感染试剂。随后，准备实验所需细胞培养皿。先取1mg/ml抗人CD3抗体和0.5mg/ml抗人CD28抗体先用PBS缓冲液按1∶1000的体积比稀释并混匀，然后用retronectin(1mg/ml)试剂按1∶40的体积比稀释并混匀，然后将其均匀铺至细胞培养皿中，于室温孵育2小时。2小时后，用PBS进行洗涤，细胞培养皿准备完成。
用所配感染试剂稀释已激活的T细胞，并按MOI＝3的比例分别加入实施例1所制备的各个慢病毒并混匀。随后均匀铺在所述细胞培养皿中，进行慢病毒感染，从而获得分别表达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和HER2-41BB-TL6的T细胞。感染后监测细胞密度，使 这些感染后的细胞的密度维持在1×10 6个细胞/ml。14天后，细胞数可扩增10-100倍。
实施例3 LRP6、LRP6截短体、LRP5或LRP5截短体促进记忆性T细胞形成
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达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和HER2-41BB-TL6的T细胞，共培养9天或13天。
用BD流式检测T细胞中CD3、CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达情况。其中，测量得到的蛋白表达结果如图2所示。
结果显示，在表达GPC3-41BB-L6或GPC3-41BB-TL6的T细胞中，记忆性干细胞样T细胞(TSCM)和中央记忆性T细胞(TCM)占总细胞比例均显著高于对照组；在表达GPC3-CD28-L6或GPC3-CD28-TL6的T细胞中，TSCM和TCM占总细胞百分比均显著高于对照组；在表达GPC3-41BB-L5或GPC3-41BB-TL5的T细胞中，TSCM和TCM占总细胞百分比均显著高于对照组；在表达BCMA-41BB-L6或BCMA-41BB-TL6的T细胞中，TSCM和TCM占总细胞百分比均显著高于对照组；在表达CD19-41BB-L6或CD19-41BB-TL6的T细胞中，TSCM和TCM占总细胞百分比均显著高于对照组；在表达MSLN-41BB-L6或MSLN-41BB-TL6的T细胞中，TSCM和TCM占总细胞百分比均显著高于对照组；在表达HER2-41BB-L6或HER2-41BB-TL6的T细胞中，TSCM和TCM占总细胞百分比也均显著高于对照组。
实施例4 LRP6或其截短体抑制Treg细胞的分化
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-41BB和GPC3-41BB-L6的T细胞，共培养9天或12天。用BD流式检测T细胞中CD3、CD4、CD25、CD127、FoxP3蛋白的表达情况，测量得到的蛋白表达结果如图3所示。结果说明，在表达GPC3-41BB-L6的T细胞中，调节性T细胞(Treg)占CD4 +T细胞百分比显著低于对照组表达GPC3-41BB的T细胞。
实施例5 LRP6、LRP6截短体、LRP5或LRP5截短体促进肿瘤抗原诱导的特异性CAR-T细胞扩增
将实施例2中所得的表达GPC3-41BB、GPC3-41BB-L6或GPC3-41BB-TL6的T细胞与 辐照的Huh7或HepG2细胞(中科院细胞库购得)按1∶1的细胞个数比用Xvivo15培养基进共培养，每5天重新补加辐照的Huh7或HepG2进行刺激，刺激3次，每次用台盼蓝染色进行计数；将表达GPC3-41BB、GPC3-41BB-L5或GPC3-41BB-TL5的T细胞与辐照的Huh7或HepG2细胞(中科院细胞库购得)按1∶1的细胞个数比用Xvivo15培养基进共培养，每5天重新补加辐照的Huh7或HepG2进行刺激，刺激3次，每次用台盼蓝染色进行计数；将表达HER2-41BB、HER2-41BB-L6或HER2-41BB-TL6的T细胞与辐照的SKOV3细胞(中科院细胞库购得)按1∶1的细胞个数比用Xvivo15培养基进共培养，每5天重新补加辐照的SKOV3进行刺激，刺激3次，每次用台盼蓝染色进行计数；各组细胞增殖情况如图4所示。结果显示，表达GPC3-41BB-L6或GPC3-41BB-TL6的T细胞的扩增倍数远高于对照组(表达GPC3-41BB的T细胞)，表达GPC3-41BB-L5或GPC3-41BB-TL5的T细胞的扩增倍数远高于对照组，表达HER2-41BB-L6或HER2-41BB-TL6的T细胞的扩增倍数也远高于对照组(表达HER2-41BB的T细胞)。
实施例6 LRP6或其截短体促进CAR-T细胞体内增殖以及细胞因子的释放
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约100mm 3。此时将小鼠分为4组，分别为T细胞组、GPC3-41BB组、GPC3-41BB-L6组和GPC3-41BB-TL6组，每组6只。然后通过尾静脉分别向其注射细胞(即每组分别注射未经修饰的T细胞、表达GPC3-41BB的T细胞、表达GPC3-41BB-L6的T细胞和表达GPC3-41BB-TL6的T细胞，剂量为3×10 6个/只)，于第8天从小鼠尾部取血50μl。用BD流式检测每组中人CD8、CD4蛋白的表达。结果如图5A所示。图5A的结果说明，GPC3-41BB-L6或GPC3-41BB-TL6组中表达CD8、CD4蛋白的细胞占总细胞数的比例远高于其他对照组。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约100mm 3。此时将小鼠分为5组，分别为T细胞组、GPC3-41BB组和GPC3-41BB-L6组，每组6只。然后通过尾静脉分别向T细胞组注射T细胞(即每注射未经修饰的T细胞，剂量为2×10 6个/只)，向GPC3-41BB组注射表达GPC3-41BB的T细胞(剂量为8×10 5个/只)，向GPC3-41BB-L6组注射表达GPC3-41BB-L6的T细胞(剂量为3×10 5个/只、8×10 5个/只或2×10 6个/只)，于第8天从小鼠尾部取血50μl。用BD流式检测每组中人IL-2、IL4、IL6、IL10、TNF-α和IFNγ细胞因子的表达。结果如图5B所示。图5B的结果说明，GPC3-41BB-L6组中IFNγ细胞因子的表达显著高于其他对照组。
实施例7 LRP6或其截短体增强CAR-T细胞抗肿瘤效果
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(剂量为1×10 7个/只)，14天后，测得小鼠瘤体积约100mm 3。此时将小鼠分为4组，分别为T细胞组、GPC3-41BB组和GPC3-41BB-L6组，每组8只。然后通过尾静脉分别向T细胞组注射T细胞(即每注射未经修饰的T细胞，剂量为8×10 5个/只)，向GPC3-41BB组注射表达GPC3-41BB的T细胞(剂量为8×10 5个/只)，向GPC3-41BB-L6组注射表达GPC3-41BB-L6的T细胞(剂量为3×10 5个/只或8×10 5个/只)。
于每周的周一和周四分别测量瘤体积大小，并记录小鼠死亡情况。结果如图6所示，结果显示GPC3-41BB-L6组抑瘤效果和小鼠存活率均显著高于其他对照组，约是对照组GPC3-41BB组的3倍。
实施例8 LRP6或其截短体促进CAR-T细胞在体内长期存活且预防肿瘤复发
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(剂量为1×10 7个/只)，14天后，测得小鼠瘤体积约100mm 3。此时将小鼠分为3组，分别为T细胞组、GPC3-41BB组和GPC3-41BB-L6组，每组9只。然后通过尾静脉分别向T细胞组注射T细胞(剂量为2×10 6个/只)，向GPC3-41BB组注射表达GPC3-41BB的T细胞(剂量为2×10 6个/只)，向GPC3-41BB-L6组注射表达GPC3-41BB-L6的T细胞(剂量为2×10 6个/只)。
待肿瘤完全消85天后，NSG小鼠皮下再次接种Huh7细胞(剂量为3×10 7个/只)。于第99天从小鼠尾部取血50μl，用BD流式检测每组中人CD8、CD4和CD3蛋白的表达；于第120天，取小鼠骨髓，用BD流式检测每组中人CD8、CD4和CD3蛋白的表达。
结果如图7所示。结果显示GPC3-41BB-L6组外周血中表达CD8或CD4蛋白的细胞占总细胞数的比例以及骨髓中表达CD3蛋白的细胞占总细胞数的比例均远高于GPC3-41BB组，而且GPC3-41BB-L6组小鼠即使再次接种肿瘤细胞，也无法形成肿瘤。这就说明LRP6或其截短体促进CAR-T细胞在体内长期存活且具有预防肿瘤复发的特点。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (154)

  1. 一种促进免疫细胞增殖的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  2. 一种促进记忆性免疫细胞产生的方法，其包含以下步骤：使免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而促进所述免疫细胞分化为记忆性免疫细胞。
  3. 一种抑制免疫细胞分化的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调，从而抑制所述免疫细胞分化为分化型免疫细胞。
  4. 根据权利要求3所述的方法，其中所述分化型免疫细胞包含调节性T细胞(Treg)。
  5. 一种增强免疫细胞释放细胞因子的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  6. 根据权利要求5所述的方法，其中所述细胞因子包含白介素、干扰素和/或肿瘤坏死因子。
  7. 根据权利要求5-6中任一项所述的方法，其中所述细胞因子包含IL-2、IL4、IL6、IL7、IL10、IL21、TNF-α和/或IFNγ。
  8. 一种增强免疫细胞对肿瘤杀伤能力的方法，其包含以下步骤：使所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  9. 一种预防受试者中肿瘤复发的方法，所述方法包含：向易患肿瘤的受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  10. 一种治疗有需要的受试者中的肿瘤的方法，其包含以下步骤：向所述受试者施用免疫细胞，其中所述免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  11. 根据权利要求8-10中任一项所述的方法，其中所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
  12. 根据权利要求8-11中任一项所述的方法，其中所述方法为体外或离体方法。
  13. 根据权利要求1-12中任一项所述的方法，其中所述免疫细胞包含淋巴细胞。
  14. 根据权利要求1-13中任一项所述的方法，其中所述免疫细胞包含T细胞。
  15. 根据权利要求14所述的方法，其中所述T细胞包含记忆性干细胞样T细胞(TSCM)和/或中央记忆性T细胞(TCM)。
  16. 根据权利要求15所述的方法，其中所述TSCM为CCR7 +和/或CD62L +。
  17. 根据权利要求15-16中任一项所述的方法，其中所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。
  18. 根据权利要求1-17中任一项所述的方法，其中所述免疫细胞包含经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。
  19. 根据权利要求18所述的方法，其中所述经遗传修饰的免疫细胞包括经遗传修饰的T细胞。
  20. 根据权利要求19所述的方法，其中所述CAR包含细胞内结构域，所述细胞内结构域包含信号传导结构域和/或共刺激结构域。
  21. 根据权利要求20所述的方法，其中所述信号传导结构域包含选自下组的部分：CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。
  22. 根据权利要求20-21中任一项所述的方法，其中所述信号传导结构域包含SEQ ID NO：18所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  23. 根据权利要求20-22中任一项所述的方法，其中编码所述信号传导结构域的核酸分子包含SEQ ID NO：17所示的核酸序列或与其具备至少80％同源性的核酸序列。
  24. 根据权利要求20-23中任一项所述的方法，其中所述共刺激结构域包含选自下组的部分：CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。
  25. 根据权利要求20-24中任一项所述的方法，其中所述共刺激结构域包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：14和SEQ ID NO：16。
  26. 根据权利要求20-25中任一项所述的方法，其中编码所述共刺激结构域的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：13和SEQ ID NO：15。
  27. 根据权利要求18-26中任一项所述的方法，其中所述CAR包含铰链区。
  28. 根据权利要求27所述的方法，其中所述铰链区包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。
  29. 根据权利要求27-28中任一项所述的方法，其中所述铰链区包含SEQ ID NO：31所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  30. 根据权利要求27-29中任一项所述的方法，其中编码所述铰链区的核酸分子包含SEQ ID NO：32所示的核酸序列或与其具备至少80％同源性的核酸序列。
  31. 根据权利要求18-30中任一项所述的方法，其中所述CAR包含跨膜区。
  32. 根据权利要求31所述的方法，其中所述跨膜区包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和/或CD24的跨膜区。
  33. 根据权利要求31-32中任一项所述的方法，其中所述跨膜区包含SEQ ID NO：33所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  34. 根据权利要求31-33中任一项所述的方法，其中编码所述跨膜区的核酸分子包含SEQ ID NO：34所示的核酸序列或与其具备至少80％同源性的核酸序列。
  35. 根据权利要求18-34中任一项所述的方法，其中所述CAR包含靶向部分。
  36. 根据权利要求35所述的方法，其中所述靶向部分包含ScFv。
  37. 根据权利要求35-36中任一项所述的方法，其中所述靶向部分特异性结合和/或识别肿瘤抗原。
  38. 根据权利要求35-37中任一项所述的方法，其中所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。
  39. 根据权利要求35-38中任一项所述的方法，其中所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
  40. 根据权利要求35-39中任一项所述的方法，其中所述靶向部分为特异性结合和/或识别CD19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：46所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：47所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：48所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  41. 根据权利要求40所述的方法，其中所述轻链可变区包含SEQ ID NO：50所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  42. 根据权利要求40-41中任一项所述的方法，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：43所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：44所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：45所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  43. 根据权利要求42所述的方法，其中所述重链可变区包含SEQ ID NO：49所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  44. 根据权利要求35-39中任一项所述的方法，其中所述靶向部分为特异性结合和/或识 别BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：54所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：55所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：56所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  45. 根据权利要求44所述的方法，其中所述轻链可变区包含SEQ ID NO：58所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  46. 根据权利要求44-45中任一项所述的方法，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：51所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：52所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：53所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  47. 根据权利要求46所述的方法，其中所述重链可变区包含SEQ ID NO：57所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  48. 根据权利要求35-39中任一项所述的方法，其中所述靶向部分为特异性结合和/或识别HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：70所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：71所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：72所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  49. 根据权利要求48所述的方法，其中所述轻链可变区包含SEQ ID NO：74所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  50. 根据权利要求48-49中任一项所述的方法，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：67所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：68所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：69所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  51. 根据权利要求50所述的方法，其中所述重链可变区包含SEQ ID NO：73所示的氨基 酸序列或与其具备至少80％同源性的氨基酸序列。
  52. 根据权利要求35-39中任一项所述的方法，其中所述靶向部分为特异性结合和/或识别Mesothelin的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：63所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：64所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  53. 根据权利要求52所述的方法，其中所述轻链可变区包含SEQ ID NO：66所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  54. 根据权利要求52-53中任一项所述的方法，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：59所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：60所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：61所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  55. 根据权利要求54所述的方法，其中所述重链可变区包含SEQ ID NO：65所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  56. 根据权利要求35-39中任一项所述的方法，其中所述靶向部分为特异性结合和/或识别GPC3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：38所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：39所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：40所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  57. 根据权利要求56所述的方法，其中所述轻链可变区包含SEQ ID NO：42所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  58. 根据权利要求56-57中任一项所述的方法，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：35所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：36所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：37所示的氨基酸序列或与其具备至少80％同源性的氨基酸 序列。
  59. 根据权利要求58所述的方法，其中所述重链可变区包含SEQ ID NO：41所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  60. 根据权利要求35-59中任一项所述的方法，其中所述靶向部分包含下述任一项所示的氨基酸序列：SEQ ID NO：2、4、6、8和10或与其具备至少80％同源性的氨基酸序列。
  61. 根据权利要求35-60中任一项所述的方法，其中编码所述靶向部分的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：1、3、5、7和9。
  62. 据权利要求1-61所述的方法，所述方法还包含以下步骤：分离获得外周血单核细胞PBMC、CD3 +T淋巴细胞、CD8 +T淋巴细胞、CD4 +T淋巴细胞或调节T细胞。
  63. 根据权利要求62所述的方法，所述方法还包含：向经分离的所述PBMC加入一种或多种T细胞刺激因子。
  64. 根据权利要求63所述的方法，所述T细胞刺激因子选自以下组：B淋巴细胞表面抗原抗体、TNF抗体、细胞内聚酯和抗生素。
  65. 根据权利要求63-64中任一项所述的方法，其中所述T细胞刺激因子选自以下组：CD3抗体、CD28抗体、4-1BB抗体、CD80抗体、CD86抗体、PHA、PMA和离子霉素。
  66. 根据权利要求63-65中任一项所述的方法，其中所述T细胞刺激因子包含CD3抗体，且所述CD3抗体的浓度为1-10000ng/mL。
  67. 根据权利要求63-66中任一项所述的方法，其中所述T细胞刺激因子包含CD28抗体，且所述CD28抗体的浓度为1-10000ng/mL。
  68. 根据权利要求63-67中任一项所述的方法，所述方法还包含：向经分离的所述PBMC加入一种或多种细胞因子剂。
  69. 根据权利要求68所述的方法，所述细胞因子剂包含白细胞介素。
  70. 根据权利要求69所述的方法，所述白细胞介素包含选自下组的一种或多种：IL2、IL21、IL7和IL15。
  71. 根据权利要求69-70中任一项所述的方法，所述白细胞介素包含IL2，且所述IL2的浓度为0.1-10000U/mL。
  72. 根据权利要求69-71中任一项所述的方法，所述白细胞介素包含IL21，且所述IL21 的浓度为0.01-1000ng/mL。
  73. 根据权利要求69-72中任一项所述的方法，所述白细胞介素包含IL7，且所述IL7的浓度为0.01-1000ng/mL。
  74. 根据权利要求69-73中任一项所述的方法，所述白细胞介素包含IL15，且所述IL15的浓度为0.01-1000ng/mL。
  75. 根据权利要求1-74中任一项所述的方法，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  76. 根据权利要求1-75中任一项所述的方法，其中所述低密度脂蛋白受体相关蛋白或其片段来源于人。
  77. 根据权利要求75-76中任一项所述的方法，其中所述功能性片段包含具备所述低密度脂蛋白受体相关蛋白活性的所述低密度脂蛋白受体相关蛋白的片段或截短体。
  78. 根据权利要求1-77中任一项所述的方法，其中所述低密度脂蛋白受体相关蛋白包含低密度脂蛋白受体相关蛋白6和其截短体，和/或低密度脂蛋白受体相关蛋白5和其截短体。
  79. 根据权利要求78所述的方法，其中所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的胞内区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的胞内区。
  80. 根据权利要78-79中任一项所述的方法，其中所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的跨膜区和所述低密度脂蛋白受体相关蛋白6的LDLR区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的跨膜区和所述低密度脂蛋白受体相关蛋白5的LDLR区。
  81. 根据权利要求1-80中任一项所述方法，其中所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
  82. 根据权利要求1-81中任一项所述的方法，其中编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：21、23、25和27。
  83. 一种经遗传修饰的免疫细胞，其中所述遗传修饰使得该免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  84. 根据权利要求83所述的免疫细胞，其中所述经遗传修饰的免疫细胞包含淋巴细胞。
  85. 根据权利要求83-84中任一项所述的免疫细胞，其中所述经遗传修饰的免疫细胞包括经遗传修饰的T细胞。
  86. 根据权利要求83-85中任一项所述的免疫细胞，其中所述经遗传修饰的免疫细胞包含经遗传修饰的记忆性干细胞样T细胞(TSCM)和/或经遗传修饰的中央记忆性T细胞(TCM)。
  87. 根据权利要求86所述的免疫细胞，其中所述TSCM为CCR7+和/或CD62L+。
  88. 根据权利要求86-87中任一项所述的免疫细胞，其中所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD3 +、CD4 +和CD8 +。
  89. 根据权利要求83-88中任一项所述的免疫细胞，其中所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。
  90. 根据权利要求89所述的免疫细胞，其中所述CAR包含细胞内结构域，所述细胞内结构域包含信号传导结构域和/或共刺激结构域。
  91. 根据权利要求90所述的免疫细胞，其中所述信号传导结构域包含选自下组的部分：CD3ζ的信号传导结构域、CD3δ的信号传导结构域和CD3ε的信号传导结构域。
  92. 根据权利要求90-91中任一项所述的免疫细胞，其中所述信号传导结构域包含SEQ ID NO：18所示的氨基酸序列。
  93. 根据权利要求90-92中任一项所述的免疫细胞，其中编码所述信号传导结构域的核酸分子包含SEQ ID NO：17所示的核酸序列或与其具备至少80％同源性的核酸序列。
  94. 根据权利要求90-93中任一项所述的免疫细胞，其中所述共刺激结构域包含选自下组的部分：CD27的共刺激结构域、CD28的共刺激结构域和4-1BB的共刺激结构域。
  95. 根据权利要求90-94中任一项所述的免疫细胞，其中所述共刺激结构域包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：14和SEQ ID NO：16。
  96. 根据权利要求90-95中任一项所述的免疫细胞，其中编码所述共刺激结构域的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：13和SEQ ID NO：15。
  97. 根据权利要求89-96中任一项所述的免疫细胞，其中所述CAR包含铰链区。
  98. 根据权利要求97所述的免疫细胞，其中所述铰链区包含选自下组的部分：IgG4的铰 链区、IgG1的铰链区和CD8的铰链区。
  99. 根据权利要求97-98中任一项所述的免疫细胞，其中所述铰链区包含SEQ ID NO：31所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  100. 根据权利要求97-99中任一项所述的免疫细胞，其中编码所述铰链区的核酸分子包含SEQ ID NO：32所示的核酸序列或与其具备至少80％同源性的核酸序列。
  101. 根据权利要求98-100中任一项所述的免疫细胞，其中所述CAR包含跨膜区。
  102. 根据权利要求101所述的免疫细胞，其中所述跨膜区包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和/或CD24的跨膜区。
  103. 根据权利要求101-102中任一项所述的免疫细胞，其中所述跨膜区包含SEQ ID NO：33所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  104. 根据权利要求101-103中任一项所述的免疫细胞，其中编码所述跨膜区的核酸分子包含SEQ ID NO：34所示的核酸序列或与其具备至少80％同源性的核酸序列。
  105. 根据权利要求89-104中任一项所述的免疫细胞，其中所述CAR包含靶向部分。
  106. 根据权利要求105所述的免疫细胞，其中所述靶向部分包含ScFv。
  107. 根据权利要求105-106中任一项所述的免疫细胞，其中所述靶向部分特异性结合和/或识别肿瘤抗原。
  108. 根据权利要求105-107中任一项所述的免疫细胞，其中所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。
  109. 根据权利要求105-108中任一项所述的免疫细胞，其中所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
  110. 根据权利要求105-109中任一项所述的免疫细胞，其中所述靶向部分为特异性结合和/或识别CD19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：46所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：47所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：48所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  111. 根据权利要求110所述的免疫细胞，其中所述轻链可变区包含SEQ ID NO：50所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  112. 根据权利要求110-111中任一项所述的免疫细胞，其中所述抗体或其抗原结合片段包 含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：43所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：44所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：45所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  113. 根据权利要求112所述的免疫细胞，其中所述重链可变区包含SEQ ID NO：49所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  114. 根据权利要求105-109中任一项所述的免疫细胞，其中所述靶向部分为特异性结合和/或识别BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：54所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：55所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：56所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  115. 根据权利要求114所述的免疫细胞，其中所述轻链可变区包含SEQ ID NO：58所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  116. 根据权利要求114-115中任一项所述的免疫细胞，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：51所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：52所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：53所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  117. 根据权利要求116所述的免疫细胞，其中所述重链可变区包含SEQ ID NO：57所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  118. 根据权利要求105-109中任一项所述的免疫细胞，其中所述靶向部分为特异性结合和/或识别HER2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：70所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：71所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：72所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  119. 根据权利要求118所述的免疫细胞，其中所述轻链可变区包含SEQ ID NO：74所示的 氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  120. 根据权利要求118-119中任一项所述的免疫细胞，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：67所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：68所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：69所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  121. 根据权利要求120所述的免疫细胞，其中所述重链可变区包含SEQ ID NO：73所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  122. 根据权利要求105-109中任一项所述的免疫细胞，其中所述靶向部分为特异性结合和/或识别Mesothelin的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：62所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：63所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3包含SEQ ID NO：64所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  123. 根据权利要求122所述的免疫细胞，其中所述轻链可变区包含SEQ ID NO：66所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  124. 根据权利要求122-123中任一项所述的免疫细胞，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：59所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：60所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：61所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  125. 根据权利要求124所述的免疫细胞，其中所述重链可变区包含SEQ ID NO：65所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  126. 根据权利要求105-109中任一项所述的免疫细胞，其中所述靶向部分为特异性结合和/或识别GPC3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含LCDR1-LCDR3，其中所述LCDR1包含SEQ ID NO：38所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR2包含SEQ ID NO：39所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述LCDR3 包含SEQ ID NO：40所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  127. 根据权利要求126所述的免疫细胞，其中所述轻链可变区包含SEQ ID NO：42所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  128. 根据权利要求126-127中任一项所述的免疫细胞，其中所述抗体或其抗原结合片段包含重链可变区，所述重链可变区包含HCDR1-HCDR3，其中所述HCDR1包含SEQ ID NO：35所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR2包含SEQ ID NO：36所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列；所述HCDR3包含SEQ ID NO：37所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  129. 根据权利要求128所述的免疫细胞，其中所述重链可变区包含SEQ ID NO：41所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列。
  130. 根据权利要求105-129中任一项所述的免疫细胞，其中所述靶向部分包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：2、4、6、8和10。
  131. 根据权利要求105-130中任一项所述的免疫细胞，其中编码所述靶向部分的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：1、3、5、7和9。
  132. 根据权利要求83-131中任一项所述的免疫细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  133. 根据权利要求83-132中任一项所述的免疫细胞，其中所述低密度脂蛋白受体相关蛋白或其片段来源于人。
  134. 根据权利要求132-133中任一项所述的免疫细胞，其中所述功能性片段包含具备所述低密度脂蛋白受体相关蛋白活性的所述低密度脂蛋白受体相关蛋白的片段或截短体。
  135. 根据权利要求83-134中任一项所述的免疫细胞，其中所述低密度脂蛋白受体相关蛋白包含低密度脂蛋白受体相关蛋白6和其截短体，和/或低密度脂蛋白受体相关蛋白5和其截短体。
  136. 根据权利要求135所述的免疫细胞，其中所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的胞内区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的胞内区。
  137. 根据权利要135-136中任一项所述的免疫细胞，其中所述低密度脂蛋白受体相关蛋白6的截短体包含所述低密度脂蛋白受体相关蛋白6的跨膜区和所述低密度脂蛋白受体相关蛋白6的LDLR区；和/或，所述低密度脂蛋白受体相关蛋白5的截短体包含所述低密度脂蛋白受体相关蛋白5的跨膜区和所述低密度脂蛋白受体相关蛋白5的LDLR区。
  138. 根据权利要求83-137中任一项所述免疫细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
  139. 根据权利要求83-138中任一项所述的免疫细胞，其中编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子包含下述任一项所示的核酸序列或与其具备至少80％同源性的核酸序列：SEQ ID NO：21、23、25和27。
  140. 组合物，其包含权利要求83-139中任一项所述的经遗传修饰的免疫细胞。
  141. 根据权利要求140所述的组合物，其还包含任选地药学上可接受的载体。
  142. 权利要求83-139中任一项所述的经遗传修饰的免疫细胞和/或权利要求140-141中任一项所述的组合物在制备药物中的用途，其中所述药物用于治疗和/或预防肿瘤。
  143. 根据权利要求142所述的用途，其中所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
  144. 使低密度脂蛋白受体相关蛋白或其片段的表达量上调的试剂在制备药剂中的用途，所述药剂用于促进免疫细胞增殖、促进记忆性免疫细胞产生、抑制免疫细胞分化、增强免疫细胞释放细胞因子，和/或，增强免疫细胞对肿瘤的杀伤能力。
  145. 根据权利要求144所述的用途，其中所述低密度脂蛋白受体相关蛋白或其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
  146. 制备权利要求83-139中任一项所述经遗传修饰的免疫细胞的方法，所述方法包括以下的步骤，使所述经遗传修饰的免疫细胞中低密度脂蛋白受体相关蛋白或其片段的表达量上调。
  147. 根据权利要求146所述的方法，其包括以下步骤：向所述经遗传修饰的免疫细胞中引入使得所述低密度脂蛋白受体相关蛋白或其片段的表达量上调的载体。
  148. 根据权利要求147所述的方法，其中所述载体选自以下组：逆转录病毒载体、慢病毒载体和转座子质粒。
  149. 根据权利要求146-148中任一项所述的方法，其中所述低密度脂蛋白受体相关蛋白或 其片段包含下述任一项所示的氨基酸序列或与其具备至少80％同源性的氨基酸序列：SEQ ID NO：22、24、26和28。
  150. 根据权利要求146-149中任一项所述的方法，其中所述经遗传修饰的免疫细胞包含淋巴细胞。
  151. 根据权利要求146-150中任一项所述的方法，其中所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。
  152. 根据权利要求151所述的方法，其包括分离并激活所述经遗传修饰的免疫细胞，其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基。
  153. 根据权利要求152所述的方法，其中所述T细胞培养基选自以下组中的一种或多种：DMEM培养基、1640培养基、MEM培养基和X-VIVO培养基。
  154. 根据权利要求151-153中任一项所述的方法，其还包括向所述经遗传修饰的免疫细胞施用T细胞刺激因子。
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