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WO2018121679A1 - 表达cxcr4的嵌合抗原受体修饰的淋巴细胞及制备方法和用途 - Google Patents

表达cxcr4的嵌合抗原受体修饰的淋巴细胞及制备方法和用途 Download PDF

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WO2018121679A1
WO2018121679A1 PCT/CN2017/119451 CN2017119451W WO2018121679A1 WO 2018121679 A1 WO2018121679 A1 WO 2018121679A1 CN 2017119451 W CN2017119451 W CN 2017119451W WO 2018121679 A1 WO2018121679 A1 WO 2018121679A1
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chimeric antigen
antigen receptor
seq
nucleotide sequence
protein
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French (fr)
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王永生
魏于全
郭福春
王玮
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Sichuan University
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4219—Receptors for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the field of cells, in particular to genetically engineering non-specific lymphocytes to be specifically transformed into a tumor surface-associated antigen and expressing a chemokine receptor CXCR4, thereby enriching and killing cells at specific sites. Preparation method of sex lymphocytes and its use in the treatment of malignant tumors.
  • Immunotherapy is considered to be the most promising treatment for cancer.
  • immunological checkpoint inhibitors such as CTLA-4, PD-1, etc.
  • CTLA-4, PD-1, etc. have shown superior effects over existing therapies in various tumor clinical trials, and are approved for malignancy such as non-small cell lung cancer and bladder cancer.
  • Treatment of tumors Unlike CTLA-4 and PD-1 antibodies, which exert anti-tumor effects by re-activating the body's immune response, chimeric antigen receptor-modified immune cell therapy does not depend on the immune status of the body, and the chimeric antigen receptor is genetically engineered. It is expressed on the surface of immune cells, recognizes tumor antigens, activates immune effector cells to produce direct anti-tumor effects.
  • chimeric antigen receptor-modified lymphocytes targeting CD19 have shown good results in clinical trials for the treatment of acute lymphoblastic leukemia and non-Hodgkin's lymphoma, with an effective rate of up to 90%, showing this The enormous therapeutic potential of therapies.
  • chimeric antigen receptor-modified lymphocytes have attractive prospects in tumor immunotherapy, there are still several problems: 1. Tumor-specific antigen deficiency. Currently, there are few tumor-specific surface antigens, and most tumors lack specific antigens, and most of them are tumor-associated antigens that are highly expressed in tumors but also expressed in normal tissues, thereby limiting their lymphocyte therapy as a chimeric antigen receptor. Target. For example, in the clinical study of chimeric antigen receptor-modified lymphocytes targeting HER2, since human lung tissue also expresses HER2, a non-specific cytotoxic killing reaction is caused, resulting in death of the patient. 2. Enrichment of chimeric antigen receptor-modified lymphocytes at the tumor site.
  • CXCL12/SDF-1 ⁇ is an important chemotactic regulator of T cells and myeloid monocytes, and by binding to its receptor CXCR4, drives cells to reach relevant sites along the concentration gradient.
  • CXCL12/SDF-1 ⁇ was elevated at the irradiation site, and the myeloid mononuclear cells were recruited to migrate to the irradiation site. This phenomenon was also found in some glial tumors that highly express CXCL12.
  • CXCR4 can be expressed in CXCL12/SDF-1 ⁇ -expressing gliomas or multiple myeloma by expressing CXCR4 on chimeric antigen receptor-modified lymphocytes, thereby enhancing anti-tumor effects.
  • the expression of CXCL12/SDF-1 ⁇ in the tumor site can be increased by radiotherapy and the like, and the chimeric antigen-modified lymphocytes can be induced to produce a homing effect, thereby producing a better anti-tumor effect.
  • the technical problem to be solved by the present invention is to provide a new and effective means for the treatment of malignant tumors.
  • the technical solution to solve the technical problem of the present invention is to provide a chimeric antigen receptor comprising an extracellular antigen binding region, a transmembrane region and an intracellular signal region.
  • the extracellular antigen-binding region is a single-chain variable region, receptor or ligand of a monoclonal antibody that binds to a tumor antigen.
  • the tumor antigen includes but not limited to: (EGFR), epithelial cell adhesion molecule (EpCAM), mesothelin, interleukin 13 receptor ⁇ 2 (IL-13R ⁇ 2), epidermal growth factor receptor 2 (ERBB2), epidermal growth factor receptor 3 (ERBB3), epidermal growth factor receptor 4 (ERBB4), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), ganglioside Antigen G D2 (GD2), folate receptor 1 (FOLR1), prostate specific membrane antigen (PSMA), melanin precursor protein (gp100), mucin 1 (MUC1), mucin 16 (MUC16), carbonic anhydrase 9 (CA9), L1 cell adhesion molecule (CD171), tumor antigen 125 (CA125), tumor antigen 15-3 (CA15-3), tumor antigen 19-9 (CA19-9), NY-ESO-1, MART-1 , MAGE4, B cell mature
  • the transmembrane region is a leukocyte differentiation antigen 8 (CD8) transmembrane region or a leukocyte differentiation antigen 28 (CD28) transmembrane region.
  • CD8 leukocyte differentiation antigen 8
  • CD28 leukocyte differentiation antigen 28
  • the intracellular signal region is leukocyte differentiation antigen 28 (CD28), tumor necrosis factor receptor superfamily member 4 (TNFRSF9/CD134/OX40), tumor necrosis factor receptor superfamily Member 9 (TNFRSF9/CD137/4-1BB), lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory molecule (ICOS), CD40, CD27 or DAP10 (DNAX activator protein 10) Or a variety of intracellular domains.
  • CD28 leukocyte differentiation antigen 28
  • TNFRSF9/CD134/OX40 tumor necrosis factor receptor superfamily member 4
  • TNFRSF9/CD137/4-1BB tumor necrosis factor receptor superfamily Member 9
  • LCK lymphocyte-specific protein tyrosine kinase
  • IFN inducible T cell costimulatory molecule
  • CD40 CD27 or DAP10 (DNAX activator protein 10)
  • DAP10 DNAX activator protein 10
  • the monoclonal antibody single-chain variable region is a scFv single-chain antibody variable region.
  • amino acid sequence of the variable region of the scFv single-chain antibody is represented by SEQ ID No. 1.
  • the coding nucleotide sequence of the variable region of the scFv single-chain antibody is represented by SEQ ID No. 2.
  • SEQ ID No. 2 Encoding nucleotide sequence of the variable region of scFv single-chain antibody
  • the above chimeric antigen receptor is obtained by sequentially linking a signal peptide, an scFv single chain antibody, a CD8 ⁇ transmembrane region, a CD28 intracellular segment, a 4-1BB intracellular segment, and a CD3 ⁇ chain through a nitrogen terminal to a carbon terminal.
  • amino acid sequence of the signal peptide is shown in SEQ ID No. 3.
  • the coding nucleotide sequence of the signal peptide is shown in SEQ ID No. 4.
  • amino acid sequence of the CD8 transmembrane region is represented by SEQ ID No. 5.
  • the coding nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID No. 6.
  • the amino acid sequence of the intracellular domain of CD28 is shown in SEQ ID No. 7.
  • the coding nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID No. 8.
  • amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID No. 9.
  • the coding nucleotide sequence of the 4-1BB intracellular domain is shown in SEQ ID No. 10.
  • the amino acid sequence of the CD3 ⁇ domain is shown in SEQ ID No. 11.
  • the coding nucleotide sequence of the CD3 ⁇ domain is shown in SEQ ID No. 12.
  • SEQ ID No. 12 The coding nucleotide sequence of the CD3 ⁇ domain
  • the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 13;
  • the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 14 or a degenerate sequence thereof;
  • nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.
  • SEQ ID NO: 14 Nucleotide sequence of gene encoding chimeric antigen receptor
  • the ligand is an APRIL ligand.
  • the APRIL amino acid sequence is represented by SEQ ID NO.
  • the coding nucleotide sequence of the APRIL is represented by SEQ ID NO.
  • the chimeric antigen receptor is obtained by sequentially linking a signal peptide, an APRIL, a CD8 ⁇ transmembrane region, an CD28 intracellular segment, a 4-1BB intracellular segment, and a CD3 ⁇ chain through a nitrogen terminal to a carbon terminal.
  • the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 17;
  • the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 18 or a degenerate sequence thereof;
  • nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.
  • SEQ ID NO: 18 Nucleotide sequence of gene encoding chimeric antigen receptor
  • the present invention also provides a chimeric antigen receptor-modified lymphocyte, which coexpresses the chemokine CXCR4.
  • the CXCR4 receptor is (1) a protein having the amino acid sequence of SEQ ID No. 19;
  • the nucleotide sequence of the coding gene of CXCR4 is (1): the nucleotide sequence shown in SEQ ID No: 20 or a degenerate sequence thereof;
  • nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO: 20. Or a similar protein.
  • the CXCR4 is co-expressed with a chimeric antigen receptor by P2A, and the amino acid sequence of the P2A is represented by SEQ ID No. 21.
  • nucleotide sequence of the coding gene of P2A is represented by SEQ ID No. 22.
  • the P2A may be replaced by T2A, and the amino acid sequence of the T2A is represented by SEQ ID No. 23.
  • nucleotide sequence of the coding gene of T2A is represented by SEQ ID No. 24.
  • SEQ ID No. 24 The coding nucleotide sequence of T2A
  • both P2A and T2A can be used for co-expression of genes, and the advantage is that the shear efficiency is relatively high.
  • the chimeric antigen receptor comprises: an extracellular antigen binding region, a transmembrane region and an intracellular signal region, wherein the extracellular antigen binding region is a ligand that binds to a tumor surface specific antigen or a related antigen, and a transmembrane region
  • the intracellular signal region is an intracellular domain of one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, CD40, CD27 or DAP10.
  • the tumor surface specific antigen or related antigen includes, but not limited to, EGFR, EpCAM, mesothelin, IL-13R ⁇ 2, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2, GD2, FR, PSMA, gp100, MUC1, MUC16, CA9, CD171, CD125, CD15-3, CD19-9, NY-ESO-1, MART-1, MAGE4, CD19, CD20, CD22, CD30, CD33, CEA, CD38, CD138, CD123, EphA2.
  • the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 13;
  • the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 14 or a degenerate sequence thereof;
  • nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.
  • the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 17;
  • nucleotide sequence of the coding gene of the chimeric antigen receptor in the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 18 or a degenerate sequence thereof;
  • nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.
  • the above chimeric antigen receptor and CXCR4 are co-expressed by one vector.
  • the invention also provides an expression vector.
  • the expression vector is an expression vector for simultaneously expressing the chimeric antigen receptor of SEQ ID NO: 13 and CXCR4 of SEQ ID NO: 19. or
  • the expression vector is an expression vector for simultaneously expressing the chimeric antigen receptor of SEQ ID NO: 17 and CXCR4 of SEQ ID NO: 19.
  • the expression vector is a viral vector. Further, the expression vector is a lentiviral vector.
  • the invention also provides a virus.
  • the virus comprises the expression vector described above.
  • the invention also provides a host cell comprising the expression vector or virus described above.
  • the above host cell is a lymphocyte.
  • the lymphocytes are T cells or monocytes, and the T cells are ⁇ T cells, NKT cells or ⁇ T cells.
  • the invention also provides the use of a chimeric antigen receptor, chimeric antigen receptor modified lymphocyte, as described above, alone or in combination with radiation therapy for the treatment of a malignant tumor. It is mainly used to prepare drugs for treating malignant tumors or to inhibit tumors in combination with radiation therapy.
  • the malignant tumor is lung cancer, liver cancer, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, Multiple myeloma or prostate cancer.
  • the present invention includes three aspects, which are described as follows:
  • a first aspect of the invention relates to a chimeric antigen receptor that coexpresses CXCR4.
  • the chimeric antigen receptor is composed of a ligand capable of binding to a tumor-specific or related antigen, a transmembrane region and an intracellular signal region, and is linked to the CXCR4 expression gene by a P2A sequence or a T2A sequence. Co-expressed on the same carrier. (for example, the structure shown in Figure 1).
  • a second aspect of the invention relates to obtaining a chimeric antigen receptor modified lymphocyte expressing CXCR4.
  • the human peripheral lymphocytes are transfected with a vector which co-expresses CXCR4 and a chimeric antigen receptor provided by the first aspect of the present invention, thereby obtaining a chimeric antigen receptor-modified lymphocyte expressing CXCR4.
  • a high titer of lentivirus is generated and transfected into human peripheral lymphocytes.
  • a third aspect of the invention relates to the use of a chimeric antigen receptor which coexpresses CXCR4 according to any one of the first aspects of the invention, or a lymphocyte according to the second aspect, alone or in combination with radiation therapy for the treatment of a malignant tumor.
  • a gene sequence similar to the expression "the nucleotide sequence in SEQ ID NO: 14 is substituted, deleted or added with at least one nucleotide-derived sequence” generally means that the encoding is encoded by SEQ ID NO: 14.
  • the degenerate sequence refers to a sequence produced by the substitution of one or more codons in the sequence by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, a degenerate sequence having a homology of less than about 89% to SEQ ID NO: 14 can also encode the sequence set forth in SEQ ID NO: 14.
  • nucleotide sequence of the nucleotide sequence of SEQ ID NO: 14 hybridization.
  • the term also encompasses nucleotide sequences having at least 80%, preferably at least 89%, more preferably at least 90%, and optimally at least 95% homology to the nucleotide sequence of SEQ ID NO: 14.
  • nucleotide sequence in SEQ ID NO: 14 is substituted, deleted or added with at least one nucleotide-derived sequence
  • SEQ ID NO: 14 also includes SEQ ID encoding a protein having the same function as the protein encoded by SEQ ID NO: 14.
  • the variant form of the open reading frame sequence in 14. include, but are not limited to, a plurality of (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, optimally 1 to 10) nucleotide deletions. , inserts and/or substitutions, and adding a few at 5' and / or 3' ends (usually within 60, preferably within 30, more preferably within 10, optimally within 5) ) nucleotides.
  • the expression "the amino acid sequence obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence” includes but is not limited to several (usually 1-50, compared Preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually Within 20, preferably less than 10, more preferably less than 5 amino acids.
  • the function of the protein is usually not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the protein.
  • the amino acid sequence obtained by substituting, deleting or adding at least one amino acid in the amino acid sequence also includes, but is not limited to, having up to 10 (ie one or several), preferably up to 8, More preferably, at most 5 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide, ie a conservative variant polypeptide.
  • conservative variant polypeptides are preferably produced by substitution according to Table 1.
  • the present invention produces a lentivirus and transduces lymphocytes by constructing a viral expression vector which co-expresses a chimeric antigen receptor and a chemokine receptor CXCR4, thereby obtaining a chimeric antigen receptor T lymphocyte which coexpresses CXCR4, which can be reversed.
  • the expression of CXCR4 in T cells decreased by virus modification, enhanced the chemotactic activity of chimeric antigen receptor T cells, and promoted the aggregation of lymphocytes to high concentration areas of SDF-1 (CXCL12), and the chemotaxis increased from 3% to 9%. It is beneficial to enhance the anti-tumor effect of chimeric antigen receptor T lymphocytes. When used in combination with radiation therapy, it also induces a homing effect on chimeric antigen receptor T lymphocytes, resulting in better anti-tumor effects.
  • Figure 1 is a schematic diagram showing the chimeric antigen receptor that coexpresses CXCR4; A indicates that the extracellular antigen binding region is a chimeric antigen receptor of scFv, B indicates that the extracellular antigen binding region is a chimeric antigen receptor of APRIL, and C indicates The extracellular antigen binding region is a chimeric antigen receptor of the receptor; wherein SP is a GM-CFSR signal peptide, scFv represents an antigen binding region, and APRIL represents a ligand binding region.
  • CD8 ⁇ hinge indicates the CD8 ⁇ transmembrane region
  • CD28 and 4-1BB indicate the costimulatory signal domain
  • CD3 ⁇ indicates the T cell activation domain
  • P2A is the ligated fragment
  • CXCR4 is the chemokine receptor
  • Figure 2 shows the expression of CXCR4 in T cells after lentivirus infection
  • Figure 3 shows the chemotactic activity of T cells after lentivirus infection
  • FIG. 4 shows CXCR4 expression in CAR-T cells after coexpression of CXCR4
  • Figure 5 shows the chemotactic activity of CAR-T cells after coexpression of CXCR4; A indicates the CAR-T chemotaxis activity induced by RPMI-8226 cell culture supernatant, and B indicates the CAR-T trend induced by SDF-1 (ie CXCL12). Activity
  • Figure 6 shows CAR expression of CAR-T and CAR/CXCR4-T cells
  • Figure 7 shows the killing activity of CAR-T and CAR/CXCR4-T cells.
  • Example 1 The full-length gene of CAR, CAR/CXCR4 was obtained, and the construction of recombinant plasmid vector was completed.
  • the scFv-CAR, scFv-CAR-P2A-CXCR4 gene fragment was obtained by whole gene synthesis.
  • the reaction conditions are as follows:
  • PCR reaction denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.
  • the reaction conditions are as follows:
  • PCR reaction denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 2 minutes. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.
  • the APRIL-CAR, APRIL-CAR-P2A-CXCR4 gene fragment was obtained by whole-genome synthesis.
  • the reaction conditions are as follows:
  • PCR reaction denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.
  • the reaction conditions are as follows:
  • PCR reaction denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 2 minutes. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.
  • the lentiviral vector pWPXLd was linearized by restriction endonucleases EcoR1 and BamH1, and the full-length CAR gene amplified fragment was inserted into the EcoR I/ of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201).
  • the Tiangen EasyGeno Rapid Recombination Cloning Kit Tiangen Biochemical Technology Co., Ltd., VI201.
  • the recombinant product pWPXLd-CAR was transformed into E. coli Stbl3, and 30 clones were randomly selected for sequencing. The sequencing results were consistent with the designed full-length CAR sequence.
  • the lentiviral vector pWPXLd was linearized by restriction endonucleases EcoR1 and BamH1, and the full-length CAR/CXCR4 gene amplified fragment was inserted into the ecoR of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201).
  • the I/BamH I site, the recombinant product pWPXLd-CAR/CXCR4 was transformed into E. coli Stbl3, and 30 clones were randomly selected for sequencing. The sequencing results were consistent with the designed full length CAR/CXCR4 sequence.
  • Figure 1A The schematic diagram of the constructed lentiviral recombinant vector is shown in Figure 1.
  • Figure 1B and Figure 1C show the recombinant vector structure when the extracellular antigen binding region of the chimeric antigen receptor is scFv, ligand and receptor binding region, respectively. .
  • 293T cells were cultured in DMEM + 10% fetal bovine serum (FBS) and used for virus packaging when grown to 70-90% density.
  • FBS fetal bovine serum
  • the procedure was as follows: fresh pre-warmed DMEM medium (containing 10% FBS) 2 h before transfection
  • the transfection system was prepared in a 15 ml centrifuge tube: the experimental conditions are shown in Tables 2 and 3.
  • the transfection solution was evenly added to the dish, and the cultivation was continued at 37 ° C in a 5% CO 2 incubator. After 12h, it was replaced with DMEM+2% FBS medium. The virus supernatant was collected at 48 h and 72 h, respectively, and the virus solution was filtered through a 0.45 ⁇ m filter, centrifuged at 4 ° C, 160,000 g, ultracentrifuged for 90 min, resuspended in PBS, and stored in -80 °.
  • PBMC Human peripheral blood mononuclear cells
  • the lentivirus-infected or uninfected T cells and peripheral blood mononuclear cells constructed in Example 1 were exchanged after 24 hours, and X-VIVO 15 (04-418Q, LONZA) complete medium + 10% human AB serum was used ( H4522, sigma) continued to culture for 5 days while adding recombinant human interleukin 2 (IL-2) cytokine 100 U/ml (C013, novaprotein). The cells were then collected, washed twice with PBS, stained with APC-labeled anti-human CXCR4 antibody (Cat. No. 306510, biolegend), and analyzed for cell membrane CXCR4 expression by flow cytometry.
  • IL-2 human interleukin 2
  • Lymphocyte chemotactic activity assays were assessed by cell migration assays.
  • the transwell polycarbonate membrane was soaked in PBS containing 20 ⁇ g/ml BSA at 4 ° C overnight at 5 ⁇ m pore size (Cat. No. 3421; Costar, Cambridge, MA) transwell, and placed in a 24-well plate to dry before use.
  • CAR-T group infected (CAR-T group) or uninfected (control group) lentiviral cell T cells and peripheral blood mononuclear cells in RPMI1640 complete medium (containing 1% BSA, 10 mM HEPES buffer, pH 6.9) serum Hunger for 4 hours.
  • CXCR4 was co-expressed in CAR-T cells.
  • Lentivirus-infected T cells containing full-length CAR (CAR-T group) or CAR/CXCR4 (CAR/CXCR4-T group) were collected 48 h later, washed twice with PBS, APC-labeled anti-human CXCR4 antibody (Cat. No. 306510, Biolegend staining and flow cytometry were used to detect the expression of CXCR4 in cell membrane.
  • APC-labeled anti-human CXCR4 antibody Cat. No. 306510, Biolegend staining and flow cytometry were used to detect the expression of CXCR4 in cell membrane.
  • the results showed that compared with CAR-T group, the expression of CXCR4 in T cells of CAR/CXCR4 group was significantly increased, and the average fluorescence intensity (logarithm) increased from 108 to 229. (As shown in Figure 4).
  • Example 4 the chemotaxis activity was tested in the same manner as in Example 4: using a 5 ⁇ m pore size (Cat. No. 3421; Costar, Cambridge, MA) transwell, and the polycarbonate membrane in transwell was soaked overnight at 4 ° C in PBS containing 20 ⁇ g/ml BSA. Store in a 24-well plate before use.
  • 5 ⁇ m pore size Cat. No. 3421; Costar, Cambridge, MA
  • T cells expressing (CAR/CXCR4-T group) or not expressing (CAR-T group) CXCR4 were serum starved for 4 hours in RPMI1640 complete medium (containing 1% BSA, 10 mM HEPES buffer, pH 6.9). .
  • T cells were infected with lentivirus containing full-length CAR or CAR/CXCR4. After 5 days of culture, cells were harvested and stained with FITC-labeled recombinant human BCMA protein (novaprotein) to detect T cell CAR expression. The results showed that compared with the control group (less than 5%), the CAR expression was significantly increased (up to 40%) in the CAR group and the CAR/CXCR4 group, and the fluorescence peak shifted (Fig. 6).
  • CAR-T cells or CAR/CXCR4-T cells were co-cultured with CSFE-labeled human myeloma cell line RPMI-8226 cells (target cells, BCMA high expression), 24 h later.
  • a total of 1 ⁇ 10 5 Far red-labeled K562 cells were added to each group, washed twice with PBS, and the ratio of fluorescent cells was detected by flow cytometry FITC and APC channels. Because the number of Far red-labeled K562 cells in each group is the same, K562 cells can be used as a reference. Among them, the negative group did not add T cells).
  • the above assay shows that the present invention can eliminate the chemotaxis of T cells expressing CAR-T by constructing a co-expressing chimeric antigen receptor and a CXCR4 lentiviral expression vector and introducing into T cells.
  • the defect provides a lymphocyte with improved killing activity and chemotactic activity, which is beneficial to enhance the antitumor effect of chimeric antigen receptor-modified lymphocytes.

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Abstract

细胞治疗领域，特别是一种表达趋化因子受体CXCR4的嵌合抗原受体修饰的淋巴细胞，通过基因工程手段，将非特异性的淋巴细胞改造为能够识别肿瘤表面相关抗原并表达趋化因子受体CXCR4，从而在特定部位富集并对特定细胞进行靶向杀伤的特异性淋巴细胞的制备方法及其在恶性肿瘤治疗中的用途。

Description

表达CXCR4的嵌合抗原受体修饰的淋巴细胞及制备方法和用途 技术领域
本发明涉及细胞领域，具体涉及通过基因工程手段，将非特异性的淋巴细胞定向改造为能够识别肿瘤表面相关抗原并表达趋化因子受体CXCR4，从而在特定部位富集并对细胞进行杀伤的特异性淋巴细胞的制备方法及其在恶性肿瘤治疗中的用途。
背景技术
免疫治疗被认为是最具前景的肿瘤治疗方法。其中，免疫检查点抑制剂(如CTLA-4、PD-1等)在多种肿瘤临床试验中显示出了明显优于现有疗法的效果，被批准用于非小细胞肺癌、膀胱癌等恶性肿瘤的治疗。不同于CTLA-4和PD-1抗体等通过重新激活机体免疫反应发挥抗肿瘤作用，嵌合抗原受体修饰的免疫细胞疗法不依赖于机体免疫状态，通过基因工程技术，将嵌合抗原受体表达于免疫细胞表面，识别肿瘤抗原，激活免疫效应细胞从而产生直接抗肿瘤作用。目前，以CD19为靶点的嵌合抗原受体修饰的淋巴细胞在治疗急性淋巴细胞白血病、非霍奇金淋巴瘤临床试验中显示出良好的效果，有效率最高达90％，显示出这种疗法巨大的治疗潜力。目前，超出300项与嵌合抗原受体修饰的淋巴细胞治疗相关的临床试验正在进行。
尽管嵌合抗原受体修饰的淋巴细胞在肿瘤免疫治疗中有着诱人的前景，但仍然存在几个问题：1.肿瘤特异性抗原缺乏。目前发现的肿瘤特异性表面抗原很少，且多数肿瘤缺乏特异性抗原，大部分为肿瘤高表达但正常组织也表达的肿瘤相关抗原，从而限制了其作为嵌合抗原受体修饰的淋巴细胞治疗靶点。如在靶向HER2的嵌合抗原受体修饰的淋巴细胞临床研究中，由于人体正常肺组织同样表达HER2，引起了非特异性细胞毒性杀伤反应，导致患者死亡。2.嵌合抗原受体修饰的淋巴细胞在肿瘤部位的富集。目前关于嵌合抗原受体修饰的淋巴细胞治疗进展均发生在血液系统肿瘤中，实体瘤尚无报道，其中一个重要原因是体外扩增后的嵌合抗原受体修饰的淋巴细胞不能大量、有效进入实体瘤内，导致抗肿瘤作用不明显。3.肿瘤微环境对嵌合抗原受体修饰的淋巴细胞的抑制作用。相对于血液系统肿瘤，实体瘤内存在复杂的肿瘤免疫微环境，使嵌合抗原受体修饰的淋巴细胞无法发挥功能，而且最新的文献提示，坏死的肿瘤细胞还可以通过释放K +使淋巴细胞衰竭。因此，仍需要加强本领域的研究，以期进一步提高嵌合抗原受体修饰 的淋巴细胞的疗效。
淋巴细胞向肿瘤聚集迁移是一个依赖趋化因子的复杂过程，涉及循环淋巴细胞与内皮细胞间的相互作用。研究显示，CXCL12/SDF-1α是重要的T细胞和髓源单核细胞的趋化调节因子，通过与其受体CXCR4结合，驱使细胞沿着浓度梯度达到相关部位。放射治疗后，照射部位CXCL12/SDF-1α表达升高，募集髓源单核细胞向照射部位迁移。在一些高表达CXCL12的胶质细胞肿瘤中也发现了这个现象。研究证实，HIV感染可导致T细胞CXCR4表达下调，而且随时间延长，下降更明显。我们发现，T细胞经慢病毒介导的基因修饰后，趋化因子受体CXCR4表达明显下降，趋化活性受限。
基于以上现象，我们设想通过在嵌合抗原受体修饰的淋巴细胞表达CXCR4，可以使其在CXCL12/SDF-1α高表达的脑胶质瘤或多发性骨髓瘤中聚集，增强抗肿瘤作用，也可以通过放疗等手段使肿瘤部位CXCL12/SDF-1α表达升高，诱导嵌合抗原受体修饰的淋巴细胞产生归巢效应，从而产生更好的抗肿瘤作用。
发明内容
本发明要解决的技术问题是为恶性肿瘤的治疗提供一种新的有效手段。
本发明解决技术问题的技术方案是提供了一种嵌合抗原受体，包括胞外抗原结合区，跨膜区和胞内信号区。
其中，上述嵌合抗原受体中，所述的胞外抗原结合区是与肿瘤抗原结合的单克隆抗体单链可变区，受体或配体。
进一步的，所述的肿瘤抗原包括但不限于：(EGFR)、上皮细胞粘附分子(EpCAM)、间皮素(mesothelin)、白介素13受体α2(IL-13Rα2)、表皮生长因子受体2(ERBB2)、表皮生长因子受体3(ERBB3)、表皮生长因子受体4(ERBB4)、血管内皮生长因子受体1(VEGFR1)、血管内皮生长因子受体2(VEGFR2)、神经节苷脂抗原G D2(GD2)、叶酸受体1(FOLR1)、前列腺特异性膜抗原(PSMA)、黑色素前体蛋白(gp100)、粘蛋白1(MUC1)、粘蛋白16(MUC16)、碳酸酐酶9(CA9)、L1细胞黏附分子(CD171)、肿瘤抗原125(CA125)、肿瘤抗原15-3(CA15-3)、肿瘤抗原19-9(CA19-9)、NY-ESO-1、MART-1、MAGE4、B细胞成熟抗原(BCMA)、白细胞分化抗原19(CD19)、白细胞分化抗原20(CD20)、白细胞分化抗原22(CD22)、白细胞分化抗原30(CD30)、白细胞分化抗原33(CD33)、癌胚抗原(CEA)、白细胞分化抗原38(CD38)、白细胞分化抗原133(CD133)、白细胞分化抗原138(CD138)、白细胞分化抗原123(CD123)、EPH受体A2(EPHA2)、EPH受体A3(EPHA3)。
其中，上述嵌合抗原受体中，所述的跨膜区为白细胞分化抗原8(CD8)跨膜区或白细胞分化抗原28(CD28)跨膜区。
其中，上述嵌合抗原受体中，所述的胞内信号区为白细胞分化抗原28(CD28)、肿瘤坏死因子受体超家族成员4(TNFRSF9/CD134/OX40)、肿瘤坏死因子受体超家族成员9(TNFRSF9/CD137/4-1BB)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、诱导性T细胞共刺激分子(ICOS)、CD40、CD27或DAP10(DNAX激活蛋白10)中一种或多种的胞内结构域。
其中，上述嵌合抗原受体中，所述的单克隆抗体单链可变区为scFv单链抗体可变区。
其中，上述嵌合抗原受体中，所述的scFv单链抗体可变区的氨基酸序列为SEQ ID No.1所示。
SEQ ID No.1 scFv单链抗体可变区的氨基酸序列
Figure PCTCN2017119451-appb-000001
其中，上述嵌合抗原受体中，所述的scFv单链抗体可变区的编码核苷酸序列为SEQ ID No.2所示。
SEQ ID No.2 scFv单链抗体可变区的编码核苷酸序列
Figure PCTCN2017119451-appb-000002
Figure PCTCN2017119451-appb-000003
更具体的，上述嵌合抗原受体为通过氮端到碳端顺次联接信号肽、scFv单链抗体、CD8α跨膜区、CD28胞内段、4-1BB胞内段、CD3ζ链得到的。
所述的信号肽的氨基酸序列为SEQ ID No.3所示。
MVLLVTSLLLCELPHPAFLLIP。SEQ ID No.3
所述信号肽的编码核苷酸序列为SEQ ID No.4所示。
Figure PCTCN2017119451-appb-000004
其中，所述的CD8跨膜区氨基酸序列为SEQ ID No.5所示。
Figure PCTCN2017119451-appb-000005
所述的CD8跨膜区的编码核苷酸序列为SEQ ID No.6所示。
Figure PCTCN2017119451-appb-000006
所述CD28胞内域的氨基酸序列为SEQ ID No.7所示。
Figure PCTCN2017119451-appb-000007
所述CD28胞内域的编码核苷酸序列为SEQ ID No.8所示。
Figure PCTCN2017119451-appb-000008
所述4-1BB胞内域的氨基酸序列为SEQ ID No.9所示。
Figure PCTCN2017119451-appb-000009
所述4-1BB胞内域的编码核苷酸序列为SEQ ID No.10所示。
Figure PCTCN2017119451-appb-000010
所述CD3ζ域的氨基酸序列为SEQ ID No.11所示。
Figure PCTCN2017119451-appb-000011
所述CD3ζ域的编码核苷酸序列为SEQ ID No.12所示。
SEQ ID No.12 CD3ζ域的编码核苷酸序列
Figure PCTCN2017119451-appb-000012
进一步的，上述嵌合抗原受体中，所述的嵌合抗原受体为(1)具有SEQ ID No.13所示的氨基酸序列的蛋白；
或：(2)在SEQ ID No.13所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.13所示的蛋白的功能相同或相似的蛋白。
SEQ ID No.13嵌合抗原受体的氨基酸序列
Figure PCTCN2017119451-appb-000013
Figure PCTCN2017119451-appb-000014
进一步的，上述嵌合抗原受体中，所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.14所示的核苷酸序列或其简并序列；
或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.14的核苷酸序列编码功能相同或相似的蛋白。
SEQ ID NO:14编码嵌合抗原受体的基因的核苷酸序列
Figure PCTCN2017119451-appb-000015
Figure PCTCN2017119451-appb-000016
特别的，上述嵌合抗原受体中，所述的配体为APRIL配体。
进一步的，上述嵌合抗原受体中，所述APRIL氨基酸序列为SEQ ID NO.15所示。
Figure PCTCN2017119451-appb-000017
所述APRIL的编码核苷酸序列为SEQ ID NO.16所示。
Figure PCTCN2017119451-appb-000018
进一步的，上述嵌合抗原受体为通过氮端到碳端顺次联接信号肽、APRIL、CD8α跨膜区、CD28胞内段、4-1BB胞内段、CD3ζ链得到的。
特别的，上述嵌合抗原受体中，所述的嵌合抗原受体为(1)具有SEQ ID No.17所示的氨基酸序列的蛋白；
或：(2)在SEQ ID No.17所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.17所示的蛋白的功能相同或相似的蛋白。
SEQ ID No.17嵌合抗原受体的氨基酸序列
Figure PCTCN2017119451-appb-000019
Figure PCTCN2017119451-appb-000020
进一步的，上述嵌合抗原受体中，所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.18所示的核苷酸序列或其简并序列；
或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.18的核苷酸序列编码功能相同或相似的蛋白。
SEQ ID NO:18编码嵌合抗原受体的基因的核苷酸序列
Figure PCTCN2017119451-appb-000021
本发明还提供了一种上述嵌合抗原受体修饰的淋巴细胞，共表达趋化因子CXCR4。
进一步的，所述的CXCR4受体为(1)具有SEQ ID No.19所示的氨基酸序列的蛋白；
或：(2)在SEQ ID No.19所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.19所示的蛋白的功能相同或相似的蛋白。
SEQ ID No.19 CXCR4的氨基酸序列
Figure PCTCN2017119451-appb-000022
进一步的，上述嵌合抗原受体修饰的淋巴细胞中，所述CXCR4的编码基因核苷酸序列为(1)：如SEQ ID No：20所示的核苷酸序列或其简并序列；
或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO：20的核苷酸序列编码功能相同或相似的蛋白。
SEQ ID No：20编码CXCR4基因的核苷酸序列
Figure PCTCN2017119451-appb-000023
Figure PCTCN2017119451-appb-000024
其中，所述的CXCR4通过P2A与嵌合抗原受体共表达，所述的P2A的氨基酸序列为SEQ ID No.21所示。
SEQ ID No.21 P2A的氨基酸序列
进一步的，所述的P2A的编码基因的核苷酸序列为SEQ ID No.22所示。
SEQ ID No.22 P2A的编码核苷酸序列
Figure PCTCN2017119451-appb-000026
进一步的，所述的P2A可以有T2A替代，所述的T2A的氨基酸序列为SEQ ID No.23所示。
SEQ ID No.23 T2A的氨基酸序列
Figure PCTCN2017119451-appb-000027
进一步的，所述T2A的编码基因的核苷酸序列为SEQ ID No.24所示。
SEQ ID No.24 T2A的编码核苷酸序列
Figure PCTCN2017119451-appb-000028
在本发明中，P2A和T2A均可用于基因的共表达，其优势是剪切效率比较高。
其中，所述的嵌合抗原受体包括：胞外抗原结合区，跨膜区和胞内信号区，其中胞外抗原结合区是结合肿瘤表面特异性抗原或相关抗原的配体，跨膜区为CD8跨膜区或CD28跨膜区，胞内信号区为CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS、CD40、CD27或DAP10中的一种或几种的胞内结构域。
其中，所述的肿瘤表面特异性抗原或相关抗原包括但不限于：EGFR、EpCAM、mesothelin、IL-13Rα2、ERBB2、ERBB3、ERBB4、VEGFR1、VEGFR2、GD2、FR、PSMA、gp100、MUC1、MUC16、CA9、CD171、CD125、CD15-3、CD19-9、NY-ESO-1、MART-1、MAGE4、CD19、CD20、CD22、CD30、CD33、CEA、CD38、CD138、CD123、EphA2。
进一步的，上述嵌合抗原受体中，所述的嵌合抗原受体为(1)具有SEQ ID No.13所示的氨基酸序列的蛋白；
或：(2)在SEQ ID No.13所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.13所示的蛋白的功能相同或相似的蛋白。
其中，上述嵌合抗原受体中，所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.14所示的核苷酸序列或其简并序列；
或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.14的核苷酸序列编码功能相同或相似的蛋白。
或者，上述嵌合抗原受体中，所述的嵌合抗原受体为(1)具有SEQ ID No.17所示的氨基酸序列的蛋白；
或：(2)在SEQ ID No.17所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.17所示的蛋白的功能相同或相似的蛋白。
其中，上述嵌合抗原受体中，所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.18所示的核苷酸序列或其简并序列；
或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.18的核苷酸序列编码功能相同或相似的蛋白。
其中，上述的嵌合抗原受体和CXCR4，由一个载体共同表达。
本发明还提供了一种表达载体。
所述的表达载体为同时表达SEQ ID NO：13所述的嵌合抗原受体和SEQ ID NO：19所述的CXCR4的表达载体。或
所述的表达载体为同时表达SEQ ID NO：17所述的嵌合抗原受体和SEQ ID NO：19所述的CXCR4的表达载体。
进一步的，所述的表达载体为病毒载体。更进一步的，所述的表达载体为慢病毒载体。
本发明还提供了一种病毒。该病毒包含上述的表达载体。
本发明还提供了含有上述的表达载体或病毒的宿主细胞。
进一步的，上述宿主细胞为淋巴细胞。更进一步的，所述淋巴细胞为T细胞或单核细胞，所述的T细胞为αβT细胞、NKT细胞或γδT细胞。
本发明还提供了一种上述嵌合抗原受体，嵌合抗原受体修饰的淋巴细胞在单独或与放疗联合用于治疗恶性肿瘤的用途。主要用于制备治疗恶性肿瘤的药物或与放射治疗联合抑制肿瘤。所述的恶性肿瘤为肺癌、肝癌、淋巴瘤、结肠癌、直肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、多发性骨髓瘤或前列腺癌。
更具体的，本发明包括三个方面，分述如下：
本发明的第一个方面涉及一种共表达CXCR4的嵌合抗原受体。其中嵌合抗原受体由能够结合肿瘤特异性或相关抗原的配体、跨膜区和胞内信号区组成，通过P2A序列或T2A序列与CXCR4表达基因相连。共同表达在同一个载体上。(例如如图1所示的结构)。
本发明的第二个方面涉及获得一种表达CXCR4的嵌合型抗原受体修饰的淋巴细胞。利用本发明第一个方面提供的共表达CXCR4和嵌合型抗原受体的载体，转染人外周淋巴细胞，从而获得表达CXCR4的嵌合型抗原受体修饰的淋巴细胞。一般通过慢病毒包装体系，产生高滴度的慢病毒，转染人外周淋巴细胞，
本发明的第三个方面涉及本发明第一方面任一所述的共表达CXCR4的嵌合抗原受体或第二方面所述的淋巴细胞单独或与放疗联合用于治疗恶性肿瘤的用途。
在本发明中，类似于“在SEQ ID NO：14中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”表述的基因序列一般是指编码具有SEQ ID NO：14所编码的蛋白活性的多肽的核苷酸序列及其简并序列。该简并序列是指所述序列中有一个或多个密码子被编码相同氨基酸的简并密码子所取代后而产生的序列。由于密码子的简并性，所以与SEQ ID NO：14同源性低至约89％的简并序列也能编码出SEQ ID NO：14所述的序列。另外,“在SEQ ID NO：14中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO：14核苷酸序列杂交的核苷酸序列。该术语还包括与SEQ ID NO：14中的核苷酸序列的同源性至少80％，较佳地至少89％，更佳地至少90％，最佳地至少95％的核苷酸序列。
该术语“在SEQ ID NO：14中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”还包括能编码具有与SEQ ID NO：14所编码蛋白功能相同的蛋白的SEQ ID NO：14中开放阅读框序列的变异形式。这些变异形式包括(但并不限于)：若干个(通常为1～90个，较佳地1～60个，更佳地1～20个，最佳地1～10个)核苷酸的缺失、插入和/或取代，以及在5’和/或3’端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。
在本发明中，“在所述的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸衍生所得的氨基酸序列”的表述包括但并不限于若干个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变 蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括所述蛋白的活性片段和活性衍生物。
“在所述的氨基酸序列中经过取代、缺失或添加至少一个氨基酸衍生所得的氨基酸序列”的表述还包括但并不限于有至多10个(即一个或几个)，较佳地至多8个，更佳地至多5个氨基酸被性质相似或相近的氨基酸所替换而形成多肽，即保守性变异多肽。这些保守性变异多肽最好根据表1进行替换而产生。
表1氨基酸替换表
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的有益效果为：
本发明通过构建共表达嵌合抗原受体和趋化因子受体CXCR4的病毒表达载体，生产慢病毒，转导淋巴细胞，得到一种共表达CXCR4的嵌合抗原受体T淋巴细胞，能够逆转病毒修饰导致的T细胞CXCR4表达下降，增强嵌合抗原受体T细胞的趋化活性，促进该淋巴细胞向SDF-1(CXCL12)高浓度区域聚集，趋化性从3％提高至9％，有利于增强嵌合抗原受体T淋巴细胞的抗肿瘤效应。当其与放射治疗联合使用时，还能诱导嵌合抗原受体T淋巴细胞产生归巢效应，从而产生更好的抗肿瘤作用。
附图说明
图1所示为共表达CXCR4的嵌合抗原受体示意图；A表示胞外抗原结合区为scFv的嵌合抗原受体，B表示胞外抗原结合区为APRIL的嵌合抗原受体，C表示胞外抗原结合区为受体的嵌合抗原受体；其中，SP为GM-CFSR信号肽，scFv表示抗原结合区，APRIL表示配体结合区。CD8αhinge表示CD8α跨膜区，CD28和4-1BB表示共刺激信号域，CD3ζ表示T细胞激活域，P2A为连接片段，CXCR4为趋化因子受体；
图2所示为慢病毒感染后T细胞CXCR4表达；
图3所示为慢病毒感染后T细胞的趋化活性；
图4所示为共表达CXCR4后CAR-T细胞CXCR4表达；
图5所示为共表达CXCR4后CAR-T细胞趋化活性；A表示RPMI-8226细胞培养上清诱导的CAR-T趋化活性，B表示SDF-1(即CXCL12)诱导的CAR-T趋化活性；
图6所示为CAR-T和CAR/CXCR4-T细胞CAR表达；
图7所示为CAR-T和CAR/CXCR4-T细胞杀伤活性。
具体实施方式
以下通过具体实施方式结合附图对本发明进行详细说明。下述实施例中，凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件，例如Sambrook J,Russell D.W.,2001,Molecular Cloning:A laboratory manual(3 rd ed),Spring Harbor Laboratory Press中所述的条件，或按照制造厂商所建议的条件。
实施例1 获得CAR、CAR/CXCR4全长基因，完成重组质粒载体的构建
一、全长scFv-CAR和scFv-CAR/CXCR4基因的获得
采用全基因合成的方法得到scFv-CAR，scFv-CAR-P2A-CXCR4基因片段。
同时，设计如下引物扩增scFv-CAR片段：
5’引物：5’AGGTTTAAACTACGGATGGTGCTGCTGGTGACCTCCC3’SEQ ID NO：25
3’引物：5’ATGACTAGTCCCGGGTTAGCGAGGGGGCAGGGCCTGC3’SEQ ID NO：26
反应条件如下：
PCR反应：94℃变性30秒；60℃退火30秒；68℃延伸1分钟。反应25个循环。然后72℃再延伸10分钟。
设计如下引物扩增scFv-CAR/CXCR4片段：
5’引物：5’AGGTTTAAACTACGGATGGTGCTGCTGGTGACCTCCC3’SEQ ID NO：27
3’引物：5’ATGACTAGTCCCGGGTCAGCTGGAGTGGAAGCTGGAG 3’SEQ ID NO：28
反应条件如下：
PCR反应：94℃变性30秒；60℃退火30秒；68℃延伸2分钟。反应25个循环。然后72℃再延伸10分钟。
二、全长APRIL-CAR和APRIL-CAR/CXCR4基因的获得
采用全基因合成的方法得到APRIL-CAR，APRIL-CAR-P2A-CXCR4基因片段。
同时，设计如下引物扩增APRIL-CAR片段：
5’引物：5’aggtttaaactacggATGGAGACCGACACCCTGCTGC3’SEQ ID NO：29
3’引物：5’atgactagtcccgggTTAGCGAGGGGGCAGGGCCTGC3’SEQ ID NO：30
反应条件如下：
PCR反应：94℃变性30秒；60℃退火30秒；68℃延伸1分钟。反应25个循环。然后72℃再延伸10分钟。
设计如下引物扩增APRIL-CAR/CXCR4片段：
5’引物：5’aggtttaaactacggATGGAGACCGACACCCTGCTGC3’SEQ ID NO：31
3’引物：5’atgactagtcccgggTCAGCTGGAGTGGAAGCTGGAG 3’SEQ ID NO：32
反应条件如下：
PCR反应：94℃变性30秒；60℃退火30秒；68℃延伸2分钟。反应25个循环。然后72℃再延伸10分钟。
三、含全长CAR和CAR/CXCR4基因的慢病毒重组质粒的构建
如上所述，在全基因合成片段中的没有酶切位点，我们采用同源重组的方法将片段直接插入真核表达载体pWPXLd(购自美国addgene公司)的多克隆位点EcoR I/BamH I中，重组后酶切位点将会消失。其构建过程如下：
1.重组慢病毒质粒pWPXLd-CAR的构建
通过限制性内切酶EcoR1和BamH1将慢病毒载体pWPXLd线性化，利用天根EasyGeno快速重组克隆试剂盒(天根生化科技有限公司、VI201)将全长CAR基因扩增 片段插入pWPXLd的EcoR I/BamH I位点，重组产物pWPXLd-CAR转化大肠杆菌Stbl3，随机挑选30个克隆进行测序鉴定。测序结果与设计的CAR全长序列一致。
2.重组慢病毒质粒pWPXLd-CAR/CXCR4的构建
通过限制性内切酶EcoR1和BamH1将慢病毒载体pWPXLd线性化，利用天根EasyGeno快速重组克隆试剂盒(天根生化科技有限公司、VI201)将全长CAR/CXCR4基因扩增片段插入pWPXLd的EcoR I/BamH I位点，重组产物pWPXLd-CAR/CXCR4转化大肠杆菌Stbl3，随机挑选30个克隆进行测序鉴定。测序结果与设计的CAR/CXCR4全长序列一致。
构建的慢病毒重组载体结构示意图如图1所示，图1A、图1B和图1C分别表示嵌合抗原受体的胞外抗原结合区为scFv、配体和受体结合区时的重组载体结构。
实施例2 慢病毒包装及CAR-T细胞的生产
1、慢病毒包装
293T细胞采用DMEM+10％胎牛血清(FBS)培养，待生长至70-90％密度时用于病毒包装，步骤如下：转染前2h更换新鲜预热的DMEM培养基(含10％FBS)，在15ml离心管中配制转染体系：实验条件如表2和3所示。
表2质粒体系
Figure PCTCN2017119451-appb-000029
表3转染体系:
Figure PCTCN2017119451-appb-000030
混匀后，将转染液均匀滴加至平皿中，37℃，5％CO 2培养箱中继续培养。12h后更换为DMEM+2％FBS培养基。分别于48h和72h收集病毒上清，0.45μm滤膜过滤病毒液，4℃，160000g，超速离心90min，PBS重悬，分装保存于-80°。
2、T细胞感染
采用Ficoll-Hypaque(sigma)分离人外周血单核细胞(PBMC)，步骤如下：将等体 积的外周血缓慢加入Ficoll-Hypaque中，1000g，18°离心30分钟，吸取中间层白膜，红细胞裂解液作用2分钟，300g离心5分钟，1640培养基洗两次，计数，加入CD3/CD28 Dynabeads(life technologies 11131D)(cells:beads＝1：3)共培养。24h后，慢病毒感染激活的PBMC，步骤如下：离心细胞，用含病毒上清的培养基(感染复数MOI＝1：10)重悬细胞，1×10 6/ml细胞，加入凝聚胺(polybrene)，终浓度为8mg/ml，混匀，32°条件下1000g离心90分钟，然后37℃5％CO 2培养箱中继续培养。
实施例3 T细胞CXCR4表达检测
将实施例1构建的慢病毒感染或未感染的T细胞和外周血单核细胞，24h后换液，用X-VIVO 15(04-418Q，LONZA公司)完全培养基+10％人AB血清(H4522，sigma公司)继续培养5天，同时加入重组人白介素2(IL-2)细胞因子100U/ml(C013，novaprotein公司)。然后收集细胞，PBS洗两次，APC标记的抗人CXCR4抗体(货号306510，biolegend公司)染色，流式细胞术检测细胞膜CXCR4表达，结果显示：感染了含CAR全长基因的慢病毒后，外周血单核细胞CXCR4表达明显下降，其荧光强度(对数值)由238下降至174(图4)，T细胞感染慢病毒后CXCR4表达下降更明显，其荧光强度(对数值)由239降至108，降低超过一半(如图2所示)。
实施例4 慢病毒感染的T细胞趋化活性检测
在实施例3中感染的外周血单核细胞和T细胞观察到CXCR4表达下降后，为评估其影响，进一步对其介导的趋化活性进行检测。具体操作步骤如下：
淋巴细胞趋化活性检测通过细胞迁移试验评估。采用5μm孔径的(货号3421；Costar，Cambridge，MA)transwell检测，transwell中聚碳酸酯膜在含20μg/ml BSA的PBS中4℃浸泡过夜，使用前置于24孔板中晾干。
试验前，感染(CAR-T组)或未感染(对照组)慢病毒细胞T细胞和外周血单核细胞在RPMI1640完全培养基(含有1％BSA，10mM HEPES缓冲液，pH6.9)中血清饥饿4小时。
试验时，在24孔板中加入500ul含100ng/mlSDF-1的无血清RPMI1640完全培养基；晾干的transwell放入孔内，然后将200ul Far red标记的血清饥饿细胞(5×10 5)加入transwell上室。在37℃，5％CO 2条件下孵育4h，收集下室的迁细胞，离心沉淀，并重悬于300ul预冷的FACS缓冲液(含10％小牛血清和0.2％叠氮钠)，每个样品中加入1×10 5数量的9微米的乳胶珠(BCR167，sigma公司)和25微升浓度为50ug/ml的Propidium Iodide/碘化丙啶工作液(ST511，碧云天生物技术有限公司)，流式细胞仪检测样品。
因各组中乳胶珠数量相同，以其作为参照。统计PI染色阴性细胞，可以计算出
Figure PCTCN2017119451-appb-000031
Figure PCTCN2017119451-appb-000032
分析发现：感染慢病毒后，在SDF-1趋化作用下，外周单核细胞的迁移率由16.7％下降至9.2％，T细胞迁移率由19.3％降至4.1％(图3)，结果表明随着CXCR4表达的降低，T细胞和外周单核细胞的趋化活性也明显下降。
实施例5 表达CXCR4增强嵌合抗原受体修饰T细胞趋化活性
为逆转T细胞的趋化活性，我们在T细胞中过表达CXCR4以增强慢病毒感染的CAR-T细胞趋化活性。
首先，在CAR-T细胞中共表达CXCR4。含全长CAR(CAR-T组)或CAR/CXCR4(CAR/CXCR4-T组)的慢病毒感染T细胞，48h后收集细胞，PBS洗两次，APC标记的抗人CXCR4抗体(货号306510，biolegend公司)染色，流式细胞术检测细胞膜CXCR4表达，结果显示：与CAR-T组相比，CAR/CXCR4组T细胞CXCR4表达明显升高，平均荧光强度(对数值)由108升高至229(如图4所示)。
其次，趋化活性检测，试验方法同实施例4：采用5μm孔径的(货号3421；Costar，Cambridge，MA)transwell检测，transwell中聚碳酸酯膜在含20μg/ml BSA的PBS中4℃浸泡过夜，使用前置于24孔板中晾干。
试验前，表达(CAR/CXCR4-T组)或不表达(CAR-T组)CXCR4的T细胞在RPMI1640完全培养基(含有1％BSA，10mM HEPES缓冲液，pH6.9)中血清饥饿4小时。
试验时，在24孔板中加入500ul含100ng/mlSDF-1的无血清RPMI1640完全培养基或RPMI-8226细胞培养上清；晾干的transwell放入孔内，然后将200ul Far red标记的血清饥饿细胞(5×10 5)加入transwell上室。在37℃，5％CO 2条件下孵育4h，收集下室的迁细胞，离心沉淀，并重悬于300ul预冷的FACS缓冲液(含10％小牛血清和0.2％叠氮钠)，每个样品中加入1×10 5数量的9微米的乳胶珠(BCR167，sigma公司)和25微升浓度为50ug/ml的Propidium Iodide/碘化丙啶工作液(ST511，碧云天生物技术有限公司)，流式细胞仪检测样品。
因各组中乳胶珠数量相同，以其作为参照。统计PI染色阴性细胞，可以计算出
Figure PCTCN2017119451-appb-000033
Figure PCTCN2017119451-appb-000034
结果显示：RPMI-8226细胞(高表达并分泌SDF-1)培养上清可促进CAR/CXCR4-T 细胞迁移，从不足2.9％增加到4.15％(图5A)；在趋化因子SDF-1(CXCL12)浓度为100ng/ml时，可明显增加共表达CXCR4的CAR-T细胞趋化效率，由CAR-T组的3％左右升高到9％，提升了三倍(图5B)；充分说明共表达CXCR4可以增强CAR-T细胞向SDF-1高浓度部位迁移的趋化活性。
实施例6 CAR表达和活性检测
含全长CAR或CAR/CXCR4的慢病毒感染T细胞，培养5天后，收集细胞，采用FITC标记的重组人BCMA蛋白(novaprotein公司)染色，检测T细胞CAR表达。结果显示，相对于对照组(小于5％)，CAR组和CAR/CXCR4组中CAR表达明显升高(达40％)，荧光峰值发生偏移(图6)。
不同浓度的上述T细胞、CAR-T细胞或CAR/CXCR4-T细胞(即效应细胞)分别与CSFE标记的人骨髓瘤细胞系RPMI-8226细胞(靶细胞，BCMA高表达)共培养，24h后，每组中加入数量为1×10 5的Far red标记K562细胞，PBS洗2次，流式细胞仪FITC和APC通道检测荧光细胞比例。因各组中Far red标记的K562细胞数量相同，因此，以K562细胞为参照，可以计算出
Figure PCTCN2017119451-appb-000035
Figure PCTCN2017119451-appb-000036
其中阴性组未加T细胞)。
结果显示：在效应细胞数量/靶细胞数量(E/T)＝1:1时，对照组细胞对RPMI-8226细胞的杀伤效率为23.7％，而CAR-T细胞和CAR/CXCR4-T细胞分别达45.3％和51％；在效应细胞数量/靶细胞数量(E/T)＝5:1时，对照组细胞对RPMI-8226细胞的杀伤效率约为44.7％，而CAR-T细胞和CAR/CXCR4-T细胞分别达75.1％和82.3％(图7)，结果表明，嵌合抗原受体(CAR)可以增强T细胞对RPMI-8226的杀伤活性，而共表达CXCR4可以增强CAR-T细胞的杀伤活性。
综上所述，由上述试验可知，本发明通过构建共表达嵌合抗原受体和CXCR4慢病毒表达载体，并导入T细胞中，能够消除T细胞表达CAR-T时存在的趋化性降低的缺陷，提供一种杀伤活性和趋化活性都提高的淋巴细胞，有利于增强嵌合抗原受体修饰的淋巴细胞的抗肿瘤效应。

Claims (33)

  1. 一种嵌合抗原受体修饰的淋巴细胞，共表达趋化因子CXCR4。
  2. 根据权利要求1所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的CXCR4受体为(1)具有SEQ ID No.19所示的氨基酸序列的蛋白；
    或：(2)在SEQ ID No.19所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.19所示的蛋白的功能相同或相似的蛋白。
  3. 根据权利要求2所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的CXCR4的编码核苷酸序列为(1)：如SEQ ID No.20所示的核苷酸序列或其简并序列；
    或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.20的核苷酸序列编码功能相同或相似的蛋白。
  4. 根据权利要求1所述的嵌合抗原受体修饰的淋巴细胞，其特征在于，所述的CXCR4通过P2A与嵌合抗原受体共表达。
  5. 根据权利要求4所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的P2A的氨基酸序列为SEQ ID No.21所示。
  6. 根据权利要求4所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的P2A的编码基因的核苷酸序列为SEQ ID No.22所示。
  7. 根据权利要求1所述的嵌合抗原受体修饰的淋巴细胞，其特征在于，所述的嵌合抗原受体包括：胞外抗原结合区，跨膜区和胞内信号区。
  8. 根据权利要求1所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：
    所述的胞外抗原结合区是与肿瘤抗原结合的单克隆抗体单链可变区，受体或配体；
    所述跨膜区为CD8跨膜区或CD28跨膜区；
    所述胞内信号区为CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS、CD40、CD27或DAP10中一种或多种的胞内结构域。
  9. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于，所述的肿瘤抗原包括但不限于：EGFR、EpCAM、mesothelin、IL-13Rα2、ERBB2、 ERBB3、ERBB4、VEGFR1、VEGFR2、GD2、FR、PSMA、gp100、MUC1、MUC16、CA9、CD171、CD125、CD15-3、CD19-9、NY-ESO-1、MART-1、MAGE4、CD19、CD20、CD22、CD30、CD33、CEA、CD38、CD138、CD123、EphA2。
  10. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的单克隆抗体单链可变区为scFv单链抗体。
  11. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的scFv单链抗体的氨基酸序列为SEQ ID No.1所示。
  12. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的scFv单链抗体的编码核苷酸序列为SEQ ID No.2所示。
  13. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的嵌合抗原受体为通过氮端到碳端顺次联接信号肽、scFv单链抗体、CD8α跨膜区、CD28胞内段、4-1BB胞内段、CD3ζ链得到的。
  14. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的嵌合抗原受体为(1)具有SEQ ID No.13所示的氨基酸序列的蛋白；
    或：(2)在SEQ ID No.13所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.13所示的蛋白的功能相同或相似的蛋白。
  15. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：
    所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.14所示的核苷酸序列或其简并序列；
    或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.14的核苷酸序列编码功能相同或相似的蛋白。
  16. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的配体为APRIL。
  17. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的APRIL的氨基酸序列为SEQ ID NO.15所示。
  18. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所述的APRIL的编码核苷酸序列为SEQ ID NO.16所示。
  19. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：所 述的嵌合抗原受体为通过氮端到碳端顺次联接信号肽、APRIL、CD8α跨膜区、CD28胞内段、4-1BB胞内段、CD3ζ链得到的。
  20. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：
    所述的嵌合抗原受体为(1)具有SEQ ID No.17所示的氨基酸序列的蛋白；
    或：(2)在SEQ ID No.17所示的蛋白的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的与SEQ ID No.17所示的蛋白的功能相同或相似的蛋白。
  21. 根据权利要求8所述的嵌合抗原受体修饰的淋巴细胞，其特征在于：
    所述嵌合抗原受体的编码基因的核苷酸序列为(1)：如SEQ ID No.18所示的核苷酸序列或其简并序列；
    或(2)：在(1)限定的核苷酸序列中经过取代、缺失或添加至少一个核苷酸衍生所得的核苷酸序列，且与SEQ ID NO.18的核苷酸序列编码功能相同或相似的蛋白。
  22. 权利要求1-21中任一项的嵌合抗原受体，其与CXCR4由一个载体共同表达。
  23. 一种表达载体，含有权利要求1-21任一项所述的编码核酸的表达载体。
  24. 根据权利要求23所述的表达载体，其特征在于，所述表达载体为慢病毒载体。
  25. 一种病毒系统，其特征在于，所述病毒系统中包含权利要求23或24所述的表达载体。
  26. 权利要求23或24所述的表达载体的用途，其特征在于：用于制备靶向肿瘤细胞的嵌合抗原受体修饰的淋巴细胞。
  27. 含有权利要求23～24任一项所述的表达载体或病毒的宿主细胞。
  28. 根据权利要求27所述的宿主细胞，其特征在于：所述宿主细胞为T淋巴细胞。
  29. 根据权利要求28所述的宿主细胞，其特征在于：所述的T淋巴细胞为αβT细胞、NKT细胞或γδT细胞。
  30. 一种制备权利要求28或29所述的宿主细胞的方法，其包括在淋巴细胞表达权利要求1-21任一所述的嵌合抗原受体的步骤。
  31. 权利要求1-21所述的嵌合抗原受体修饰的淋巴细胞的用途，用于制备 治疗恶性肿瘤药物。
  32. 权利要求1-21任一项所述的嵌合抗原受体修饰的淋巴细胞的用途，用于与放射治疗联合抑制肿瘤。
  33. 根据权利要求32的用途，其特征在于：所述的恶性肿瘤为肺癌、肝癌、淋巴瘤、结肠癌、直肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、食管癌、肾癌、神经胶质瘤、黑色素瘤、胰腺癌、多发性骨髓瘤或前列腺癌。
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