WO2021057932A1 - 一种经修饰的免疫细胞及其用途 - Google Patents

一种经修饰的免疫细胞及其用途 Download PDF

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WO2021057932A1
WO2021057932A1 PCT/CN2020/117936 CN2020117936W WO2021057932A1 WO 2021057932 A1 WO2021057932 A1 WO 2021057932A1 CN 2020117936 W CN2020117936 W CN 2020117936W WO 2021057932 A1 WO2021057932 A1 WO 2021057932A1
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Prior art keywords
receptor
leptin
modified immune
seq
immune cell
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PCT/CN2020/117936
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English (en)
French (fr)
Inventor
凌有国
郭昊
陈思晔
李慧姣
杨月
李霄培
杨棋
杨焕凤
何晓文
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上海原能细胞医学技术有限公司
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Application filed by 上海原能细胞医学技术有限公司 filed Critical 上海原能细胞医学技术有限公司
Priority to EP20867583.5A priority Critical patent/EP4036222A4/en
Priority to US17/764,128 priority patent/US20220389076A1/en
Priority to JP2022518931A priority patent/JP2022549468A/ja
Priority to CN202080067420.5A priority patent/CN114502723A/zh
Publication of WO2021057932A1 publication Critical patent/WO2021057932A1/zh

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    • 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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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • A61K2039/515—Animal cells
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • This application relates to the field of biomedicine, in particular to a modified immune cell and its use.
  • Tumor is a disease that seriously threatens human health.
  • CTL cytotoxic lymphocytes
  • TCR T lymphocyte receptor
  • scFv is fused with the T lymphocyte receptor to form a chimeric antigen receptor (CAR), which is genetically modified on the surface of T lymphocytes by means of lentivirus infection for tumor treatment.
  • CAR-T lymphocytes can selectively direct T lymphocytes to tumor cells and specifically kill tumor cells in a non-limiting manner of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • adoptive immunotherapy based on immune effector cells has achieved certain results in some tumors, but the efficacy in most tumors is still unsatisfactory.
  • the present application provides a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element, and the modified immune cell further comprises:
  • Leptin receptor and/or functional fragments thereof and the expression amount of the leptin receptor and/or functional fragments thereof is increased in the modified immune cells compared with immune cells without corresponding modification .
  • the modified immune cells include lymphocytes.
  • the modified immune cells include: T cells.
  • the leptin and/or functional fragments thereof are derived from humans.
  • the leptin receptor and/or functional fragments thereof are derived from humans.
  • the leptin is secreted leptin or membrane-bound leptin.
  • the leptin and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NO: 13-14.
  • the functional fragment of the leptin receptor comprises a domain of the leptin receptor selected from the group consisting of intracellular region, BOX 1 region, FN3 region, C2 region and transmembrane region .
  • the leptin receptor and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-17.
  • the chimeric antigen receptor does not include the leptin and/or functional fragments thereof.
  • the chimeric antigen receptor does not include the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor comprises an intracellular domain, and the intracellular domain includes a signal transduction domain and/or a costimulatory domain.
  • the signaling domain comprises the signaling domain of CD3 ⁇ .
  • the costimulatory domain comprises the costimulatory domain of 4-1BB.
  • the chimeric antigen receptor comprises a hinge region.
  • the hinge region comprises the hinge region of CD8.
  • the chimeric antigen receptor comprises a transmembrane region.
  • the transmembrane region comprises the transmembrane region of CD8.
  • the chimeric antigen receptor comprises a targeting moiety.
  • the targeting moiety includes scFv.
  • the targeting moiety specifically binds and/or recognizes tumor antigens.
  • the targeting moiety specifically binds and/or recognizes a target selected from the group consisting of CD19, HER2, Mesothelin, GPC3 and BCMA.
  • the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • the application also provides a pharmaceutical composition, which includes the modified immune cell described in the application and optionally a pharmaceutically acceptable carrier.
  • this application also provides the use of the modified immune cell described in this application or the pharmaceutical composition described in this application in the preparation of drugs for treating tumors.
  • this application also provides a method for promoting the proliferation of immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • this application also provides a method for promoting the production of memory immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • this application also provides a method for enhancing the ability of immune cells to kill tumors, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • the present application also provides a method for inhibiting the expression of immune negative regulatory proteins by immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • step 2) of the method described in this application includes:
  • the polynucleotide encoding the leptin and/or functional fragments thereof is introduced into the modified immune cell, and/or the leptin receptor encoding and/or is introduced into the modified immune cell The functional fragment of the polynucleotide.
  • the polynucleotide encoding the leptin and/or functional fragments thereof, and/or the polynucleotide encoding the leptin receptor and/or functional fragments thereof are contained in a plasmid in.
  • the plasmid includes a viral vector.
  • the modified immune cells include T cells.
  • the increase in the expression level includes an increase in the expression level of the leptin receptor and/or functional fragments thereof in the modified immune cell compared to an immune cell that has not been modified accordingly.
  • the leptin and/or functional fragments thereof are derived from humans.
  • the leptin receptor and/or functional fragments thereof are derived from humans.
  • the leptin is secreted leptin or membrane-bound leptin.
  • the leptin and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NO: 13-14.
  • the functional fragment of the leptin receptor comprises a domain of the leptin receptor selected from the group consisting of intracellular region, BOX 1 region, FN3 region, C2 region and transmembrane region .
  • the leptin receptor and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-17.
  • the chimeric antigen receptor does not include the leptin and/or functional fragments thereof and/or the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor comprises an intracellular domain, and the intracellular domain includes a signal transduction domain and/or a costimulatory domain.
  • the signaling domain comprises the signaling domain of CD3 ⁇ .
  • the costimulatory domain comprises the costimulatory domain of 4-1BB.
  • the chimeric antigen receptor comprises a hinge region.
  • the hinge region in includes the hinge region of CD8.
  • the chimeric antigen receptor comprises a transmembrane region.
  • the transmembrane region comprises the transmembrane region of CD8.
  • the chimeric antigen receptor comprises a targeting moiety.
  • the targeting moiety includes scFv.
  • the targeting moiety specifically binds and/or recognizes tumor antigens.
  • the targeting moiety specifically binds and/or recognizes a target selected from the group consisting of CD19, HER2, Mesothelin, GPC3 and BCMA.
  • the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • this application also provides a method for inhibiting tumor cell proliferation and/or killing tumor cells, the method comprising: contacting the modified immune cells described in this application with tumor cells.
  • the method includes an in vitro method or an ex vivo method.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • the tumor includes B lymphoid leukemia, breast cancer, gastric cancer, pancreatic cancer, multiple myeloma, mesothelioma, lung cancer, and/or liver cancer.
  • the present application provides a modified immune cell comprising leptin and/or functional fragments thereof, and/or leptin receptor and/or functional fragments thereof, and low-density lipoprotein receptor Related proteins or fragments thereof.
  • the modified immune cells include T cells.
  • the leptin and/or functional fragments thereof are derived from humans.
  • the leptin receptor and/or functional fragments thereof are derived from humans.
  • the leptin is secreted leptin or membrane-bound leptin.
  • the leptin and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NO: 13-14.
  • the functional fragment of the leptin receptor comprises a domain of the leptin receptor selected from the group consisting of: intracellular region, BOX 1 region, FN3 region, C2 region and transmembrane region .
  • the leptin receptor and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-17.
  • the density lipoprotein receptor-related protein or a fragment thereof is selected from the following group: low-density lipoprotein receptor-related protein 6 or a truncated body thereof, and low-density lipoprotein receptor-related protein 5 or Its truncated body.
  • the truncated body of the low-density lipoprotein receptor-associated protein 6 comprises the intracellular region of the low-density lipoprotein receptor-associated protein 6; and/or, the low-density lipoprotein receptor-associated protein 6
  • the truncated body-associated protein 5 contains the intracellular region of the low-density lipoprotein receptor-associated protein 5.
  • the truncated body of the low-density lipoprotein receptor-related protein 6 comprises a functional domain selected from the group consisting of: the transmembrane region of the low-density lipoprotein receptor-related protein 6 and the low-density lipoprotein receptor-related protein 6 The LDLR region of density lipoprotein receptor-related protein 6; and/or, wherein the truncated body of low-density lipoprotein receptor-related protein 5 comprises a functional domain selected from the group consisting of: the low-density lipoprotein receptor-related protein 5 The transmembrane region of protein 5 and the LDLR region of the low-density lipoprotein receptor-related protein 5.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 18-21.
  • the modified immune cell further comprises a chimeric antigen receptor or a T cell receptor.
  • the chimeric antigen receptor does not include the leptin and/or functional fragments thereof.
  • the chimeric antigen receptor does not include the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor does not include the density lipoprotein receptor-related protein or fragments thereof.
  • the chimeric antigen receptor comprises an intracellular domain, and the intracellular domain includes a signal transduction domain and/or a costimulatory domain.
  • the signaling domain comprises the signaling domain of CD3 ⁇ .
  • the costimulatory domain comprises the costimulatory domain of 4-1BB.
  • the chimeric antigen receptor comprises a hinge region.
  • the hinge region comprises the hinge region of CD8.
  • the chimeric antigen receptor comprises a transmembrane region.
  • the transmembrane region comprises the transmembrane region of CD8.
  • the chimeric antigen receptor comprises a targeting moiety.
  • the targeting moiety includes scFv.
  • the targeting moiety specifically binds and/or recognizes tumor antigens.
  • the targeting moiety specifically binds and/or recognizes a target selected from the group consisting of CD19, HER2, Mesothelin, GPC3 and BCMA.
  • the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • the present application also provides a pharmaceutical composition, which includes the modified immune cell described in the present application and optionally a pharmaceutically acceptable carrier.
  • this application also provides the use of the modified immune cell described in this application or the pharmaceutical composition described in this application in the preparation of drugs for treating tumors.
  • this application also provides a method for promoting the proliferation of immune cells, which includes the following steps:
  • modified immune cells To cause the modified immune cells to express the leptin and/or functional fragments thereof, and/or to increase the expression of the leptin receptor and/or functional fragments thereof in the modified immune cells,
  • modified immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • this application also provides a method for promoting the production of memory immune cells, which includes the following steps:
  • modified immune cells To cause the modified immune cells to express the leptin and/or functional fragments thereof, and/or to increase the expression of the leptin receptor and/or functional fragments thereof in the modified immune cells,
  • modified immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • this application also provides a method for enhancing the ability of immune cells to kill tumors, which includes the following steps:
  • modified immune cells To cause the modified immune cells to express the leptin and/or functional fragments thereof, and/or to increase the expression of the leptin receptor and/or functional fragments thereof in the modified immune cells,
  • modified immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • the present application also provides a method for inhibiting the expression of immune negative regulatory proteins by immune cells, which includes the following steps:
  • modified immune cells To cause the modified immune cells to express the leptin and/or functional fragments thereof, and/or to increase the expression of the leptin receptor and/or functional fragments thereof in the modified immune cells,
  • modified immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • the method described in this application includes the following steps: introducing a polynucleotide encoding the leptin and/or a functional fragment thereof into the modified immune cell, and/or introducing the polynucleotide encoding the leptin and/or functional fragment thereof into the modified immune cell; A polynucleotide encoding the leptin receptor and/or a functional fragment thereof is introduced into the modified immune cell.
  • the method described in the present application includes the following steps: introducing a polynucleotide encoding the low-density lipoprotein receptor-related protein or a fragment thereof into the modified immune cell.
  • the polynucleotide encoding the leptin and/or functional fragments thereof, and/or the polynucleotide encoding the leptin receptor and/or functional fragments thereof are contained in a plasmid in.
  • the polynucleotide encoding the low-density lipoprotein receptor-related protein or fragment thereof is contained in a plasmid.
  • the plasmid includes a viral vector.
  • the modified immune cells include T cells.
  • the leptin and/or functional fragments thereof are derived from humans.
  • the leptin receptor and/or functional fragments thereof are derived from humans.
  • the leptin is secreted leptin or membrane-bound leptin.
  • the leptin and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NO: 13-14.
  • the functional fragment of the leptin receptor comprises a domain of the leptin receptor selected from the group consisting of intracellular region, BOX 1 region, FN3 region, C2 region and transmembrane region .
  • the leptin receptor and/or functional fragments thereof comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-17.
  • the density lipoprotein receptor-related protein or a fragment thereof is selected from the following group: low-density lipoprotein receptor-related protein 6 or a truncated body thereof, and low-density lipoprotein receptor-related protein 5 or Its truncated body.
  • the truncated body of the low-density lipoprotein receptor-associated protein 6 comprises the intracellular region of the low-density lipoprotein receptor-associated protein 6; and/or, the low-density lipoprotein receptor-associated protein 6
  • the truncated body-associated protein 5 contains the intracellular region of the low-density lipoprotein receptor-associated protein 5.
  • the truncated body of the low-density lipoprotein receptor-related protein 6 comprises a functional domain selected from the group consisting of: the transmembrane region of the low-density lipoprotein receptor-related protein 6 and the low-density lipoprotein receptor-related protein 6 The LDLR region of density lipoprotein receptor-related protein 6; and/or, wherein the truncated body of low-density lipoprotein receptor-related protein 5 comprises a functional domain selected from the group consisting of: the low-density lipoprotein receptor-related protein 5 The transmembrane region of protein 5 and the LDLR region of the low-density lipoprotein receptor-related protein 5.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 18-21.
  • the modified immune cell further comprises a chimeric antigen receptor or a T cell receptor.
  • the chimeric antigen receptor does not include the leptin and/or functional fragments thereof, and/or, the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor does not include the density lipoprotein receptor-related protein or fragments thereof.
  • the chimeric antigen receptor comprises an intracellular domain, and the intracellular domain includes a signal transduction domain and/or a costimulatory domain.
  • the signaling domain comprises the signaling domain of CD3 ⁇ .
  • the costimulatory domain comprises the costimulatory domain of 4-1BB.
  • the chimeric antigen receptor comprises a hinge region.
  • the hinge region comprises the hinge region of CD8.
  • the chimeric antigen receptor comprises a transmembrane region.
  • the transmembrane region comprises the transmembrane region of CD8.
  • the chimeric antigen receptor comprises a targeting moiety.
  • the targeting moiety includes scFv.
  • the targeting moiety specifically binds and/or recognizes tumor antigens.
  • the targeting moiety specifically binds and/or recognizes a target selected from the group consisting of CD19, HER2, Mesothelin, GPC3 and BCMA.
  • the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • the present application provides a method for inhibiting tumor cell proliferation and/or killing tumor cells, the method comprising: contacting the modified immune cells described in the present application with tumor cells.
  • the method includes an in vitro method or an ex vivo method.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • the tumor includes B lymphoid leukemia, breast cancer, gastric cancer, pancreatic cancer, multiple myeloma, mesothelioma, lung cancer, and/or liver cancer.
  • Figures 1A-1H show the results of flow cytometric detection of memory T cells (CCR7+CD45RO-) of the immune cells targeted and modified by GPC3 described in this application.
  • Figures 2A-2H show the results of flow cytometric detection of memory T cells (CCR7+CD62L+) of the immune cells targeted and modified by GPC3 described in this application.
  • Figures 3A-3H show the results of flow cytometric detection of memory T cells (CCR7+CD45RO-) of immune cells targeted and modified by BCMA described in this application.
  • Figures 4A-4H show the results of flow cytometric detection of memory T cells (CCR7+CD62L+) of immune cells targeted and modified by BCMA described in this application.
  • Figures 5A-5H show the results of flow cytometric detection of memory T cells (CCR7+CD45RO-) of CD8+ immune cells modified by GPC3 targeting described in this application in CD4 cells.
  • Figures 6A-6H show the results of flow cytometric detection of memory T cells (CCR7+CD45RO-) of CD4+ immune cells targeted and modified by GPC3 described in this application in CD8 cells.
  • FIGS 7A-7F show the expression of the modified immune cell negative regulatory protein described in this application.
  • Figure 8 shows the amplification of GPC3/MSLN/HER2 related CAR-T.
  • Figure 9 shows the results of flow cytometric detection of memory T cells of immune cells targeted and modified by MSLN with different structures described in the present application.
  • Figure 10 shows the results of flow cytometric detection of memory T cells of the HER2-targeted modified immune cells described in this application.
  • Figure 11 shows the results of flow cytometric detection of memory T cells of the immune cells with different structures targeted and modified by CD19 as described in the present application.
  • Figure 12 shows the repeated stimulation and expansion results of immune cells modified with different targets and different structures as described in this application.
  • Figure 13 shows the results of suppressing tumors and recurrence in mice of immune cells modified by GPC3 targeting described in the present application.
  • Figure 14 shows the results of mouse body weight changes of the immune cells modified by GPC3 targeting described in this application.
  • Figure 15 shows the results of cytokine changes in mice of the immune cells modified by GPC3 targeting described in this application.
  • Figure 16 shows the results of in vivo cell changes of the immune cells modified by GPC3 targeting described in this application.
  • Fig. 17 shows the detection results of the modified cell content in the spleen (A) and bone marrow (B) of the mouse after the second tumor-bearing of the immune cells modified by GPC3 targeting described in the present application.
  • Figure 18 shows the results of suppressing tumors and suppressing recurrence in mice with BCMA-targeted and modified immune cells described in the present application.
  • Figure 19 shows the cytokine results of the immune cells targeted and modified by BCMA described in this application in mice.
  • immune cell generally refers to a cell that participates in an immune response, such as promoting an immune effector response.
  • immune cells may include lymphocytes.
  • immune cells may include T cells, B cells, natural killer (NK) cells, mast cells, and phagocytes derived from bone marrow.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • TAA tumor-associated antigen
  • the CAR can be combined with the T cell receptor activation intracellular domain based on the antigen specificity of the antibody.
  • Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • T cell receptor usually refers to the T cell antigen receptor, which is the molecular structure of the T cell that specifically recognizes and binds to the antigen peptide-MHC molecule, usually with the CD3 molecule Exist in the form of a complex on the surface of T cells.
  • the TCR of most T cells is composed of ⁇ and ⁇ peptide chains, and the TCR of a few T cells is composed of ⁇ and ⁇ peptide chains.
  • the term "encoding" generally refers to the inherent characteristics of polynucleotides such as specific sequences of nucleotides in genes, cDNAs, or mRNAs used as templates for the synthesis of other polymers and macromolecules in biological processes.
  • the polymer and the macromolecule have a definite nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a definite amino acid sequence and the biological properties derived therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein.
  • Leptin generally refers to a class of protein hormones secreted by adipose tissue.
  • the precursor is composed of 167 amino acid residues, and the N-terminal has a signal peptide of 21 amino acid residues.
  • the signal peptide of this precursor is cut off in the blood to become 146 amino acids with a molecular weight of 16KD, forming Leptin.
  • Leptin has a wide range of biological effects, the more important of which is to act on the metabolic regulation center of the hypothalamus, to suppress appetite, reduce energy intake, increase energy consumption, and inhibit fat synthesis.
  • the term "leptin receptor” generally refers to a type of transmembrane receptor, which is mainly composed of an extracellular region, a transmembrane region, and an intracellular region.
  • leptin receptor There are many forms of human leptin receptor, which are divided into long type (OB-RL) and short type (OB-RS). The extracellular and transmembrane regions of the two are completely the same. The main difference is the intracellular region. The length and amino acid sequence composition are different.
  • the long-type leptin receptor is mainly expressed in the hypothalamus, including arcuate nucleus, paraventricular nucleus, dorsal medial hypothalamic nucleus, and lateral hypothalamus.
  • Short-type receptors are mostly distributed in adipose tissue, myocardium, lung, ovary and hematopoietic tissue. .
  • the term "functional fragment” generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same activity or ability as its larger counterpart.
  • the level of activity of the functional fragment may be the same as the activity of the larger counterpart, or smaller or greater than it.
  • a functional fragment of leptin may be a polypeptide that consists of fewer amino acids than full-length leptin protein, but still retains the activity of full-length leptin.
  • NK cells are a type of cytotoxic lymphocytes, which represent the main components of the innate immune system. NK cells reject tumors and virus-infected cells. NK cells work through apoptosis or programmed cell death methods. NK cells are called "natural killer” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (no antibodies involved). The T cell receptor (TCR) of T cells makes T cells different from other lymphocyte types. The thymus (a specialized organ of the immune system) is the main factor in the maturation of T cells.
  • TCR T cell receptor
  • T cells There are six types of T cells: helper T cells (such as CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells) , Or killer T cells), memory T cells ((i) stem memory TSCM cells (similar to initial cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7R ⁇ +, However, it also shows a large amount of CD95, IL-2R ⁇ , CXCR3, and LFA-1, and shows many functional properties with memory cell characteristics); (ii) Central memory TCM cells express L-selectin and CCR7, which secrete IL -2, but does not produce IFN ⁇ or IL-4; and (iii) Effector memory TEM cells do not express L-selectin or CCR7, but produce effector cytokines like IFN ⁇ and IL-4
  • intracellular domain generally refers to any truncated intracellular domain sufficient to transduce an activation signal.
  • intracellular domains may include signal transduction domains and/or costimulatory domains.
  • the term "signal transduction domain” generally refers to a domain located inside a cell capable of transducing signals.
  • the signal transduction domain can transmit signals into cells.
  • the signal transduction domain may be the signal transduction domain of the chimeric antigen receptor.
  • the signaling domain may be selected from the signaling domain of CD3 ⁇ , the signaling domain of CD3 ⁇ , and the signaling domain of CD3 ⁇ .
  • costimulatory domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
  • the costimulatory domain may include the costimulatory domain of 4-1BB, the costimulatory domain of CD27, and/or the costimulatory domain of CD28.
  • the term "hinge region” generally refers to the part of the heavy chain molecule that connects the CH1 domain and the CH2 domain.
  • the hinge region may be the hinge region of the CAR.
  • the hinge region may be selected from the hinge region of CD8, the hinge region of IgG1, and the hinge region of IgG4.
  • the term "transmembrane region” generally refers to a peptide, polypeptide, or protein domain that can span the plasma membrane of a cell.
  • the transmembrane region may be the transmembrane region of the CAR.
  • the transmembrane region may be selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD24, and the transmembrane region of CD28.
  • targeting moiety generally refers to a type of moiety that acts on certain special tissues and cells.
  • the targeting moiety can specifically target tumor antigens.
  • the targeting moiety may include an antibody or an antigen-binding fragment thereof.
  • the term "specific binding and/or recognition” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can exist in a heterogeneous population of molecules (including biomolecules)
  • the situation can determine the existence of the target.
  • an antibody that specifically binds a target (which may be an epitope) is an antibody that binds to the target with greater affinity, affinity, easier, and/or longer duration than it binds to other targets.
  • the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • tumor antigen generally refers to an antigen molecule present on tumor cells or normal cells.
  • the tumor antigen may include: embryonic protein, glycoprotein antigen and squamous cell antigen.
  • the tumor antigen may be selected from the group consisting of the EDB domain of fibronectin, the EDA domain of fibronectin, and necrotic regions.
  • the term "antigen-binding fragment” generally refers to a fragment having antigen-binding activity.
  • the antigen-binding fragment may be selected from the following group: Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to patients, human patients.
  • the pharmaceutical composition described in this application may include a modified immune cell and optionally a pharmaceutically acceptable carrier.
  • the acceptable ingredients of the pharmaceutical composition are non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally refers to one of the components of the pharmaceutical composition, which may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids , Sugars, chelating agents, counter ions, metal complexes and/or non-ionic surfactants, etc.
  • the pharmaceutically acceptable carrier may include excipients, for example, the excipients may be selected from the following group: starch, dextrin, sucrose, lactose, magnesium stearate, calcium sulfate, carboxymethyl , Talcum powder, calcium alginate gel, chitosan and nano microspheres.
  • the pharmaceutically acceptable carrier may also be selected from the group consisting of pH adjusters, osmotic pressure adjusters, solubilizers and bacteriostatic agents.
  • the term "tumor” generally refers to a tumor or solid lesion formed by abnormal cell growth.
  • the tumor may be a solid tumor or hematoma.
  • the tumor may be selected from the following group: B lymphoid leukemia, breast cancer, gastric cancer, pancreatic cancer, multiple myeloma, mesothelioma, lung cancer, and liver cancer.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • administration generally refers to a method of administering a certain dose of a substance to a subject (eg, a patient).
  • Administration can be carried out by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if local treatment is required) intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Administration may be by any suitable route, for example by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is short-lived or long-term.
  • Various dosing schedules are covered herein, including but not limited to a single administration or multiple administrations at various time points, bolus administrations, and pulse infusions.
  • low density lipoprotein receptor-related protein (lowdensity lipoprotein receptor-related protein, LRP) generally refers to a type of cell surface protein, which belongs to an endocytic receptor, which is a low density lipoprotein receptor ( A member of the lowde nsitylipoprotein receptor (LDLR) gene family.
  • the density lipoprotein receptor-related protein or fragments thereof may be selected from the following group: low-density lipoprotein receptor-related protein 6 or truncated body thereof, and low-density lipoprotein receptor-related protein 5 or its fragment Truncated body.
  • LRP6 low-density lipoprotein receptor-related protein 6
  • LRP6 generally refers to any vertebrate source (including mammals such as primates (e.g., Any natural LRP6 of humans) and rodents (e.g., mice and rats), unless otherwise specified.
  • the term encompasses "full-length” unprocessed LRP6 as well as any form of LRP6 produced by intracellular processing.
  • the term also includes naturally occurring variants of LRP6, for example, splice variants or allelic variants.
  • LRP5 low-density lipoprotein receptor-related protein 5"
  • LRP5 generally refers to any vertebrate source (including mammals such as primates (e.g., Any natural LRP5 of humans) and rodents (e.g., mice and rats), unless otherwise specified.
  • the term encompasses "full-length” unprocessed LRP5 as well as any form of LRP5 produced by intracellular processing.
  • the term also includes naturally occurring variants of LRP5, for example, splice variants or allelic variants.
  • the term "truncated" generally refers to anything less than the whole.
  • the amino acid sequence of the truncated body of low-density lipoprotein receptor-related protein 6 may include any amino acid sequence shorter than the full-length or complete low-density lipoprotein receptor-related protein 6.
  • the truncated body of the low-density lipoprotein receptor-related protein 6 may include the intracellular region, the transmembrane region or the LDLR region of the low-density lipoprotein receptor-related protein 6.
  • the truncated body of the low-density lipoprotein receptor-related protein 5 may include the intracellular region, the transmembrane region, or the LDLR region of the low-density lipoprotein receptor-related protein 5.
  • Plasmid generally refers to DNA molecules other than chromosomes or nucleoids in organisms such as bacteria and yeasts. Plasmids can exist in the cytoplasm and have the ability to replicate autonomously, enabling them to maintain a constant copy number in progeny cells and express the genetic information they carry. Plasmids are used as gene carriers in genetic engineering research.
  • the polynucleotide encoding the leptin and/or functional fragments thereof, and/or the polynucleotide encoding the leptin receptor and/or functional fragments thereof may be contained in a plasmid .
  • the plasmid may include a viral vector.
  • retroviral vector generally refers to a virus particle that can clone and express foreign genes, but cannot self-package into virus particles that have the ability to proliferate. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains genes for the proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase, and env, which contains genes that make up the virus shell. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome. For example, the CAR molecule can be integrated into the host cell.
  • the term "lentiviral vector” generally refers to a diploid RNA viral vector belonging to retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus. Many of the sequence structures related to the viral activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome skeleton And expression structure prepared into a vector.
  • the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome.
  • the CAR molecule can be integrated into the host cell.
  • transposon generally refers to a discrete DNA fragment containing a transposase gene. It is flanked by terminal inverted repeats (TIR) containing transposase binding sites. Transposase can bind to TIR and transfer the transposon to a new site.
  • TIR terminal inverted repeats
  • homolog generally refers to having at least about 80% (for example, having at least about 80%, about 83%, about 85%, about 85%) with the amino acid sequence of the protein and/or the polypeptide. 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology protein or Peptides.
  • identity or “homology” are used interchangeably, which generally refers to the similarity, similarity, or association between two or more sequences.
  • percentage of sequence homology can be calculated in the following way: the two sequences to be aligned are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G, I) exists in the two sequences.
  • the same amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met
  • the sequence homology percentage e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met
  • the alignment to determine the percent sequence homology can be achieved 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.
  • the homology can also be determined by the following methods: PASTA and BLAST.
  • PASTA a description of the PASTA algorithm, please refer to WRPearson and DJLipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; And DJL ipman and WRPearson's "Fast and Sensitive Protein Similarity Search", Science, 227:1435-1441, 1989.
  • BLAST see S. Altschul, W. Gish, W. Miller, EW Myers, and DL ipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215:403-410, 1990.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation 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%.
  • the present application provides a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element, and the modified immune cell further Comprising: leptin and/or functional fragments thereof; and/or, leptin receptor and/or functional fragments thereof, and compared with immune cells without corresponding modification, the modified immune cells The expression of the leptin receptor and/or its functional fragments is increased.
  • the expression level of the leptin receptor and/or its functional fragments can be measured by Western blotting (WB), ELISA, flow cytometry, and PCR methods.
  • the modified immune cells may include lymphocytes.
  • the modified immune cells may include T cells.
  • the T cells may comprise memory stem cell-like T cells (TSCM) and/or central memory T cells (TCM).
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • the TSCM may be CCR7 + and/or CD62L + .
  • the TSCM also be selected from the group: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD95 +, CD3 +, CD4 + And CD8 + .
  • the leptin and/or functional fragments thereof may be derived from humans.
  • the leptin receptor and/or functional fragments thereof can also be derived from humans.
  • the leptin may be secreted leptin or membrane-bound leptin.
  • the secreted leptin may include the amino acid sequence shown in SEQ ID NO: 13.
  • the membrane-bound leptin may include the amino acid sequence shown in SEQ ID NO: 14, which may be determined by the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, and SEQ ID NO: 15. The sequence is connected in sequence.
  • the leptin and/or functional fragments thereof may comprise the amino acid sequence shown in any one of SEQ ID NO: 13-14.
  • the leptin receptor may include a sequence with a self-activation function, so that the downstream signaling pathway of the leptin receptor can be continuously activated without binding leptin.
  • the functional fragment of the leptin receptor may comprise a structural domain of the leptin receptor selected from the group consisting of intracellular region, BOX1 region, FN3 region, C2 region and transmembrane region.
  • the functional fragment of the leptin receptor may comprise the transmembrane region of the leptin receptor (for example, it may comprise the amino acid sequence shown in SEQ ID NO: 15).
  • the functional fragment of the leptin receptor may include a leptin receptor signal peptide (for example, it may include the amino acid sequence shown in SEQ ID NO: 16).
  • the intracellular region of the leptin receptor may be the amino acid sequence at positions 865-1165 in the full length sequence of the leptin receptor, and the BOX 1 region of the leptin receptor may be the full length sequence of the leptin receptor.
  • the amino acid sequence of positions 871-878 in the leptin receptor, the FN3 region of the leptin receptor may be the position 732-820 or 636-723 or 536-620 or 235-317 in the full-length sequence of the leptin receptor
  • the amino acid sequence, the C2 region of the leptin receptor may be the amino acid sequence of positions 329-424 in the full length sequence of the leptin receptor
  • the transmembrane region of the leptin receptor may be the full length sequence of the leptin receptor
  • the leptin receptor and/or functional fragments thereof may comprise the amino acid sequence shown in any one of SEQ ID NO: 15-17.
  • sequence of the leptin and/or functional fragments thereof, and the sequence of the leptin receptor and/or functional fragments thereof may include one or more amino acid substitutions, additions, and / Or missing.
  • sequence of the leptin and/or functional fragments thereof may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the source sequence.
  • sequence of the leptin receptor and/or its functional fragment may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the source sequence.
  • the chimeric antigen receptor may not include the leptin and/or functional fragments thereof.
  • the chimeric antigen receptor may also not include the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor may contain the leptin and/or functional fragments thereof to form a protein complex.
  • the chimeric antigen receptor may also include the leptin receptor and/or functional fragments thereof to form a protein complex.
  • the chimeric antigen receptor includes an intracellular domain, and the intracellular domain may include a signal transduction domain and/or a costimulatory domain.
  • the signal transduction domain may comprise one or more selected from the group consisting of: a signal transduction domain of CD3 ⁇ , a signal transduction domain of CD3 ⁇ , and a signal transduction domain of CD3 ⁇ .
  • the signaling domain may include the signaling domain of CD3 ⁇ (for example, it may include the amino acid sequence shown in SEQ ID NO: 10).
  • the costimulatory domain includes one or more selected from the group consisting of a costimulatory domain of 4-1BB, a costimulatory domain of CD27, and a costimulatory domain of CD28.
  • the costimulatory domain may include the costimulatory domain of 4-1BB (for example, it may include the amino acid sequence shown in SEQ ID NO: 9).
  • the costimulatory domain may include the costimulatory domain of CD28 (for example, it may include the amino acid sequence shown in SEQ ID NO: 8).
  • the nucleotide sequence encoding the costimulatory domain of 4-1BB may be as shown in SEQ ID NO: 33, and the nucleotide sequence encoding the costimulatory domain of CD28 may be as shown in SEQ ID NO: 32.
  • the chimeric antigen receptor may include a hinge region.
  • the hinge region may include one or more selected from the group consisting of a hinge region of CD8, a hinge region of IgG1, and a hinge region of IgG4.
  • the hinge region may include the hinge region of CD8 (for example, it may include the amino acid sequence shown in SEQ ID NO: 6).
  • the chimeric antigen receptor may comprise a transmembrane region.
  • the transmembrane region may include one or more 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 include the transmembrane region of CD8 (for example, it may include the amino acid sequence shown in SEQ ID NO: 7).
  • the chimeric antigen receptor may include a targeting moiety.
  • the targeting moiety may include an antibody or an antigen-binding fragment thereof.
  • the targeting moiety may include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, Fv Fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab') 2 fragments and camelized single domain antibodies.
  • the antibody may include a murine antibody.
  • the murine antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chain or a variant thereof, or comprise murine IgGl, IgG2, IgG3 or IgG4 or its The heavy chain constant region of the variant.
  • the antibody may be a humanized antibody.
  • the targeting moiety may be a framework (FR) region that immunospecifically binds to the relevant antigen and contains substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) that substantially has the amino acid sequence of a non-human antibody.
  • CDR complementarity determining region
  • substantially in the case of CDR means that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. .
  • the humanized antibody may basically comprise all at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human
  • the CDR regions of immunoglobulins (ie, antibodies) and all or substantially all of the framework regions are framework regions with human immunoglobulin consensus sequences.
  • the humanized antibody also contains at least a portion of an immunoglobulin constant region (e.g., Fc), usually that of a human immunoglobulin.
  • a humanized antibody contains at least one variable domain of a light chain and a heavy chain.
  • the antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • humanized antibodies contain only humanized light chains. In some embodiments, a humanized antibody contains only a humanized heavy chain. In a specific embodiment, a humanized antibody only contains a humanized variable domain of a light chain and/or a humanized heavy chain.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the targeting moiety may include scFv.
  • the targeting moiety may include the scFv of GPC3, and its amino acid sequence may be as shown in SEQ ID NO:1.
  • the targeting moiety may include L1H2 (targeting GPC3) scFv, and its amino acid sequence may be as shown in SEQ ID NO:47.
  • the targeting moiety may include a scFv of CD19, and its amino acid sequence may be as shown in SEQ ID NO: 2.
  • the targeting moiety may include scFv of BCMA, and its amino acid sequence may be as shown in SEQ ID NO: 3.
  • the targeting moiety may include Mesothelin (or MSLN) scFv, and its amino acid sequence may be as shown in SEQ ID NO:4.
  • the targeting moiety may include scFv of HER2, and its amino acid sequence may be as shown in SEQ ID NO: 5.
  • the nucleotide sequence of the scFv encoding GPC3 may be as shown in SEQ ID NO: 25.
  • the nucleotide sequence of the scFv encoding CD19 may be as shown in SEQ ID NO: 26.
  • the nucleotide sequence of the scFv encoding BCMA may be as shown in SEQ ID NO: 27.
  • the nucleotide sequence of the scFv encoding Mesothelin may be as shown in SEQ ID NO: 28.
  • the nucleotide sequence of the scFv encoding HER2 may be as shown in SEQ ID NO: 29.
  • the targeting moiety can specifically bind and/or recognize tumor antigens.
  • the targeting moiety can specifically bind to and/or recognize a target selected from the group consisting of CD19, HER2, Mesothelin, GPC3 and BCMA.
  • the chimeric antigen receptor can be connected to a splicing protein.
  • the splicing protein can be T2A or P2A.
  • the splicing protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 11-12.
  • the chimeric antigen receptor can also be connected to a ribosome skip site.
  • the ribosome skip site may be IRES.
  • the ribosome skip site may include the nucleotide sequence shown in SEQ ID NO: 46.
  • the chimeric antigen receptor may sequentially include a targeting moiety, a hinge region, a transmembrane region, and an intracellular domain from N-terminus to C-terminus.
  • the chimeric antigen receptor may sequentially include a targeting moiety, a hinge region, a transmembrane region, a costimulatory domain, and a signal transduction domain from N-terminal to C-terminal.
  • the chimeric antigen receptor may sequentially include the scFv of GPC3, the hinge region of CD8, the transmembrane region of CD8, the costimulatory domain of 4-1BB, and the signal transduction domain of CD3 ⁇ from N-terminal to C-terminal.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the chimeric antigen receptor may be GPC3-41BB.
  • the chimeric antigen receptor may sequentially include the scFv of BCMA, the hinge region of CD8, the transmembrane region of CD8, the costimulatory domain of 4-1BB, and the signal transduction domain of CD3 ⁇ in sequence from N-terminus to C-terminus.
  • the chimeric antigen receptor may include the amino acid sequence shown in SEQ ID NO: 24.
  • the chimeric antigen receptor may be BCMA-41BB.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • sequence of the chimeric antigen receptor may include one or more amino acid substitutions, additions and/or deletions.
  • the sequence of the chimeric antigen receptor may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the source sequence.
  • sequence of the T cell receptor may include one or more amino acid substitutions, additions and/or deletions.
  • sequence of the T cell receptor may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the source sequence.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element and leptin and/or a functional fragment thereof.
  • the modified immune cell may be GPC3-41BB-Li, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the amino acid sequence of leptin is shown in SEQ ID NO: 13, The amino acid sequence of the spliced protein is shown in SEQ ID NO: 11.
  • the modified immune cell may be BCMA-41BB-Li, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the amino acid sequence of leptin is shown in SEQ ID NO: 13 , The amino acid sequence of the spliced protein is shown in SEQ ID NO: 11.
  • the modified immune cell may include a chimeric antigen receptor and/or its coding element and a leptin receptor and/or a functional fragment thereof.
  • the modified immune cell may be GPC3-41BB-LiR, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the amino acid sequence of the leptin receptor is shown in SEQ ID NO: 17.
  • the amino acid sequence of the leptin receptor signal peptide is shown in SEQ ID NO: 16
  • the amino acid sequence of the spliced protein is shown in SEQ ID NO: 11.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, leptin and/or functional fragments thereof, and leptin receptor and/or functional fragments thereof.
  • the modified immune cell may be GPC3-41BB-LiM, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the amino acid sequence of leptin is shown in SEQ ID NO: 13, The amino acid sequence of the transmembrane region of the leptin receptor is shown in SEQ ID NO: 15, the amino acid sequence of the splicing protein is shown in SEQ ID NO: 11, and the amino acid sequence of the linker is shown in SEQ ID NO: 22.
  • the modified immune cell may be BCMA-41BB-LiM, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the amino acid sequence of leptin is shown in SEQ ID NO: 13
  • the amino acid sequence of the transmembrane region of the leptin receptor is shown in SEQ ID NO: 15, the amino acid sequence of the splicing protein is shown in SEQ ID NO: 11, and the amino acid sequence of the linker is shown in SEQ ID NO: 22.
  • the modified immune cells may include T cell receptors and/or coding elements thereof, and leptin and/or functional fragments thereof.
  • the modified immune cells may include T cell receptors and/or coding elements thereof, and leptin receptors and/or functional fragments thereof.
  • the modified immune cells may include T cell receptors and/or coding elements thereof, leptin and/or functional fragments thereof, and leptin receptors and/or functional fragments thereof.
  • sequence of the modified immune cell described in the present application may include one or more amino acid substitutions, additions and/or deletions.
  • the sequence of the modified immune cell may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to its source sequence.
  • the present application provides a modified immune cell comprising leptin and/or functional fragments thereof, and/or leptin receptor and/or functional fragments thereof, and low-density lipoprotein receptor Related proteins or fragments thereof.
  • the modified immune cells may include lymphocytes.
  • the modified immune cells may include T cells.
  • the T cells may comprise memory stem cell-like T cells (TSCM) and/or central memory T cells (TCM).
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • the TSCM may be CCR7 + and/or CD62L + .
  • the TSCM also be selected from the group: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD95 +, CD3 +, CD4 + And CD8 + .
  • the structure and properties of the leptin and/or its functional fragments and the leptin receptor and/or its functional fragments can be as described above, and will not be repeated here.
  • the density lipoprotein receptor-related protein or fragments thereof may be selected from the following group: low-density lipoprotein receptor-related protein 6 or truncated body thereof, and low-density lipoprotein receptor-related protein 5 or its fragment Truncated body.
  • the truncated body of the low-density lipoprotein receptor-related protein 6 may comprise the intracellular region of the low-density lipoprotein receptor-related protein 6; and/or, the low-density lipoprotein receptor-related protein 5
  • the truncated body may include the intracellular region of the low-density lipoprotein receptor-related protein 5.
  • the truncated body of the low-density lipoprotein receptor-associated protein 6 may further comprise a functional domain selected from the group consisting of: the transmembrane region of the low-density lipoprotein receptor-associated protein 6 and the low-density lipoprotein The LDLR region of receptor-associated protein 6; and/or, wherein the truncated body of low-density lipoprotein receptor-related protein 5 may further comprise a functional domain selected from the group consisting of: the low-density lipoprotein receptor-related protein The transmembrane region of 5 and the LDLR region of the low-density lipoprotein receptor-related protein 5.
  • the density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 (ie, LRP6), and its amino acid sequence may be as shown in SEQ ID NO: 18.
  • the density lipoprotein receptor-related protein or a fragment thereof may be a truncated body of low-density lipoprotein receptor-related protein 6 (ie, TL6), and its amino acid sequence may be as shown in SEQ ID NO: 19.
  • the density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 (ie LRP5), and its amino acid sequence may be as shown in SEQ ID NO: 20.
  • the density lipoprotein receptor-related protein or a fragment thereof may be a truncated body of low-density lipoprotein receptor-related protein 5 (ie, TL5), and its amino acid sequence may be as shown in SEQ ID NO:21.
  • the nucleotide sequence encoding LRP6 may be as shown in SEQ ID NO: 41
  • the nucleotide sequence encoding TL6 may be as shown in SEQ ID NO: 42
  • the nucleotide sequence encoding LRP5 may be as shown in SEQ ID NO: 43.
  • the nucleotide sequence encoding TL5 may be as shown in SEQ ID NO:44.
  • the density lipoprotein receptor related protein or a fragment thereof may be low density lipoprotein receptor related protein 6 or a truncated body thereof. In other cases, the density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 or a truncated body thereof. In other cases, the density lipoprotein receptor-related protein or fragments thereof may also be low-density lipoprotein receptor-related protein 6 or its truncation, and low-density lipoprotein receptor-related protein 5 or its truncation. Short body.
  • the low-density lipoprotein receptor-related protein or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 18-21.
  • sequence of the low-density lipoprotein receptor-related protein or a fragment thereof may include one or more amino acid substitutions, additions and/or deletions.
  • sequence of the low-density lipoprotein receptor-related protein or fragment thereof may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the source sequence.
  • the modified immune cell may also include a chimeric antigen receptor or a T cell receptor.
  • the chimeric antigen receptor may not include the leptin and/or functional fragments thereof.
  • the chimeric antigen receptor may also not include the leptin receptor and/or functional fragments thereof.
  • the chimeric antigen receptor may not include the density lipoprotein receptor-related protein or fragments thereof.
  • the chimeric antigen receptor may contain the leptin and/or functional fragments thereof to form a protein complex.
  • the chimeric antigen receptor may also include the leptin receptor and/or functional fragments thereof to form a protein complex.
  • the chimeric antigen receptor may also include the density lipoprotein receptor-related protein or a fragment thereof to form a protein complex.
  • the specific structure and properties of the chimeric antigen receptor can be as described above, and will not be repeated here.
  • the modified immune cell may include a chimeric antigen receptor and/or its coding element and a low-density lipoprotein receptor related protein or a fragment thereof.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element and low-density lipoprotein receptor-related protein 6.
  • the modified immune cell may be GPC3-41BB-L6, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the amino acid sequence of the low-density lipoprotein receptor-related protein 6 As shown in SEQ ID NO: 18, the amino acid sequence of the spliced protein is shown in SEQ ID NO: 11.
  • the modified immune cell may be BCMA-41BB-L6, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the amino acid of the low-density lipoprotein receptor-related protein 6 The sequence is shown in SEQ ID NO: 18, and the amino acid sequence of the spliced protein is shown in SEQ ID NO: 11.
  • the modified immune cell may include a chimeric antigen receptor and/or its coding element, a low-density lipoprotein receptor related protein or a fragment thereof, and leptin and/or a functional fragment thereof.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, low-density lipoprotein receptor-related protein 6, and leptin and/or a functional fragment thereof.
  • the modified immune cell may be GPC3-41BB-L6-Li, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence is shown in SEQ ID NO: 18, the amino acid sequence of leptin is shown in SEQ ID NO: 13, and the amino acid sequence of the spliced protein is shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the modified immune cell may be BCMA-41BB-L6-Li, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence of leptin is shown in SEQ ID NO: 18, the amino acid sequence of leptin is shown in SEQ ID NO: 13, and the amino acid sequence of splicing protein is shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, a low-density lipoprotein receptor related protein or its fragment, and a leptin receptor and/or its functional fragment.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, low-density lipoprotein receptor-related protein 6, and a leptin receptor and/or functional fragments thereof.
  • the modified immune cell may be GPC3-41BB-L6-LiR, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence is shown in SEQ ID NO: 18, the amino acid sequence of the leptin receptor signal peptide is shown in SEQ ID NO: 16, and the amino acid sequence of the leptin receptor is shown in SEQ ID NO: 17, the amino acid of the cut protein
  • the sequence is shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the modified immune cell may be BCMA-41BB-L6-LiR, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence of the leptin receptor is shown in SEQ ID NO: 18, the amino acid sequence of the leptin receptor signal peptide is shown in SEQ ID NO: 16, and the amino acid sequence of the leptin receptor is shown in SEQ ID NO: 17.
  • the amino acid sequence is shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, a low-density lipoprotein receptor related protein or its fragment, leptin and/or its functional fragment, and leptin Receptors and/or functional fragments thereof.
  • the modified immune cell may comprise a chimeric antigen receptor and/or its coding element, low-density lipoprotein receptor-related protein 6, leptin and/or functional fragments thereof, and leptin Receptors and/or functional fragments thereof.
  • the modified immune cell may be GPC3-41BB-L6-LiM, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 23, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence is shown in SEQ ID NO: 18, the amino acid sequence of leptin is shown in SEQ ID NO: 13, the amino acid sequence of the leptin receptor transmembrane region is shown in SEQ ID NO: 15, the amino acid sequence of the cut protein As shown in SEQ ID NO: 11 and SEQ ID NO: 12, the amino acid sequence of the linker is shown in SEQ ID NO: 22.
  • the modified immune cell may be BCMA-41BB-L6-LiM, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 24, and the low-density lipoprotein receptor-related protein 6
  • the amino acid sequence of the leptin is shown in SEQ ID NO: 18, the amino acid sequence of leptin is shown in SEQ ID NO: 13, and the amino acid sequence of the leptin receptor transmembrane region is shown in SEQ ID NO: 15, the amino acid sequence of the cut protein
  • the sequence is shown in SEQ ID NO: 11 and SEQ ID NO: 12, and the amino acid sequence of the linker is shown in SEQ ID NO: 22.
  • the modified immune cells may include T cell receptors and/or their coding elements and low-density lipoprotein receptor-related proteins or fragments thereof.
  • the modified immune cells may include T cell receptors and/or their coding elements, low-density lipoprotein receptor-related proteins or fragments thereof, and leptin and/or functional fragments thereof.
  • the modified immune cells may include T cell receptors and/or their coding elements, low-density lipoprotein receptor related proteins or fragments thereof, and leptin receptors and/or functional fragments thereof.
  • the modified immune cells may include T cell receptors and/or their coding elements, low-density lipoprotein receptor-related proteins or fragments thereof, leptin and/or functional fragments thereof, and leptin receptors. Body and/or functional fragments thereof.
  • sequence of the modified immune cell described in the present application may include one or more amino acid substitutions, additions and/or deletions.
  • the sequence of the modified immune cell may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to its source sequence.
  • the present application also provides a pharmaceutical composition, which may include the modified immune cells described in the present application and optionally a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surface activity. ⁇ etc.
  • the pharmaceutically acceptable carrier may include excipients, for example, the excipients may be selected from the following group: starch, dextrin, sucrose, lactose, magnesium stearate, calcium sulfate, carboxymethyl , Talcum powder, calcium alginate gel, chitosan and nano microspheres.
  • the pharmaceutically acceptable carrier may also be selected from the following group: pH adjusters, osmotic pressure adjusters, solubilizers and bacteriostatic agents.
  • the application also provides the use of the modified immune cells or the pharmaceutical composition in the preparation of drugs for treating tumors.
  • this application provides a method for promoting the proliferation of immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • the present application provides a method for promoting the production of memory immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • this application provides a method for enhancing the ability of immune cells to kill tumors, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • the present application provides a method for inhibiting the expression of immune negative regulatory proteins by immune cells, which includes the following steps:
  • a modified immune cell comprising a chimeric antigen receptor and/or its coding element, or a T cell receptor and/or its coding element;
  • the negative immunomodulatory protein may be TIM3, LAG3, PD1 or PDL1.
  • the increase in the expression level may include an increase in the expression level of the leptin receptor and/or functional fragments thereof in the modified immune cells compared to immune cells that have not been modified accordingly.
  • the expression level of the leptin receptor and/or its functional fragments can be measured by Western blot (WB), ELISA, flow cytometry, and PCR methods.
  • the step 2) may include:
  • the polynucleotide encoding the leptin and/or functional fragments thereof is introduced into the modified immune cell, and/or the leptin receptor encoding and/or is introduced into the modified immune cell The functional fragment of the polynucleotide.
  • the polynucleotide encoding the leptin and/or functional fragments thereof, and/or the polynucleotide encoding the leptin receptor and/or functional fragments thereof may be contained in a plasmid .
  • the plasmid may include a viral vector.
  • the plasmid may also include a retroviral vector, a lentiviral vector or a transposon plasmid.
  • the modified immune cells may include lymphocytes.
  • the modified immune cells may include T cells.
  • the T cells may comprise memory stem cell-like T cells (TSCM) and/or central memory T cells (TCM).
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • the TSCM may be CCR7 + and/or CD62L + .
  • the TSCM also be selected from the group: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD95 +, CD3 +, CD4 + And CD8 + .
  • the structure and properties of the leptin and/or its functional fragments and the leptin receptor and/or its functional fragments can be as described above, and will not be repeated here.
  • this application also provides a method for promoting the proliferation of immune cells, which includes the following steps:
  • the modified immune cells express the leptin and/or functional fragments thereof, and/or the expression amount of the leptin receptor and/or functional fragments thereof in the modified immune cells is increased, and the modified immune cells Of immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • this application also provides a method for promoting the production of memory immune cells, which includes the following steps:
  • the modified immune cells express the leptin and/or functional fragments thereof, and/or the expression amount of the leptin receptor and/or functional fragments thereof in the modified immune cells is increased, and the modified immune cells Of immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • this application also provides a method for enhancing the ability of immune cells to kill tumors, which includes the following steps:
  • the modified immune cells express the leptin and/or functional fragments thereof, and/or the expression amount of the leptin receptor and/or functional fragments thereof in the modified immune cells is increased, and the modified immune cells Of immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • the present application also provides a method for inhibiting the expression of immune negative regulatory proteins by immune cells, which includes the following steps:
  • the modified immune cells express the leptin and/or functional fragments thereof, and/or the expression amount of the leptin receptor and/or functional fragments thereof in the modified immune cells is increased, and the modified immune cells Of immune cells express low-density lipoprotein receptor-related proteins or fragments thereof.
  • the negative immunomodulatory protein may be TIM3, LAG3, PD1 or PDL1.
  • the increase in the expression level may include an increase in the expression level of the leptin receptor and/or functional fragments thereof in the modified immune cells compared to immune cells that have not been modified accordingly.
  • the expression level of the leptin receptor and/or its functional fragments can be measured by Western blot (WB), ELISA, flow cytometry, and PCR methods.
  • the method of promoting the proliferation of immune cells can also be It includes the following steps: introducing a polynucleotide encoding the leptin and/or a functional fragment thereof into the modified immune cell, and/or introducing the polynucleotide encoding the leptin receptor into the modified immune cell Body and/or polynucleotides of functional fragments thereof.
  • the method of promoting the proliferation of immune cells can also be The method includes the following steps: introducing a polynucleotide encoding the low-density lipoprotein receptor-related protein or a fragment thereof into the modified immune cell.
  • the polynucleotide encoding the leptin and/or functional fragments thereof, and/or the polynucleotide encoding the leptin receptor and/or functional fragments thereof may be contained in a plasmid .
  • the polynucleotide encoding the low-density lipoprotein receptor-related protein or fragment thereof is contained in a plasmid.
  • the plasmid may include a viral vector.
  • the plasmid may also include a retroviral vector, a lentiviral vector or a transposon plasmid.
  • the modified immune cells may include lymphocytes.
  • the modified immune cells may include T cells.
  • the T cells may comprise memory stem cell-like T cells (TSCM) and/or central memory T cells (TCM).
  • TSCM memory stem cell-like T cells
  • TCM central memory T cells
  • the TSCM may be CCR7 + and/or CD62L + .
  • the TSCM also be selected from the group: CD45RA + or CD45RA -, CD45RO +, or CD45RO -, CD27 +, CD28 + , CD127 +, CD122 +, CD95 +, CD3 +, CD4 + And CD8 + .
  • the structure and properties of the leptin and/or functional fragments thereof, the leptin receptor and/or functional fragments thereof, and the low-density lipoprotein receptor-related protein or fragments thereof may be as described above The description of the article will not be repeated here.
  • this application also provides a method for inhibiting tumor cell proliferation and/or killing tumor cells, the method comprising: contacting the modified immune cells described in this application with tumor cells.
  • the contact may be administration, that is, a dose of the modified immune cells described in this application is administered to the subject (eg, patient).
  • Administration can be carried out by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if local treatment is required) intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Administration may be by any suitable route, for example by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is short-lived or long-term.
  • Various dosing schedules are covered herein, including but not limited to a single administration or multiple administrations at various time points, bolus administrations, and pulse infusions.
  • the method for inhibiting tumor cell proliferation and/or killing tumor cells may include an in vitro method or an ex vivo method.
  • the tumor includes solid tumors and/or non-solid tumors.
  • the tumor may include B lymphoid leukemia, breast cancer, gastric cancer, pancreatic cancer, multiple myeloma, mesothelioma, lung cancer, and/or liver cancer.
  • the following examples are only used to illustrate the protein molecules, preparation methods, and uses of the present application, and are not used to limit the scope of the present application.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.
  • the CAR-T targets GPC3 and BCMA as examples.
  • Fragments containing the CAR structure are artificially synthesized and constructed into a lentiviral vector (LV100A, System Biosciences), and then transfected to obtain a lentivirus according to the method described in its instructions.
  • LV100A lentiviral vector
  • GPC3-41BB GPC3-41BB-L6, GPC3-41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6 -LiR; BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-Li, BCMA-41BB-LiM, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM, BCMA-41BB -L6-LiR lentivirus.
  • the amino acid sequence of GPC3-41BB (from N-terminal to C-terminal) is composed of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, which are sequentially spliced and synthesized.
  • amino acid sequence of GPC3-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO:18 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 13 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, SEQ ID NO: 15 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 16, SEQ ID NO: 17 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-L6-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, SEQ ID NO: 15 are spliced and synthesized in sequence.
  • amino acid sequence of GPC3-41BB-L6-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17 are spliced and synthesized in sequence.
  • amino acid sequence of L1H2-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 47, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO:18 are spliced and synthesized in sequence.
  • amino acid sequence of L1H2-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 47, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 16, SEQ ID NO: 17 are spliced and synthesized in sequence.
  • amino acid sequence of L1H2-41BB-L6-Li (from N-terminus to C-terminus) is represented by SEQ ID NO: 47, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13 are spliced and synthesized in sequence.
  • amino acid sequence of L1H2-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 47, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, SEQ ID NO: 15 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB (from N-terminal to C-terminal) is sequenced from SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 Stitching and synthesis.
  • the nucleotide sequence of GPC3-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 41 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 37 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 38 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-L6-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 38 are spliced and synthesized in sequence.
  • the nucleotide sequence of GPC3-41BB-L6-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40 are spliced and synthesized in sequence.
  • the nucleotide sequence of L1H2-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 48, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 41 are spliced and synthesized in sequence.
  • the nucleotide sequence of L1H2-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 48, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40 are spliced and synthesized in sequence.
  • the nucleotide sequence of L1H2-41BB-L6-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 48, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37 are spliced and synthesized in sequence.
  • the nucleotide sequence of L1H2-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 48, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 38 are spliced and synthesized in sequence.
  • amino acid sequence of BCMA-41BB (from N-terminus to C-terminus) is synthesized by sequential splicing of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.
  • the amino acid sequence of BCMA-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO:18 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 13 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, SEQ ID NO: 15 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 16, SEQ ID NO: 17 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-L6-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 6, SEQ ID NO: 15 are spliced and synthesized in sequence.
  • the amino acid sequence of BCMA-41BB-L6-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17 are spliced and synthesized in sequence.
  • nucleotide sequence of BCMA-41BB (from N-terminal to C-terminal) is sequenced from SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 Stitching and synthesis.
  • the nucleotide sequence of BCMA-41BB-L6 (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 41 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 37 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 38 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-L6-Li (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-L6-LiM (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 38 are spliced and synthesized in sequence.
  • the nucleotide sequence of BCMA-41BB-L6-LiR (from N-terminal to C-terminal) is represented by SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40 are spliced and synthesized in sequence.
  • the infection experiment was carried out according to a conventional method known to those skilled in the art. A brief description of the infection steps are as follows:
  • peripheral blood mononuclear lymphocytes obtained> 1x10 7 cells by blood apheresis system.
  • T lymphocyte culture medium (Xvivo15 medium + 5% FBS + 200U/mlIL2 or Xvivo15 medium + 5% FBS+20ng/mlIL21+10ng/mlIL7) to make the final concentration 1*10 6 cells/ml, and cultured in a petri dish treated in 2 steps, the culture condition is 37°C + 5% CO 2 , and the culture time is 24 hours.
  • T cell culture medium Take a certain amount of T cell culture medium, add synperonic F108 with a final concentration of 1mg/ml, mix well, and heat the water bath to 37°C for later use.
  • GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6 -LiR T cells were cultured in vitro, BD Canto2 flow cytometer was used to detect the expression of T cells CD45RO, CCR7, CD62L and other memory-related proteins, as shown in Figure 1A-1H and Figure 2A-2H, expressing GPC3-41BB-L6, GPC3 -41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6-LiR Among T cells, memory stem cell-like T cells ( TSCM) and central memory T
  • BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-Li, BCMA-41BB-LiM, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM, BCMA-41BB-L6 -LiR T cells were cultured in vitro, and the BD Canto2 flow cytometer was used to detect the expression of T cells CD45RO, CCR7, CD62L and other memory-related proteins, as shown in Figure 3A-3H and Figure 4A-4H, expressing BCMA-41BB-L6, BCMA -41BB-Li, BCMA-41BB-LiM, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM, BCMA-41BB-L6-LiR among T cells memory stem cell-like T cells ( TSCM) and central memory T cells (TCM) (CD45RO-CCR7+;
  • Example 5 Flow cytometric detection of memory T cells in CD4 cells
  • GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6 -LiR T cells were cultured in vitro, and the CD45RO protein expression of T cells was detected by BD Canto2 flow cytometer.
  • TSCM memory stem cell-like T cells
  • GPC3-41BB, GPC3-41BB-L6, GPC3-41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6 -LiR T cells were cultured in vitro, and BD Canto2 flow cytometry was used to detect the expression of T cells CD3, CD4, CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28, as shown in Figure 6A-6H , Express GPC3-41BB-L6, GPC3-41BB-Li, GPC3-41BB-LiM, GPC3-41BB-LiR, GPC3-41BB-L6-Li, GPC3-41BB-L6-LiM, GPC3-41BB-L6-LiM, GPC3-41BB
  • Example 7 Flow cytometric detection of T cell negative regulatory protein expression
  • BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-Li, BCMA-41BB-LiM, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM, BCMA-41BB-L6 -LiR T cells were cultured in vitro, BD Canto2 flow cytometer was used to detect the expression of T cells CD3, CD4, CD8, PD1, PDL1, TIM3, LAG3, as shown in Figure 7A-7F, expressing BCMA-41BB-L6, BCMA- 41BB-Li, BCMA-41BB-LiM, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM, BCMA-41BB-L6-LiR The expression of negative regulatory proteins in T cells was significantly lower In the BCMA-41BB control group.
  • T cells expressing MSLN-41BB, MSLN-41BB-L6, MSLN-41BB-LiR, MSLN-41BB-L6-Li, MSLN-41BB-L6-LiM flow cytometric detection of T cells CD45RO, CCR7, CD62L and other memories
  • sex-related proteins as shown in Figure 9A and Figure 9B, among T cells expressing MSLN-41BB-L6, MSLN-41BB-LiR, MSLN-41BB-L6-Li, MSLN-41BB-L6-LiM
  • TSCM T-like T cells
  • T cells expressing HER2-41BB, HER2-41BB-L6, HER2-41BB-LiR, HER2-41BB-L6-Li, HER2-41BB-L6-LiM are cultured in vitro, and the memory of T cells CD45RO, CCR7, CD62L, etc.
  • T-like T cells CD45RO-CCR7+; CD45RO-CD62L+
  • T cells expressing CD19-41BB, CD19-41BB-L6, CD19-41BB-LiR, CD19-41BB-L6-Li, CD19-41BB-L6-LiM flow cytometric detection of T cells CD45RO, CCR7, CD62L and other memories
  • the proportion of T-like T cells (TSCM) (CD45RO-CCR7+; CD45RO-CD62L+) in the total cells was significantly higher than that of the CD19-41BB control group, especially CD19-41BB-LiR, CD19-41BB-L6-Li and CD19-41BB-L6 -LiM group.
  • T cells expressing GPC3-41BB, GPC3-41BB-L6, L1H2-41BB-L6, L1H2-41BB-LiR, L1H2-41BB-L6-Li, L1H2-41BB-L6-LiM and T cells not infected with CAR-T At the dose of 4x10e5 and 8x10e5 CAR-positive cells, 6-week-old subcutaneous tumor-bearing Huh7 tumors (1x10e7/ mouse) to 80-150mm 3 size NSG mice were given a single tail vein injection of CAR-T, 11 mice in each group.
  • Figures 14A-14G are injections of 8x10e5 uninfected CAR-T T cells, 8x10e5 GPC3-41BB T cells, 4x10e5 GPC3-41BB-L6 T cells, and 4x10e5 L1H2-41BB-infected T cells, respectively.
  • T cells infected with 8x10e5 L1H2-41BB-L6, T cells infected with 8x10e5 L1H2-41BB-LiR, weight changes in mice infected with T cells 8x10e5 L1H2-41BB-L6-Li) and tumors Body size measurement (Figure 13A-13G are T cells injected with uninfected 8x10e5 CAR-T, T cells infected with 8x10e5 GPC3-41BB, T cells infected with 4x10e5 GPC3-41BB-L6, T cells infected with 4x10e5 L1H2-41BB-L6-LiM, respectively T cells infected with 8x10e5 L1H2-41BB-L6, T cells infected with 8x10e5 L1H2-41BB-LiR, and mice infected with T cells 8x10e5 L1H2-41BB-L6-Li were measured).
  • mice in each group were randomly selected to take anticoagulant blood (sodium heparin anticoagulation) from the tail vein for blood IFNg detection (BD human th1/th2 CBA kit) ( Figure 15A-15C are the results on the 4th, 11th, and 17th days).
  • anticoagulant blood sodium heparin anticoagulation
  • mice in each group were randomly selected to take anticoagulant blood (sodium heparin anticoagulation) from the tail vein for flow cytometric detection of human CD8 cell content in the blood ( Figure 16A-16C in sequence) It is the results of the 11th, 17th, and 26th days).
  • mice receiving the second tumor in each group randomly selected 5 tail veins to take anticoagulant blood (sodium heparin anticoagulation) for flow cytometric detection of the human CD8 cell content in the blood ( Figure 16D-16F in turn The results are the 91st, 98th, and 105th days).
  • L1H2-41BB -LiR and L1H2-41BB-L6-Li are the most significant ( Figure 15A-15C); in vivo cell proliferation test results show GPC3-41BB-L6, L1H2-41BB-L6, L1H2-41BB-LiR, L1H2-41BB-L6-Li , L1H2-41BB-L6-LiM group has higher cell proliferation ability, among which L1H2-41BB-LiR and L1H2-41BB-L6-Li are the most significant (Figure 16A-16C).
  • the spleen and bone marrow examinations after the second tumor-bearing showed that the L1H2-41BB-L6, L1H2-41BB-LiR, L1H2-41BB-L6-Li, L1H2-41BB-L6-LiM groups had more memory T cells.
  • T cells expressing BCMA-41BB, BCMA-41BB-L6, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM and T cells not infected with CAR-T press 6x10e5 and 2x10e6CAR positive
  • the dose of cells was injected into the tail vein of 6-week-old subcutaneously tumor-bearing MM1.S tumor (1x10e7/ mouse) to 80-150mm 3 size NSG mice with CAR-T in a single tail vein injection, 11 rats in each group.
  • the tumor size was measured twice a week (Figure 18A-18J).
  • mice in each 2x10e6 dose group were randomly selected to take anticoagulant blood (sodium heparin anticoagulation) from the tail vein for blood IFNg detection (BD human th1/th2 CBA kit) ( Figure 19) .
  • anticoagulant blood sodium heparin anticoagulation
  • the other back of the mice without recurrence after tumor elimination was subjected to a second tumor-bearing MM1.S (1x10e7/mouse) to simulate tumor recurrence and observe tumor formation (Figure 18B-18J).
  • Figure 18A The results of the control group inoculated with uninfected CAR-T T cells are shown in Figure 18A.
  • the BCMA-41BB-LiR group is the most significant ( Figure 19).
  • All groups caused significant recurrence, but the number of recurrences in each dose group of BCMA-41BB-L6, BCMA-41BB-LiR, BCMA-41BB-L6-Li, BCMA-41BB-L6-LiM was significantly lower than that of the high-dose BCMA-41BB
  • BCMA-41BB-L6-Li and BCMA-41BB-L6-LiM are slightly better than BCMA-41BB-LiR, and both are significantly better than BCMA-41BB-L6, while the low-dose BCMA-41BB is completely suppressed Can't help the tumor.

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Abstract

提供了一种经修饰的免疫细胞，其包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件，且该免疫细胞还包含：瘦素和/或其功能性片段；和/或，瘦素受体和/或其功能性片段，且与未经相应经修饰的免疫细胞相比，该免疫细胞中的瘦素受体和/或其功能性片段的表达量增加。此外，还提供了一种经修饰的免疫细胞，其包含瘦素和/或其功能性片段，和/或，瘦素受体和/或其功能性片段，以及低密度脂蛋白受体相关蛋白或其片段。

Description

一种经修饰的免疫细胞及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种经修饰的免疫细胞及其用途。
背景技术
肿瘤是一种严重威胁人类健康的疾病，近年来，随着分子生物学、基因组学及蛋白质组学的进展，陆续问世了一系列的分子靶向药物，用于肿瘤的救治。而随着免疫学的快速发展，根据细胞毒性淋巴细胞(CTL)对靶细胞的识别特异性依赖于T淋巴细胞受体(TCR)这一发现，进一步研究了将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体融合成嵌合抗原受体(CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面用于肿瘤的治疗。这种CAR-T淋巴细胞能够以主要组织兼容性复合物(MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤细胞。然而基于免疫效应细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，但在大多数肿瘤的疗效仍不能令人满意。
发明内容
本申请提供一种经修饰的免疫细胞，其包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件，且所述经修饰的免疫细胞还包含：
瘦素和/或其功能性片段；和/或，
瘦素受体和/或其功能性片段，且与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。
在某些实施方式中，所述经修饰的免疫细胞包括淋巴细胞。
在某些实施方式中，所述经修饰的免疫细胞包括：T细胞。
在某些实施方式中，所述瘦素和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素受体和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素为分泌型瘦素或膜结合型瘦素。
在某些实施方式中，所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
在某些实施方式中，所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
在某些实施方式中，所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所 示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素受体和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体包含细胞内结构域，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CD3ζ的信号传导结构域。
在某些实施方式中，所述共刺激结构域包含4-1BB的共刺激结构域。
在某些实施方式中，所述嵌合抗原受体包含铰链区。
在某些实施方式中，所述铰链区包含CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体包含跨膜区。
在某些实施方式中，所述跨膜区包含CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体包含靶向部分。
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：CD19、HER2、Mesothelin、GPC3和BCMA。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
另一方面，本申请还提供药物组合物，其包括本申请所述的经修饰的免疫细胞及任选地药学上可接受的载体。
另一方面，本申请还提供本申请所述的经修饰的免疫细胞或本申请所述的药物组合物在制备治疗肿瘤的药物中的用途。
另一方面，本申请还提供一种促进免疫细胞增殖的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请还提供一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修 饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请还提供一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请还提供一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
在某些实施方式中，本申请所述方法的步骤2)包括：
向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
在某些实施方式中，所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸包含于质粒中。
在某些实施方式中，所述质粒包括病毒载体。
在某些实施方式中，所述经修饰的免疫细胞包括T细胞。
在某些实施方式中，所述表达量增加包括与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。
在某些实施方式中，所述瘦素和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素受体和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素为分泌型瘦素或膜结合型瘦素。
在某些实施方式中，所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
在某些实施方式中，所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
在某些实施方式中，所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素和/或其功能性片段和/或所述瘦素 受体和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体包含细胞内结构域，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CD3ζ的信号传导结构域。
在某些实施方式中，所述共刺激结构域包含4-1BB的共刺激结构域。
在某些实施方式中，所述嵌合抗原受体包含铰链区。
在某些实施方式中，中所述铰链区包含CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体包含跨膜区。
在某些实施方式中，所述跨膜区包含CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体包含靶向部分。
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：CD19、HER2、Mesothelin、GPC3和BCMA。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
另一方面，本申请还提供一种抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，所述方法包括：使本申请所述的经修饰的免疫细胞与肿瘤细胞接触。
在某些实施方式中，所述方法包括体外方法或离体方法。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和/或肝癌。
另一方面，本申请提供一种经修饰的免疫细胞，其包含瘦素和/或其功能性片段，和/或，瘦素受体和/或其功能性片段，以及低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述经修饰的免疫细胞包括T细胞。
在某些实施方式中，所述瘦素和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素受体和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素为分泌型瘦素或膜结合型瘦素。
在某些实施方式中，所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
在某些实施方式中，所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域： 胞内区，BOX 1区，FN3区，C2区和跨膜区。
在某些实施方式中，所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
在某些实施方式中，所述密度脂蛋白受体相关蛋白或其片段选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
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的截短体包含选自以下组的功能域：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，其中所述低密度脂蛋白受体相关蛋白5的截短体包含选自以下组的功能域：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SEQ ID NO:18-21中任一项所示的氨基酸序列。
在某些实施方式中，所述经修饰的免疫细胞还包含嵌合抗原受体或者T细胞受体。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素受体和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体不包含所述密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述嵌合抗原受体包含细胞内结构域，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CD3ζ的信号传导结构域。
在某些实施方式中，所述共刺激结构域包含4-1BB的共刺激结构域。
在某些实施方式中，所述嵌合抗原受体包含铰链区。
在某些实施方式中，所述铰链区包含CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体包含跨膜区。
在某些实施方式中，所述跨膜区包含CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体包含靶向部分。
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：CD19、HER2、Mesothelin、GPC3和BCMA。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
另一方面，本申请还提供一种药物组合物，其包括本申请所述的经修饰的免疫细胞及任选地药学上可接受的载体。
另一方面，本申请还提供本申请所述的经修饰的免疫细胞或本申请所述的药物组合物在制备治疗肿瘤的药物中的用途。
另一方面，本申请还提供一种促进免疫细胞增殖的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，本申请所述的方法包括以下步骤：向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
在某些实施方式中，本申请所述的方法包括以下步骤：向所述经修饰的免疫细胞中引入编码所述低密度脂蛋白受体相关蛋白或其片段的多核苷酸。
在某些实施方式中，所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸包含于质粒中。
在某些实施方式中，所述编码所述低密度脂蛋白受体相关蛋白或其片段的多核苷酸包含 于质粒中。
在某些实施方式中，所述质粒包括病毒载体。
在某些实施方式中，所述经修饰的免疫细胞包括T细胞。
在某些实施方式中，所述瘦素和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素受体和/或其功能性片段来源于人。
在某些实施方式中，所述瘦素为分泌型瘦素或膜结合型瘦素。
在某些实施方式中，所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
在某些实施方式中，所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
在某些实施方式中，所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
在某些实施方式中，所述密度脂蛋白受体相关蛋白或其片段选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
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的截短体包含选自以下组的功能域：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，其中所述低密度脂蛋白受体相关蛋白5的截短体包含选自以下组的功能域：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SEQ ID NO:18-21中任一项所示的氨基酸序列。
在某些实施方式中，所述经修饰的免疫细胞还包含嵌合抗原受体或者T细胞受体。
在某些实施方式中，所述嵌合抗原受体不包含所述瘦素和/或其功能性片段，和/或，所述瘦素受体和/或其功能性片段。
在某些实施方式中，所述嵌合抗原受体不包含所述密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述嵌合抗原受体包含细胞内结构域，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CD3ζ的信号传导结构域。
在某些实施方式中，所述共刺激结构域包含4-1BB的共刺激结构域。
在某些实施方式中，所述嵌合抗原受体包含铰链区。
在某些实施方式中，所述铰链区包含CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体包含跨膜区。
在某些实施方式中，所述跨膜区包含CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体包含靶向部分。
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：CD19、HER2、Mesothelin、GPC3和BCMA。
在某些实施方式中，所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
另一方面，本申请提供一种抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，所述方法包括：使本申请所述的经修饰的免疫细胞与肿瘤细胞接触。
在某些实施方式中，所述方法包括体外方法或离体方法。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和/或肝癌。
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申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请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
图1A-1H显示本申请所述的经GPC3靶向修饰的免疫细胞的记忆性T细胞流式检测结果(CCR7+CD45RO-)。
图2A-2H显示本申请所述的经GPC3靶向修饰的免疫细胞的记忆性T细胞流式检测结果(CCR7+CD62L+)。
图3A-3H显示本申请所述的经BCMA靶向修饰的免疫细胞的记忆性T细胞流式检测结果(CCR7+CD45RO-)。
图4A-4H显示本申请所述的经BCMA靶向修饰的免疫细胞的记忆性T细胞流式检测结果(CCR7+CD62L+)。
图5A-5H显示CD4细胞中本申请所述的经GPC3靶向修饰的CD8+免疫细胞的记忆性T细胞流式检测结果(CCR7+CD45RO-)。
图6A-6H显示CD8细胞中本申请所述的经GPC3靶向修饰的CD4+免疫细胞的记忆性T细胞流式检测结果(CCR7+CD45RO-)。
图7A-7F显示本申请所述的经修饰的免疫细胞负调控蛋白表达情况。
图8显示GPC3/MSLN/HER2相关CAR-T的扩增情况。
图9显示本申请所述的不同结构的经MSLN靶向修饰的免疫细胞的记忆性T细胞流式检测结果。
图10显示本申请所述的不同结构的经HER2靶向修饰的免疫细胞的记忆性T细胞流式检测结果。
图11显示本申请所述的不同结构的经CD19靶向修饰的免疫细胞的记忆性T细胞流式检测结果。
图12显示本申请所述的经不同靶点不同结构修饰的免疫细胞的反复刺激扩增结果。
图13显示本申请所述的经GPC3靶向修饰的免疫细胞的小鼠抑瘤消瘤和抑制复发的结果。
图14显示本申请所述的经GPC3靶向修饰的免疫细胞的小鼠体重变化结果。
图15显示本申请所述的经GPC3靶向修饰的免疫细胞的小鼠体内细胞因子变化结果。
图16显示本申请所述的经GPC3靶向修饰的免疫细胞的小鼠体内细胞变化结果。
图17显示本申请所述的经GPC3靶向修饰的免疫细胞的小鼠二次荷瘤后脾脏(A)和骨髓(B)修饰细胞含量检测结果。
图18显示本申请所述的经BCMA靶向修饰的免疫细胞的小鼠抑瘤消瘤和抑制复发的结果。
图19显示本申请所述的经BCMA靶向修饰的免疫细胞的小鼠体内细胞因子结果。
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。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分 子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“免疫细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。在本申请中，免疫细胞可以包括淋巴细胞。此外，在本申请中，免疫细胞可以包括T细胞、B细胞、自然杀伤(NK)细胞、肥大细胞、以及来源于骨髓的吞噬细胞。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor,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。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括靶向部分(例如，结合肿瘤相关抗原(tumor-associatedantigen,TAA)的部分)、铰链区、跨膜区和细胞内结构域。在本申请中，所述CAR可以基于抗体的抗原特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。
在本申请中，术语“T细胞受体”(T cell receptor，TCR)通常是指T细胞抗原受体，其为T细胞特异性识别和结合抗原肽-MHC分子的分子结构，通常与CD3分子呈复合物形式存在于T细胞表面。大多数T细胞的TCR由α和β肽链组成，少数T细胞的TCR由γ和δ肽链组成。
在本申请中，术语“编码”通常是指多核苷酸比如基因、cDNA、或mRNA中的核苷酸的特异性序列在生物学过程中作为合成其它聚合物和大分子的模板的固有特性，该聚合物以及大分子具有确定的核苷酸序列(即，rRNA、tRNA和mRNA)或确定的氨基酸序列及由其得到的生物学特性。因此，如果与所述基因对应的mRNA的转录和翻译在细胞或其它生物系统中产生蛋白质，则基因、cDNA或RNA编码蛋白质。
在本申请中，术语“瘦素”(Leptin)通常是指一类由脂肪组织分泌的蛋白质类激素。其前体由167个氨基酸残基组成，N末端有21个氨基酸残基信号肽，该前体的信号肽在血液中被切掉而成为146氨基酸，分子量为16KD，形成Leptin。Leptin具有广泛的生物学效应，其中较重要的是作用于下丘脑的代谢调节中枢，发挥抑制食欲，减少能量摄取，增加能量消耗，抑制脂肪合成的作用。
在本申请中，术语“瘦素受体”通常是指一类跨膜受体，主要由胞外区、跨膜区和胞内区组成。人类瘦素受体有多种形式，分为长型(OB-RL)和短型(OB-RS)两种，两者的胞外区和跨膜区完全一致，主要区别是胞内区的长度及氨基酸序列组成不同。瘦素长型受体主要在下丘脑表达，包括弓型核、室旁核、下丘脑背内侧核及侧下丘脑区，短型受体多分布于脂肪组织、心肌、肺、卵巢和造血组织等。
在本申请中，术语“功能性片段”通常是指保留与其更大对应物相同活性或能力的较大多肽或多核苷酸的片段。功能性片段的活性水平可以与所述更大对应物的活性相同，或比它更小或更大。例如，瘦素的功能性片段可以是这样的多肽，其由比全长瘦素蛋白更少的氨基酸组成，但是仍然可以保留全长瘦素的活性。
在本申请中，术语“淋巴细胞”通常主要包括自然杀伤(NK)细胞、T细胞、或B细胞。NK细胞是一类细胞毒性淋巴细胞，其代表先天免疫系统的主要成分。NK细胞排斥肿瘤与受病毒感染的细胞。NK细胞通过细胞凋亡或程序性细胞死亡方法来起作用。NK细胞被称为“自然杀伤”是因为其不需要活化就能杀伤细胞。T细胞在细胞介导免疫(不涉及抗体)中起着主要作用。T细胞的T细胞受体(TCR)使得T细胞与其他淋巴细胞型不同。胸腺(免疫系统的特化器官)是T细胞成熟的主要因素。T细胞有下列六种类型：辅助T细胞(例如CD4+细胞)、细胞毒性T细胞(也称为TC、细胞毒性T淋巴细胞、CTL、T-杀伤细胞、溶细胞性T细胞、CD8+T细胞、或杀伤T细胞)、记忆T细胞((i)干记忆TSCM细胞(类似初始细胞)是CD45RO-、CCR7+、CD45RA+、CD62L+(L-选凝素)、CD27+、CD28+、与IL-7Rα+，但是也表现大量的CD95、IL-2Rβ、CXCR3、与LFA-1，及显出很多有记忆细胞特色的功能属性)；(ii)中央记忆TCM细胞表达L-选凝素和CCR7，其分泌IL-2，但不产生IFNγ或IL-4；与(iii)效应记忆TEM细胞不表达L-选凝素或CCR7，但产生像IFNγ与IL-4的效应细胞因子)、调节T细胞(Treg、抑制T细胞、或CD4+CD25+调节T细胞)、自然杀伤T细胞(NKT)及γδT细胞。另一方面，B细胞在体液免疫(涉及抗体)中起着主要作用。B细胞产生抗体与抗原及具有抗原呈递细胞(APC)作用和在被抗原相互作用活化后转变成记忆B细胞。
在本申请中，术语“细胞内结构域”通常是指足以转导活化信号的任何截短部分的细胞内结构域。在本申请中，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。
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，所述信号传导结构域可以将信号传导至细胞内。例如，所述信号传导结构域可以是所述嵌合抗原受体的信号传导结构域。例如，在某些实施方式中，所述信号传导结构域可选自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。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。在本申请中，所述共刺激结构域可包括4-1BB的共刺激结构域、CD27的共刺激结构域和/或CD28的共刺激结构域。
在本申请中，术语“铰链区”通常是指使CH1结构域与CH2结构域连接的重链分子的部分。在本申请中，所述铰链区可以是CAR的铰链区。例如，所述铰链区可以选自CD8的 铰链区、IgG1的铰链区和IgG4的铰链区。
在本申请中，术语“跨膜区”通常是指能够跨越细胞质膜的肽、多肽或蛋白质的结构域。在本申请中，所述跨膜区可以是CAR的跨膜区。例如，所述跨膜区可以选自下组：CD8的跨膜区、CD24的跨膜区和CD28的跨膜区。
在本申请中，术语“靶向部分”通常是指一类针对某一些特殊组织、细胞起作用的部分。例如，靶向部分能够特异性靶向肿瘤抗原。在本申请中，所述靶向部分可以包括抗体或其抗原结合片段。
在本申请中，术语“特异性结合和/或识别”通常是指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在一个实施方案中，抗体结合无关靶标的程度小于抗体对靶标的结合的约10％，如例如通过放射免疫分析(RIA)测量的。在某些实施方案中，特异性结合靶标的抗体具有<1x10 -6M、<1x10 -7M、<1x10 -8M、<1x10 -9M或<1x10 - 10M的解离常数(KD)。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“肿瘤抗原”，通常是指在肿瘤细胞或正常细胞上存在的抗原分子。所述肿瘤抗原可以包括：胚胎性蛋白、糖蛋白抗原和鳞状细胞抗原。所述肿瘤抗原可以选自下组：纤连蛋白的EDB结构域、纤连蛋白的EDA结构域和细胞坏死区域(necrotic regions)。
在本申请中，术语“抗原结合片段”通常是指具有抗原结合活性的片段。在本申请中，所述抗原结合片段可以选自下组：Fab，Fab’，F(ab’) 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。
在本申请中，术语“药物组合物”通常指涉及适合施用于患者、人患者的组合物。例如，本申请所述的药物组合物，其可以包含经修饰的免疫细胞及任选地药学上可接受的载体。在某些实施方式中，药物组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载体”通常是指药物组合物的组分之一，其可以包括缓冲剂、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。例如，所述药学上可接受的载体可以包括赋形剂，例如，所述赋形剂可以选自下组：淀粉、糊精、蔗糖、乳糖、硬脂酸镁、硫酸钙、羧甲基素、滑石粉、海藻酸钙凝胶、壳聚糖和纳米微球等。例如，所述药学上可接受的载体还可以 选自下组：pH调节剂、渗透压调节剂、增溶剂和抑菌剂。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤。例如，肿瘤可选自以下组：B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和肝癌。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的物质的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。肠胃外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。可以通过任何合适的途径，例如通过注射(诸如静脉内或皮下注射)来给药，这部分地取决于施用是短暂的或长期的。本文涵盖各种给药排程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
在本申请中，术语“低密度脂蛋白受体相关蛋白(lowdensitylipoproteinreceptor-relatedpro tein,LRP)”通常是指一类细胞表面蛋白，属于一种内吞性受体，系低密度脂蛋白受体(lowde nsitylipoproteinreceptor,LDLR)基因家族中的一员。在本申请中，所述密度脂蛋白受体相关蛋白或其片段可以选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
在本申请中，术语“低密度脂蛋白受体相关蛋白6(Low-density lipoproteinreceptor-related protein 6)”或“LRP6”通常是指来自任何脊椎动物来源(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠))的任何天然LRP6，除非另有说明。该术语涵盖“全长”未加工的LRP6以及由细胞内加工产生的任何形式的LRP6。该术语还包括天然存在的LRP6的变体，例如，剪接变体或等位变体。
在本申请中，术语“低密度脂蛋白受体相关蛋白5(Low-density lipoproteinreceptor-related protein 5)”或“LRP5”通常是指来自任何脊椎动物来源(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠))的任何天然LRP5，除非另有说明。该术语涵盖“全长”未加工的LRP5以及由细胞内加工产生的任何形式的LRP5。该术语还包括天然存在的LRP5的变体，例如，剪接变体或等位变体。
在本申请中，术语“截短体”通常是指少于整体的任何事物。例如，低密度脂蛋白受体相关蛋白6的截短体的氨基酸序列可以包括比全长或完整低密度脂蛋白受体相关蛋白6短的任何氨基酸序列。例如，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、跨膜区或LDLR区。又例如，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、跨膜区或LDLR区。
在本申请中，术语“质粒”通常是指细菌、酵母菌等生物中染色体或拟核以外的DNA分子。质粒可存在于细胞质中，具有自主复制能力，使其能够在子代细胞中保持恒定的拷贝数，并表达所携带的遗传信息。质粒在遗传工程研究中被用作基因的载体。例如，在本申请中，编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸可以包含于质粒中。在本申请中，所述质粒可以包括病毒载体。
在本申请中，术语“逆转录病毒载体”通常是指可以克隆并表达外源基因，但不能自我包装成有增殖能力的病毒颗粒。此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构制备成的载体。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“转座子”通常是指含有转座酶基因的离散DNA片段。侧翼是含有转座酶结合位点的末端反向重复序列(TIR)。转座酶可与TIR结合并使转座子转移到新的位点。
在本申请中，术语“同源物”通常是指与所述蛋白质和/或所述多肽的氨基酸序列具有至少约80％(例如，具有至少约80％、约83％、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，术语“同一性”或“同源性”可互换地使用，其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：PASTA和BLAST。对PASTA算法的描述可以参见W.R.Pearson和D.J.L ipman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.L ipman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.L ipman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，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
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。
经修饰的免疫细胞
一方面，本申请提供一种经修饰的免疫细胞，其包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件，且所述经修饰的免疫细胞还包含：瘦素和/或其功能性片段；和/或，瘦素受体和/或其功能性片段，且与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。
所述瘦素受体和/或其功能性片段的表达量可以通过通过免疫印迹(WB)，ELISA，流式，PCR的方法进行测定。
在本申请中，所述经修饰的免疫细胞可以包括淋巴细胞。在某些实施方式中，所述经修饰的免疫细胞可以包括T细胞。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)。在某些实施方式中，所述TSCM可以为CCR7 +和/或CD62L +。在其他的一些实施方式中，所述TSCM还可以选自下组：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
瘦素和瘦素受体
在本申请中，所述瘦素和/或其功能性片段可以来源于人。所述瘦素受体和/或其功能性片段也可以来源于人。
在本申请中，所述瘦素可以为分泌型瘦素或膜结合型瘦素。例如，所述分泌性瘦素可以包含SEQ ID NO:13所示的氨基酸序列。所述膜结合型瘦素可以包含SEQ ID NO:14所示的氨基酸序列，其可以通过SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15所示的氨基酸序列依次连接合成。
在本申请中，所述瘦素和/或其功能性片段可以包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
在本申请中，所述瘦素受体可以包含具有自激活功能的序列，以便不需要结合瘦素就可以持续激活瘦素受体的下游信号通路。所述瘦素受体的功能性片段可以包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。例如，所述瘦素受体的功能性片段可以包含瘦素受体跨膜区(例如可包含SEQ ID NO:15所示的氨基酸序列)。又例如，所述瘦素受体的功能性片段可以包含瘦素受体信号肽(例如可包含SEQ ID NO:16所示的氨基酸序列)。此外，所述瘦素受体的胞内区可以是瘦素受体全长序列中第865-1165位的氨基酸序列，所述瘦素受体的BOX 1区可以是瘦素受体全长序列中第871-878位的氨基酸序列，所述瘦素受体的FN3区可以是瘦素受体全长序列中第732-820位或636-723位或536-620位或235-317位的氨基酸序列，所述瘦素受体的C2区可以是瘦素受体全长序列中第329-424位的氨基酸序列，所述瘦素受体的跨膜区可以是瘦素受体全长序列中第841-864位的氨基酸序列，所述瘦素受体的上述结构域在瘦素信号传导、蛋白二聚体或多聚体形成中具有重要功能。
在本申请中，所述瘦素受体和/或其功能性片段可以包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
此外，需要说明的是，所述瘦素和/或其功能性片段的序列，以及所述瘦素受体和/或其功能性片段的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述瘦素和/或其功能性片段的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。又例如，所述瘦素受体和/或其功能性片段的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
嵌合抗原受体
在本申请中，所述嵌合抗原受体可以不包含所述瘦素和/或其功能性片段。所述嵌合抗原受体还可以不包含所述瘦素受体和/或其功能性片段。在某些情形中，所述嵌合抗原受体可以包含所述瘦素和/或其功能性片段，以形成一个蛋白复合物。所述嵌合抗原受体还可以包含所述瘦素受体和/或其功能性片段，以形成一个蛋白复合物。
在本申请中，所述嵌合抗原受体包含细胞内结构域，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。所述信号传导结构域可以包含选自下组的一个或多个：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CD3ζ的信号传导结构域(例如可包含SEQ ID NO:10所示的氨基酸序列)。所述共刺激结构域包含选自下组的一个或多个：4-1BB的共刺激结构域、CD27的共刺激结构 域和CD28的共刺激结构域。所述共刺激结构域可以包含4-1BB的共刺激结构域(例如可包含SEQ ID NO:9所示的氨基酸序列)。又例如，所述共刺激结构域可以包含CD28的共刺激结构域(例如可包含SEQ ID NO:8所示的氨基酸序列)。编码4-1BB的共刺激结构域的核苷酸序列可以如SEQ ID NO:33所示，编码CD28的共刺激结构域的核苷酸序列可以如SEQ ID NO:32所示。
在本申请中，所述嵌合抗原受体可以包含铰链区。所述铰链区可以包含选自下组的一个或多个：CD8的铰链区、IgG1的铰链区和IgG4的铰链区。例如，所述铰链区可以包含CD8的铰链区(例如可包含SEQ ID NO:6所示的氨基酸序列)。
在本申请中，所述嵌合抗原受体可以包含跨膜区。所述跨膜区可以包含选自下组的一个或多个：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CD8的跨膜区(例如可包含SEQ ID NO:7所示的氨基酸序列)。
在本申请中，所述嵌合抗原受体可以包含靶向部分。所述靶向部分可以包括抗体或其抗原结合片段。例如，所述靶向部分可以包括但不限于重组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段、scFv片段、Fab片段、Fab'片段、F(ab') 2片段和骆驼化单结构域抗体。
在本申请中，所述抗体可以包括鼠源抗体。在某些实施方案中，所述鼠源抗体或其抗原结合片段，还可以包含鼠源κ、λ链或其变体的轻链恒定区，或包含鼠源IgGl，IgG2，IgG3或IgG4或其变体的重链恒定区。
在本申请中，所述抗体可以为人源化抗体。换句话说，所述靶向部分可以为免疫特异性结合至相关抗原且包含基本上具有人类抗体的氨基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。此处的“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一。所述人源化抗体基本上可以包含所有至少一个且通常两个可变域(Fab、Fab′、F(ab′)2、FabC、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白(即抗体)的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。人源化抗体还包含至少一部分免疫球蛋白恒定区(例如，Fc)，通常为人类免疫球蛋白的恒定区。在一些实施例中，人源化抗体含有轻链以及重链的至少一个可变域。抗体还可包括重链的CH1、铰链、CH2、CH3及CH4区。在一些实施例中，人源化抗体仅含人源化轻链。在一些实施例中，人源化抗体仅含人源化重链。在特定实施例中，人源化抗体仅含轻链和/或人源化重链的人源化可变域。
在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv， di-scFv和/或dAb。
在本申请中，所述靶向部分可以包括scFv。例如，所述靶向部分可以包括GPC3的scFv，其氨基酸序列可以如SEQ ID NO:1所示。例如，所述靶向部分可以包括L1H2(靶向GPC3)的scFv，其氨基酸序列可以如SEQ ID NO:47所示。例如，所述靶向部分可以包括CD19的scFv，其氨基酸序列可以如SEQ ID NO:2所示。例如，所述靶向部分可以包括BCMA的scFv，其氨基酸序列可以如SEQ ID NO:3所示。例如，所述靶向部分可以包括Mesothelin(或MSLN)的scFv，其氨基酸序列可以如SEQ ID NO:4所示。例如，所述靶向部分可以包括HER2的scFv，其氨基酸序列可以如SEQ ID NO:5所示。编码GPC3的scFv的核苷酸序列可以如SEQ ID NO:25所示。编码CD19的scFv的核苷酸序列可以如SEQ ID NO:26所示。编码BCMA的scFv的核苷酸序列可以如SEQ ID NO:27所示。编码Mesothelin(或MSLN)的scFv的核苷酸序列可以如SEQ ID NO:28所示。编码HER2的scFv的核苷酸序列可以如SEQ ID NO:29所示。
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。在某些实施方式中，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、HER2、Mesothelin、GPC3和BCMA。
在本申请中，所述嵌合抗原受体可以连接剪切蛋白。所述剪切蛋白可以为T2A或P2A。例如，所述剪切蛋白可以包含SEQ ID NO:11-12中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可以连接核糖体跳跃位点。所述核糖体跳跃位点可以为IRES。例如，所述核糖体跳跃位点可以包含SEQ ID NO:46所示的核苷酸序列。
在本申请中，所述嵌合抗原受体从N端至C端可以依次包含靶向部分、铰链区、跨膜区和细胞内结构域。例如，所述嵌合抗原受体从N端至C端可以依次包含靶向部分、铰链区、跨膜区、共刺激结构域和信号传导结构域。例如，所述嵌合抗原受体从N端至C端可以依次包含GPC3的scFv、CD8的铰链区、CD8的跨膜区、4-1BB的共刺激结构域和CD3ζ的信号传导结构域。例如，所述嵌合抗原受体可以包含SEQ ID NO:23所示的氨基酸序列。例如，所述嵌合抗原受体可以为GPC3-41BB。又例如，所述嵌合抗原受体从N端至C端可以依次包含BCMA的scFv、CD8的铰链区、CD8的跨膜区、4-1BB的共刺激结构域和CD3ζ的信号传导结构域。例如，所述嵌合抗原受体可以包含SEQ ID NO:24所示的氨基酸序列。例如，所述嵌合抗原受体可以为BCMA-41BB。
在本申请中，所述嵌合抗原受体可以包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
此外，需要说明的是，所述嵌合抗原受体的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述嵌合抗原受体的序列可以与其来源序列至少80％、85％、90％、 95％、96％、97％、98％或99％同源。
同样地，所述T细胞受体的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述T细胞受体的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
经修饰的免疫细胞
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件以及瘦素和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-Li，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，瘦素的氨基酸序列如SEQ ID NO:13所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-Li，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，瘦素的氨基酸序列如SEQ ID NO:13所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件以及瘦素受体和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-LiR，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，瘦素受体的氨基酸序列如SEQ ID NO:17所示，瘦素受体信号肽的氨基酸序列如SEQ ID NO:16所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-LiR，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，瘦素受体的氨基酸序列如SEQ ID NO:17所示，瘦素受体信号肽的氨基酸序列如SEQ ID NO:16所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、瘦素和/或其功能性片段以及瘦素受体和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-LiM，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，瘦素的氨基酸序列如SEQ ID NO:13所示，瘦素受体跨膜区的氨基酸序列如SEQ ID NO:15所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示，连接子的氨基酸序列如SEQ ID NO:22所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-LiM，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，瘦素的氨基酸序列如SEQ ID NO:13所示，瘦素受体跨膜区的氨基酸序列如SEQ ID NO:15所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示，连接子的氨基酸序列如SEQ ID NO:22所示。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件以及瘦素和/或其功能性片段。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件以及瘦素受体和/或其功能性片段。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件、瘦素和/或其功能性片段以及瘦素受体和/或其功能性片段。
此外，需要说明的是，本申请所述经修饰的免疫细胞的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述经修饰的免疫细胞的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
经修饰的免疫细胞
另一方面，本申请提供一种经修饰的免疫细胞，其包含瘦素和/或其功能性片段，和/或，瘦素受体和/或其功能性片段，以及低密度脂蛋白受体相关蛋白或其片段。
在本申请中，所述经修饰的免疫细胞可以包括淋巴细胞。在某些实施方式中，所述经修饰的免疫细胞可以包括T细胞。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)。在某些实施方式中，所述TSCM可以为CCR7 +和/或CD62L +。在其他的一些实施方式中，所述TSCM还可以选自下组：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
在本申请中，所述瘦素和/或其功能性片段以及所述瘦素受体和/或其功能性片段的结构和性质可以如上文所述，此处不再赘述。
在本申请中，所述密度脂蛋白受体相关蛋白或其片段可以选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。其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。此外，所述低密度脂蛋白受体相关蛋白6的截短体还可以包含选自以下组的功能域：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，其中所述低密度脂蛋白受体相关蛋白5的截短体还可以包含选自以下组的功能域：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。例如，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6(即LRP6)，其氨基酸序列可以如SEQ ID NO:18所示。例如，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6的截短体(即TL6)，其氨基酸序列可以如SEQ ID NO:19所示。又例如，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白5(即LRP5)，其氨基酸序列可以如SEQ ID NO:20所示。又例如，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白5的截短体(即TL5)，其氨基酸序列可以如SEQ ID NO:21所示。此外，编码LRP6的核苷酸序列可以如SEQ ID NO:41所示，编码TL6的核苷酸序列可以如SEQ ID NO:42所示，编码LRP5的核苷酸序列可以如SEQ ID NO:43所示， 编码TL5的核苷酸序列可以如SEQ ID NO:44所示。
在本申请中，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6或其截短体。在其他情形中，所述密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白5或其截短体。在另外的一些情形中，所述密度脂蛋白受体相关蛋白或其片段还可以为低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
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包含SEQ ID NO:18-21所示的氨基酸序列。
此外，需要说明的是，所述低密度脂蛋白受体相关蛋白或其片段的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述低密度脂蛋白受体相关蛋白或其片段的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
在本申请中，所述经修饰的免疫细胞还可以包含嵌合抗原受体或者T细胞受体。所述嵌合抗原受体可以不包含所述瘦素和/或其功能性片段。所述嵌合抗原受体还可以不包含所述瘦素受体和/或其功能性片段。此外，所述嵌合抗原受体还可以不包含所述密度脂蛋白受体相关蛋白或其片段。在某些情形中，所述嵌合抗原受体可以包含所述瘦素和/或其功能性片段，以形成一个蛋白复合物。所述嵌合抗原受体还可以包含所述瘦素受体和/或其功能性片段，以形成一个蛋白复合物。此外，所述嵌合抗原受体还可以包含所述密度脂蛋白受体相关蛋白或其片段，以形成一个蛋白复合物。所述嵌合抗原受体的具体结构和性质可以如上文所述，此处不再赘述。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件以及低密度脂蛋白受体相关蛋白或其片段。在某些实施方式中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件以及低密度脂蛋白受体相关蛋白6。例如，所述经修饰的免疫细胞可以为GPC3-41BB-L6，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-L6，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段以及瘦素和/或其功能性片段。在某些实施方式中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白6以及瘦素和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-L6-Li，其中，嵌合抗原 受体的氨基酸序列如SEQ ID NO:23所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素的氨基酸序列如SEQ ID NO:13所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-L6-Li，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素的氨基酸序列如SEQ ID NO:13所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段以及瘦素受体和/或其功能性片段。在某些实施方式中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白6以及瘦素受体和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-L6-LiR，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素受体信号肽的氨基酸序列如SEQ ID NO:16所示，瘦素受体的氨基酸序列如SEQ ID NO:17所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-L6-LiR，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素受体信号肽的氨基酸序列如SEQ ID NO:16所示，瘦素受体的氨基酸序列如SEQ ID NO:17所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示。
在本申请中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段、瘦素和/或其功能性片段以及瘦素受体和/或其功能性片段。在某些实施方式中，所述经修饰的免疫细胞可以包含嵌合抗原受体和/或其编码元件、低密度脂蛋白受体相关蛋白6、瘦素和/或其功能性片段以及瘦素受体和/或其功能性片段。例如，所述经修饰的免疫细胞可以为GPC3-41BB-L6-LiM，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:23所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素的氨基酸序列如SEQ ID NO:13所示，瘦素受体跨膜区的氨基酸序列如SEQ ID NO:15所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示，连接子的氨基酸序列如SEQ ID NO:22所示。又例如，所述经修饰的免疫细胞可以为BCMA-41BB-L6-LiM，其中，嵌合抗原受体的氨基酸序列如SEQ ID NO:24所示，所述低密度脂蛋白受体相关蛋白6的氨基酸序列如SEQ ID NO:18所示，瘦素的氨基酸序列如SEQ ID NO:13所示，瘦素受体跨膜区的氨基酸序列如SEQ ID NO:15所示，剪切蛋白的氨基酸序列如SEQ ID NO:11所示和SEQ ID NO:12所示，连接子的氨基酸序列如SEQ ID NO:22所示。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件以及低密度脂蛋白受体相关蛋白或其片段。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段以及瘦素和/或其功能性片段。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段以及瘦素受体和/或其功能性片段。
在本申请中，所述经修饰的免疫细胞可以包含T细胞受体和/或其编码元件、低密度脂蛋白受体相关蛋白或其片段、瘦素和/或其功能性片段以及瘦素受体和/或其功能性片段。
此外，需要说明的是，本申请所述经修饰的免疫细胞的序列可以包含一个或更多个氨基酸的替换、添加和/或缺失。例如，所述经修饰的免疫细胞的序列可以与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
药物组合物
另一方面，本申请还提供药物组合物，其可以包括本申请所述的经修饰的免疫细胞以及任选地药学上可接受的载体。其中，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。例如，所述药学上可接受的载体可以包括赋形剂，例如，所述赋形剂可以选自下组：淀粉、糊精、蔗糖、乳糖、硬脂酸镁、硫酸钙、羧甲基素、滑石粉、海藻酸钙凝胶、壳聚糖和纳米微球等。例如，所述药学上可接受的载体还可以选自下组：pH调节剂、渗透压调节剂、增溶剂和抑菌剂。
应用和用途
另一方面，本申请还提供所述的经修饰的免疫细胞或所述的药物组合物在制备治疗肿瘤的药物中的用途。
另一方面，本申请提供一种促进免疫细胞增殖的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请提供一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经 修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请提供一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
另一方面，本申请提供一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
在本申请中，所述免疫负调控蛋白可以是TIM3、LAG3、PD1或PDL1。
在本申请中，所述表达量增加可以包括与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。所述瘦素受体和/或其功能性片段的表达量可以通过免疫印迹(WB)，ELISA，流式，PCR的方法进行测定。
在本申请中，所述步骤2)可以包括：
向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
在本申请中，所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸可以包含于质粒中。所述质粒可以包括病毒载体。所述质粒还可以包括逆转录病毒载体、慢病毒载体或转座子质粒。
在本申请中，所述经修饰的免疫细胞可以包括淋巴细胞。在某些实施方式中，所述经修饰的免疫细胞可以包括T细胞。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)。在某些实施方式中，所述TSCM可以为CCR7 +和/或CD62L +。在其他的一些实施方式中，所述TSCM还可以选自下组：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
在本申请中，所述瘦素和/或其功能性片段以及所述瘦素受体和/或其功能性片段的结构和性质可以如上文所述，此处不再赘述。
另一方面，本申请还提供一种促进免疫细胞增殖的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
在本申请中，所述免疫负调控蛋白可以是TIM3、LAG3、PD1或PDL1。
在本申请中，所述表达量增加可以包括与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。所述瘦素受体和/或其功能性片段的表达量可以通过免疫印迹(WB)，ELISA，流式，PCR的方法进行测定。
在本申请中，所述促进免疫细胞增殖的方法、促进记忆性免疫细胞产生的方法、增强免疫细胞对肿瘤的杀伤能力的方法或抑制免疫细胞表达免疫负调控蛋白的方法的方法，其还可以包括以下步骤：向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
在本申请中，所述促进免疫细胞增殖的方法、促进记忆性免疫细胞产生的方法、增强免疫细胞对肿瘤的杀伤能力的方法或抑制免疫细胞表达免疫负调控蛋白的方法的方法，其还可以包括以下步骤：向所述经修饰的免疫细胞中引入编码所述低密度脂蛋白受体相关蛋白或其片段的多核苷酸。
在本申请中，所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸可以包含于质粒中。
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多核苷酸包含于质粒 中。
所述质粒可以包括病毒载体。所述质粒还可以包括逆转录病毒载体、慢病毒载体或转座子质粒。
在本申请中，所述经修饰的免疫细胞可以包括淋巴细胞。在某些实施方式中，所述经修饰的免疫细胞可以包括T细胞。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)。在某些实施方式中，所述TSCM可以为CCR7 +和/或CD62L +。在其他的一些实施方式中，所述TSCM还可以选自下组：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
在本申请中，所述瘦素和/或其功能性片段、所述瘦素受体和/或其功能性片段以及所述低密度脂蛋白受体相关蛋白或其片段的结构和性质可以如上文所述，此处不再赘述。
另一方面，本申请还提供一种抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，所述方法包括：使本申请所述的经修饰的免疫细胞与肿瘤细胞接触。在某些实施方式中，所述接触可以为施用，即向受试者(例如，患者)给予一定剂量的本申请所述经修饰的免疫细胞。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。肠胃外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。可以通过任何合适的途径，例如通过注射(诸如静脉内或皮下注射)来给药，这部分地取决于施用是短暂的或长期的。本文涵盖各种给药排程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
在本申请中，抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，其中所述方法可以包括体外方法或离体方法。
在本申请中，所述肿瘤包括实体瘤和/或非实体瘤。例如，所述肿瘤可以包括B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和/或肝癌。
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蛋白质分子、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。
实施例
实施例1.慢病毒载体的构建
所述CAR-T以靶向GPC3、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，System Biosciences公司)，随后依照其说明书记载的方式转染获得慢病毒，分别得到GPC3-41BB、GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR；BCMA-41BB、BCMA-41BB-L6、BCMA-41BB-Li、BCMA-41BB-LiM、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM、BCMA-41BB-L6-LiR慢病毒。
GPC3-41BB的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10依次拼接合成。
GPC3-41BB-L6的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18依次拼接合成。
GPC3-41BB-Li的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13依次拼接合成。
GPC3-41BB-LiM的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15依次拼接合成。
GPC3-41BB-LiR的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:17依次拼接合成。
GPC3-41BB-L6-Li的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13依次拼接合成。
GPC3-41BB-L6-LiM的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15依次拼接合成。
GPC3-41BB-L6-LiR的氨基酸序列(从N端到C端)由SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:17依次拼接合成。
L1H2-41BB-L6的氨基酸序列(从N端到C端)由SEQ ID NO:47、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18依次拼接合成。
L1H2-41BB-LiR的氨基酸序列(从N端到C端)由SEQ ID NO:47、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:17依次拼接合成。
L1H2-41BB-L6-Li的氨基酸序列(从N端到C端)由SEQ ID NO:47、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13依次拼接合成。
L1H2-41BB-L6-LiM的氨基酸序列(从N端到C端)由SEQ ID NO:47、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15依次拼接合成。相应的，GPC3-41BB的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34依次拼接合成。
GPC3-41BB-L6的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41依次拼接合成。
GPC3-41BB-Li的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:37依次拼接合成。
GPC3-41BB-LiM的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:30、SEQ ID NO:38依次拼接合成。
GPC3-41BB-LiR的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:40依次拼接合成。
GPC3-41BB-L6-Li的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37依次拼接合成。
GPC3-41BB-L6-LiM的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:30、SEQ ID NO:38依次拼接合成。
GPC3-41BB-L6-LiR的核苷酸序列(从N端到C端)由SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40依次拼接合成。
L1H2-41BB-L6的核苷酸序列(从N端到C端)由SEQ ID NO:48、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41依次拼接合成。
L1H2-41BB-LiR的核苷酸序列(从N端到C端)由SEQ ID NO:48、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:40 依次拼接合成。
L1H2-41BB-L6-Li的核苷酸序列(从N端到C端)由SEQ ID NO:48、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37依次拼接合成。
L1H2-41BB-L6-LiM的核苷酸序列(从N端到C端)由SEQ ID NO:48、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:30、SEQ ID NO:38依次拼接合成。此外，BCMA-41BB的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10依次拼接合成。
BCMA-41BB-L6的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18依次拼接合成。
BCMA-41BB-Li的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13依次拼接合成。
BCMA-41BB-LiM的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15依次拼接合成。
BCMA-41BB-LiR的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:17依次拼接合成。
BCMA-41BB-L6-Li的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13依次拼接合成。
BCMA-41BB-L6-LiM的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:22、SEQ ID NO:6、SEQ ID NO:15依次拼接合成。
BCMA-41BB-L6-LiR的氨基酸序列(从N端到C端)由SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:17依次拼接合成。
相应的，BCMA-41BB的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34依次拼接合成。
BCMA-41BB-L6的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ  ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41依次拼接合成。
BCMA-41BB-Li的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:37依次拼接合成。
BCMA-41BB-LiM的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:30、SEQ ID NO:38依次拼接合成。
BCMA-41BB-LiR的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:40依次拼接合成。
BCMA-41BB-L6-Li的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37依次拼接合成。
BCMA-41BB-L6-LiM的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:45、SEQ ID NO:30、SEQ ID NO:38依次拼接合成。
BCMA-41BB-L6-LiR的核苷酸序列(从N端到C端)由SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:36、SEQ ID NO:39、SEQ ID NO:40依次拼接合成。
实施例2.慢病毒感染T细胞
感染实验按照本领域技术人员已知的常规方法进行。简述感染步骤如下：
1.外周血单核淋巴细胞(PBMC)的获得，通过血液单采系统获得>1x10 7的细胞。
2.实验抗人CD3/CD28抗体处理细胞培养皿。
用PBS稀释抗人CD3及抗人CD28抗体，终浓度为1ug/ml，向细胞培养皿中加入稀释后的抗体混合液，使其铺满培养皿，室温孵育2小时。2小时后用PBS洗一次，备用。
3.对T细胞进行激活处理
将分离的PBMC用T淋巴细胞培养液(Xvivo15培养基+5％FBS+200U/mlIL2或Xvivo15培养基+5％FBS+20ng/mlIL21+10ng/mlIL7)进行重悬，使终浓度为1*10 6个细胞/ml，并放入2步骤中处理过的培养皿中培养，培养条件为37℃+5％CO 2，培养时间为24小时。
4.对激活的T细胞进行感染
1)感染试剂配制。
取一定量的T细胞培养液，加入终浓度为1mg/ml的synperonic F108，混匀，水浴锅加热至37℃待用。
2)培养板处理。
取1mg/ml抗人CD3抗体及0.5mg/ml抗人CD28抗体按1:1000体积比稀释至适量的PBS缓冲液中，并取retronectin(1mg/ml)试剂，按1:40体积比稀释至该PBS缓冲液中，混匀后均匀铺至细胞皿，室温孵育2小时。2小时后用PBS进行洗涤并待用。
3)慢病毒感染T细胞
用1)中所配感染试剂稀释已激活的T细胞，并按MOI＝3加入慢病毒，并混匀。均匀铺在2)中所处理的培养皿中。
感染后监测细胞密度，使细胞维持在1*10 6个细胞/ml，一般14天，可扩增30-1000倍。
实施例3.记忆性T细胞流式检测(GPC3)
表达GPC3-41BB、GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR T细胞体外培养，BD Canto2流式细胞仪检测T细胞CD45RO、CCR7、CD62L等记忆性相关蛋白的表达，如图1A-1H和图2A-2H所示，表达GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR 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)(CD45RO-CCR7+；CCR7+CD62L+)占总细胞比例均显著高于GPC3-41BB对照组,并且以GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR最显著。
实施例4.记忆性T细胞流式检测(BCMA)
表达BCMA-41BB、BCMA-41BB-L6、BCMA-41BB-Li、BCMA-41BB-LiM、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM、BCMA-41BB-L6-LiR T细胞体外培养，BD Canto2流式细胞仪检测T细胞CD45RO、CCR7、CD62L等记忆性相关蛋白的表达，如图3A-3H和图4A-4H所示，表达BCMA-41BB-L6、BCMA-41BB-Li、BCMA-41BB-LiM、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM、BCMA-41BB-L6-LiR 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)(CD45RO-CCR7+；CCR7+CD62L+)占总细胞比例均显著高于BCMA-41BB对照组。
实施例5.CD4细胞中记忆性T细胞流式检测
表达GPC3-41BB、GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR T细胞体外培养，BD Canto2流式细胞仪检测T细胞CD45RO蛋白的表达，如图5A-5H所示，表达GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR CD4T细胞中记忆性干细胞样T细胞(TSCM)(CD45RO-CCR7+)占总细胞比例均显著高于GPC3-41BB对照组。
实施例6.CD8细胞中记忆性T细胞流式检测
表达GPC3-41BB、GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR T细胞体外培养，BD Canto2流式细胞仪检测T细胞CD3、CD4、CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图6A-6H所示，表达GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR CD8T细胞中记忆性干细胞样T细胞(TSCM)(CD45RO-CCR7+)占总细胞比例均显著高于GPC3-41BB对照组。
实施例7：T细胞负调控蛋白表达流式检测
表达BCMA-41BB、BCMA-41BB-L6、BCMA-41BB-Li、BCMA-41BB-LiM、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM、BCMA-41BB-L6-LiR T细胞体外培养，BD Canto2流式细胞仪检测T细胞CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达，如图7A-7F所示，表达BCMA-41BB-L6、BCMA-41BB-Li、BCMA-41BB-LiM、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM、BCMA-41BB-L6-LiR T细胞中负调控蛋白的表达均显著低于BCMA-41BB对照组。
实施例8：T细胞扩增检测
表达GPC3-41BB、GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB-L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR、MSLN-41BB、MSLN-41BB-L6、MSLN-41BB-LiR、MSLN-41BB-L6-Li、MSLN-41BB-L6-LiM、HER2-41BB、HER2-41BB-L6、HER2-41BB-LiR、HER2-41BB-L6-Li、HER2-41BB-L6-LiM的T细胞体外培养，台盼蓝(GIBICO)染色后用Cellometer Mini全自动细胞计数仪计数记录细胞扩增能力，如图8A-8C所示，表达GPC3-41BB-L6、GPC3-41BB-Li、GPC3-41BB-LiM、GPC3-41BB-LiR、GPC3-41BB- L6-Li、GPC3-41BB-L6-LiM、GPC3-41BB-L6-LiR、MSLN-41BB-L6、MSLN-41BB-LiR、MSLN-41BB-L6-Li、MSLN-41BB-L6-LiM、HER2-41BB-L6、HER2-41BB-LiR、HER2-41BB-L6-Li、HER2-41BB-L6-LiM的T细胞扩增能力均显著高于GPC3-41BB、MSLN-41BB以及HER2-41BB对照组。
实施例9.记忆性T细胞流式检测(MSLN)
表达MSLN-41BB、MSLN-41BB-L6、MSLN-41BB-LiR、MSLN-41BB-L6-Li、MSLN-41BB-L6-LiM的T细胞体外培养，流式检测T细胞CD45RO、CCR7、CD62L等记忆性相关蛋白的表达，如图9A和图9B所示，表达MSLN-41BB-L6、MSLN-41BB-LiR、MSLN-41BB-L6-Li、MSLN-41BB-L6-LiM的T细胞中记忆性干细胞样T细胞(TSCM)(CD45RO-CCR7+；CD45RO-CD62L+)占总细胞比例均显著高于MSLN-41BB对照组，尤其是MSLN-41BB-LiR和MSLN-41BB-L6-LiM组。
实施例10.记忆性T细胞流式检测(HER2)
表达HER2-41BB、HER2-41BB-L6、HER2-41BB-LiR、HER2-41BB-L6-Li、HER2-41BB-L6-LiM的T细胞体外培养，流式检测T细胞CD45RO、CCR7、CD62L等记忆性相关蛋白的表达，如图10A和图10B所示，表达HER2-41BB-L6、HER2-41BB-LiR、HER2-41BB-L6-Li、HER2-41BB-L6-LiM的T细胞中记忆性干细胞样T细胞(TSCM)(CD45RO-CCR7+；CD45RO-CD62L+)占总细胞比例均显著高于HER2-41BB对照组，尤其是HER2-41BB-LiR、HER2-41BB-L6-Li和HER2-41BB-L6-LiM组。
实施例11.记忆性T细胞流式检测(CD19)
表达CD19-41BB、CD19-41BB-L6、CD19-41BB-LiR、CD19-41BB-L6-Li、CD19-41BB-L6-LiM的T细胞体外培养，流式检测T细胞CD45RO、CCR7、CD62L等记忆性相关蛋白的表达，如图11A和图11B所示，表达CD19-41BB-L6、CD19-41BB-LiR、CD19-41BB-L6-Li、CD19-41BB-L6-LiM的T细胞中记忆性干细胞样T细胞(TSCM)(CD45RO-CCR7+；CD45RO-CD62L+)占总细胞比例均显著高于CD19-41BB对照组，尤其是CD19-41BB-LiR、CD19-41BB-L6-Li和CD19-41BB-L6-LiM组。
实施例12.反复刺激检测(MSLN、L1H2[GPC3靶向]、HER2、CD19)
表达L1H2-41BB、L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6- LiM、MSLN-41BB、MSLN-41BB-L6、MSLN-41BB-LiR、MSLN-41BB-L6-Li、MSLN-41BB-L6-LiM、HER2-41BB、HER2-41BB-L6、HER2-41BB-LiR、HER2-41BB-L6-Li、HER2-41BB-L6-LiM、CD19-41BB、CD19-41BB-L6、CD19-41BB-LiR、CD19-41BB-L6-Li、CD19-41BB-L6-LiM的T细胞体外培养，第9天检测阳性率后用未感染的T细胞调整至阳性率一致，各取3x10e5 CAR阳性细胞与3x10e5辐照过的靶细胞进行第一轮共孵育刺激(L1H2 CAR-T对应靶细胞为Huh7、MSLN CAR-T对应靶细胞为OVCAR3、HER2 CAR-T对应靶细胞为SKOV3、CD19 CAR-T对应靶细胞为Raji)，每3-5天计数一次，并取3x10e5总细胞与3x10e5辐照过的靶细胞进行第二轮共孵育刺激，3-5天后重复上述操作进行第三轮共孵育刺激，3-5天后重复上述操作进行第四轮共孵育刺激。将计数结果换算后形成反复刺激扩增曲线。如图12A-12D，结果显示，各组带L6、LiR、L6-Li、L6-LiM结构组的反复刺激扩增能力均显著高于41BB对照组，暗示在体内具有更优的扩增能力和肿瘤治疗能力。
实施例13.CAR-T抑瘤消瘤实验(GPC3靶向)
表达GPC3-41BB、GPC3-41BB-L6、L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM的T细胞以及未感染CAR-T的T细胞，按4x10e5和8x10e5 CAR阳性细胞的剂量对6周龄的皮下荷瘤Huh7肿瘤(1x10e7/只)至80-150mm 3大小的NSG小鼠进行单次尾静脉注射CAR-T，每组11只。每周进行2次体重(图14A-14G分别为注射8x10e5未感染CAR-T的T细胞、感染8x10e5 GPC3-41BB的T细胞、感染4x10e5 GPC3-41BB-L6的T细胞、感染4x10e5 L1H2-41BB-L6-LiM的T细胞、感染8x10e5 L1H2-41BB-L6的T细胞、感染8x10e5 L1H2-41BB-LiR的T细胞、感染8x10e5 L1H2-41BB-L6-Li的T细胞的小鼠的体重变化)和瘤体大小测量(图13A-13G分别为注射未感染8x10e5 CAR-T的T细胞、感染8x10e5 GPC3-41BB的T细胞、感染4x10e5 GPC3-41BB-L6的T细胞、感染4x10e5 L1H2-41BB-L6-LiM的T细胞、感染8x10e5 L1H2-41BB-L6的T细胞、感染8x10e5 L1H2-41BB-LiR的T细胞、感染8x10e5 L1H2-41BB-L6-Li的T细胞的小鼠的瘤体大小测量)。CAR-T注射后第4、11、17天，各组随机选5只小鼠尾静脉取抗凝血(肝素钠抗凝)进行血液内IFNg检测(BD human th1/th2 CBA试剂盒)(图15A-15C依次为第4、11、17天的结果)。CAR-T注射后第11、17、26天，各组随机选5只小鼠尾静脉取抗凝血(肝素钠抗凝)进行血液内人CD8细胞含量的流式检测(图16A-16C依次为第11、17、26天的结果)。CAR-T注射后85天(肿瘤注射后98天)对消瘤后无复发的小鼠另侧背部进行二次荷瘤Huh7(1x10e7/只)，模拟肿瘤复发，观察肿瘤形成情况(图13)。第91、98、105天，各组二次接瘤的小鼠随机选5只尾静脉取抗凝血(肝素钠抗凝)进行血液内人CD8细胞含量的流式检测(图16D-16F 依次为第91、98、105天的结果)。第132天，各组二次荷瘤小鼠随机选5只取脾脏和骨髓，进行人CD8细胞含量的流式检测(图17A-17B)，实验结束。实验结果显示，与GC33-41BB对照组相比，GPC3-41BB-L6、L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM组均具有更显著的抑瘤消瘤效果和抗复发能力(图13)，同时均未表现出明显的毒副作用(图14，体重未出现明显下降)；体内细胞因子检测结果显示GPC3-41BB-L6、L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM组均具有更高的因子分泌，有利于促进CAR-T在体内的扩增，其中L1H2-41BB-LiR和L1H2-41BB-L6-Li最显著(图15A-15C)；体内细胞增殖检测结果显示GPC3-41BB-L6、L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM组均具有更高的细胞增殖能力，其中L1H2-41BB-LiR和L1H2-41BB-L6-Li最显著(图16A-16C)。另外，在二次荷瘤后，L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM组恢复到基值的体内CAR-T细胞水平有明显二次增殖，与其更优的抗复发能力成正相关，其中L1H2-41BB-LiR和L1H2-41BB-L6-Li最显著(图16D-16F)。同时，二次荷瘤后的脾脏和骨髓检测显示L1H2-41BB-L6、L1H2-41BB-LiR、L1H2-41BB-L6-Li、L1H2-41BB-L6-LiM组具有更多的记忆性T细胞归巢到淋巴结，其中以L1H2-41BB-L6-Li最显著。
实施例14.CAR-T抑瘤消瘤实验(BCMA靶向)
表达BCMA-41BB、BCMA-41BB-L6、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM的T细胞以及未感染CAR-T的T细胞，按6x10e5和2x10e6CAR阳性细胞的剂量对6周龄的皮下荷瘤MM1.S肿瘤(1x10e7/只)至80-150mm 3大小的NSG小鼠进行单次尾静脉注射CAR-T，每组11只。每周进行2次瘤体大小测量(图18A-18J)。CAR-T注射后第8天，各2x10e6剂量组随机选6只小鼠尾静脉取抗凝血(肝素钠抗凝)进行血液内IFNg检测(BD human th1/th2 CBA试剂盒)(图19)。CAR-T注射后80天对消瘤后无复发的小鼠另侧背部进行二次荷瘤MM1.S(1x10e7/只)，模拟肿瘤复发，观察肿瘤形成情况(图18B-18J)。接种未感染CAR-T的T细胞的对照组结果如图18A所示。实验结果显示，与接种2x10e6 BCMA-41BB阳性细胞的对照组(图18B)和接种6x10e5 BCMA-41BB阳性细胞的对照组相比(图18G)，接种2x10e6 BCMA-41BB-L6阳性细胞(图18C)和接种6x10e5 BCMA-41BB-L6阳性细胞(图18H)的实验组、接种2x10e6 BCMA-41BB-LiR阳性细胞(图18E)和接种6x10e5 BCMA-41BB-LiR阳性细胞(图18J)的实验组、接种2x10e6 BCMA-41BB-L6-Li阳性细胞(图18D)和接种6x10e5 BCMA-41BB-L6-Li阳性细胞(图18I)的实验组和接种6x10e5 BCMA-41BB-L6-LiM阳性细胞(图18F)的实验组均具有更显著的抑瘤消瘤效果和抗 复发能力；体内细胞因子检测结果显示现对于BCMA-41BB组，BCMA-41BB-L6、BCMA-41BB-LiR、BCMA-41BB-L6-Li组均具有更高的因子分泌，有利于促进CAR-T在体内的扩增，其中BCMA-41BB-LiR组最显著(图19)；另外，在二次荷瘤后，由于荷瘤剂量较高，各组均引起了明显复发，但是BCMA-41BB-L6、BCMA-41BB-LiR、BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM各剂量组复发数量均显著低于BCMA-41BB高剂量组，低剂量组中，BCMA-41BB-L6-Li、BCMA-41BB-L6-LiM略优于BCMA-41BB-LiR，均显著好于BCMA-41BB-L6，而低剂量BCMA-41BB则完全压不住肿瘤。
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。目前本申请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 (126)

  1. 经修饰的免疫细胞，其包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件，且所述经修饰的免疫细胞还包含：
    瘦素和/或其功能性片段；和/或，
    瘦素受体和/或其功能性片段，且与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。
  2. 根据权利要求1所述的经修饰的免疫细胞，其中所述经修饰的免疫细胞包括淋巴细胞。
  3. 根据权利要求1-2中任一项所述的经修饰的免疫细胞，其中所述经修饰的免疫细胞包括：T细胞。
  4. 根据权利要求1-3中任一项所述的经修饰的免疫细胞，其中所述瘦素和/或其功能性片段来源于人。
  5. 根据权利要求1-4中任一项所述的经修饰的免疫细胞，其中所述瘦素受体和/或其功能性片段来源于人。
  6. 根据权利要求1-5中任一项所述的经修饰的免疫细胞，其中所述瘦素为分泌型瘦素或膜结合型瘦素。
  7. 根据权利要求1-6中任一项所述的经修饰的免疫细胞，其中所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的经修饰的免疫细胞，其中所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
  9. 根据权利要求1-8中任一项所述的经修饰的免疫细胞，其中所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体不包含所述瘦素和/或其功能性片段。
  11. 根据权利要求1-10中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体不包含所述瘦素受体和/或其功能性片段。
  12. 根据权利要求1-11中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含细胞内结构域，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。
  13. 根据权利要求12所述的经修饰的免疫细胞，其中所述信号传导结构域包含CD3ζ的信号传导结构域。
  14. 根据权利要求12-13中任一项所述的经修饰的免疫细胞，其中所述共刺激结构域包含4-1BB的共刺激结构域。
  15. 根据权利要求1-14中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含铰链 区。
  16. 根据权利要求15所述的经修饰的免疫细胞，其中所述铰链区包含CD8的铰链区。
  17. 根据权利要求1-16中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含跨膜区。
  18. 根据权利要求17所述的经修饰的免疫细胞，其中所述跨膜区包含CD8的跨膜区。
  19. 根据权利要求1-18中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含靶向部分。
  20. 根据权利要求19所述的经修饰的免疫细胞，其中所述靶向部分包括scFv。
  21. 根据权利要求19-20中任一项所述的经修饰的免疫细胞，其中所述靶向部分特异性结合和/或识别肿瘤抗原。
  22. 根据权利要求19-21中任一项所述的经修饰的免疫细胞，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、HER2、Mesothelin、GPC3和BCMA。
  23. 根据权利要求1-22中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
  24. 药物组合物，其包括权利要求1-23中任一项所述的经修饰的免疫细胞及任选地药学上可接受的载体。
  25. 权利要求1-23中任一项所述的经修饰的免疫细胞或权利要求24所述的药物组合物在制备治疗肿瘤的药物中的用途。
  26. 一种促进免疫细胞增殖的方法，其包括以下步骤：
    1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
    2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
  27. 一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
    1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
    2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
  28. 一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
    1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
    2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
  29. 一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
    1)提供包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件的经修饰的免疫细胞；以及
    2)使1)的所述经修饰的免疫细胞表达瘦素和/或其功能性片段，和/或使1)的所述经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加。
  30. 根据权利要求26-29中任一项所述的方法，其中2)包括：
    向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
  31. 根据权利要求30所述的方法，其中所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸包含于质粒中。
  32. 根据权利要求31所述的方法，其中所述质粒包括病毒载体。
  33. 根据权利要求26-32中任一项所述的方法，其中所述经修饰的免疫细胞包括T细胞。
  34. 根据权利要求26-33中任一项所述的方法，其中所述表达量增加包括与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。
  35. 根据权利要求26-34中任一项所述的方法，其中所述瘦素和/或其功能性片段来源于人。
  36. 根据权利要求26-35中任一项所述的方法，其中所述瘦素受体和/或其功能性片段来源于人。
  37. 根据权利要求26-36中任一项所述的方法，其中所述瘦素为分泌型瘦素或膜结合型瘦素。
  38. 根据权利要求26-37中任一项所述的方法，其中所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
  39. 根据权利要求26-38中任一项所述的方法，其中所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
  40. 根据权利要求26-39中任一项所述的方法，其中所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
  41. 根据权利要求26-40中任一项所述的方法，其中所述嵌合抗原受体不包含所述瘦素和/或其功能性片段和/或所述瘦素受体和/或其功能性片段。
  42. 根据权利要求26-41中任一项所述的方法，其中所述嵌合抗原受体包含细胞内结构域，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。
  43. 根据权利要求42所述的方法，其中所述信号传导结构域包含CD3ζ的信号传导结构域。
  44. 根据权利要求42-43中任一项所述的方法，其中所述共刺激结构域包含4-1BB的共刺激结构域。
  45. 根据权利要求26-44中任一项所述的方法，其中所述嵌合抗原受体包含铰链区。
  46. 根据权利要求45所述的方法，其中所述铰链区包含CD8的铰链区。
  47. 根据权利要求26-46中任一项所述的方法，其中所述嵌合抗原受体包含跨膜区。
  48. 根据权利要求47所述的方法，其中所述跨膜区包含CD8的跨膜区。
  49. 根据权利要求26-48中任一项所述的方法，其中所述嵌合抗原受体包含靶向部分。
  50. 根据权利要求49所述的方法，其中所述靶向部分包括scFv。
  51. 根据权利要求49-50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。
  52. 根据权利要求49-51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、HER2、Mesothelin、GPC3和BCMA。
  53. 根据权利要求26-52中任一项所述的方法，其中所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
  54. 一种抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，所述方法包括：使权利要求1-23中任一项所述的经修饰的免疫细胞与肿瘤细胞接触。
  55. 根据权利要求54所述的方法，其中所述方法包括体外方法或离体方法。
  56. 根据权利要求54-55中任一项所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  57. 根据权利要求54-56中任一项所述的方法，其中所述肿瘤包括B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和/或肝癌。
  58. 经修饰的免疫细胞，其包含瘦素和/或其功能性片段，和/或，瘦素受体和/或其功能性片段，以及低密度脂蛋白受体相关蛋白或其片段。
  59. 根据权利要求58所述的经修饰的免疫细胞，其中所述经修饰的免疫细胞包括T细胞。
  60. 根据权利要求58-59中任一项所述的经修饰的免疫细胞，其中所述瘦素和/或其功能性片段来源于人。
  61. 根据权利要求58-60中任一项所述的经修饰的免疫细胞，其中所述瘦素受体和/或其功能性片段来源于人。
  62. 根据权利要求58-61中任一项所述的经修饰的免疫细胞，其中所述瘦素为分泌型瘦素或膜结合型瘦素。
  63. 根据权利要求58-62中任一项所述的经修饰的免疫细胞，其中所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
  64. 根据权利要求58-63中任一项所述的经修饰的免疫细胞，其中所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
  65. 根据权利要求58-64中任一项所述的经修饰的免疫细胞，其中所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
  66. 根据权利要求58-65中任一项所述的经修饰的免疫细胞，其中所述密度脂蛋白受体相关蛋白或其片段选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
  67. 根据权利要求66所述的经修饰的免疫细胞，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。
  68. 根据权利要求66-67中任一项所述的经修饰的免疫细胞，其中所述低密度脂蛋白受体相关蛋白6的截短体包含选自以下组的功能域：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，其中所述低密度脂蛋白受体相关蛋白5的截短体包含选自以下组的功能域：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。
  69. 根据权利要求58-68中任一项所述的经修饰的免疫细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:18-21中任一项所示的氨基酸序列。
  70. 根据权利要求58-69中任一项所述的经修饰的免疫细胞，其还包含嵌合抗原受体或者T细胞受体。
  71. 根据权利要求70所述的经修饰的免疫细胞，其中所述嵌合抗原受体不包含所述瘦素和/或其功能性片段。
  72. 根据权利要求70-71中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体不包含所述瘦素受体和/或其功能性片段。
  73. 根据权利要求70-72中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体不包含所述密度脂蛋白受体相关蛋白或其片段。
  74. 根据权利要求70-73中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含细胞内结构域，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。
  75. 根据权利要求74所述的经修饰的免疫细胞，其中所述信号传导结构域包含CD3ζ的信号传导结构域。
  76. 根据权利要求74-75中任一项所述的经修饰的免疫细胞，其中所述共刺激结构域包含4-1BB的共刺激结构域。
  77. 根据权利要求70-76中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含铰链区。
  78. 根据权利要求77所述的经修饰的免疫细胞，其中所述铰链区包含CD8的铰链区。
  79. 根据权利要求70-78中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含跨膜区。
  80. 根据权利要求79所述的经修饰的免疫细胞，其中所述跨膜区包含CD8的跨膜区。
  81. 根据权利要求70-80中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含靶向部分。
  82. 根据权利要求81所述的经修饰的免疫细胞，其中所述靶向部分包括scFv。
  83. 根据权利要求81-82中任一项所述的经修饰的免疫细胞，其中所述靶向部分特异性结合和/或识别肿瘤抗原。
  84. 根据权利要求81-83中任一项所述的经修饰的免疫细胞，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、HER2、Mesothelin、GPC3和BCMA。
  85. 根据权利要求70-84中任一项所述的经修饰的免疫细胞，其中所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
  86. 药物组合物，其包括权利要求58-85中任一项所述的经修饰的免疫细胞及任选地药学上可接受的载体。
  87. 权利要求58-85中任一项所述的经修饰的免疫细胞或权利要求86所述的药物组合物在制备治疗肿瘤的药物中的用途。
  88. 一种促进免疫细胞增殖的方法，其包括以下步骤：
    使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
    以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
  89. 一种促进记忆性免疫细胞产生的方法，其包括以下步骤：
    使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
    以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
  90. 一种增强免疫细胞对肿瘤的杀伤能力的方法，其包括以下步骤：
    使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
    以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
  91. 一种抑制免疫细胞表达免疫负调控蛋白的方法，其包括以下步骤：
    使经修饰的免疫细胞表达所述瘦素和/或其功能性片段，和/或，使经修饰的免疫细胞中瘦素受体和/或其功能性片段的表达量增加，
    以及使经修饰的免疫细胞表达低密度脂蛋白受体相关蛋白或其片段。
  92. 根据权利要求88-91中任一项所述的方法，其包括以下步骤：向所述经修饰的免疫细胞中引入编码所述瘦素和/或其功能性片段的多核苷酸，和/或向所述经修饰的免疫细胞中引入编码所述瘦素受体和/或其功能性片段的多核苷酸。
  93. 根据权利要求88-92中任一项所述的方法，其包括以下步骤：向所述经修饰的免疫细胞中引入编码所述低密度脂蛋白受体相关蛋白或其片段的多核苷酸。
  94. 根据权利要求88-93中任一项所述的方法，其中所述编码所述瘦素和/或其功能性片段的多核苷酸，和/或编码所述瘦素受体和/或其功能性片段的多核苷酸包含于质粒中。
  95. 根据权利要求88-94中任一项所述的方法，其中所述编码所述低密度脂蛋白受体相关蛋白或其片段的多核苷酸包含于质粒中。
  96. 根据权利要求95所述的方法，其中所述质粒包括病毒载体。
  97. 根据权利要求88-96中任一项所述的方法，其中所述经修饰的免疫细胞包括T细胞。
  98. 根据权利要求88-97中任一项所述的方法，其中所述瘦素和/或其功能性片段来源于人。
  99. 根据权利要求88-98中任一项所述的方法，其中所述瘦素受体和/或其功能性片段来源于人。
  100. 根据权利要求88-99中任一项所述的方法，其中所述瘦素为分泌型瘦素或膜结合型瘦素。
  101. 根据权利要求88-100中任一项所述的方法，其中所述瘦素和/或其功能性片段包含SEQ ID NO:13-14中任一项所示的氨基酸序列。
  102. 根据权利要求88-101中任一项所述的方法，其中所述瘦素受体的功能性片段包含所述瘦素受体的选自下组的结构域：胞内区，BOX 1区，FN3区，C2区和跨膜区。
  103. 根据权利要求88-102中任一项所述的方法，其中所述瘦素受体和/或其功能性片段包含SEQ ID NO:15-17中任一项所示的氨基酸序列。
  104. 根据权利要求88-103中任一项所述的方法，其中所述密度脂蛋白受体相关蛋白或其片段选自以下组：低密度脂蛋白受体相关蛋白6或其截短体，和低密度脂蛋白受体相关蛋白5或其截短体。
  105. 根据权利要求104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。
  106. 根据权利要求104-105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的截短体包含选自以下组的功能域：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，其中所述低密度脂蛋白受体相关蛋白5的截短体包含选自以下组的功能域：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。
  107. 根据权利要求88-106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SEQ ID NO:18-21中任一项所示的氨基酸序列。
  108. 根据权利要求88-107中任一项所述的方法，其中所述经修饰的免疫细胞还包含嵌合抗原受体或者T细胞受体。
  109. 根据权利要求108所述的方法，其中所述嵌合抗原受体不包含所述瘦素和/或其功能性片段，和/或，所述瘦素受体和/或其功能性片段。
  110. 根据权利要求108-109中任一项所述的方法，其中所述嵌合抗原受体不包含所述密度脂蛋白受体相关蛋白或其片段。
  111. 根据权利要求108-110中任一项所述的方法，其中所述嵌合抗原受体包含细胞内结构域，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。
  112. 根据权利要求111所述的方法，其中所述信号传导结构域包含CD3ζ的信号传导结构域。
  113. 根据权利要求111-112中任一项所述的方法，其中所述共刺激结构域包含4-1BB的共刺激结构域。
  114. 根据权利要求108-113中任一项所述的方法，其中所述嵌合抗原受体包含铰链区。
  115. 根据权利要求114所述的方法，其中所述铰链区包含CD8的铰链区。
  116. 根据权利要求108-115中任一项所述的方法，其中所述嵌合抗原受体包含跨膜区。
  117. 根据权利要求116所述的方法，其中所述跨膜区包含CD8的跨膜区。
  118. 根据权利要求108-117中任一项所述的方法，其中所述嵌合抗原受体包含靶向部分。
  119. 根据权利要求118所述的方法，其中所述靶向部分包括scFv。
  120. 根据权利要求118-119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。
  121. 根据权利要求118-120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、HER2、Mesothelin、GPC3和BCMA。
  122. 根据权利要求108-121中任一项所述的方法，其中所述嵌合抗原受体包含SEQ ID NO:23-24中任一项所示的氨基酸序列。
  123. 一种抑制肿瘤细胞增殖和/或杀伤肿瘤细胞的方法，所述方法包括：使权利要求58-85中 任一项所述的经修饰的免疫细胞与肿瘤细胞接触。
  124. 根据权利要求123所述的方法，其中所述方法包括体外方法或离体方法。
  125. 根据权利要求123-124中任一项所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  126. 根据权利要求123-125中任一项所述的方法，其中所述肿瘤包括B淋巴白血病、乳腺癌、胃癌、胰腺癌、多发性骨髓瘤、间皮瘤、肺癌和/或肝癌。
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