WO2018058432A1 - 一种多基因重组嵌合抗原受体分子及其应用 - Google Patents

一种多基因重组嵌合抗原受体分子及其应用 Download PDF

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WO2018058432A1
WO2018058432A1 PCT/CN2016/100797 CN2016100797W WO2018058432A1 WO 2018058432 A1 WO2018058432 A1 WO 2018058432A1 CN 2016100797 W CN2016100797 W CN 2016100797W WO 2018058432 A1 WO2018058432 A1 WO 2018058432A1
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cells
peptide
chimeric antigen
antigen receptor
receptor molecule
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French (fr)
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李华顺
韩昆昆
薛亚男
王保垒
任宝永
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李华顺
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to the field of molecular biology, in particular to a multi-gene recombinant chimeric antigen receptor molecule and application thereof.
  • a malignant tumor is a disease that seriously threatens human life.
  • the pathogenesis of malignant tumors or cancers is diverse.
  • the common manifestation is that the mutant tumor cells are not cleared by the body's immune system, can propagate and spread without restriction, and destroy the normal cells and functions of surrounding tissues.
  • the medical profession has made a lot of efforts in trying to heal and control the course of tumor diseases, but the results have been minimal.
  • the clinical adjuvant treatment of mainstream medical circles is still radiation therapy, chemical drug therapy and antibody therapy.
  • the recognition and killing effect of immunocompetent cells on tumor cells depends on the expression of receptor molecules on the surface of tumor cell membranes. At least two factors lead to the inability of T lymphocytes in vivo to recognize cancer cells well: (1) Downregulation of antigens by cancer cells Presentation of the expression of the molecule, (2) the presented antigen is associated with the T cell receptor The force is very weak. Although there are highly specific T lymphocytes in cancer cells in cancer patients, the number of patients is too small to treat cancer.
  • the chimeric antigen receptor is mainly composed of two parts, one end is located outside the cell and can specifically recognize an antigen on the surface of the cancer cell, and the other end is located in the cell containing a signal activation element (such as a Zeta chain of T cell receptor) to transmit a signal. Activate the role of T cells.
  • a signal activation element such as a Zeta chain of T cell receptor
  • CAR-T cell therapy developed with CD19 has achieved remarkable results in the clinical treatment of both primary and secondary hematological tumors, but the progress of CAR-T cell therapy in solid tumor research is slow. There is no significant breakthrough.
  • One of the reasons is that 95% of lymphocytic leukemia cells express the B cell antigen CD19, while the specific antigen expression of other solid tumor cells is between 40-70%; therefore, in the treatment of solid tumors, a single monoclonal antibody CAR - T cells are unlikely to kill cancer cells that express other tumor-specific antigens.
  • the second reason is the rate and number of immune killer cells infiltrating into the solid tumor through the blood/lymph circulation.
  • the third reason is the negative regulation of tumor tissue cells on immunocompetent cells.
  • T-regulated cells mainly controlled by Regulatory T cells (Treg).
  • T-regulated cells When the control of T-regulated cells is weakened, it may cause hyperimmune or autoimmune diseases. When the control is enhanced, the immune function will be low. , breeding tumors or other viral skin diseases.
  • CTL4 Cytotoxic T lymphocyte-associated antigen-4
  • programmed death protein 1 programmed yellow cell-associated antigen 1 (Programmed cell death protein) 1/Programmed cell death protein 1 ligand 1, PD-1/PD-L1) and other molecules.
  • Inhibitory T-regulated cells express CTLA4, which interacts with B7-type protein subunits on the surface of immune cells DC and T cells to inhibit immune cell function.
  • B7 protein family include B7-H1 (PD-L1), B7-H2 (PD-1L2) and the like.
  • B7-H1 (PD-L1) can also further inhibit the active function of target cells by binding to lymphocyte PD-1 molecules.
  • the inhibition of T cell function by PD-1 and PD-L1 interaction is a major obstacle to the treatment of tumors by immune cells.
  • cancer cells express The increase of PD-L1/PD-1 level directly inhibits the activated lymphocytes infiltrating into cancer tissues, which is one of the mechanisms of tumor immune evasion; the expression of PD-1 on activated T cells is increased, which is easier. Under the inhibition of negative regulation.
  • the latest antibody drugs, keytruda and opdivo, have been approved by the US FDA for the clinical treatment of malignant tumors.
  • the principle of action is to block the binding of CTLA4/B7 and PD-1/PD-L1, thereby releasing T-regulated cells or cancer cells in vivo.
  • the inhibition of killer cells allows the immune-active cells in the body to kill and clear the tumor cells.
  • an object of the present invention is to provide a polygenic recombinant chimeric antigen receptor molecule having higher specificity and killing property and use thereof.
  • a first aspect of the present invention provides a multi-gene recombinant chimeric antigen receptor molecule comprising a sequence of extracellular domain peptides, a transmembrane region peptide and an intracellular domain peptide, which are linked in series a LIGHT protein peptide and an immunobinding protein peptide, wherein the LIGHT protein peptide comprises at least one extracellular domain peptide of a human LIGHT protein or a fragment thereof, the immunobinding protein peptide comprising a binding to a tumor cell surface and/or a tumor stromal cell An extracellular domain peptide of a protein or a fragment thereof of a surface immunosuppressive molecule.
  • LIGHT also known as TNFSF14 or CD258, is a member of the ligand TNF superfamily (TNFSF). Its name is derived from lymphoid toxins, exhibits inducible expression, and competes with HSV glycoprotein D for herpesvirus invasive mediators (HVEMs) (receptors expressed by T lymphocytes). LIGHT is expressed on the surface of T cells by activation in a tightly regulated manner (Castellano et al. (2002) J. Biol. Chem. 27742441-51). LIGHT regulates its biological effects by binding to one of three TNF superfamily receptors, including the lymphotoxin beta receptor (LT[beta]R) (Crowe et al. (1994) Science 264707).
  • LT[beta]R lymphotoxin beta receptor
  • LIGHT exhibits a variety of immunostimulatory activities when interacting with its receptors, including regulation of chemokine expression and cell adhesion molecules (Wang, J. et al. (2002) Eur. J. Immunol. 32: 1969- 1979).
  • LIGHT and LT ⁇ 1 ⁇ 2 act synergistically in lymphoid organogenesis and lymphoid development (Scheu, S. et al. (2002) J. Exp. Med. 195:1613-1624; Wang, J. et al. (2002) ) above). Signaling of LT ⁇ R via LIGHT transgenes is shown to be sufficient to induce chemokines and adhesion Upregulation of expression of attached molecules (Wang, J. et al. (2004) J. Clin. Invest. 113: 826-835).
  • LIGHT has also been shown to modulate T cell-initiated and swelled CD28-independent costimulatory activity, which results in enhanced T cell immunity and/or enhanced autoimmunity against tumors (Tamada, K. et al. (2000) Nat. Med. 6: 283-289; Ware, CF (2005) Annu. Rev. Immunol. 23: 787-819; Wang, J. et al. (2005) J. Immunol. 174: 8173-8182).
  • the LIGHT protein peptide has one or more LIGHT-related activities including, but not limited to: (i) with one or more LIGHT receptors (eg, lymphotoxin beta receptor (LT ⁇ R), herpes Viral invasive mediator (HVEM) and/or anti-trapping receptor 3 (DcR3) binding; (ii) induction of chemokines or cytokines (eg CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 And/or TNF), expression of one or more of chemokines or cytokine receptors (eg IL-10RA), adhesion molecules and/or costimulatory molecules; (iii) activated T cells, such as lymphocytes (eg, cytotoxic T lymphocytes), T cells expressing CD4 or CD8 and/or regulatory T cells; (iv) cells or tissues that recruit T cells into hyperproliferation (eg, tumors); (v) activation and/or enhancement Proliferation of tumor-reactive T cells; (
  • sequence of the LIGHT protein peptide is as shown in SEQ ID NO: 1.
  • the LIGHT protein peptide and the immunobinding protein peptide can be linked with or without a linker peptide.
  • the sequence of the linker peptide is shown in SEQ ID NO: 2.
  • the immuno-binding protein peptide segment comprises an extracellular domain peptide of PD-1 or HAC.
  • the extracellular region peptide sequence of PD-1 is represented by SEQ ID NO: 3.
  • the mutant HAC of PD-1 has higher affinity with the tumor surface PDL-1 molecule; the extracellular domain peptide of the HAC The sequence is shown in SEQ ID NO: 13.
  • the multi-gene recombinant chimeric antigen receptor molecule further comprises a signal peptide.
  • the signal peptide can enhance the secretion of the fusion protein of the multi-gene recombinant chimeric antigen receptor molecule, and after the signal peptide is expressed together with other amino acid sequences of the fusion protein, it is finally cleaved by the protease.
  • the protease has a certain recognition sequence, and the signal peptide is fused with the peptide after it to form a new amino acid sequence, so if you choose Improper signal peptides may cause mis-cleavage of the protease and inactivation of the protein.
  • the signal peptide may be selected from a signal peptide of an immunoglobulin light chain or a signal peptide secreted by a PD-1 protein, the sequences of which are shown in SEQ ID NOS: 4 and 5, respectively.
  • the transmembrane region peptide is a region spanning a cell membrane in a protein sequence, usually an ⁇ -helical structure, about 20-25 amino acid residues, and the amino acid is mostly a hydrophobic amino acid; wherein
  • the transmembrane region peptide includes, but is not limited to, a CD8 transmembrane region peptide or a PD-1 transmembrane region peptide, and when the transmembrane region peptide is a CD8 transmembrane region peptide, the extracellular region peptide and CD8 cross
  • the membrane region peptides are also linked by the hinge region peptide of CD8.
  • the sequence of the CD8 transmembrane region peptide is as shown in SEQ ID NO: 6, the sequence of the hinge region of CD8 is shown in SEQ ID NO: 7, and the sequence of the PD-1 transmembrane region is SEQ ID NO: 8. Shown.
  • the intracellular domain peptide is a costimulatory signal molecule selected from one or more of the intracellular domain peptides of 4-1BB (also known as CD137), CD28, and CD3 ⁇ .
  • the intracellular domain peptide is selected from the group consisting of interconnected 4-1BB and CD3 sputum intracellular domain peptides, the sequences of which are set forth in SEQ ID NOs: 9 and 10, respectively.
  • the multi-gene recombinant chimeric antigen receptor molecule comprises the sequence set forth in SEQ ID NO: 3.
  • the multi-gene recombinant chimeric antigen receptor molecule of the present invention comprises a signal peptide of an immunoglobulin light chain, a LIGHT protein peptide, a linker peptide, an extracellular domain peptide of PD-1, and a hinge region peptide of CD8.
  • the transmembrane region peptide segment of CD8, the 4-1BB intracellular domain peptide segment and the CD3 intracellular domain peptide segment are sequentially joined, and the sequence thereof is shown in SEQ ID NO:11.
  • a second aspect of the invention provides a nucleotide encoding a chimeric antigen receptor molecule provided by the first aspect of the invention.
  • a third aspect of the invention provides a recombinant vector comprising the nucleotide provided by the second aspect of the invention.
  • the vector is a lentiviral vector, which can efficiently integrate a foreign gene or an exogenous shRNA into a host chromosome, thereby achieving the effect of persistently expressing the sequence of interest. Effectively infects neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stems in terms of infectious ability Cells and other types of cells, in order to achieve good gene therapy results.
  • the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or the target shRNA, and the target gene or the target shRNA is integrated into the host cell. The probability of genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be realized conveniently and quickly.
  • the lentiviral vector used in the present invention should include, but is not limited to, a pRRSLIN lentiviral expression vector and a pLVX vector, preferably a pRRSLIN lentiviral expression vector.
  • a fourth aspect of the invention provides a recombinant cell comprising the recombinant vector provided by the third aspect of the invention.
  • the recombinant cell is preferably a T cell or an NK cell.
  • the gene encoding the chimeric antigen receptor can be transferred to T cells or NK cells by the aforementioned vector, used to modify T cells or NK cells to become CAR-T or CAR-NK cells; and the chimeric antigen receptor is modified by the chimeric antigen receptor T cells or NK cells can kill tumor cells by identifying tissue factors on the surface of tumor cells for tumor treatment.
  • tumor stromal cells refers to cells in the tumor microenvironment that support the malignant proliferation, anti-apoptosis, invasion, metastasis, escape immune surveillance and other life activities of tumor cells, including fibroblasts and tumor-associated macrophages (TAMs). ), regulatory T cells (Treg), undifferentiated bone marrow cells, endothelial cells, pericytes and platelets, endothelial cells, and the like.
  • immunosuppressive molecule refers to a molecule produced by a tumor cell or a tumor stromal cell, which acts as an immunosuppressive agent, allows tumor cells to escape from the immune surveillance, and induces immune tolerance.
  • co-stimulating molecule refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., which activate immune cells by binding to their ligands.
  • the second signal enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
  • extracellular domain refers to a segment of the membrane protein that is located outside the cell.
  • domain refers to a region of a protein biomacromolecule having a specific structure and an independent function.
  • the number of amino acid residues in a common domain is between 100 and 400, and the smallest domain is only 40 to 50 amino acid residues. Large domains can exceed 400 amino acid residues.
  • single-chain antibody variable region fragment means The antibody VL region amino acid sequence and the VH region amino acid sequence are linked by Linker, and have an antibody fragment capable of binding antigen.
  • PD-1 refers to programmed cell death protein 1 and the gene name PDCD1_HUMAN.
  • the corresponding protein sequence number is UniProtKB-Q15116, which is a T cell immunosuppressive molecule whose extracellular domain resembles an immunoglobulin.
  • the variable region (V-section) of the protein has the property of specifically binding to its ligands PD-L1 and PD-L2 (Programmed cell death protein 1 ligand 1/2).
  • PD-1 is normally expressed in activated T lymphocytes and is also expressed in a variety of malignant cells.
  • P-L1 and P-L2 refer to the currently discovered human programmed death factor 1 ligand 1/2. Its extracellular domain domain has immunoglobulin-like V and C1 regions, which are combined with the V region of PD-1 via the V region (4zqk Structure 232341-2348, 2015). It is usually expressed in a small amount in dendritic cells DC, T regulatory cells and Th cells, macrophages, Mast cells and bone marrow, and is also expressed in various malignant cells.
  • the present invention has at least the following advantages: immunological checkpoint therapy such as PD-1 antibody is not effective for a large number of tumor patients, and how to increase the objective response rate becomes an urgent challenge.
  • immunological checkpoint therapy such as PD-1 antibody is not effective for a large number of tumor patients, and how to increase the objective response rate becomes an urgent challenge.
  • Studies have shown that there is sufficient T cell infiltration in tumor tissue, which is a prerequisite for the blocking of PD-L1 and the onset of PD-1 antibody.
  • Targeting the tumor necrosis factor superfamily member LIGHT molecule is a tumor-activated lymphotoxin B-receptor signal that results in the production of a large number of chemokines to recruit T cells.
  • This patent combines the characteristics of LIGHT molecules with the killing properties of CAR-T cells to simultaneously introduce LIGHT and PD-1 molecules into the extracellular segment to prepare LIGHT-PD1 CAR-T cells with higher specificity and lethality to treat entities. Tumor.
  • Figure 1 is a schematic diagram showing the construction of a lentiviral expression vector
  • Figure 2 is a flow chart showing the results of LIGHT-PD-1 CAT-T cell infection for 7 days in the present invention
  • Figure 3 is a graph showing the killing results of LIGHT-PD-1 CAT-T cells against different target cells in the present invention
  • Figure 4 is a graph showing the results of in vitro proliferation of LIGHT-PD-1 CAT-T cells against different target cells in the present invention
  • Figure 5 is a graph showing the results of detection of cytokines of different kinds of target cells by LIGHT-PD-1 CAT-T cells in the present invention.
  • Figure 6 is a graph showing the results of detection of CD3/CD8 of MCF7/MCF7-PDL1/HeLa/SMC7721 cell lines by CAR-LIGHT CAT-T cells in the present invention
  • Fig. 7 is a graph showing the results of the killing effect of PD-1 CAR-T and LIGHT-PD1 CAR-T in the present invention.
  • the invention provides a preparation method of a lentiviral expression vector for expressing a chimeric antigen receptor molecule, comprising the following steps:
  • the LIGHT gene fragment is synthesized according to the LIGHT molecular sequence information reported in the article "Facilitating T Cell Infiltration in Tumor Microenvironment Overcomes Resistance".
  • the known human PD-1 sequence, CD8 transmembrane region gene sequence, human 4-1BB intracellular region gene sequence and CD3 intracellular region gene sequence were searched from the GenBank database.
  • LIGHT-PD1-CAR LIGHT-PD1-CD8-4-1BB-CD3 ⁇
  • the gene sequence of LIGHT-PD1-CD8-4-1BB-CD3 ⁇ is ligated into the pRRSLIN vector by restriction enzyme digestion, and the upstream of the gene is the EP-1 ⁇ promoter.
  • the vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, and clones were identified by restriction enzyme digestion.
  • the vector was successfully constructed by sequencing to obtain pRRSLIN-LIGHT-
  • the PD1 lentiviral expression vector and the lentiviral expression vector are constructed as shown in Figure 1.
  • the present invention provides a method for producing a lentivirus by expressing a lentiviral expression vector in Example 1, comprising the following step:
  • 293T cells were seeded into a 15 cm culture dish at approximately 8 x 10 6 per dish. Ensure that the cells are confluent at 80% and evenly distributed in the culture dish during transfection.
  • Solution B A mixture of the following plasmids was added: 112.5 ⁇ g pRRSLIN-LIGHT-PD1 (target plasmid); 39.5 ⁇ g pMD2.G (VSV-G envelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ L 2M Calcium ion solution. Total volume of solution B: 6.25 mL.
  • centrifuge at 25,000 rpm (82,700g), temperature 4 ° C for 2h; carefully remove the centrifuge tube, pour off the supernatant, invert the centrifuge tube to remove residual liquid; add 100 ⁇ L PBS, seal the centrifuge tube, and place at 4 ° C for 2 h, every 20 min The mixture was gently vortexed, centrifuged at 500 g for 1 min (25 ° C), and the virus supernatant was collected; after cooling on ice, it was stored at -80 ° C.
  • the present invention provides a method for preparing CAR-T cells by lentiviral infection of cells in Example 2, comprising the steps of:
  • S1 take 0.5mL blood for rapid detection of pathogenic microorganisms, exclude microbial infections such as HBV, HCV, HDV and HEV, HIV-1/2, Treponema pallidum and parasites; under sterile conditions, use blood collection of heparin bottle 50mL (heparin antibiotic) Condensed), immediately (4 ° C, within 24 hours) to the cell preparation laboratory to ensure that this process is free of pathogenic microorganisms. After obtaining the patient's blood, in the GMP preparation room, the surface of the heparin bottle is wiped with an alcohol cotton ball for disinfection and then placed in a biological safety cabinet.
  • heparin bottle 50mL (heparin antibiotic) Condensed immediately (4 ° C, within 24 hours
  • PBMC peripheral blood mononuclear cells
  • V-VIVO15 added autologous AB (FBS) concentration of 5%, interleukin-2 (IL-2) concentration of 40 ng / mL, and the isolated PBMC was diluted to 2 ⁇ with the culture medium 10 6 /mL, 50 ⁇ L flow detection of the purity of T cells in PBMC.
  • FBS autologous AB
  • IL-2 interleukin-2
  • the CAR-T cells prepared in Example 3 were subjected to flow analysis, and the specific steps were as follows:
  • FIG. 2 The effect of CAR-T cell flow dip staining is shown in Figure 2.
  • the ordinate is the flow SSC-H lateral scatter signal
  • the abscissa is the FITC fluorescence signal.
  • a and B are control groups, which are T cells that do not infect the virus; FITC-conjugated antibodies for detecting CAR molecules do not detect CAR molecule expression;
  • C and D maps are transfected with PRRSLIN-LIGHT-PD1 lentivirus The T cells were flow-detected and compared with the A and B images, and the cells were successfully transfected. After the virus infects the T cells, the flow detection efficiency can reach 47%, indicating the successful preparation of LIGHT-PD1 CAR-T. cell.
  • LDH release assay was used to detect the killing effect of LIGHT-PD1 CAR-T cells on engineered cell lines MCF-7/PDL1 and PDL1 high expressing cells. LDH release was detected by ELISA, including the following steps:
  • the target cells were adjusted to 5 ⁇ 10 4 /mL with RPMI-1640 medium containing 5% calf serum.
  • target cells were added to a 96-well cell culture plate, and 100 ⁇ L per well was added. Three wells were used as effector cells (LIGHT-PD1 CAR-T cells) to naturally release control wells, and no target cells were added, and only 100 ⁇ L of the culture solution was added.
  • effector cells 100 ⁇ L were added to each well, and the ratio of effector cells to target cells was 10:1; 5:1; 1:1.
  • Natural release wells were added with effector cells plus only 100 ⁇ L of culture medium, and effector cells were incubated with target cells for 6 hours. Three replicate wells were placed for each experiment.
  • the optical density (OD value) of each well was measured on an enzyme-linked detector, and the detection wavelength was 490 nm or 492 nm, and the measurement was completed within 1 hour.
  • Killing rate experimental group LDH (OD) / maximum LDH release group (OD).
  • the cytokine secretion was measured by CBA kit, and the proliferation of each group of CAR-T cells was calculated, and the ratio of CD8-positive T cells in the proliferating T cells was confirmed by staining with CD3 and CD8 antibodies.
  • the abscissa indicates the different target ratio of CAR-T cells to tumor cells, and the ordinate indicates the killing efficiency.
  • Different types of histograms indicate different tumor cells, for MCF-7, MCF-1/ LIGHT-PD1 CAR-T can significantly kill tumor cells under different target-to-target ratios of PDL-1, SMCC7721 and Hela.
  • the SMCC7721 and Hela cells are most obvious; correspondingly, as shown in Fig. 4.
  • the abscissa indicates the ratio of T cells to tumor or CAR-T cells to tumors.
  • the ordinate indicates the number of cells, T stands for T cells, and LIG stands for LIGHT-PD1 CAR-T cells. Compared with normal T cells, CAR-T cells can undergo specific activation and proliferation after exposure to tumor cells, and the high-efficiency target is more significant than in the case.
  • the abscissa indicates the different target ratio of CAR-T cells to tumor cells
  • the ordinate indicates the cytokine content
  • the cytokines in the culture supernatant were detected
  • the CAR-T killing experimental group IL-2 was found.
  • the secretion of Figure 5A) and TNF-[alpha] (Figure 5B) was significantly elevated.
  • the CD3/CD8 flow antibody was used to detect the ratio of CART to CD8T cells after killing MCF7/MCF7-PDL1/HeLa/SMC7721 cells.
  • the ordinate PE signal indicates the specific detection of CD3 molecule expression
  • the abscissa FITC signal indicates detection of CD8 molecule expression, detection by flow cytometry, LIGHT-PD1 CAR-T cell activation After that, mainly CD8T cells undergo specific proliferation.
  • CAR-PD-1 indicates that the extracellular segment of the expressed CAR molecule is a common PD-1 molecule CAR-T cell
  • CAR-LIGHT-PD-1 indicates the LIGHT-PD1 CAR-T constructed by the present invention.
  • the results showed that the LIGHT-PD1 CAR-T cells had better killing effect on SMC7721 tumor cells than PD-1 CAR-T.
  • LIGHT-PD1-CART cells can specifically activate and proliferate after exposure to tumor cells, release cytokines and kill tumor cells, and CD8-positive T cells play a major role.
  • the killing effect of PD-1 CAR-T and LIGHT-PD1CAR-T on tumors was compared.
  • the results showed that LIGHT-PD1 CAR-T was significantly better than PD-1 CAR-T.

Abstract

一种多基因重组嵌合抗原受体分子，所述多基因重组嵌合抗原受体分子包括依次串联的胞外区肽段、跨膜区肽段和胞内结构域肽段，胞外区肽段包括相连的LIGHT蛋白肽段和免疫结合蛋白肽段，其中，LIGHT蛋白肽段包括人LIGHT蛋白质或其片段的至少一个胞外区肽段，免疫结合蛋白肽段包括结合肿瘤细胞表面和/或肿瘤基质细胞表面免疫抑制分子的蛋白质或其片段的胞外区肽段；同时还提供该多基因重组嵌合抗原受体分子的应用，包括编码核苷酸、重组载体和重组细胞；所述多基因重组嵌合抗原受体分子的特异性和杀伤性更高。

Description

一种多基因重组嵌合抗原受体分子及其应用
本申请要求了申请日为2016年9月28日，申请号为201610855799.1，发明名称为“一种多基因重组嵌合抗原受体分子及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及分子生物学领域，尤其涉及一种多基因重组嵌合抗原受体分子及其应用。
背景技术
恶性肿瘤是一种严重威胁人类生命的疾病。恶性肿瘤或癌症的发病机理多种多样，其共同的表现在于变异的肿瘤细胞不受机体免疫***的清除，能够无限制的繁殖和扩散，破坏周围组织的正常细胞和功能。长期以来，医药学界在尝试医治和控制肿瘤疾病的病程方面做了大量的努力，但收效甚微。目前，在恶性肿瘤的治疗上，除根除手术外，主流医学界的临床辅助治疗手段仍然是放射线治疗、化学药物治疗和抗体治疗。
1985年，美国科学家尝试分离病人的单个核细胞，并在体外用各种细胞因子诱导、激活、产生杀伤性T细胞(Cytokine-induced killer cells，CIK)，继而发现这些细胞通过静脉滴注回输给病人后，对肿瘤的生长有杀伤作用。经过近三十年的临床应用和技术发展，用免疫杀伤细胞***已经成为第四种公认的恶性肿瘤辅助治疗手段。常见的用于临床治疗的免疫杀伤细胞：自然杀伤细胞(Natural killer cells，NK)、γδT细胞、细胞毒性T淋巴细胞(Cytotoxic T lymphocytes，CTL)、NK样T淋巴细胞(Natural killer-like T cells，NKT)、Th1效应细胞(Effector cells)等。抗原提呈细胞之一树突状细胞(Dentritic cells，DC)也常用于免疫细胞治疗的应用，DC可以提高免疫杀伤细胞的特异性、加强免疫杀伤细胞的活力。
免疫活性细胞对肿瘤细胞的识别和杀伤效果，取决于肿瘤细胞膜表面的受体分子表达，至少有两方面的因素导致体内的T淋巴细胞不能很好地识别癌细胞：(1)癌细胞下调抗原呈递分子的表达，(2)被呈递的抗原与T细胞受体亲和 力很弱。虽然癌症患者体内存在癌细胞高度特异性的T淋巴细胞，但是数量太少起不到治疗癌症的作用。为了克服免疫杀伤细胞特异性低下的缺点，美国科学家发明一种转基因的方法：将识别肿瘤表面特异性抗原的单克隆抗体高变区序列在体外重组、亚克隆为单链抗体片段(Single chain antibody fragment of variable regions，scFv)，再与其他基因的跨膜蛋白片段和胞内信号肽融合形成人造的嵌合抗原受体(Chimeric antigen receptor，CAR)，转染至T细胞内而形成嵌合抗原受体T细胞(Chimeric antigen receptor，CAR-T)。嵌合抗原受体主要由两部分构成，一端位于细胞外能够特异性识别癌细胞表面的某一抗原，另一端位于胞内含有信号激活元件(如T细胞受体的Zeta链)，起传递信号激活T细胞的作用。
目前以CD19为靶点开发的CAR-T细胞疗法，在一期和二期血液***肿瘤临床治疗上都取得了明显的效果，但是CAR-T细胞疗法在实体瘤的研究中的进展缓慢，目前还没有显著性的突破。原因之一是淋巴性白血病癌细胞中95％都表达B细胞抗原CD19，而其他实体瘤细胞的特异性抗原表达在40-70％之间；所以在实体瘤的治疗中，单一的单抗CAR-T细胞不可能杀伤表达其他肿瘤特异性抗原的癌细胞。原因之二是免疫杀伤细胞通过血液/淋巴液循环浸润到实体肿瘤的速度和数目。原因之三是肿瘤组织细胞对免疫活性细胞的负调节作用。
在正常情况下，人体的血液循环中存在少量的活性杀伤细胞，如NK，γδT细胞等，其作用在于清除衰老、变异的组织细胞或抵御病毒入侵。免疫细胞的活性主要受到T调节细胞(Regulatory T cells，Treg)的控制，当T调节细胞的控制减弱时，会造成免疫功能亢进或产生自身免疫性疾病；当控制增强时，会使免疫功能低下，滋生肿瘤或其他病毒性皮肤病。目前确认的参与负调节的蛋白因子有细胞毒T淋巴细胞相关抗原4(Cytotoxic T lymphocyte-associated antigen-4,CTLA4)和程序性死亡蛋白1/程序性死亡蛋白1配基1(Programmed cell death protein 1/Programmed cell death protein 1 ligand 1，PD-1/PD-L1)等分子。抑制性T调节细胞表达CTLA4，可以与免疫细胞DC和T细胞表面的B7族蛋白亚基相互作用，抑制免疫细胞功能。B7蛋白家族成员有B7-H1(PD-L1)，B7-H2(PD-1L2)等。B7-H1(PD-L1)也可以通过结合淋巴细胞PD-1分子，进一步抑制靶细胞的活性功能。PD-1和PD-L1相互作用对T细胞功能的抑制是免疫细胞***的一个主要障碍。在大多数实体瘤组织中，癌细胞表达 PD-L1/PD-1的水平增高，对浸润到癌组织的活化淋巴细胞产生直接的抑制，这也是肿瘤逃逸机体免疫监管的机制之一；活化T细胞表面PD-1的表达增加，更容易受到负调节的抑制。最新的抗体药物keytruda和opdivo已被美国FDA批准临床治疗恶性肿瘤，其作用原理是阻断CTLA4/B7以及PD-1/PD-L1的相互结合，从而解除体内T调节细胞或癌细胞对活性T杀伤细胞的抑制，让体内免疫活性细胞对肿瘤细胞进行杀伤、清除。
发明内容
为解决上述技术问题，本发明的目的是提供一种特异性和杀伤性更高的多基因重组嵌合抗原受体分子及其应用。
本发明第一方面提供一种多基因重组嵌合抗原受体分子，包括依次串联的胞外区肽段、跨膜区肽段和胞内结构域肽段，所述胞外区肽段包括相连的LIGHT蛋白肽段和免疫结合蛋白肽段，其中，LIGHT蛋白肽段包括人LIGHT蛋白质或其片段的至少一个胞外区肽段，免疫结合蛋白肽段包括结合肿瘤细胞表面和/或肿瘤基质细胞表面免疫抑制分子的蛋白质或其片段的胞外区肽段。
LIGHT，也称为TNFSF14或CD258，为配体TNF超家族(TNFSF)的成员。它的名字衍生自类淋巴毒素，表现为可诱导的表达，并且与HSV糖蛋白D竞争疱疹病毒侵入介质(HVEM)(T淋巴细胞所表达的受体)。LIGHT通过活化以紧密调节的方式在T细胞的表面上表达(Castellano et al.(2002)J.Biol.Chem.27742841-51)。LIGHT通过结合3种TNF超家族受体中的一者来调控其生物学作用，所述的3种TNF超家族受体包括淋巴毒素β受体(LTβR)(Crowe et al.(1994)Science 264707-10，Browning et al.(1997)JImmunol 159：3288-98)、疱疹病毒侵入介质(HVEM)(Montgomery et al.(1996)Cell 87(3)：427-36)以及抗诱捕受体3(DeR3)(Yu et al.(1999)J.Biol.Chem.274 13733-6)。LIGHT在与其受体相互作用时，表现出多种免疫刺激活性，包括调节趋化因子的表达以及细胞粘附分子(Wang，J.et al.(2002)Eur.J.Immunol.32：1969-1979)。例如，LIGHT和LTα1β2在淋巴的器官形成以及***构的发育中协同作用(Scheu，S.et al.(2002)J.Exp.Med.195：1613-1624；Wang，J.et al.(2002)上文)。LTβR通过LIGHT转基因的信号传递显示出足以诱导对趋化因子和粘 附分子的表达的上调节(Wang，J.et al.(2004)J.Clin.Invest.113：826-835)。此外，LIGHT还显示出调控T细胞引发和膨胀的CD28非依赖性共刺激活性，这可以得到对抗肿瘤的增强的T细胞免疫和/或增强的自身免疫(Tamada，K.et al.(2000)Nat.Med.6：283-289；Ware，C.F.(2005)Annu.Rev.Immunol.23：787-819；Wang，J.et al.(2005)J.Immunol.174：8173-8182)。
进一步的，所述的LIGHT蛋白肽段具有一种或多种LIGHT相关的活性，包括但不限于：(i)与一种或多种LIGHT受体(例如淋巴毒素β受体(LTβR)、疱疹病毒侵入介质(HVEM)和/或抗诱捕受体3(DcR3))结合；(ii)诱导趋化因子或细胞因子(例如CXCL10(IP-10)，CCL21，CXCL9，IL-5，IL-8和/或TNF)、趋化因子或细胞因子受体(例如IL-10RA)、粘附分子和/或共刺激分子中的一种或多种的表达；(iii)活化T细胞，例如淋巴细胞(例如细胞毒素T淋巴细胞)、表达CD4或CD8的T细胞和/或调节T细胞；(iv)募集T细胞进入到过度增殖(例如肿瘤)的细胞或组织；(v)活化和/或增强肿瘤反应性T细胞的增殖；(vi)例如，在过度增殖(例如肿瘤细胞或组织)下，创建类淋巴微环境；(vii)诱导过度增殖(例如肿瘤)细胞或组织的凋亡；和/或(viii)刺激受试对象中的免疫应答，例如刺激受试对象对抗过度增殖(例如肿瘤或癌症)的细胞或组织的免疫***。
进一步的，所述LIGHT蛋白肽段的序列如SEQ ID NO：1所示。
进一步的，所述LIGHT蛋白肽段和免疫结合蛋白肽段可以使用或不使用连接肽相连。具体的，所述连接肽的序列如SEQ ID NO：2所示。
进一步的，所述免疫结合蛋白肽段包括PD-1或HAC的胞外区肽段。具体的，所述PD-1的胞外区肽段序列如SEQ ID NO：3所示。据《Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging》文章报道，PD-1的突变体HAC具有与肿瘤表面PDL-1分子更高亲和力；所述HAC的胞外区肽段序列如SEQ ID NO：13所示。
进一步的，所述多基因重组嵌合抗原受体分子还包括信号肽。信号肽可以提高多基因重组嵌合抗原受体分子这种融合蛋白分泌的效果，在信号肽与融合蛋白其它氨基酸序列一起被表达后，最终被蛋白酶切除。蛋白酶具有一定的识别序列，而信号肽与其后面的肽段融合后构成新的氨基酸序列，所以如果选择 的信号肽不当，可能会导致蛋白酶的误切，蛋白失活。信号肽可选自免疫球蛋白轻链的信号肽、或是PD-1蛋白分泌的信号肽，其序列分别如SEQ ID NO：4和5所示。
应当说明的是，所述跨膜区肽段为蛋白质序列中跨越细胞膜的区域，通常为α-螺旋结构，约20～25个氨基酸残基，其氨基酸大部分是疏水性氨基酸；其中，所述跨膜区肽段包括但不限于CD8跨膜区肽段或PD-1跨膜区肽段，当跨膜区肽段为CD8跨膜区肽段时，所述胞外区肽段与CD8跨膜区肽段还通过CD8的hinge区肽段相连。CD8跨膜区肽段的序列如SEQ ID NO：6所示，CD8的hinge区肽段的序列如SEQ ID NO：7所示，PD-1跨膜区肽段的序列如SEQ ID NO：8所示。
进一步的，所述胞内结构域肽段为共刺激信号分子，选自4-1BB(又称CD137)、CD28、CD3ζ的胞内结构域肽段中的一种或多种。优选的，所述胞内结构域肽段选自相互连接的4-1BB和CD3ζ胞内结构域肽段，其序列分别如SEQ ID NO：9和10所示。
进一步的，所述多基因重组嵌合抗原受体分子包含如SEQ ID NO：3所示的序列。
优选的，本发明的多基因重组嵌合抗原受体分子，由免疫球蛋白轻链的信号肽、LIGHT蛋白肽段、连接肽、PD-1的胞外区肽段、CD8的hinge区肽段、CD8的跨膜区肽段、4-1BB胞内结构域肽段和CD3ζ胞内结构域肽段依次连接而成，其序列如SEQ ID NO：11所示。
本发明第二方面提供一种核苷酸，其编码本发明第一方面提供的嵌合抗原受体分子。
进一步的，包含或为如SEQ ID NO：12所示的核苷酸序列。
本发明第三方面提供一种重组载体，其包含本发明第二方面提供的核苷酸。
进一步的，所述载体为慢病毒载体，其可以将外源基因或外源的shRNA有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干 细胞等多种类型的细胞，从而达到良好的的基因治疗效果。对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，使用慢病毒载体，能大大提高目的基因或目的shRNA的转导效率，且目的基因或目的shRNA整合到宿主细胞基因组的几率大大增加，能够比较方便快捷地实现目的基因或目的shRNA的长期、稳定表达。
应当说明的是，本发明中所用的慢病毒载体，应当包括但不限于pRRSLIN慢病毒表达载体和pLVX载体，优选为pRRSLIN慢病毒表达载体。
本发明第四方面提供一种重组细胞，其包含本发明第三方面提供的重组载体。所述重组细胞优选为T细胞或NK细胞。该嵌合抗原受体的编码基因能够通过前述载体被转移至T细胞或NK细胞内，用于修饰T细胞或NK细胞，成为CAR-T或CAR-NK细胞；利用该嵌合抗原受体修饰的T细胞或NK细胞，能够通过识别肿瘤细胞表面的组织因子，杀死肿瘤细胞，进行肿瘤治疗。
术语“肿瘤基质细胞”是指肿瘤微环境内一些支撑肿瘤细胞的恶性增殖、抗凋亡、侵袭、转移，逃脱免疫监控等生命活动的细胞，主要包括成纤维细胞、肿瘤相关巨噬细胞(TAMs)、调节性T细胞(Treg)、未分化的骨髓细胞、内皮细胞、周细胞和血小板、内皮细胞等。
术语“免疫抑制分子”是指由肿瘤细胞或肿瘤基质细胞产生，起抑制免疫作用，使肿瘤细胞逃脱机体免疫监控，诱导免疫耐受的分子。
术语“共刺激信号分子”(Co-stimulating molecule)是指免疫细胞表面的一些粘附分子，如CD28、CD134/OX40、CD137/4-1BB、CD40等，通过与其配体结合，激活免疫细胞的第二信号，增强免疫细胞的增殖能力及细胞因子的分泌功能，延长活化免疫细胞的存活时间。
术语“胞外区”是指膜蛋白位于细胞外的区段。
术语“结构域”是指蛋白质生物大分子中具有特异结构和独立功能的区域，常见结构域的氨基酸残基数在100～400个之间，最小的结构域只有40～50个氨基酸残基，大的结构域可超过400个氨基酸残基。
术语“单链抗体”(single-chain antibody variable region fragment,scFv)是指 由抗体VL区氨基酸序列和VH区氨基酸序列经Linker连接而成，具有结合抗原能力的抗体片段。
术语“PD-1”是指人类程序性死亡因子1(programmed cell death protein1)，基因名称PDCD1_HUMAN，对应蛋白序列编号有UniProtKB-Q15116，是T细胞免疫抑制分子，其胞外区结构域类似免疫球蛋白的可变区(V-section)，具有特异性结合其配体PD-L1和PD-L2(Programmed cell death protein 1 ligand 1/2)的特性。PD-1通常在活化的T淋巴细胞中表达，在多种恶性肿瘤细胞中也有表达。
术语“PD-L1”、“PD-L2”是指目前发现的人类程序性死亡因子1配基1和配基2(programmed cell death protein 1 ligand 1/2)。其胞外区结构域具有类似免疫球蛋白的V和C1区，通过V区与PD-1的V区相结合(4zqk Structure 232341-2348，2015)。通常在树突状细胞DC、T调节细胞和Th细胞、巨噬细胞、Mast细胞和骨髓中少量表达，在多种恶性肿瘤细胞中也有表达。
借由上述方案，本发明至少具有以下优点：免疫检查点疗法如PD-1抗体并不是对广大肿瘤患者均有效，如何增加客观缓解率成为一个紧迫的挑战。研究证明有足够的T细胞浸润在肿瘤组织，是PD-L1的封闭且PD-1抗体起效的先决条件。靶向肿瘤坏死因子超家族成员LIGHT分子是肿瘤激活淋巴毒素B-受体信号，它导致生产数量庞大的趋化因子以招募T细胞。本专利将结合LIGHT分子的特点和CAR-T细胞的杀伤特性，将LIGHT和PD-1分子同时引入胞外段，制备特异性和杀伤性更高的LIGHT-PD1 CAR-T细胞，以治疗实体肿瘤。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是慢病毒表达载体构建示意图；
图2是本发明中LIGHT-PD-1 CAT-T细胞侵染7天的流式结果图；
图3是本发明中LIGHT-PD-1 CAT-T细胞对不同种靶细胞的杀伤结果图；
图4是本发明中LIGHT-PD-1 CAT-T细胞对不同种靶细胞的体外增殖的检测结果图；
图5是本发明中LIGHT-PD-1 CAT-T细胞对不同种靶细胞的细胞因子的检测结果图；
图6是本发明中CAR-LIGHT CAT-T细胞对MCF7/MCF7-PDL1/HeLa/SMC7721 4种细胞株CD3/CD8的检测结果图；
图7是本发明中PD-1 CAR-T和LIGHT-PD1 CAR-T的杀伤效果结果对比图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1慢病毒表达载体制备
本发明提供一种用于表达嵌合抗原受体分子的慢病毒表达载体的制备方法，包括以下步骤：
S1、根据《Facilitating T Cell Infiltration in Tumor Microenvironment Overcomes Resistance》文章报道的LIGHT分子序列信息，合成LIGHT基因片段。，从GenBank数据库中搜寻已知的人PD-1序列、CD8跨膜区基因序列、人4-1BB胞内区基因序列和CD3ζ胞内区基因序列。
S2、将上述基因序列依次按人LIGHT基因、PD-1基因、CD8膜区基因、人4-1BB胞内区基因和CD3ζ胞内区基因进行连接，在各序列连接处引入不同的酶切位点，形成完整的LIGHT-PD1-CD8-4-1BB-CD3ζ(简称LIGHT-PD1-CAR)基因序列信息，
S3、将LIGHT-PD1-CD8-4-1BB-CD3ζ的基因序列通过酶切转化连接到pRRSLIN载体中，基因上游为EP-1α启动子。将载体转化到Stbl3大肠杆菌菌株后，转种到含有氨苄青霉素的固体培养基中进行繁殖，筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，通过测序确认载体构建成功，获得pRRSLIN-LIGHT-PD1慢病毒表达载体，慢病毒表达载体构建示意图如图1所示。
实施例2慢病毒制备
本发明提供实施例1中慢病毒表达载体表达制备慢病毒的方法，包括以下 步骤：
S1、转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
S2、准备溶液A和溶液B
溶液A：6.25mL 2×HEPES buffer缓冲液,.
溶液B：分以加入以下质粒的混合物：112.5μg pRRSLIN-LIGHT-PD1(target plasmid)；39.5μg pMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μL2M钙离子溶液。溶液B总体积：6.25mL。
S3、充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液B，静置2-3分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，在转速500g，温度25℃下离心10min，使用PES膜(0.45μm)过滤；以70％乙醇消毒离心管(贝克曼库尔特ultra-clearSW28centrifugetubes)，并置于紫外灯下消毒30min；将已过滤的含慢病毒的上清液转移至离心管中，在离心管底部小心铺上一层20％蔗糖(每8mL上清液加1mL蔗糖)，以PBS平衡离心管，在转速25,000rpm(82,700g)，温度4℃下离心2h；小心取出离心管，倒掉上清液，倒置离心管去掉残余液体；加入100μLPBS，密封离心管，在4℃放置2h，每20min轻轻涡旋一次，500g离心1min(25℃)，收集病毒上清；冰上冷却后，置于-80℃保存。
实施例3 CAR-T细胞制备
本发明提供实施例2中慢病毒侵染细胞制备CAR-T细胞的方法，包括以下步骤：
S1、取0.5mL血进行快速的病原微生物检测，排除HBV、HCV、HDV和HEV、HIV-1/2、***及寄生虫等微生物感染；无菌条件下，用肝素瓶采血50mL(肝素抗凝)，立即(4℃，24小时内)送至细胞制备实验室，保证此过程无病原微生物污染。得到患者血液后，在GMP制备室，用酒精棉球擦拭肝素瓶表面进行消毒后放入生物安全柜。
S2、预先打开2个50mL离心管，将血液转入两个50mL离心管中，旋紧； 将上述装好血液的两个50mL离心管放入离心机离心，400g(2000rpm)离心10min，室温离心后收集上层血浆，留下沉淀层；收集的自体血浆经56℃，30min灭活，4℃放置15min后，900g，离心30min(4℃)，取上清备用。
S3、将上述富集的血细胞用生理盐水稀释至30mL/管，打开2个新的50mL离心管，每个离心管分别加入15mL人淋巴细胞分离液，用移液管把稀释后的血细胞液缓缓加入到盛有人淋巴分离液的离心管中，旋紧。注意血液要加到淋巴分离液的上层，勿打破人淋巴分离液的界面。将加好的血细胞液放入离心机，调至最小的升降速率，400g(2000rpm)离心20min(常温)。收集两管的中层白细胞层于一支15mL无菌离心管中，加入5mL生理盐水，洗两次(400g，离心10min)，得外周血单核细胞(PBMC)。
S4、配置完全生长培养基，V-VIVO15添加自体AB(FBS)浓度为5％，白细胞介素-2(IL-2)浓度为40ng/mL，将分离得到的PBMC用培养基稀释成2×106/mL，取50μL流式检测PBMC中T细胞的纯度。
S5、Day 0，配置缓冲液(在PBS缓冲液中添加1％的胎牛血清(FBS))，选用微珠作为细胞培养载体，将微珠振荡30s或手动上下摇匀5min，按照微珠与T细胞的用量比为3：1取CD3/CD28微珠置于1.5mL EP管中，添加1mL缓冲液清洗微珠，之后使用磁铁从EP管向外吸微珠1min，弃洗液，重复两次，再使用培养基将微珠重悬到原体积，将细胞和微珠混合后按2×106PBMC/mL加到合适的培养瓶中。
S6、Day 2将细胞密度调整至3-5×106/mL，按病毒载体与细胞的比例为1:5添加实施例2制备得到的PRRSLIN-LIGHT-PD1慢病毒载体，同时添加聚凝胺(polybrene)4μg/mL和40ng/mL IL-2。4h之后，补加新鲜的完全培养基将细胞密度调整至1×106/mL继续培养。将所有的细胞离心，加入新鲜的培养基，继续培养。
S7、每隔2-3天进行半量换液，维持细胞密度在0.5-1×106/mL。
S8、Day 10-12，细胞数量达到109级别，在400g下离心5min得到免疫细胞，再用预冷的PBS洗涤两遍(400g，5min)。
S9、用血球计数板计数，流式细胞仪检测细胞类群，CAR-T细胞比例。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。逐步扩大培养过 程中，加入总体积所需的白细胞介素-2。
实施例4 CAR-T细胞流式分析
对实施例3制备的CAR-T细胞进行流式分析，其具体步骤如下：
S1、取5×104细胞(包括T细胞、CAR-T细胞)用于染色；
S2、细胞与抗体(抗体可与PD-1分子识别结合，偶联FITC荧光分子)共孵育45min，50μl，置于冰上；
S3、PBS洗脱两次；
S4、用120μl FACS试剂重悬细胞；
S5、流式细胞仪器测量FITC荧光信号，如果与对照T细胞对比，CAR细胞FITC荧光信号增强，表面CAR细胞构建成功。
CAR-T细胞流式浸染效果分别如图2所示，图中，纵坐标为流式SSC-H侧向角散射信号，横坐标为FITC荧光信号，该信号值越强，表明PD-1分子在膜上表达的越多，CAR-T细胞转染成功的比例越高。A图和B图为对照组，为不侵染病毒的T细胞；FITC偶联的检测CAR分子的抗体检测不到CAR分子表达；C图和D图，为转染PRRSLIN-LIGHT-PD1慢病毒的T细胞，经流式检测，与A图和B图对比，有细胞成功转染；病毒侵染T细胞后，流式检测侵染效率可以达到47％，说明成功制备LIGHT-PD1 CAR-T细胞。
实施例5 LIGHT-PD1 CAR-T细胞体外活性检测
采用LDH释放法检测LIGHT-PD1 CAR-T细胞对工程细胞株MCF-7/PDL1和高表达PDL1的肿瘤细胞的杀伤效应，通过ELISA方法检测LDH释放，包括以下步骤：
S1、用含5％小牛血清的RPMI-1640培养液将靶细胞调整到5×104/mL。
S2、在96孔细胞培养板中加入靶细胞，每孔加100μL。取3个孔作为效应细胞(LIGHT-PD1 CAR-T细胞)自然释放对照孔，不加靶细胞，仅加100μL培养液。
S3、向各孔加100μL效应细胞，效应细胞与靶细胞的比例10:1；5:1；1:1。自然释放孔不加效应细胞只加100μL培养液，效应细胞与靶细胞共孵育6小时， 每个实验置三个复孔。
S4、最大释放孔中(阳性对照)加10μL Lysis Solution(10×)，孵育45min-60min，每个实验置三个复孔。
S5、取上述3和4中待测样品和对照样品各50μL，加入新鲜的96孔酶标板中，再加入反应液和底物，避光30min。
S6、加入50μL终止液。
S7、在酶联检测仪上测定各孔的光密度(OD值)，检测波长490nm或492nm，在1小时内测完。
S8、特异性杀伤效率计算
杀伤率＝实验组LDH(OD)/最大LDH释放组(OD)。
计算公式：杀伤效率＝(实验组-效应自然释放-靶自然释放)/(靶最大释放-靶自然释放)×100％。
9.通过CBA试剂盒测定细胞因子分泌情况，同时计算CAR-T细胞各组中的增殖情况，并利用CD3和CD8抗体染色，确认增殖的T细胞中CD8阳性的T细胞的比例。
如图3所示，图中，横坐标表示CAR-T细胞与肿瘤细胞不同的效靶比，纵坐标表示杀伤效率，不同类型的柱状图表示不同肿瘤细胞，针对MCF-7，MCF-1/PDL-1,SMCC7721和Hela四种肿瘤细胞，在不同的效靶比条件下，LIGHT-PD1 CAR-T可以显著杀伤肿瘤细胞，且对其中SMCC7721和Hela细胞最为明显；相应的，如图4所示，横坐标表示T细胞与肿瘤或CAR-T细胞与肿瘤的效靶比，纵坐标表示细胞数目，T代表T细胞，LIG代表LIGHT-PD1 CAR-T细胞，可见与普通T细胞相比，CAR-T细胞在接触到肿瘤细胞刺激后，可发生特异性激活和增殖，且高效靶比情况下更为显著。
如图5杀伤实验中，横坐标表示CAR-T细胞与肿瘤细胞不同的效靶比，纵坐标表示细胞因子含量，检测培养上清中的细胞因子，发现CAR-T杀伤实验组IL-2(图5A)和TNF-α(图5B)的分泌显著升高。如图6所示，为CD3/CD8流式抗体检测CART对MCF7/MCF7-PDL1/HeLa/SMC7721 4种细胞株杀伤后CD8T细胞的比例，纵坐标PE信号表示特异性检测CD3分子表达，横坐标FITC信号表示检测CD8分子表达，通过流式细胞仪检测，LIGHT-PD1 CAR-T细胞激活 后，主要是CD8T细胞发生特异性增殖。
如图7所示，CAR-PD-1表示表达的CAR分子的胞外段为普通PD-1分子的CAR-T细胞，CAR-LIGHT-PD-1表示本发明构建的LIGHT-PD1 CAR-T细胞，结果表明对SMC7721肿瘤细胞，LIGHT-PD1 CAR-T细胞与PD-1 CAR-T相比，其杀伤效果更好。
上述结果证明，LIGHT-PD1-CART细胞在接触肿瘤细胞后，可以特异性的发生活化和增殖，释放细胞因子，杀伤肿瘤细胞，其中CD8阳性T细胞起到主要作用。同时比较了PD-1 CAR-T和LIGHT-PD1CAR-T对肿瘤的杀伤效果，结果显示LIGHT-PD1 CAR-T明显优于PD-1 CAR-T.
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2016100797-appb-000001
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Claims (15)

  1. 一种多基因重组嵌合抗原受体分子，其特征在于：包括依次串联的胞外区肽段、跨膜区肽段和胞内结构域肽段，所述胞外区肽段包括相连的LIGHT蛋白肽段和免疫结合蛋白肽段，其中，LIGHT蛋白肽段包括人LIGHT蛋白质或其片段的至少一个胞外区肽段，免疫结合蛋白肽段包括结合肿瘤细胞表面和/或肿瘤基质细胞表面免疫抑制分子的蛋白质或其片段的胞外区肽段。
  2. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述的LIGHT蛋白肽段具有一种或多种LIGHT相关的活性，包括但不限于：与一种或多种LIGHT受体结合；(ii)诱导趋化因子或细胞因子、趋化因子或细胞因子受体、粘附分子和/或共刺激分子中的一种或多种的表达；(iii)活化T细胞；(iv)募集T细胞进入到肿瘤细胞或组织中；(v)活化和/或增强肿瘤反应性T细胞的增殖；(vi)在肿瘤细胞或组织中，创建类淋巴微环境；(vii)诱导肿瘤细胞或组织的凋亡；或(viii)刺激受试对象中的免疫应答。
  3. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述LIGHT蛋白肽段的序列如SEQ ID NO：1所示。
  4. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述LIGHT蛋白肽段和免疫结合蛋白肽段使用连接肽相连。
  5. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述免疫结合蛋白肽段包括PD-1或HAC的胞外区肽段。
  6. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述多基因重组嵌合抗原受体分子还包括信号肽。
  7. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述跨膜区肽段为CD8跨膜区肽段，所述胞外区肽段与CD8跨膜区肽段通过CD8的hinge区肽段相连。
  8. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：所述胞内结构域肽段为共刺激信号分子，选自4-1BB、CD28、CD3ζ的胞内结构域肽段中的一种或多种。
  9. 根据权利要求1所述的多基因重组嵌合抗原受体分子，其特征在于：包含如SEQ ID NO：3所示的序列。
  10. 一种核苷酸，其特征在于：其编码根据权利要求1至9任一项所述的 多基因重组嵌合抗原受体分子。
  11. 根据权利要求10所述的核苷酸，其特征在于：包含或为SEQ ID NO：4所示的核苷酸序列。
  12. 一种重组载体，其特征在于：其包含根据权利要求10或11所述的的核苷酸。
  13. 根据权利要求12所述的重组载体，其特征在于：所述重组载体为pRRSLIN慢病毒表达载体。
  14. 一种重组细胞，其特征在于：其包含根据权利要求12或13任一项所述的重组载体。
  15. 根据权利要求14所述的重组细胞，其特征在于：所述重组细胞优选为T细胞或NK细胞。
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