WO2022007804A1 - T淋巴细胞及其应用 - Google Patents

T淋巴细胞及其应用 Download PDF

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WO2022007804A1
WO2022007804A1 PCT/CN2021/104802 CN2021104802W WO2022007804A1 WO 2022007804 A1 WO2022007804 A1 WO 2022007804A1 CN 2021104802 W CN2021104802 W CN 2021104802W WO 2022007804 A1 WO2022007804 A1 WO 2022007804A1
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cancer
fusion protein
lymphocytes
nucleic acid
lymphocyte
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PCT/CN2021/104802
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English (en)
French (fr)
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王保垒
都晓龙
彭亮
王先进
叶立军
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深圳市菲鹏生物治疗股份有限公司
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Priority to US18/014,479 priority Critical patent/US20230257706A1/en
Publication of WO2022007804A1 publication Critical patent/WO2022007804A1/zh

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Definitions

  • the present disclosure relates to the field of biopharmaceuticals, in particular, the present disclosure relates to T lymphocytes and applications thereof, and more particularly, the present disclosure relates to T lymphocytes, lentiviruses, transgenic lymphocytes, constructs, preparation of T lymphocytes or transgenic lymphocytes Methods, therapeutic compositions for treating cancer, and methods for reducing expression of immune checkpoints on the surface of T lymphocytes.
  • Chimeric antigen receptor T-cell immunotherapy is a method of transforming the patient's T cells in vitro, so that the patient's T cells have the ability to recognize tumor cells, and then re-infused into the patient for treatment after expansion in vitro. a method.
  • CAR-T targeting CD19 has achieved great results in the treatment of B-cell hematological tumors, but according to clinical research results, the efficacy of CD19 CAR-T in the treatment of B-cell lymphoma is far less than that in the treatment of B-cell lymphoma.
  • B-cell acute lymphoblastic leukemia may be due to the fact that B-cell lymphoma is a solid tumor, and its cell surface expresses a large number of PD-L1 molecules.
  • L1 molecule The expression level of L1 molecule was reported, but in clinical studies on diffuse large B-cell lymphoma, it was found that the expression level of PD-L1 molecule on the surface of B-cell lymphoma of patients was directly related to the clinical efficacy, and the expression of PD-L1 on the surface of B-cell lymphoma was directly related to the clinical efficacy.
  • tumor cells will also evade CAR-T killing them by overexpressing PD-L1, suggesting that in the process of solid tumor treatment, blocking PD-1 /PD-L1 signaling pathway will maximize the clinical efficacy of CAR-T and benefit patients.
  • IL-21 is produced by CD4T cells and NKT cells, stimulates the maturation of CD8T cells and NK cells and enhances their cytotoxicity, and has functions such as promoting the differentiation of memory CD8T cells.
  • the many potencies of IL-21 make it a potential target for immunotherapy, but since IL-21R is widely expressed in T cells, B cells, NK cells and myeloid cells, how to make IL-21 specific for CART? cells to control their toxicity has become the focus of immune cell therapy.
  • the half-life of IL-21 itself is very short, and effective modification is also required to improve the half-life.
  • the present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.
  • the inventor has developed a CART that secretes a PD-1 antibody and IL-21 fusion protein.
  • PD-1 is mainly expressed on the surface of T cells, mainly CD8+ T cells, and the PD-1 antibody and IL-21 fusion protein secreted It selectively binds to the surface of T cells and CART cells.
  • the PD-1 antibody and IL-21 fusion protein secreted It selectively binds to the surface of T cells and CART cells.
  • it blocks the PD-1/PD-L1 signaling pathway, and on the other hand, it enables IL-21 to specifically act on T cells and CART cells, and then exercise its Dual function; at the same time, the fusion protein greatly increases the half-life of the drug due to its larger molecular weight.
  • the present disclosure proposes a T lymphocyte.
  • the T lymphocytes co-express a fusion protein and a chimeric antigen receptor, wherein the chimeric antigen receptor includes: an extracellular region, and the extracellular region includes a heavy chain of a single-chain antibody Variable region and light chain variable region and CD8 hinge region, the single-chain antibody specifically recognizes tumor antigen; transmembrane region, the transmembrane region is connected to the extracellular region, and the transmembrane region includes CD8 Transmembrane segment, and embedded in the cell membrane of the T lymphocyte; intracellular region, the intracellular region is connected to the transmembrane region, and the intracellular region includes the intracellular segment of 4-1BB and the CD3 ⁇ chain ;
  • the fusion protein includes: immune checkpoint single chain antibody and T cell activation molecule.
  • T lymphocytes secrete a fusion protein comprising an immune checkpoint single-chain antibody and a T cell activating molecule, and the double superiority of the immune checkpoint single-chain antibody and T cell activating molecule is specifically applied to CAR- T cells, while reducing the inhibitory effect of the tumor microenvironment on CART, make the effect of CART cells more long-term; at the same time, the inventors unexpectedly and pleasantly found that the cell surface of CART cells according to the embodiments of the present disclosure expresses a very low proportion of immune detection Compared with CART cells that secrete immune checkpoint single-chain antibodies or T cell activating molecules alone, the CART cells according to the embodiments of the present disclosure greatly improve the effect of T cell activating molecules and T lymphocytes. The specificity of cell binding reduces the toxicity of the drug.
  • the above-mentioned T lymphocytes may further include at least one of the following additional technical features:
  • the immune checkpoint includes at least one selected from PD-1, PD-L1, CTLA-4, TIM3, LAG3, BTLA and TIGIT.
  • the T cell activating molecule includes at least one selected from the group consisting of IL2, IL7, IL9, IL12, IL15, IL18 and IL21.
  • the immune checkpoint is PD-1
  • the T cell activating molecule is IL21
  • the C-terminus of the IL21 is connected to the N-terminus of the PD-1 single-chain antibody; preferably, the C-terminus of the PD-1 single-chain antibody is connected to the N-terminus of the IL21.
  • the inventors found that when the C-terminus of the PD-1 single-chain antibody is connected to the N-terminus of the IL21, the expression of PD-1 on the surface of T lymphocytes is lower, and the killing effect of T lymphocytes on tumors is more significant.
  • the fusion protein further includes a linker peptide, and the linker peptide is disposed between the immune checkpoint single-chain antibody and the T cell activating molecule.
  • the connecting peptide has the amino acid sequence shown in SEQ ID NO: 1. .
  • the N-terminus of the connecting peptide is connected to the C-terminus of the immune checkpoint single-chain antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the T cell activating molecule.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.
  • the fusion protein scFV-IL21 has the amino acid sequence shown in SEQ ID NO: 2
  • the fusion protein IL21-scFV has the amino acid sequence shown in SEQ ID NO: 3, wherein, scFV represents immune checkpoint single-chain antibody, and IL21 represents T
  • the cell activation molecule is IL21
  • the connection sequence of scFV and IL21 in the fusion protein scFV-IL21 is that the C-terminus of the immune checkpoint single-chain antibody is connected to the N-terminus of IL21
  • the connection sequence of scFV and IL21 in the fusion protein IL21-scFV is that of IL21.
  • the C-terminus is linked to the N-terminus of the immune checkpoint single chain antibody.
  • the present disclosure proposes a lentivirus.
  • the lentivirus carries the following nucleic acid molecules: (a) a nucleic acid molecule encoding a fusion protein, the fusion protein comprising: an immune checkpoint single-chain antibody and a T cell activating molecule; (b) a nucleic acid molecule encoding a chimeric A nucleic acid molecule of an antigen receptor whose extracellular region recognizes a tumor antigen.
  • the recipient cells can express and secrete fusion proteins including immune checkpoint single-chain antibodies and T cell activating molecules, as well as chimeric antigen receptors.
  • the expression of immune checkpoints on the surface of T lymphocytes reduces the inhibitory effect of the tumor microenvironment on T lymphocytes, and the effect of T cells on tumors is more effective, long-lasting, and safer.
  • the above lentivirus may further include at least one of the following additional technical features:
  • the immune checkpoint is PD-1
  • the T cell activation molecule is IL21
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 2 or 3.
  • the tumor antigen is CD19
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:4.
  • the nucleic acid molecule encoding the fusion protein has the nucleotide sequence shown in any one of SEQ ID NO: 5 or 6.
  • the fusion protein encoded by the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:5 has the amino acid sequence shown in SEQ ID NO:2
  • the fusion protein encoded by the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:6 has the amino acid sequence shown in SEQ ID NO:3.
  • the nucleic acid molecule encoding the chimeric antigen receptor has the nucleotide sequence shown in SEQ ID NO:7.
  • the present disclosure proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 8 or 9.
  • nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:8 encodes CD19CAR-scFV-IL21
  • nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:9 encodes CD19CAR-IL21-scFV.
  • the present disclosure provides a transgenic lymphocyte.
  • the transgenic lymphocytes co-express a fusion protein and a chimeric antigen receptor, and the chimeric antigen receptor recognizes a tumor antigen, wherein the chimeric antigen receptor includes: an extracellular region; Membrane region, the transmembrane region is connected with the extracellular region, and is embedded in the cell membrane of the transgenic lymphocyte; intracellular region, the intracellular region is connected with the transmembrane region, and the intracellular region is The region includes the intracellular segment of immune costimulatory molecule; the fusion protein includes: immune checkpoint single chain antibody and T cell activation molecule.
  • the transgenic lymphocytes according to the embodiments of the present disclosure express chimeric antigen receptors and secrete fusion proteins including immune checkpoint single chain antibodies and T cell activating molecules, combining the dual superiority of immune checkpoint single chain antibodies and T cell activating molecules to It acts on the transgenic lymphocytes in a single way, reducing the inhibitory effect of the tumor microenvironment on the transgenic lymphocytes, and making the transgenic lymphocytes have a longer-lasting effect.
  • the cell surface of the transgenic lymphocytes expresses a very low proportion of immune checkpoint molecules, and at the same time has a stronger tumoricidal effect; the transgenic lymphocytes according to the embodiments of the present disclosure and the single-chain antibodies secreting immune checkpoints or T cell activation molecules alone. Compared with transgenic lymphocytes, the specificity of the binding of T cell activating molecules to T lymphocytes is greatly improved, and the toxicity of drugs is reduced.
  • the above-mentioned transgenic lymphocytes may further include at least one of the following additional technical features:
  • the intracellular segment of the immunostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, CD3, and derivatives thereof.
  • the intracellular segment of the immune costimulatory molecule is the intracellular segment of 4-1BB, CD3.
  • the lymphocytes are CD3 + T lymphocytes.
  • the lymphocytes are CD8 + T lymphocytes.
  • the lymphocytes are natural killer cells.
  • the lymphocytes are natural killer T cells.
  • the immune checkpoint includes at least one selected from PD-1, PD-L1, CTLA-4, TIM3, LAG3, BTLA, and TIGIT.
  • the T cell activating molecule includes at least one selected from the group consisting of IL2, IL7, IL9, IL12, IL15, IL18 and IL21.
  • the immune checkpoint is PD-1
  • the T cell activating molecule is IL21
  • the C-terminus of the IL21 is linked to the N-terminus of the PD-1 single-chain antibody.
  • the C-terminus of the PD-1 single-chain antibody is linked to the N-terminus of the IL21. Furthermore, the expression of PD-1 on the surface of the transgenic lymphocytes according to the embodiments of the present disclosure is lower, and the killing effect of the transgenic lymphocytes on tumor cells is more significant.
  • the fusion protein further includes a linker peptide, and the linker peptide is disposed between the immune checkpoint single-chain antibody and the T cell activating molecule.
  • the connecting peptide has the amino acid sequence shown in SEQ ID NO: 1.
  • the N-terminus of the connecting peptide is connected to the C-terminus of the immune checkpoint single-chain antibody, and the C-terminus of the connecting peptide is connected to the N-terminus of the T cell activating molecule.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.
  • the present disclosure proposes a construct.
  • the construct comprises: a first nucleic acid molecule encoding a fusion protein, the fusion protein comprising: an immune checkpoint single-chain antibody and a T cell activating molecule; a second nucleic acid molecule , the second nucleic acid molecule encodes a chimeric antigen receptor, and the chimeric antigen receptor recognizes a tumor antigen; wherein, the fusion protein and the chimeric antigen receptor are as described above.
  • the chimeric antigen receptor can be expressed on the surface of the lymphocytes and the fusion protein can be secreted, and the lymphocyte has a more significant, lasting and safe killing effect on tumors.
  • the above-mentioned construct may further include at least one of the following additional technical features:
  • the first nucleic acid molecule, the second nucleic acid molecule are configured to express the fusion protein, the chimeric antigen receptor in the lymphocytes described above, and the fusion protein is combined with the chimeric antigen receptor.
  • the combined antigen receptor is in a non-fused form.
  • the construct further comprises: a first promoter operably linked to the first nucleic acid molecule; and a second promoter, the second promoter and The second nucleic acid molecule is operably linked.
  • a first promoter operably linked to the first nucleic acid molecule
  • a second promoter operably linked to the second nucleic acid molecule
  • independent expression of the first nucleic acid molecule and the second nucleic acid molecule can be achieved.
  • the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoters.
  • the construct further comprises: an internal ribosome entry site sequence, the internal ribosome entry site sequence is disposed between the first nucleic acid molecule and the second nucleic acid molecule, so The internal ribosome entry site has the nucleotide sequence shown in SEQ ID NO: 10.
  • the expressed fusion protein can be in a non-fused form with the chimeric antigen receptor.
  • the internal ribosome entry site is usually located in the 5' untranslated region (UTR) of the RNA virus genome, so that the translation of one viral protein can be independent of the 5' cap structure, and the translation of another protein is usually initiated by the 5' cap structure,
  • UTR 5' untranslated region
  • the expression of the two genes before and after IRES is usually proportional.
  • the introduction of an internal ribosome entry site sequence allows for the independent expression of the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the fusion protein, respectively.
  • the construct further comprises: a third nucleic acid molecule disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a cleavable linker peptide capable of being cleaved in the lymphocyte.
  • the fusion protein and the chimeric antigen receptor expressed in the lymphocytes are cleaved at the linking peptide, so that the chimeric antigen receptor is independently expressed on the membrane surface of the lymphocytes, and the fusion protein is independently separated from the lymphocytes.
  • the cleavable linker peptide has the amino acid sequence shown in SEQ ID NO: 11.
  • the vector of the construct is a non-pathogenic viral vector
  • the viral vector includes at least one selected from a retroviral vector, a lentiviral vector, and an adeno-associated viral vector.
  • the present disclosure provides a method for preparing the aforementioned T lymphocytes or the aforementioned transgenic lymphocytes.
  • the method comprises: introducing the aforementioned construct or the aforementioned lentivirus into lymphocytes or T lymphocytes.
  • the killing effect of T lymphocytes or transgenic lymphocytes prepared according to the methods of the embodiments of the present disclosure on tumor cells is more significant, durable and safer.
  • the present disclosure proposes a therapeutic composition for treating cancer.
  • the therapeutic composition comprises: the aforementioned construct, the aforementioned lentivirus, the aforementioned T lymphocyte, or the aforementioned transgenic lymphocyte.
  • the therapeutic composition according to the embodiments of the present disclosure has a more significant, durable and safer killing effect on tumor cells.
  • the cancer includes at least one selected from the group consisting of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, and breast cancer.
  • the present disclosure provides that the aforementioned T lymphocytes, the aforementioned lentiviruses, the aforementioned transgenic lymphocytes, the aforementioned constructs, or the aforementioned therapeutic compositions are in Use in the preparation of a medicament for the treatment of cancer.
  • the cancer includes at least one selected from the group consisting of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, stomach cancer, bowel cancer, esophageal cancer, and breast cancer.
  • the present disclosure provides a method of treating cancer.
  • the method comprises administering to a subject with cancer at least one of:
  • T lymphocytes introduced with the aforementioned lentivirus
  • transgenic lymphocytes described above;
  • T lymphocytes introduced with the previously described construct
  • the cancer includes at least one selected from the group consisting of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, and breast cancer.
  • the present disclosure provides that the aforementioned T lymphocytes, the aforementioned lentiviruses, the aforementioned transgenic lymphocytes, the aforementioned constructs, or the aforementioned therapeutic compositions are in Use in the treatment of cancer.
  • the cancer includes at least one selected from the group consisting of liver cancer, pancreatic cancer, ovarian cancer, bile duct cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, and breast cancer.
  • the present disclosure provides a method for reducing the expression of immune checkpoints on the surface of T lymphocytes.
  • the method comprises: allowing the T lymphocytes to express the chimeric antigen receptor and the fusion protein; or co-culturing the T lymphocytes with the T cells expressing the chimeric antigen receptor and the fusion protein , the fusion protein includes: immune checkpoint single-chain antibody and T cell activation molecule, wherein the chimeric antigen receptor and fusion protein are as defined above.
  • the expression of immune checkpoints on the surface of T lymphocytes can be significantly reduced, and the immune escape mechanism can be blocked.
  • the immune checkpoint is PD-1
  • the T cell activating molecule is IL21
  • PD-1 single-chain antibody first specifically selects the PD-1 molecule on the surface of T cells, so that IL-21 can specifically act on T cells Compared with the simple overexpression of IL21, it reduces the toxicity caused by the binding of IL21 to IL21 receptors on the surface of other cells;
  • Figure 1 is a schematic structural diagram of the nucleic acid constructed in Example 1 of the present disclosure, wherein “Hinge” in the figure refers to the hinge region, and “Linker” refers to the linker peptide;
  • Figure 3 shows the results of flow sorting of monoclonal cells to establish Raji-PD-L1 overexpressing target cell lines in Example 5 of the present disclosure, wherein “Multi-sample” in the figure refers to multiple sampling;
  • Fig. 4 embodiment 6 of the present disclosure results of CART killing negative control cell K562
  • Fig. 5 embodiment 6 of the present disclosure results of CART killing RAJI-PD-L1-A3;
  • Figure 6 is a graph of the results of cytokine release in the process of CART killing RAJI-PD-L1-A3 in Example 7 of the present disclosure
  • FIG. 10 Statistical chart of tumor remission and recurrence of individual mice in the group after CART in vivo treatment in Example 8 of the present disclosure
  • Figure 11 is a schematic structural diagram of the nucleic acid constructed in Example 9 of the present disclosure, wherein “Hinge” in the figure refers to the hinge region, and “Linker” refers to the connecting peptide;
  • Figure 13 Detects IL-21 in the supernatant of MSLN CAR-T in Example 10 of the present disclosure
  • Figure 15 Detects IL-2 and IFN- ⁇ secretion when MSLN CAR-T is co-cultured with AsPC-1 cells in Example 11 of the present disclosure.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless expressly and specifically defined otherwise.
  • a single-chain antibody is a genetically engineered antibody in which the VH and VL domains are linked to a flexible polypeptide linker. Compared to the Fab regions of monolithic antibodies, single-chain antibodies exhibit better tissue penetration pharmacokinetics and have full antigen-binding specificity due to the unaltered antigen-binding surface.
  • Immune checkpoints are inhibitory regulatory molecules in the immune system that are critical for maintaining self-tolerance, preventing autoimmune responses, and minimizing tissue damage by controlling the timing and intensity of immune responses.
  • the expression of immune checkpoints on immune cells will inhibit the function of immune cells, so that the body cannot produce an effective anti-tumor immune response, and tumors form immune escape.
  • the main tumor-related immune checkpoint molecules are PD1, CTLA4, Tim3 and LAG3.
  • the "immune checkpoint single-chain antibody” described in this application is a single-chain antibody that has anti-immune checkpoint activity and can specifically bind to immune checkpoints.
  • T lymphocyte activation molecules refer to molecules that can stimulate the activation or differentiation of lymphocytes and enhance the cytotoxicity of T lymphocytes. Such as IL-21.
  • Immune costimulatory molecules refer to cell surface molecules and their ligands that provide costimulatory signals for the complete activation of T lymphocytes or B lymphocytes, such as 4-1BB, OX-40, CD40L, CD27, CD30, CD28, CD3 and their derivative.
  • the inventors insert the target nucleic acid into the viral genome at the position of certain viral sequences in order to construct a lentiviral vector, thereby producing a replication-deficient virus.
  • the inventors proceeded to construct packaging cell lines (containing the gag, pol and env genes, but excluding the LTR and packaging components).
  • the inventors introduced the recombinant plasmid containing the gene of interest, along with the lentiviral LTR and packaging sequence, into a packaging cell line.
  • Packaging sequences allow recombinant plasmid RNA transcripts to be packaged into viral particles and then secreted into the culture medium.
  • the inventors further collected the matrix containing the recombinant lentivirus, selectively concentrated, and used it for gene transfer.
  • Lentiviral vectors can infect a variety of cell types, including dividing and non-dividing cells.
  • the lentiviruses of the embodiments of the present disclosure are composite lentiviruses, in addition to the common lentiviral genes gag, pol and env, other genes with regulatory and structural functions are also included.
  • Lentiviral vectors are well known to those skilled in the art and include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, such as all deletion of genes env, vif, vpr, vpu and nef, so that lentiviral vectors form biosafety vectors.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for gene transfer and nucleic acid sequence expression in vivo and in vitro.
  • suitable host cells together with two or more vectors with packaging functions (gag, pol, env, rev and tat)
  • non-dividing cells can be infected.
  • the targeting of recombinant viruses is achieved through the binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus enables the vector to have specific targeting by inserting an effective sequence (including regulatory regions) into the viral vector, together with another gene encoding a ligand for a receptor on a specific target cell.
  • a variety of useful lentiviral vectors, as well as vectors produced by various methods and manipulations, are used to alter expression in cells.
  • adeno-associated viral vectors of embodiments of the present disclosure can be constructed using the DNA of one or more well-known serotype adeno-associated viral vectors.
  • the embodiments of the present disclosure also include minigenes.
  • a minigene means that a combination (a selected nucleotide sequence and operably necessary associated linker sequences) is used to direct transformation, transcription and/or expression of a gene product in a host cell in vivo or in vitro.
  • An "operably linked" sequence is used to encompass expression control sequences that contiguously control the gene of interest, and expression control sequences that act to control the gene of interest in trans or at a distance.
  • the vectors of embodiments of the present disclosure also include conventional control elements in cell transfection with plasmid vectors or/and cell infection with viral vectors.
  • a wide variety of expression control sequences may be used.
  • the promoter is an RNA polymerase promoter selected from U6, H1, pol I, pol II and pol III.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from a selected vector-based promoter.
  • the promoter when a lentiviral vector is selected, the promoter is the U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C or phosphoglycerol kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance, among others.
  • Other components of the vector include origins of replication.
  • compositions of the embodiments of the present disclosure provided to patients are preferably applied to biocompatible solutions or acceptable pharmaceutical carriers.
  • the various therapeutic compositions as prepared are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • a pharmaceutically or physiologically acceptable carrier such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • physiological saline such as physiological saline; isotonic saline or other formulations obvious to those skilled in the art.
  • the appropriate carrier will largely depend on the route of administration.
  • Other aqueous and anhydrous isotonic sterile injectable solutions and aqueous and anhydrous sterile suspensions are pharmaceutically acceptable carriers.
  • a method of treatment includes the use of one or more drug therapies.
  • Example 1 Construction of CART19 (T cells expressing chimeric antigen receptor targeting CD19) lentiviral vector expressing PD-1 antibody and IL-21 fusion protein and its control vector
  • Example 2 Packaging and concentration of lentivirus
  • the 293T was inoculated at a density of 8 ⁇ 10 6 cells/150mm 2 petri dish, and the state of the cells was observed the next day.
  • the third-generation lentiviral packaging vector was transfected into 293T by PEI transfection. After transfection, the medium was changed 6 hours later. Add according 15ml / 150mm 2 dish of DMEM medium containing 10% fetal calf serum, were transfected 72 hours after 48 hours viral supernatant was collected, centrifuged 10min 2000rpm 4 °C, remove cellular debris, followed by a 0.45 filter to filter impurities, The filtered virus suspension was centrifuged at 25,000 rpm and 4°C for 2 hours to concentrate the lentivirus. The concentrated virus was resuspended in an appropriate amount of medium and stored at -80°C.
  • T cells were separated with Stemcell's T cell negative selection kit (Cat. No. 19051). The separated T cells were supplemented with 5% human AB serum and 300 units/mL IL-2X-VIVO.
  • FIG 2 is the detection result of the CAR positive rate on the sixth day (D6) after T cell infection, CAR19 group (66.97%), CAR19&scFv group (65.07%), CAR19&scFv&IL21 group (56.31%), CAR19&scFv-IL21 group (78.1%), CAR19&IL21-scFv group (57.78%), it can be seen from the results that the CAR19&scFv-IL21 group has the highest infection efficiency.
  • T cell group Indicates the general T cell group
  • CAR19 indicates the T cell group expressing the chimeric antigen receptor targeting CD19
  • CAR19&scFv indicates the CART19 group expressing scFv
  • CAR19&scFv&IL21 indicates the CART19 group expressing scFv and IL21 alone
  • CAR19&scFv-IL21 expresses the fusion protein scFv -IL21 in the CART19 group
  • CAR19&scFv-IL21 represents the CART19 group expressing the fusion protein IL21-scFv
  • T cells 2.7%
  • PD-1 molecule is a member of the immune checkpoint family. It has been reported in the literature that after T cells are activated by CD3/CD28 antibodies, the expression level of PD-1 molecule is up-regulated to inhibit the activation of T cells (T cells). In addition, it is also an exhausted T cell. an important marker.
  • each group of CAR-T and control T cells were labeled with CFSE dye, and then mixed with RAJI-PD-L1-A3 according to the effector-target ratio of 10:1, 5:1, 2.5:1, 1.25:1 and 1:0
  • the cell lines were co-cultured. After 4 hours, the killing supernatant was collected and reserved for cytokine detection.
  • the killed cells were stained with propidium iodide (PI) and Annexin V, and the CFSE-negative RAJI-PD-L1-A3 cell population was selected.
  • PI propidium iodide
  • cytokine Factor detection The expression of cytokines was detected according to Invitrogen IL2 detection instructions (Cat. No.: 88-7025), IL21 detection instructions (Cat. No.: 88-8218) and IFN- ⁇ (Cat. No. 88-7316), PD-1 single-chain antibody detection The method is developed by the company. The release results of cytokine molecules are shown in Figure 6: Compared with other groups, CAR19&scFv-IL21 released more IL21, PD-1 single-chain antibody (scFv) and IFN- ⁇ in the process of killing RAJI-PD-L1-A3 , released more IL2, suggesting that CAR19&scFv-IL21 may exert the killing effect through the high expression of the above factors.
  • the experimental timeline is shown in Figure 7.
  • Each NPG mouse was subcutaneously inoculated with Raji-PD-L1 cells in the amount of 2E6 in D0, the tumor volume of the mice was measured and grouped in D7, and there were 8 mice in each group, and D8 was in the tail vein according to the amount of 5E6.
  • CART cells were injected, and the efficacy of CART was assessed within one month after tumor inoculation, and the ability of the drug to control recurrence was assessed within two months.
  • the average tumor volume, tumor remission rate and tumor inhibition rate of each D26 group are shown in Table 2 and Figure 8.
  • the average tumor volume of the CART19&scFv-IL21 group was significantly different from that of the CART19&scFv group, and the difference was extremely significant compared with the CART19 group.
  • the average recurrent tumor volume and recurrence rate of each D65 group are shown in Figure 9 and Table 3.
  • the average recurrent tumor volume in the CART19&scFv-IL21 group and the CART19&scFv group was significantly different from the CART19 group, but the difference was not significant between the CART19&scFv-IL21 group and the CART19&scFv group .
  • the tumor remission and recurrence of the mice in the group are shown in Figure 10.
  • the mice in the CART19 & scFv-IL21 group have a longer tumor-free growth period.
  • $CAR-T group mean tumor volume compared with PBS group, T test (Mann-Whitney),
  • the following nucleotide sequence was synthesized, and the nucleotide fragment was constructed on the lentiviral vector according to the restriction site of the lentiviral vector, and the primers were designed.
  • the scFv sequence of Mesothelin (MSLN) antibody, MSLN CAR plasmid was named PCDHF85, MSLN CAR+Anti PD1-IL-21 plasmid was named PCDHF86.
  • Packaging lentivirus The above 2 plasmids were packaged lentivirus respectively, and the packaging system was shown in Table 4 below:
  • Embodiment 10 MSLN CAR-T supernatant detection
  • IL-21 ELISA detection kit (Invitrogen , batch number 220657), the results are shown in Figure 13, the results show that 86CART cells normally secrete IL-21 and PD1 antibodies.
  • Example 11 MSLN CAR-T tumor cell killing and cytokine release detection
  • CART cells kill tumor cells, add 2E4/100ul/well AsPC-1 cells (ATCC, CRL-1682, Mesothelin positive cells) to a flat-bottomed 96-well plate (Costar, catalog number, 25719016), and the medium is RPMI1640 (Gibco, lot number) 2215748) + 10% FBS (Gibco Lot No. 2152441P), 5% CO 2 , and incubated at 37°C for 48h. Take T cells, 85CART cells, and 86CART cells respectively.
  • the ratio of :1 was added to each well, the medium of CART cells was X-VIVO 15, 100ul/well, the CART cells and tumor cells were mixed and cultured for 16h, the supernatant was aspirated for use, and washed gently with DPBS (Hyclone, batch number AE29431662) for 2 Aspirate and discard DPBS, further aspirate and discard the suspended cells in the wells, then add RPMI1640+10% FBS (100ul) and then add the prepared Promega (CellTiter-Glo Luminescent Cell Viability Assay, Cat.
  • DPBS Hyclone, batch number AE29431662
  • IL-2/IFN- ⁇ ELISA detection kit Human IL-21 Uncoated ELISA kit, R&D, lot number 223086-004; IFN gamma Human Uncoated ELISA Kit, R&D, lot number 223086-003 was used to detect the co-culture of CART and AsPC-1 cells
  • the secretion levels of IL-2 and IFN- ⁇ in the supernatant after the test are shown in Figure 15. The results show that when CART cells are co-cultured with AsPC-1 cells, IL-2 and IFN- ⁇ are normally secreted.

Abstract

提供了一种T淋巴细胞。该T淋巴细胞共表达融合蛋白以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原，其中，所述嵌合抗原受体包括：胞外区；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子。

Description

T淋巴细胞及其应用
优先权信息
本申请请求2020年07月07日向中国国家知识产权局提交的、专利申请号为202010648451.1的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开涉及生物制药领域，具体地，本公开涉及T淋巴细胞及其应用，更具体地，本公开涉及T淋巴细胞、慢病毒、转基因淋巴细胞、构建体、制备T淋巴细胞或者转基因淋巴细胞的方法、治疗癌症的治疗组合物以及降低T淋巴细胞表面免疫检查点表达的方法。
背景技术
嵌合抗原受体T细胞免疫疗法，简称CAR-T技术，是通过体外改造病人的T细胞，使病人的T细胞具备识别肿瘤细胞的能力，体外扩大培养后回输到病人体内进行治疗的一种方法。目前，以CD19为靶点的CAR-T在治疗B细胞血液肿瘤方面取得了巨大的成果，但根据临床研究结果来看，CD19 CAR-T在治疗B细胞淋巴瘤方面的疗效远远不及在治疗B细胞急性淋巴细胞白血病方面的疗效，这可能是因为B细胞淋巴瘤是实体肿瘤，其细胞表面表达有大量的PD-L1分子造成的，虽然目前尚未有文献对B-ALL病人B细胞上PD-L1分子表达水平报道，但是在针对弥漫性大B细胞淋巴瘤的临床研究中发现病人B细胞淋巴瘤表面的PD-L1分子表达水平与临床疗效直接相关，B细胞淋巴瘤表面PD-L1表达低的病人，在综合疗法、单独化疗和PD-1抗体免疫疗法中均有着更高的生存率。与此同时，运用CART技术在对其它靶点实体肿瘤治疗的过程中，肿瘤细胞也会通过过表达PD-L1来逃避CAR-T对其进行杀伤，提示实体瘤治疗过程中，封闭PD-1/PD-L1信号通路将会最大程度的增加CAR-T的临床疗效，使患者受益。
IL-21由CD4T细胞和NKT细胞产生，刺激CD8T细胞和NK细胞的成熟并增强其细胞毒性，同时具备促进记忆性CD8T细胞的分化等功能。IL-21的诸多效能使其成为免疫治疗的潜在靶点，但由于IL-21R广泛表达于包括T细胞、B细胞、NK细胞和骨髓细胞，所以怎样使IL-21专一性的作用于CART细胞，以控制其毒性成为免疫细胞治疗所关注的焦点。此外，IL-21本身半衰期很短，也需要有效的改造以提高半衰期。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。
发明人开发了一种分泌PD-1抗体与IL-21融合蛋白的CART，PD-1主要表达在T细胞表面，主要是CD8+T细胞，分泌出的PD-1抗体与IL-21融合蛋白选择性地结合在T细胞和CART细胞表面，一方面，封闭PD-1/PD-L1信号通路，另一方面，又使得IL-21专一性的作用于T细胞和CART细胞，进而行使其双重功能；与此同时，融合蛋白由于分子量变大，大大地提高了药物的半衰期。
鉴于此，在本公开的第一方面，本公开提出了一种T淋巴细胞。根据本公开的实施例，所述T淋巴细胞共表达融合蛋白以及嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区以及CD8铰链区，所述单链抗体特异性识别肿瘤抗原；跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链；所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子。根据本公开实施例的T淋巴细胞分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白，将免疫检查点单链抗体和T细胞激活分子的双重优越性专一性的作用于CAR-T细胞，降低肿瘤微环境对CART抑制作用的同时，使CART细胞作用更为长效；同时发明人意外而惊喜地发现，根据本公开实施例的CART细胞的细胞表面表达极低比例的免疫检查点分子，同时有更强的杀瘤效果；根据本公开实施例的CART细胞与单独分泌免疫检查点单链抗体或T细胞激活分子的CART细胞相比，大大提高了T细胞激活分子与T淋巴细胞结合的特异性，降低了药物的毒性。
根据本公开的实施例，上述T淋巴细胞还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT至少之一。
根据本公开的实施例，所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的至少之一。
根据本公开的实施例，所述免疫检查点为PD-1，所述T细胞激活分子为IL21。
根据本公开的实施例，所述IL21的C端与PD-1单链抗体的N端相连；优选地，所述PD-1单链抗体的C端与所述IL21的N端相连。发明人发现，当所述PD-1单链抗体的C端与所述IL21的N端相连，T淋巴细胞表面的PD-1表达量更低，T淋巴细胞对肿瘤的杀伤效果更加显著。
根据本公开的实施例，所述融合蛋白进一步包括连接肽，所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间。
根据本公开的实施例，所述连接肽具有SEQ ID NO:1所示的氨基酸序列。.
Figure PCTCN2021104802-appb-000001
根据本公开的实施例，所述连接肽的N端与所述免疫检查点单链抗体的C端相连，所述连接肽的C端与所述T细胞激活分子的N端相连。
根据本公开的实施例，所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
Figure PCTCN2021104802-appb-000002
其中，融合蛋白scFV-IL21具有SEQ ID NO:2所示的氨基酸序列，融合蛋白IL21-scFV具有SEQ ID NO:3所示的氨基酸序列，其中，scFV表示免疫检查点单链抗体，IL21表示T细胞激活分子为IL21，融合蛋白scFV-IL21中scFV和IL21的连接顺序为免疫检查点单链抗体的C端与IL21的N端相连，融合蛋白IL21-scFV中scFV和IL21的连接顺序为IL21的C端与免疫检查点单链抗体的N端相连。
在本公开的第二方面，本公开提出了一种慢病毒。根据本公开的实施例，所述慢病毒携带下列核酸分子：(a)编码融合蛋白的核酸分子，所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子；(b)编码嵌合抗原受体的核酸分子，所述嵌合抗原受体的胞外区识别肿瘤抗原。将根据本公开实施例的慢病毒导入受体细胞T淋巴细胞中，可在受体细胞中表达和分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白以及嵌合抗原受体，降低T淋巴细胞表面免疫检查点的表达，降低肿瘤微环境对T淋巴细胞的抑制作用，T细胞作用对肿瘤的杀伤更为有效、长效，安全性更高。
根据本公开的实施例，上述慢病毒还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述免疫检查点为PD-1，所述T细胞激活分子为IL21，所述融合蛋白具有SEQ ID NO:2或3所示的氨基酸序列。
根据本公开的实施例，所述肿瘤抗原为CD19，所述嵌合抗原受体具有SEQ ID NO:4所示的氨基酸序列。
Figure PCTCN2021104802-appb-000003
根据本公开的实施例，所述编码融合蛋白的核酸分子具有SEQ ID NO:5或6任一所示的核苷酸序列。
Figure PCTCN2021104802-appb-000004
Figure PCTCN2021104802-appb-000005
其中，具有SEQ ID NO:5所示核苷酸序列的核酸分子编码的融合蛋白具有SEQ ID NO:2所示的氨基酸序列，具有SEQ ID NO:6所示核苷酸序列的核酸分子编码的融合蛋白具有SEQ ID NO:3所示的氨基酸序列。
根据本公开的实施例，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:7所示的核苷酸序列。
Figure PCTCN2021104802-appb-000006
Figure PCTCN2021104802-appb-000007
在本公开的第三方面，本公开提出了一种慢病毒。根据本公开的实施例，所述慢病毒携带具有SEQ ID NO：8或9所示核苷酸序列的核酸分子。
Figure PCTCN2021104802-appb-000008
Figure PCTCN2021104802-appb-000009
Figure PCTCN2021104802-appb-000010
其中，具有SEQ ID NO:8所示核苷酸序列的核酸分子编码CD19CAR-scFV-IL21，具有SEQ ID NO:9所示核苷酸序列的核酸分子编码CD19CAR-IL21-scFV。
在本公开的第四方面，本公开提出了一种转基因淋巴细胞。根据本公开的实施例，所述转基因淋巴细胞共表达融合蛋白以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原，其中，所述嵌合抗原受体包括：胞外区；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子。根据本公开实施例的转基因淋巴细胞表达嵌合抗原受体和分泌包括免疫检查点单链抗体和T细胞激活分子的融合蛋白，将免疫检查点单链抗体和T细胞激活分子的双重优越性专一性的作用于该转基因淋巴细胞，降低肿瘤微环境对该转基因淋巴细胞的抑制作用的同时，使该转基因淋巴细胞作用更为长效；同时发明人意外而惊喜地发现，根据本公开实施例的转基因淋巴细胞的细胞表面表达极低比例的免疫检查点分子，同时有更强的杀瘤效果；根据本公开实施例的转基因淋巴细胞与单独分泌免疫检查点单链抗体或T细胞激活分子的转基因淋巴细胞相比，大大提高了T细胞激活分子与T淋巴细胞结合的特异性，降低了药物的毒性。
根据本公开的实施例，上述转基因淋巴细胞还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种。
根据本公开的实施例，所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段。
根据本公开的实施例，所述淋巴细胞是CD3 +T淋巴细胞。
根据本公开的实施例，所述淋巴细胞是CD8 +T淋巴细胞。
根据本公开的实施例，所述淋巴细胞是自然杀伤细胞。
根据本公开的实施例，所述淋巴细胞是自然杀伤T细胞。
根据本公开的实施例，所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT的至少之一。
根据本公开的实施例，所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的至少之一。
根据本公开的实施例，所述免疫检查点为PD-1，所述T细胞激活分子为IL21。
根据本公开的实施例，所述IL21的C端与PD-1单链抗体的N端相连。
根据本公开的实施例，所述PD-1单链抗体的C端与所述IL21的N端相连。进而根据本公开实施例的转基因淋巴细胞表面的PD-1的表达更低，该转基因淋巴细胞对肿瘤细胞的杀伤效果更加显著。
根据本公开的实施例，所述融合蛋白进一步包括连接肽，所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间。
根据本公开的实施例，所述连接肽具有SEQ ID NO:1所示的氨基酸序列。
根据本公开的实施例，所述连接肽的N端与所述免疫检查点单链抗体的C端相连，所述连接肽的C端与所述T细胞激活分子的N端相连。
根据本公开的实施例，所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
在本公开的第五方面，本公开提出了一种构建体。根据本公开的实施例，所述构建体包括：第一核酸分子，所述第一核酸分子编码融合蛋白，所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子；第二核酸分子，所述第二核酸分子编码嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原；其中，所述融合蛋白、所述嵌合抗原受体是如前面所描述的。将根据本公开实施例的构建体导入受体细胞淋巴细胞后，可在淋巴细胞表面表达嵌合抗原受体和分泌融合蛋白，淋巴对肿瘤的杀伤效果更加显著、持久和安全。
根据本公开的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：
根据本公开的实施例，所述第一核酸分子、所述第二核酸分子被设置为在前面所述的淋巴细胞中表达所述融合蛋白、嵌合抗原受体，并且所述融合蛋白与嵌合抗原受体呈非融合形式。
根据本公开的实施例，所述构建体进一步包括：第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及第二启动子，所述第二启动子与所述第二核酸分子可操 作地连接。进而可实现第一核酸分子和第二核酸分子分别独立地表达。
根据本公开的具体实施例，所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子。
根据本公开的实施例，所述构建体进一步包括：内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：10所示的核苷酸序列。进而可实现所表达的融合蛋白与嵌合抗原受体呈非融合形式。
Figure PCTCN2021104802-appb-000011
内部核糖体进入位点通常位于RNA病毒基因组的5’非翻译区(UTR)，这样一个病毒蛋白的翻译就可以不依赖于5‘帽子结构，另一个蛋白通常靠5’帽子结构起始翻译，IRES前后的两个基因的表达通常是成比例的。内部核糖体进入位点序列的引入使得编码编码嵌合抗原受体的核酸分子与编码融合蛋白的核酸分子分别独立的表达。
根据本公开的实施例，所述构建体进一步包括:第三核酸分子，所述第三核酸分子设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码可切割连接肽，所述可切割连接肽能够在所述淋巴细胞中被切割。进而淋巴细胞中表达的融合蛋白和嵌合抗原受体在连接肽处被切割，使得嵌合抗原受体独立地表达于淋巴细胞膜表面，融合蛋白独立地从淋巴细胞中分离出来。
根据本公开的实施例，所述可切割连接肽具有SEQ ID NO：11所示的氨基酸序列。
Figure PCTCN2021104802-appb-000012
根据本公开的实施例，所述构建体的载体是非致病性病毒载体；
根据本公开的实施例，所述病毒载体包括选自反转录病毒载体、慢病毒载体和腺病毒相关病毒载体的至少之一。
在本公开的第六方面，本公开提出了一种制备前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞的方法。根据本公开的实施例，所述方法包括：将前面所述的构建体或者 前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。根据本公开实施例的方法所制备的T淋巴细胞或者转基因淋巴细胞对肿瘤细胞的杀伤效果更加显著、持久和安全性更高。
在本公开的第七方面，本公开提出了一种用于治疗癌症的治疗组合物。根据本公开的实施例，所述治疗组合物包括：前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞。根据本公开实施例的治疗组合物对肿瘤细胞的杀伤效果更加显著、持久和安全性更高。
根据本公开的实施例，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本公开的第八方面，本公开提出了前面所述的T淋巴细胞、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的构建体或前面所述的治疗组合物在制备治疗癌症的药物中的用途。
根据本公开的实施例，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本公开的第九方面，本公开提出了一种治疗癌症的方法。根据本公开的实施例，所述方法包括向患有癌症的受试者施用以下中的至少之一：
前面所述的T淋巴细胞；
导入前面所述的慢病毒的T淋巴细胞；
前面所述的转基因淋巴细胞；
导入前面所述的构建体的T淋巴细胞；
前面所述的治疗组合物。
根据本公开的实施例，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本公开的第十方面，本公开提出了前面所述的T淋巴细胞、前面所述的慢病毒、前面所述的转基因淋巴细胞、前面所述的构建体或前面所述的治疗组合物在癌症的治疗中的用途。
根据本公开的实施例，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
在本公开的第十一方面，本公开提出了一种降低T淋巴细胞表面免疫检查点表达的方法。根据本公开的实施例，所述方法包括：使所述T淋巴细胞表达嵌合抗原受体和融合蛋白；或使所述T淋巴细胞与表达嵌合抗原受体和融合蛋白的T细胞共培养，所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子，其中，所述嵌合抗原受体、融合蛋白是如前面所限定的。根据本公开实施例的方法，可显著降低T淋巴细胞表面的免疫检查点表达， 封闭免疫逃逸机制。
根据本公开的实施例，所述免疫检查点为PD-1，所述T细胞激活分子为IL21。
综上，本公开的创新点如下所述：
1).利用CART分泌PD-1抗体与IL-21融合蛋白，PD-1单链抗体首先特异性的选择结合T细胞表面的PD-1分子，从而使IL-21特也异性的作用于T细胞，发挥其功能，与单纯性过表达IL21相比，降低了IL21与其他细胞表面的IL21受体结合所造成的毒性；
2).与PD-1抗体和IL-21单独表达比较而言，PD-1抗体与IL-21融合表达，增加了CART药物的半衰期；
3).实验数据显示，CART分泌PD-1抗体与IL-21融合蛋白，大大降低了T细胞表面PD-1表达的比例；
4).与PD-1抗体和IL-21单独表达比较而言，PD-1抗体与IL-21融合表达，CART对实体瘤有更强的杀伤功能。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1本公开实施例1构建的核酸的结构示意图，其中，图中“Hinge”是指铰链区，“Linker”是指连接肽；
图2本公开实施例3T细胞侵染后D6的CAR19阳性率检测结果；
图3本公开实施例5流式分选单克隆建立Raji-PD-L1过表达靶细胞株的结果图，其中图中“Multi-sample”是指多重采样；
图4本公开实施例6 CART杀伤阴性对照细胞K562结果图；
图5本公开实施例6 CART杀伤RAJI-PD-L1-A3结果图；
图6本公开实施例7 CART杀伤RAJI-PD-L1-A3过程中的细胞因子释放的结果图；
图7本公开实施例8 CART体内疗效评估实验的时间轴；
图8本公开实施例8 CART体内治疗后肿瘤缓解率；
图9本公开实施例8 CART体内治疗后肿瘤复发率；
图10本公开实施例8 CART体内治疗后组内小鼠个体肿瘤缓解及复发情况统计图；
图11本公开实施例9构建的核酸的结构示意图，其中，图中“Hinge”是指铰链区，“Linker”是指连接肽；
图12本公开实施例9感染T细胞后D7的CAR-MSLN阳性率检测结果；
图13本公开实施例10MSLN CAR-T上清液中IL-21检测；
图14本公开实施例11MSLN CAR-T肿瘤细胞杀伤检测；以及
图15本公开实施例11MSLN CAR-T与AsPC-1细胞共培养时IL-2和IFN-γ分泌检测。
发明详细描述
下面通过参考附图描述的实施例是示例性的，旨在用于解释本公开，而不能理解为对本公开的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本公开的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。
单链抗体(scFv)是一种基因工程抗体，其中VH和VL域与柔性多肽连接体相连。与整体抗体的Fab区相比，单链抗体表现出更好的组织渗透药动学，并且由于抗原结合表面未被改变而具有完全的抗原结合特异性。
免疫检查点(immune cheeckpoint)是免疫系统中起抑制作用的调节分子，其对于维持自身耐受、防止自身免疫反应、以及通过控制免疫应答的时间和强度而使组织损伤最小化等至关重要。免疫检查点表达于免疫细胞上，将抑制免疫细胞功能，使机体无法产生有效的抗肿瘤免疫应答，肿瘤形成免疫逃逸。与肿瘤相关的免疫检查点分子主要有：PD1、CTLA4、Tim3和LAG3等。
本申请所述的“免疫检查点单链抗体”是具有抗免疫检查点活性，可以与免疫检查点特异性结合的单链抗体。
T淋巴细胞激活分子是指能够刺激淋巴细胞活化或分化，增强T淋巴细胞细胞毒性的分子。如IL-21。
免疫共刺激分子是指为T淋巴细胞或B淋巴细胞完全活化提供共刺激信号的细胞表面分子及其配体，如4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物。
根据本公开的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因， 但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本公开的实施例，本公开实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。
根据本公开的实施例，本公开实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。另外，根据本公开的实施例，本公开实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表达控制序列。
另外，本公开实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本公开的实施例，启动子为选自U6、H1、pol I、pol II和pol III的RNA聚合酶启动子。根据本公开的实施例，启动子为组织特异型启动子。根据本公开的实施例，启动子为诱导型启动子。根据本公开的实施例，启动子为选自基于所选载体的启动子。根据本公开的实施例，当选择慢病毒载体时，启动子为U6、H1、CMV IE基因、EF-1α、泛素C或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，
根据本公开的实施例，提供给患者的本公开实施例的组合物，较好的应用于生物兼容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。
表达和分泌融合蛋白以及表达特有的针对抗原CD19嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞，可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下，一个治疗方法的包括使用一个或多个药物疗法。
下面将结合实施例对本公开的方案进行解释。
本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1构建表达PD-1抗体与IL-21融合蛋白的CART19(表达靶向CD19的嵌合抗原受体的T细胞)慢病毒载体及其对照载体
基因合成图1所示结构的核苷酸序列，按照慢病毒载体酶切位点，将核苷酸片段构建至慢病毒载体上，设计引物，通过测序结果验证载体构建的正确性。
实施例2包装并浓缩慢病毒
将293T按照8x10 6个细胞/150mm 2培养皿的密度接种，次日观察细胞的状态，用PEI转染的方法将3代慢病毒包装载体共转至293T，转染完6小时后换液，按照15ml/150mm 2培养皿添加含有10％胎牛血清的DMEM培养基，转染完48小时与72小时收集病毒上清，2000rpm 4℃10min离心，去除细胞碎片，之后0.45微米的滤器过滤杂质，过滤后的病毒悬液在25000rpm、4℃的条件下离心2小时浓缩慢病毒，浓缩后的病毒加入适量的培养基重悬，置于-80℃保存
实施例3生产CAR-T及对照细胞
抽血20mL，Ficall梯度离心分离PBMC，用Stemcell公司T细胞阴选试剂盒(货号：19051)分离T细胞，分离后的T细胞用添加5％人AB血清及300单位/ML IL-2X-VIVO 15培养基重悬T细胞至1×10 6个细胞/ML，用含1％FBS X-VIVO 15清洗beads，按照磁珠:T细胞＝2:1比例加入预先清洗过的磁珠(Cat#40203D,10ML,Life technology)，2-3天后用新 鲜的培养基重悬T细胞至3-5×10 6个细胞/mL，按照MOI＝10值加入慢病毒，同时加入8μg/mL的Polybrene，4-6小时之后，补加培养基稀释细胞至1×10 6个细胞/mL，次日更换新鲜培养基，使细胞浓度维持在0.2-0.3×10 6PBMC/mL，之后每隔2-3天更换一次培养基，病毒侵染完72小时后流式分析细胞阳性率，图2是T细胞侵染后第六天(D6)的CAR阳性率检测结果，CAR19组(66.97％)、CAR19&scFv组(65.07％)、CAR19&scFv&IL21组(56.31％)、CAR19&scFv-IL21组(78.1％)、CAR19&IL21-scFv组(57.78％)，由结果可以看出CAR19&scFv-IL21组侵染效率最高。
实施例4 CART表型鉴定；
侵染后D6的各组细胞，流式细胞术检测其T、B、NK细胞分群，同时检测T细胞中CD4、CD8、PD-1的分群，结果如表1所示(其中，T cell组表示普通T细胞组，CAR19表示表达靶向CD19的嵌合抗原受体的T细胞组，CAR19&scFv表示表达scFv的CART19组，CAR19&scFv&IL21表示单独表达scFv、IL21的CART19组，CAR19&scFv-IL21表示表达融合蛋白scFv-IL21的CART19组，CAR19&scFv-IL21表示表达融合蛋白IL21-scFv的CART19组)，可以看到CAR19&scFv-IL21组T细胞仅有少量比例的细胞(2.87％)表达PD-1分子，提示CAR19&scFv-IL21可能会调控T细胞表面分子PD-1的表达。PD-1分子作为免疫检查站点家族的成员，文献报道T细胞受到CD3/CD28抗体活化后会上调PD-1分子的表达水平来抑制T细胞(T cell)的活化，另外它也是T细胞衰竭的一个重要标志物。
表1：
Figure PCTCN2021104802-appb-000013
实施例5构建Raji-PD-L1表达靶细胞株
构建慢病毒载体过表达人类PD-L1载体，包装浓缩慢病毒，之后按照MOI＝10的比例侵染Raji细胞，侵染72h后使用APC anti-human PD-L1抗体检测侵染效率，流式分选阳性细胞，单克隆建立Raji-PD-L1过表达靶细胞株。结果如图3所示：成功筛选出过表达PD-L1的Raji细胞株(克隆号A3表示为Raji-PD-L1-A3)，与Raji相比，该Raji-PD-L1-A3细胞株CD19抗原表达没有发生变化。
实施例6 CART体外杀伤功能评估
杀伤实验：将CAR-T各组与对照T细胞用CFSE染料标记，之后按照效靶比10:1、5:1、2.5:1、1.25:1和1:0与RAJI-PD-L1-A3细胞株共培养，4小时后，收集杀伤上清留作细胞因子检测，杀伤后的细胞用碘化丙碇(PI)和Annexin V染色，选择CFSE阴性的RAJI-PD-L1-A3 细胞群，流式检测RAJI-PD-L1-A3细胞的晚期凋亡和早期凋亡情况；同时以表达CD19阴性的K562细胞作为杀伤对照。结果如图4和图5所示：实验各组对表达CD19阴性的K562细胞无明显杀伤功能，但是CART各组对CD19表达阳性的RAJI-PD-L1-A3均有明显杀伤功能，且CAR19&scFv-IL21杀伤效果最好，在效靶比10:1的时候，CAR19&scFv-IL21杀伤RAJI-PD-L1-A3的效率为74.33％。
实施例7 CART杀伤RAJI-PD-L1-A3过程中的细胞因子释放
因子检测：检测细胞因子表达情况按照Invitrogen IL2检测说明书(货号：88-7025)、IL21检测说明书(货号：88-8218)和IFN-γ(货号88-7316)进行，PD-1单链抗体检测方法为本公司制定。细胞因子分子释放结果如图6所示：与其他各组相比，CAR19&scFv-IL21杀伤RAJI-PD-L1-A3的过程释放更多的IL21、PD-1单链抗体(scFv)与IFN-γ，释放较多的IL2，提示CAR19&scFv-IL21可能是通过高表达上述因子发挥杀伤效能的。
实施例8 CART体内疗效评估
实验方案：选择5周龄，NPG雌性小鼠，第0天(Day0)皮下接种2E5Raji-PD-L1-A3/只，共40只；第7天(DAY7)选择肿瘤体积为100mm 3的小鼠入组实验，共四组，尽量使每组小鼠肿瘤体积保持一致，剔除肿瘤体积过大或者过小的小鼠；第8天(DAY8)，每组小鼠尾静脉注射对应的CART(CAR19、CAR19&scFv和CAR19&scFv-IL21)，对照组注射PBS；给药后每周三测量肿瘤体积与体重，对照小鼠达到肿瘤测量终点评估肿瘤缓解率，二个月评估肿瘤复发率与生存曲线。实验时间轴如图7所示，D0每只NPG小鼠按照2E6的量皮下接种Raji-PD-L1细胞，D7测量小鼠肿瘤体积并进行分组，每组8只，D8按照5E6的量尾静脉注射CART细胞，肿瘤接种后一个月内评估CART药效，两个月评估药物控制复发能力。D26各组平均肿瘤体积、肿瘤缓解率和抑瘤率如表2和图8所示，CART19&scFv-IL21组平均肿瘤体积与CART19&scFv相比差异显著，与CART19组相比差异及其显著。D65各组平均复发肿瘤体积和复发率如图9和表3所示，CART19&scFv-IL21组和CART19&scFv组平均复发肿瘤体积与CART19组相比差异显著，CART19&scFv-IL21组和CART19&scFv组相比差异不显著。组内小鼠个体肿瘤缓解及复发情况如图10所示，CART19&scFv-IL21组内小鼠有更长的无瘤生长期。
表2：
Figure PCTCN2021104802-appb-000014
$CAR-T各组平均肿瘤体积与PBS组相比，T检验(Mann-Whitney)，
*P＜0.05，**P＜0.01，***P＜0.001
表3：
Figure PCTCN2021104802-appb-000015
$险PBS组外各组平均复发肿瘤体积与CART19组相比，T检验(Mann-Whitney)，
*P＜0.05，**P＜0.01，***P＜0.001
实施例9靶向Mesothelin(MSLN)的嵌合抗原受体的T细胞的构建与生产
基于图11基因合成以下核苷酸序列，按照慢病毒载体酶切位点，将核苷酸片段构建至慢病毒载体上，设计引物，通过测序结果验证载体构建的正确性，其中SS1为抗人Mesothelin(MSLN)抗体的scFv序列，MSLN CAR质粒命名为PCDHF85，MSLN CAR+Anti PD1-IL-21质粒命名为PCDHF86。
包装慢病毒将以上2个质粒分别包装慢病毒，包装体系如下表4所示：
表4：
  PCDHF-85 PCDHF-86
PCDH D(PMD2.G) 24μg 36μg
PCDH M(pMDLg/pRRE) 24μg 36μg
PCDH N(pRSV-Rev) 24μg 36μg
PCDHF85 48μg  
PCDHF86   72μg
PEI(Polysciences 636951) 240μg 360μg
OPTI-MEMI(Gibco 31985070) 12mL 18mL
接种293T细胞5E6于10cm细胞培养皿中，加入10mL含10％FBS的DMEM培养基(DMEM Gibco，11995040-1L；FBS Gibco，10091-148)，5％CO 2，37℃条件下CO 2培养箱中培养24h；按表4所示进行慢病毒包装。CART细胞制备，Ficoll淋巴分离液(达科为，AS1114546)从血液(菲鹏生物员工0068号志愿者献血50mL)中分离PBMC细胞，偶联CD3/CD28抗体的磁珠(Dynabeads，CD3/CD28 CTS，货号40203D，批号A2-011710E)阳选法分离获得T细胞，将慢病毒按MOI＝5:1感染T细胞以制备CART细胞，CART细胞培养7天后通过检测CART细胞的MSLN抗体表达来确定CART细胞CAR阳性率，如图12所示。
实施例10 MSLN CAR-T上清液检测
CART细胞分泌IL-21，首先检测CART细胞培养上清中IL-21的表达水平，细胞起始密度均为2E5个细胞/ml，培养72h后收集上清，IL-21ELISA检测试剂盒，(Invitrogen，批号220657)，结果如图13所示，结果显示86CART细胞正常分泌IL-21和PD1抗体。
实施例11 MSLN CAR-T肿瘤细胞杀伤及细胞因子释放检测
CART细胞杀伤肿瘤细胞，向平底96孔板(Costar，货号，25719016)分别加入2E4/100ul/孔AsPC-1细胞(ATCC，CRL-1682，Mesothelin表达阳性细胞)，培养基为RPMI1640(Gibco，批号2215748)+10％FBS(Gibco批号2152441P)，5％CO 2，37℃培养48h。分别取T细胞，85CART细胞，86CART细胞，计数之后用T细胞将85CART细胞稀释到CAR+阳性率为45％，按效应细胞(CAR+CART细胞)：靶细胞＝1:1，5:1，10:1的比例分别加入各孔，CART细胞的培养基为X-VIVO 15，100ul/孔，CART细胞与肿瘤细胞混合培养16h，吸上清备用，用DPBS(Hyclone，批号AE29431662)轻轻清洗2次并吸弃DPBS，进一步吸弃孔中悬浮细胞，然后加入RPMI1640+10％FBS(100ul)再加入提前准备好的Promega(CellTiter-Glo Luminescent Cell Viability Assay，货号，0000453271)底物与缓冲液混合液，100ul/孔，10min后用多功能酶标仪(MOLECRLAR DEVICES，SpectraMax i3X)检测各孔的荧光值，通过各孔的荧光值与AsPC-1细胞孔(未加T/CART细胞)荧光值进行比值，从而计算出各孔的活细胞和凋亡细胞的比值。检测结果如图14所示，结果显示，对于AsPC-1细胞的杀伤效果，86CART>85CART>T。
用IL-2/IFN-γELISA检测试剂盒(Human IL-21 Uncoated ELISA kit，R&D，批号223086-004；IFN gamma Human Uncoated ELISA Kit，R&D，批号223086-003)检测CART与AsPC-1细胞共培养后的上清中的IL-2和IFN-γ分泌水平，检测结果如图15所示，结果显示CART细胞与AsPC-1细胞共培养时，IL-2和IFN-γ正常分泌。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (28)

  1. 一种T淋巴细胞，其中，所述T淋巴细胞共表达融合蛋白以及嵌合抗原受体，其中，
    所述嵌合抗原受体包括：
    胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区以及CD8铰链区，所述单链抗体特异性识别肿瘤抗原；
    跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链；
    所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子。
  2. 根据权利要求1所述的T淋巴细胞，其中，所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT至少之一；
    任选地，所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18和IL21的至少之一。
  3. 根据权利要求1所述的T淋巴细胞，其中，所述免疫检查点为PD-1，所述T细胞激活分子为IL21。
  4. 根据权利要求3所述的T淋巴细胞，其中，所述IL21的C端与PD-1单链抗体的N端相连；优选地，所述PD-1单链抗体的C端与所述IL21的N端相连。
  5. 根据权利要求1所述的T淋巴细胞，其中，所述融合蛋白进一步包括连接肽，所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间；
    任选地，所述连接肽具有SEQ ID NO:1所示的氨基酸序列；
    优选地，所述连接肽的N端与所述免疫检查点单链抗体的C端相连，所述连接肽的C端与所述T细胞激活分子的N端相连。
  6. 根据权利要求1所述的T淋巴细胞，其中，所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
  7. 一种慢病毒，其中，所述慢病毒携带下列核酸分子：
    (a)编码融合蛋白的核酸分子，所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子；
    (b)编码嵌合抗原受体的核酸分子，所述嵌合抗原受体的胞外区识别肿瘤抗原。
  8. 根据权利要求7所述的慢病毒，其中，所述免疫检查点为PD-1，所述T细胞激活分子为IL21，所述融合蛋白具有SEQ ID NO:2或3所示的氨基酸序列；
    任选地，所述肿瘤抗原为CD19，所述嵌合抗原受体具有SEQ ID NO:4所示的氨基酸序列。
  9. 根据权利要求8所述的慢病毒，其中，所述编码融合蛋白的核酸分子具有SEQ ID NO:5或6任一所示的核苷酸序列；
    任选地，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:7所示的核苷酸序列。
  10. 一种慢病毒，其中，所述慢病毒携带具有SEQ ID NO：8或9所示核苷酸序列的核酸分子。
  11. 一种转基因淋巴细胞，其中，所述转基因淋巴细胞共表达融合蛋白以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原，
    其中，所述嵌合抗原受体包括：
    胞外区；
    跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；
    所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子。
  12. 根据权利要求11所述的转基因淋巴细胞，其中，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种；
    优选地，所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段；
    任选地，所述淋巴细胞是CD3 +T淋巴细胞；
    任选地，所述淋巴细胞是CD8 +T淋巴细胞；
    任选地，所述淋巴细胞是自然杀伤细胞；
    任选地，所述淋巴细胞是自然杀伤T细胞。
  13. 根据权利要求11所述的转基因淋巴细胞，其中，所述免疫检查点包括选自PD-1、PD-L1、CTLA-4、TIM3、LAG3、BTLA和TIGIT的至少之一；
    任选地，所述T细胞激活分子包括选自IL2、IL7、IL9、IL12、IL15、IL18与IL21的的至少之一；
    优选地，所述免疫检查点为PD-1，所述T细胞激活分子为IL21；
    任选地，所述IL21的C端与PD-1单链抗体的N端相连；
    优选地，所述PD-1单链抗体的C端与所述IL21的N端相连；
    任选地，所述融合蛋白进一步包括连接肽，所述连接肽设置于所述免疫检查点单链抗体和T细胞激活分子之间；
    任选地，所述连接肽具有SEQ ID NO:1所示的氨基酸序列；
    优选地，所述连接肽的N端与所述免疫检查点单链抗体的C端相连，所述连接肽的C端与所述T细胞激活分子的N端相连；
    任选地，所述融合蛋白具有SEQ ID NO:2或SEQ ID NO:3所示的氨基酸序列。
  14. 一种构建体，其中，所述构建体包括：
    第一核酸分子，所述第一核酸分子编码融合蛋白，所述融合蛋白包括：
    免疫检查点单链抗体和T细胞激活分子；
    第二核酸分子，所述第二核酸分子编码嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原；
    其中，所述融合蛋白、所述嵌合抗原受体是如权利要求1～6、11～13任一项中所限定的。
  15. 根据权利要求14所述的构建体，其中，所述第一核酸分子、所述第二核酸分子被设置为在权利要求11～13任一项所述的淋巴细胞中表达所述融合蛋白、嵌合抗原受体，并且所述融合蛋白与嵌合抗原受体呈非融合形式。
  16. 根据权利要求15所述的构建体，其中，进一步包括：
    第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；以及
    第二启动子，所述第二启动子与所述第二核酸分子可操作地连接；
    任选地，所述第一启动子、所述第二启动子分别独立地选自U6、H1、CMV、EF-1、LTR或RSV启动子；
    任选地，进一步包括：
    内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第二核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：10所示的核苷酸序列；
    任选地，进一步包括:
    第三核酸分子，所述第三核酸分子设置在所述第一核酸分子与所述第二核酸分子之间，并且所述第三核酸分子编码可切割连接肽，所述可切割连接肽能够在所述淋巴细胞中被切割；
    任选地，所述可切割连接肽具有SEQ ID NO：11所示的氨基酸序列。
  17. 根据权利要求14所述的构建体，其中，所述构建体的载体是非致病性病毒载体；
    任选地，所述病毒载体包括选自逆转录病毒载体、慢病毒载体和腺病毒相关病毒载体的至少之一。
  18. 一种制备权利要求1～6任一项所述的T淋巴细胞或者权利要求11～13任一项所述的转基因淋巴细胞的方法，其中，包括：
    将权利要求14～17任一项所述的构建体或者权利要求7～10任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。
  19. 一种用于治疗癌症的治疗组合物，其中，包括：
    权利要求14～17任一项所述的构建体、权利要求7～10任一项所述的慢病毒、权利要求1～6任一项所述的T淋巴细胞或者权利要求11～13任一项所述的转基因淋巴细胞。
  20. 根据权利要求19所述的治疗组合物，其中，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
  21. 权利要求1～6任一项所述的T淋巴细胞、权利要求7～10任一项所述的慢病毒、权利要求11～13任一项所述的转基因淋巴细胞、权利要求14～17任一项所述的构建体或权利要求19或20所述的治疗组合物在制备治疗癌症的药物中的用途。
  22. 根据权利要求21所述的用途，其中，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
  23. 一种治疗癌症的方法，所述方法包括向患有癌症的受试者施用以下中的至少之一：
    权利要求1～6任一项所述的T淋巴细胞；
    导入权利要求7～10任一项所述的慢病毒的T淋巴细胞；
    权利要求11～13任一项所述的转基因淋巴细胞；
    导入权利要求14～17任一项所述的构建体的T淋巴细胞；
    权利要求19或20所述的治疗组合物。
  24. 根据权利要求23所述的方法，其中，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
  25. 权利要求1～6任一项所述的T淋巴细胞、权利要求7～10任一项所述的慢病毒、权利要求11～13任一项所述的转基因淋巴细胞、权利要求14～17任一项所述的构建体或权利要求19或20所述的治疗组合物在癌症的治疗中的用途。
  26. 根据权利要求25所述的用途，其中，所述癌症包括选自肝癌、胰腺癌、卵巢癌、胆管癌、肺癌、胃癌、肠癌、食管癌和乳腺癌的至少之一。
  27. 一种降低T淋巴细胞表面免疫检查点表达的方法，其中，包括：
    使所述T淋巴细胞表达嵌合抗原受体和融合蛋白；或使所述T淋巴细胞与表达嵌合抗原受体和融合蛋白的T细胞共培养，
    所述融合蛋白包括：免疫检查点单链抗体和T细胞激活分子，
    其中，所述嵌合抗原受体、融合蛋白是如权利要求1～6、11～13任一项中所限定的。
  28. 根据权利要求27所述的方法，其中，所述免疫检查点为PD-1，所述T细胞激活分子为IL21。
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