WO2019154204A1 - 一种嵌合抗原受体基因工程载体、修饰的免疫细胞及其应用 - Google Patents

一种嵌合抗原受体基因工程载体、修饰的免疫细胞及其应用 Download PDF

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WO2019154204A1
WO2019154204A1 PCT/CN2019/073843 CN2019073843W WO2019154204A1 WO 2019154204 A1 WO2019154204 A1 WO 2019154204A1 CN 2019073843 W CN2019073843 W CN 2019073843W WO 2019154204 A1 WO2019154204 A1 WO 2019154204A1
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sequence
chimeric antigen
antigen receptor
cells
tumor
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李本尚
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上海交通大学医学院附属上海儿童医学中心
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Definitions

  • the invention belongs to the field of cell therapy, in particular to a chimeric antigen receptor genetic engineering vector, modified immune cells and applications thereof.
  • the genetically engineered vector can be stably transfected into autologous or allogeneic immune cells by virus packaging, and the target cells having the target molecule can be specifically killed by double-targeted recognition, thereby playing a clinical therapeutic role.
  • Chimeric antigen receptor abbreviated as CAR.
  • CAR-T treatment T cell-based chimeric antigen receptor cell therapy referred to as CAR-T treatment
  • NK cell-based chimeric antigen receptor cell therapy referred to as CAR-NK treatment
  • ⁇ / ⁇ T cell-based chimeric antigen receptor cell therapy Generic CART for T cell receptor alpha beta chain knockout.
  • the powerful killing ability of the corresponding immune cells to the target cells is utilized to eliminate the tumors and certain types of cells in the body.
  • CAR-T treatment technology has undergone continuous updates, from the earliest generation of CAR-T to clinical efficacy, to the second generation of CAR-T, which is a co-stimulatory molecule based on a generation of CAR-T.
  • CAR-T a co-stimulatory molecule based on a generation of CAR-T.
  • 4-1BB or CD28 a co-stimulatory molecule based on a generation of CAR-T.
  • CAR-T Three generations of CAR-T are based on the second generation of CAR-T, adding two co-stimulatory molecules, but it is generally believed that the third generation of CAR-T is no better than the second generation of CAR-T, so the current mainstream is based on the second generation CAR-T technology. Chimeric antigen receptor therapy performed.
  • the second-generation CAR-T technology is mainly directed to the tumor cell membrane surface antigen, and the light chain and heavy chain variable regions in the antibody molecule recognizing the antigen are connected in series by a linker to form a single-stranded affinity antibody structure, and then hinged
  • the region, the transmembrane region, the costimulatory molecule and the effector molecule are fused and expressed in the immune cells after the virus is prepared, so that the corresponding immune cells have the function of specifically killing the target cells.
  • the same fusion expression strategy is adopted for the CAR-T vector construction based on CD19, CD20, CD30, CD33, GD2, BCMA, EGFR VIII, Mesothelin, CD138, CD38 and the like.
  • Cytokines release syndrome is the most significant toxicity and is the number one safety risk. Cytokine release syndrome is a T cell-based activation that is a response to T cell activation activity, so side effects are clinically positively related to the therapeutic mechanism of CAR-T. Highly proliferating T cells can cause CRS, manifested as hyperthermia and myalgia, unstable hypotension, and respiratory failure.
  • the severity of CRS storms is strongly correlated with the tumor cell load in patients receiving CAR-T treatment, but it is not excluded that a small number of patients will produce more severe CRS under low load conditions.
  • a small number of patients develop acute brain edema.
  • the antigenic epitope is structurally convergent with the CD19 epitope structure, resulting in the killing of CD19-positive leukemia cells by CAR-T cells, and also the cytotoxic effect (off-target effect) against autonomic neurons.
  • CAR-T related acute necrotic encephalitis CAR-T related acute necrotic encephalitis
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and to provide a chimeric antigen receptor genetic engineering vector, modified immune cells and applications thereof.
  • the chimeric antigen receptor genetic engineering vector of the invention has good safety and high transfection efficiency, and the transfected immune cells can be rapidly expanded, and the killing effect is greatly improved.
  • the chimeric antigen receptor immune cells of the present invention are more precise in the recognition of target cells than the single target, preventing the off-target effect of a single target.
  • a first object of the present invention is to provide a chimeric antigen receptor genetic engineering vector comprising a lentiviral vector backbone, a regulatory element linked to a lentiviral vector backbone and a chimeric antigen receptor gene sequence, said regulatory elements comprising WPRE element, cPPT element and RRE element.
  • Gag encoding viral structural protein
  • Pol encodeme protein encoding the virus
  • Env encoding viral membrane protein
  • Rev encoding a protein that acts on the Rev response element, ie RRE, regulates viral mRNA.
  • the components required for lentiviruses, such as extracellular transport, are expressed in three different vectors to form a three-plasmid system, which greatly reduces the probability of virus recombination and ensures the safety of human use.
  • the lentiviral vector backbone of the present application may be a commonly used lentiviral vector, such as a viral vector derived from HIV-1.
  • the WPRE element helps to improve the stability of viral mRNA molecules.
  • the cPPT element facilitates the transfer of the virus into the nucleus, which makes it easier to integrate the genome and improve the efficiency of stable transfection.
  • the RRE element contributes to the extranuclear transport of mRNA molecules. Therefore, the virus obtained by the package has high titer and strong virus activity, which is the key to improving the transfection efficiency. Since only cells transfected with virus have the effect of specifically killing tumor cells, increasing the efficiency of transfection can greatly reduce the system of culturing cells and save the cost of pre-production.
  • a mammalian constitutive promoter preferably an EF1A promoter
  • the vector of the present application preferably adopts the EF1A promoter to initiate expression of a foreign gene
  • the EF1A promoter is a mammalian constitutive expression promoter derived from human elongation factor 1 ⁇ , so that the expression of the foreign gene is very stable and is not affected by the cell type. Widely used in different cell types such as T cells, NK cells, and macrophages.
  • the chimeric antigen receptor gene sequence comprises an independently expressed co-stimulatory signal pathway sequence and an effector signaling pathway sequence; or the chimeric antigen receptor gene sequence comprises a fusion-expressed costimulatory signaling pathway Sequence and effector signaling pathway sequences.
  • the self-shearing sequence is ligated between the independently expressed costimulatory signaling pathway sequence and the effector signaling pathway sequence; the costimulatory signaling pathway sequence and the effector signaling pathway sequence are independently expressed across the membrane, and simultaneously bind to the target site, An effect occurs when activated.
  • the costimulatory signaling pathway sequence and the effector signaling pathway sequence on the vector of the present application are two independent transmembrane signaling pathway fusion proteins after expression, one is a costimulatory signaling pathway and one is an effector signaling pathway;
  • the signaling pathway is activated, the immune cells modified by the chimeric antigen receptor genetic engineering vector can be activated to kill the target cells. When only one pathway is activated, immune cells cannot be activated or kill.
  • this method is more accurate than the recognition of the target cells by the chimeric antigen receptor immune cells of a single target; at the same time, the dual-targeted combined recognition can better prevent the off-target of a single target, and enhance the pair.
  • Target cell specificity and lethality are more accurate than the recognition of the target cells by the chimeric antigen receptor immune cells of a single target; at the same time, the dual-targeted combined recognition can better prevent the off-target of a single target, and enhance the pair.
  • the independently expressed costimulatory signaling pathway sequence and effector signaling pathway sequence may be composed of one or more costimulatory signaling pathway sequences and one or more The composition of the effector signal path sequence facilitates the synergy of multiple signal paths.
  • the chimeric antigen receptor genetic engineering vector strategy provided herein may also include a chimeric antigen receptor vector constructed using an IRES and/or a dual promoter vector and/or a multi-promoter vector.
  • the costimulatory signaling pathway sequence comprises a first leader peptide sequence, a first antibody sequence and a costimulatory molecule sequence which are sequentially linked according to a transcription direction
  • the effector signal pathway sequence comprises a sequence of linkages according to a transcription direction.
  • first leader peptide, first antibody sequence and costimulatory molecule sequence and “second leader peptide sequence second antibody sequence and effector molecule sequence” referred to in the present invention are understood to correspond to different signal pathways.
  • costimulatory signaling pathway and the effector signaling pathway herein can be composed of two, three or more independently expressed signal pathways.
  • the independently expressed costimulatory signaling pathway sequence is not limited to activating a signaling pathway sequence of an effector cell, but also a signalling pathway sequence that inhibits an effector cell.
  • the first leader peptide sequence and the second leader peptide sequence express the same or different leader peptides, respectively capable of directing the protein expressed by the costimulatory signaling pathway sequence and the protein expressed by the effector signaling pathway sequence to the cell membrane; the first antibody and the second antibody are located Cell membrane surface.
  • the two antibodies of the present application bind to it separately, resulting in a change in the structure of the antibody, and the resulting signal can activate the protein fused downstream thereof.
  • the signal generated by the first antibody activates the co-stimulatory molecule downstream thereof, and the signal generated by the second antibody activates the effector molecule downstream thereof; both the costimulatory molecule and the effector molecule are activated, causing activation of ZAP70, such that the immune cell, ie CART cells are activated and produce a variety of cytokines that produce rapid, specific, high-killing effects on target cells.
  • the first antibody sequence or the second antibody sequence includes, but is not limited to, a molecular sequence that interacts with a cell membrane surface molecule of the target cell, and an intracellular antigen or embryonic phase that is presented to the surface of the cell membrane inside the target cell.
  • the binding of the antibody to the original molecular sequence on the cell membrane surface of the target cell includes: interaction with the cell membrane surface antigen of the target cell, or interaction with a non-antigenic protein.
  • the protein expressed in the target cell is presented by a process such as degradation (including but not limited to the MHC presentation pathway) to a specific chimeric antigen receptor target formed by a protein fragment or a short peptide on the surface of the cell membrane, and the antibody may also be Activated in combination with such targets.
  • the dual targeting in the present application means that the chimeric antigen receptor gene engineering expression vector is simultaneously inserted with two ScFv antibody sequences corresponding to two identical or different molecules (antigens) on the surface of the target cell.
  • the first antibody sequence or the second antibody sequence is a ScFv antibody sequence selected from the group consisting of a molecular sequence that interacts with a cell membrane surface antigen of the target cell.
  • the first antibody sequence or the second antibody sequence is selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD13, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD36, CD37, CD38, CD40, CD41, CD42, CD43, CD44, CD45, CD56, CD58, CD64, CD66c, CD70, CD71, CD73, CD74, CD80, CD81, CD83, CD86, CD90, CD94, CD97, CD99, CD102, CD117, CD123, CD133, CD134, CD137, CD138, CD200, GD2, EGFR VIII, GD3, NG2, CA125, CA153, CA199, CA242 , CA724, HLA-DR, BDCA4, TCR ⁇ , TCR ⁇ , A33, CEA, CEACAM
  • the combination of the first antibody sequence and the second antibody sequence is selected from the group consisting of CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD13, CD14, CD15, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD36, CD37, CD38, CD40, CD41, CD42, CD43, CD44, CD45, CD56, CD58, CD64, CD66c, CD70, CD71, CD73 , CD74, CD80, CD81, CD83, CD86, CD90, CD94, CD97, CD99, CD102, CD117, CD123, CD133, CD134, CD137, CD138, CD200, GD2, EGFR VIII, GD3, NG2, CA125, CA153, CA199, CA242, CA724, HLA-DR, BDCA4, TCR ⁇ , TC ⁇ , A33, CEA, CE
  • B-line acute lymphoblastic leukemia and B-lineage lymphoma preferably, a combination of CD19/CD19 is used; for AML, preferably, a combination of CD33/CD117 or CD33/CD34 is used; for multiple myeloma, preferably, BCMA is used.
  • EPCAM/AFP or EPCAM/GPC3 combination is used for liver cancer; preferably, EPCAM/HER2 or EPCAM/MESO combination is used; for prostate cancer, preferably, EPCAM/PSMA combination is used; for colorectal cancer
  • EPCAM/AFP or EPCAM/GPC3 combination is used for breast cancer
  • EPCAM/HER2 or EPCAM/MESO combination is used for breast cancer
  • EPCAM/PSMA combination is used for prostate cancer
  • colorectal cancer preferably, a combination of EGFR/HER2 or EGFR/CEA is employed for colorectal cancer.
  • the different combinations of ScFv antibodies mentioned in the above schemes are designed according to the unique immunophenotypic characteristics of tumor cells such as acute leukemia cells, in which CD19 and CD22 are basically cell membrane antigens expressed by all B line leukemia cells, and CD20 It is a cell membrane antigen expressed by all B-line lymphoma lymphomas, and combined with the same antibody sequence or other antibody sequences, can specifically kill tumor cells, and has no effect on normal B lymphocytes.
  • the first antibody sequence and the second antibody sequence further comprise a specific pronuclear antigen receptor target formed by a protein fragment or a short peptide which is presented to the surface of the cell membrane by degradation of a protein expressed in the target cell.
  • a specific pronuclear antigen receptor target formed by a protein fragment or a short peptide which is presented to the surface of the cell membrane by degradation of a protein expressed in the target cell.
  • Dot-designed antibody sequences Specifically, some tumor-derived gene mutein products or abnormally expressed protein products in the cells are degraded by proteasome or other mechanism, and then presented to the surface of the cell membrane by MHC molecules or other molecules, and can be used as a target for identification of immune cells.
  • the mutein product or aberrantly expressed protein product includes, but is not limited to, ABL1, ALK, ASXL1, ATM, BCOR, BCORL1, BRAF, CALR, CBL, CEBPA, CSF3R, CSMD1, CUX1, DNMT3A, EP300, ETNK1, ETV6, EZH2 , FLT3, GATA1, GATA2, GNAS, IDH1, IDH2, IKZF1, JAK1, JAK2, JAK3, KIT, KMT2A, KMT2C, KMT2D, KRAS, MPL, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PRPF40B, PRPF8, PTEN Mutations in genes such as PTPN11, RAD21, ROBO1, ROBO2, RUNX1, SETBP1, SF1, SF3A1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, SUZ12, TET2, TP53, U2AF1, U2AF2, W
  • the costimulatory signaling pathway sequence comprises a first leader peptide sequence, a first antibody light chain VL sequence, a first antibody linker peptide sequence, a first antibody heavy chain VH sequence, a hinge region sequence, a transmembrane region, which are sequentially linked according to a transcription direction.
  • Sequence and costimulatory molecule sequences comprising a second leader peptide sequence, a second antibody light chain VL sequence, a second antibody linker peptide sequence, a second antibody heavy chain VH sequence, a hinge region, which are ligated in sequence according to the direction of transcription a sequence, a transmembrane region sequence, and an effector molecule sequence; a self-cleavage sequence is ligated between the costimulatory molecule sequence and the second leader peptide sequence.
  • the costimulatory molecule sequence comprises an activating co-stimulatory molecule sequence selected from the group consisting of 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 At least one of the ICOS sequences;
  • the costimulatory molecule sequence is a 4-1BB sequence.
  • the costimulatory molecule sequence comprises an inhibitory costimulatory molecule sequence
  • the inhibitory costimulatory molecule sequence comprises an effector molecule sequence of PD1, CTLA4;
  • PI3K PI3K, Akt, TCR ⁇ , ZAP70, PKC sequences
  • the inhibitory costimulatory molecule sequence comprises a PI3K, Akt sequence.
  • the costimulatory molecule sequence is selected from at least one of 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS sequence; preferably, costimulatory molecule
  • the sequence is a 4-1BB sequence.
  • the effector molecule sequence is selected from the group consisting of CD3.
  • the self-cleavage sequence is selected from at least one of the P2A, T2A, F2A, E2A, BmCPV2A, BmIFV2A sequences.
  • the self-cleavage sequence is a P2A sequence.
  • the first antibody sequence or the second antibody sequence is selected from the nucleotide sequences shown in SEQ ID NOS: 11 to 50; preferably, for CD19-positive lymphocytic leukemia and lymphoma, SEQ ID NO: 11 - Sequences in 40; preferably, for neuroblastoma, the sequences of SEQ ID NOS: 41-50 are selected.
  • SEQ ID NOS: 11 to 50 preferably, for CD19-positive lymphocytic leukemia and lymphoma
  • SEQ ID NO: 11 - Sequences in 40 preferably, for neuroblastoma
  • the same sequence described above can also be used for cellular immunotherapy of acute leukemia and lymphoma B-lineage malignancies.
  • the first leader peptide sequence or the first leader peptide sequence includes, but is not limited to, CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11a, CD11b, CD13, CD14, CD15, CD19, CD20 , CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD36, CD37, CD38, CD40, CD41, CD42, CD43, CD44, CD45, CD56, CD58, CD66c, CD70, CD73, CD74, CD80 , CD81, CD86, CD94, CD97, CD99, CD102, CD123, CD133, CD134, CD137, CD138, CD200, EGFR, GD3, NG2, CA125, A33, CEA, CEACAM6, CS1, EGFR, ERBB2, FGF19, HER3, IL3Ra Membrane protein-directed peptide sequences such as NCAM, NKG2A, BCMA, NT
  • the linker peptide sequence refers to an amino acid sequence in which VL and VH are linked in series, and a preferred sequence is the nucleotide sequence shown in SEQ ID NO: 51.
  • the hinge region sequence refers to an amino acid sequence that mediates the transfer of an activation signal from a ScFv antibody to a transmembrane region, and the preferred sequence is the nucleotide sequence set forth in SEQ ID NO:52.
  • the transmembrane region sequence refers to an amino acid sequence that mediates the transactivation of an activation signal from the hinge region to the intracellular region, and the preferred sequence is the nucleotide sequence set forth in SEQ ID NO:53.
  • the costimulatory molecule includes, but is not limited to, a sequence of a molecule such as 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS, etc., preferably the sequence is represented by SEQ ID NO:54.
  • the self-cleavage sequence refers to a sequence including, but not limited to, P2A, T2A, F2A, E2A, BmCPV2A, BmIFV2A, preferably selected from the nucleotide sequences set forth in SEQ ID NOS: 55-58.
  • the effector molecule sequence refers to a nucleotide sequence including, but not limited to, a CD3 ⁇ sequence, preferably SEQ ID NO:59.
  • a second object of the present invention is to provide a virus prepared by using the chimeric antigen receptor genetic engineering vector as described above, and a chimeric antigen receptor genetic engineering vector and a packaging plasmid are transfected into a packaging cell line to obtain a corresponding virus;
  • the virus is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, and the like.
  • a third object of the present invention is to provide a chimeric antigen receptor genetically engineered vector modified immune cell transfected with a chimeric antigen receptor genetic engineering vector as described above or a virus as described above;
  • the immune cells include T cells, NK cells, and mononuclear macrophages; and the T cells include unmodified T cells, engineered T cells, autologous T cells, and allogeneic T cells; and the NK cells include Unmodified NK cells, engineered NK cells, autologous NK cells, allogeneic NK cells; preferably, the T cells are selected from at least CD4 positive T lymphocytes, CD8 positive T lymphocytes, CD4 and CD8 double positive T lymphocytes One type;
  • the chimeric antigen receptor genetically engineered vector-modified immune cells include CD4-positive T lymphocytes and CD8-positive T lymphocytes, and the number of CD4-positive T lymphocytes is 40% to 60% of the total number of cells.
  • a fourth object of the present invention is to provide a chimeric antigen receptor genetic engineering vector as described above, or a virus as described above, or an application of the immune cell as described above for the preparation of a medicament for treating a tumor disease or an immune system disease;
  • the diseases targeted by the medicament for treating tumor diseases include acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, mast cell leukemia, plasma cell leukemia, myeloma, myeloproliferation.
  • the medicament for treating diseases of the immune system diseases includes: an autoimmune disease, a viral infectious disease, a bacterial or fungal infection disease; wherein the autoimmune disease includes rheumatoid arthritis, chronic lymphatic thyroiditis Hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary liver Hard, multi-encephalic sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, dry mouth syndrome, ankylosing spondylitis, scleroderma, nodular polyarteritis, Wegener granulomatosis.
  • the autoimmune disease includes rheumatoid arthritis, chronic lymphatic thyroiditis Hyperthyroidism, insulin-dependent diabetes mellitus, mya
  • Bacterial or fungal infections include Staphylococcus, Streptococcus, Proteus, Pseudomonas aeruginosa, B. pertussis, actinomycetes, tetanus, Clostridium perfringens, Salmonella typhi, Vibrio cholerae, meningitis Cocci, Bacillus anthracis, diphtheria, pneumococci, Klebsiella, Enterococcus, Acinetobacter, Haemophilus haemolyticus, Escherichia coli, Legionella, spores, anaerobic infections; various Candida, Aspergillus, Mucor , cryptococci, Penicillium marneffei, spores, staining buds infection; rickettsia, spirochetes, mycoplasma, chlamydia, various protozoal infections.
  • the present invention has the following beneficial effects compared with the prior art:
  • the invention can improve the safety of the vector by transforming the chimeric antigen receptor gene engineering vector, inserting the regulatory element, the promoter, optimizing the base sequence, etc., and the transfection efficiency is high, and the transfected immune cells can rapidly expand and kill. The effect is greatly improved.
  • the present invention employs a dual-targeted chimeric antigen receptor genetic engineering vector as a carrier for CAR-T treatment, and the corresponding vector can be subjected to viral packaging to perform autologous or allogeneic cells (T cells and/or NK cells and / or ⁇ / ⁇ T cells) transfection and amplification for cellular immunotherapy.
  • the dual targeting chimeric antigen receptor can be designed for one or several antigens on the surface of the cell membrane, and the same antigen can be the same or different ScFv sequences.
  • the chimeric antigen receptor After the chimeric antigen receptor binds to the target cell membrane surface antigen, signal transduction can simultaneously and simultaneously activate the costimulatory signal molecule and the effector signal molecule, thereby activating the modified immune cell to exert anti-tumor effects. Therefore, this method is more accurate than the recognition of the target cells by the chimeric antigen receptor immune cells of a single target; at the same time, the dual-targeted combined recognition can better prevent the off-target of a single target, and enhance the pair. Target cell specificity and lethality. In addition, it can reduce the binding of similar or abnormal molecules to the surface of normal cells, reduce the chance of attacking normal cells, and improve the safety of use.
  • Figure 1 is a schematic view showing the structure of a chimeric antigen genetic engineering vector of the present invention
  • Figure 2 is a core sequence of an independently expressed costimulatory molecule signaling pathway and an effector molecular signaling pathway;
  • Figure 3 is a structural diagram of a dual targeting chimeric antigen receptor of the present invention.
  • Figure 4 is a comparison of the transfection efficiency of the vector of the present application with other company lentiviral vectors; wherein a ⁇ c is the transfection efficiency of lentiviral vector packaging of other companies, and d ⁇ e is a lentivirus packaged by the carrier of the present patent Transfection efficiency, f is the untransfected control.
  • FIG. 5 is an in vitro cell killing experiment of T lymphocytes of the CD19 dual targeting specific CAR of the present invention
  • FIG. 5a to FIG. 5c are the results of in vitro cell killing experiments on the 0th day, the 1st day, and the 2nd day of the test group, respectively
  • Figure 5d - Figure 5f show the results of in vitro cell killing experiments on day 0, day 1 and day 2 of the control group;
  • 6 is a result of detection of minimal residual disease before and after CD19 dual-target specific CAR-T infusion in a refractory acute lymphoblastic leukemia of the present invention
  • 6A-6C is a residual disease of CD19 dual-target specific CAR-T cells before infusion The results of the test
  • 6D-6F is the test result on the 8th day after infusion of CD19 double-targeted specific CAR-T cells
  • Figure 7 is a graph showing the results of detection of minimal residual disease before and after CD19 dual-target specific CAR-T infusion in a secondary relapse acute lymphoblastic leukemia
  • Figure 8 is a graph showing the results of detection of minimal residual disease before and after CD19 dual-target specific CAR-T infusion in relapsed acute lymphoblastic leukemia after transplantation;
  • Figure 9 is a comparison of in vitro proliferation experiments of CD19 dual-target specific CAR-T cells with other companies; wherein the number of starting cells is 1 ⁇ 10 7 ;
  • Figure 11 is a comparison of the killing effect of the transfected cells of the other dual-vector vectors of the present invention.
  • Figure 12 is a graph showing the relationship between the appropriate ratio of CD4 and CD8 to cytotoxicity
  • Figure 13 is an estimated two-year event-free survival (EFS) for enrolled patients
  • Figure 14 is the estimated overall survival (OS) for enrolled patients over 2 years.
  • Example 1 This example illustrates a chimeric antigen receptor genetic engineering vector comprising a costimulatory signaling pathway sequence and an effector signaling pathway sequence as an example, and may also include multiple costimulatory signaling pathway sequences and multiple effectors. Signal path sequence.
  • a chimeric antigen receptor genetic engineering vector was constructed.
  • the corresponding sequences were synthesized by gene synthesis and verified by sequencing.
  • the above sequence was inserted into an appropriate position in an adenovirus, retrovirus or lentiviral vector by selection of a suitable restriction site and verified by sequencing.
  • the chimeric antigen receptor genetic engineering vector comprises:
  • the RRE sequence is inserted upstream of the multiple cloning site of the vector, the WPRE sequence and the cPPT sequence are inserted downstream of the multiple cloning site, and the EF1A promoter is inserted between the RRE sequence and the upstream of the multiple cloning site for initiation of insertion of the polyclonal Site chimeric antigen receptor gene.
  • the sequence upstream of the chimeric antigen receptor structural protein is optimized, and the optimized sequence is as shown in SEQ ID NO: 60, so that the expression of the foreign protein is stable and sustained, which is very important for improving the killing effect of CART cells in vitro.
  • the sequence of the first and second antibodies corresponding to the A or B target on the target cell that is, the ScFv antibody light chain VL sequence and the heavy chain VH sequence construction: for acute leukemia and lymphoma, we chose SEQ ID NO:11
  • sequences in ⁇ 40 the same or different two sequences can be selected for cellular immunotherapy of these diseases.
  • the sequences in SEQ ID NOs: 11-14, 19-40 can be selected, and the same basis can be selected.
  • the above sequence of CD19 is used for cellular immunotherapy of B-lineage malignancies.
  • For neuroblastoma we chose the sequences in SEQ ID NOs: 19-20, 41-50.
  • Linker peptide construction refers to the amino acid sequence in which VL and VH are ligated together. The preferred sequence is shown in SEQ ID NO: 51.
  • Hinge region sequence construction refers to the amino acid sequence that mediates the transmission of an activation signal from ScFv to the transmembrane region. The preferred sequence is the sequence provided in SEQ ID NO:52.
  • Transmembrane region sequence construction refers to the amino acid sequence that mediates the transactivation of the activation signal from the hinge region to the intracellular region. The preferred sequence is shown in the sequence provided in SEQ ID NO:53.
  • Co-stimulatory molecular sequence construction refers to a sequence including, but not limited to, 4-1BB, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS, and the like.
  • the sequence of 4-1BB is shown in the sequence provided in SEQ ID NO:54.
  • Self-cleaving sequence construction refers to, but is not limited to, P2A, T2A, F2A, E2A, BmCPV2A, BmIFV2A, etc., preferably, the P2A sequences are shown in the sequences provided in SEQ ID NOS: 55-58.
  • Effector Molecular Sequence Refers to, but is not limited to, a CD3 ⁇ sequence, and the corresponding sequence is shown in the sequence provided in SEQ ID NO:59.
  • the chimeric antigen receptor genetically engineered vector sequence targeting CD19 includes, but is not limited to, the following structure: CD8 leader peptide - CD19ScFv - hinge region - CD8 transmembrane region - 4 1BB-P2A-CD8 leader peptide-CD19ScFv-hinge region-CD8 transmembrane region-CD3zeta-TGA.
  • CD8 leader peptide - CD19ScFv - hinge region - CD8 transmembrane region - 4 1BB-P2A-CD8 leader peptide-CD19ScFv-hinge region-CD8 transmembrane region-CD3zeta-TGA There is no linker between the transmembrane region sequence and the costimulatory signal molecule or the effector signal molecule of the present application, which can shorten the signal pathway and improve the signal transduction efficiency, thereby improving the activation efficiency of immune cells such as T cells, and improving the in vitro of CART cells
  • the 293T cells were transfected for 72 hours and 96 hours, and the culture supernatant was collected and concentrated by virus.
  • 293 cells were transfected with different concentrations of virus by real-time quantitative PCR to determine viral titers.
  • the virus was transfected by collecting different immune cells from human peripheral blood to ensure transfection efficiency was above 20%, and a suitable MOI value was found for clinical treatment. It should be pointed out that each batch of virus needs to perform the above operations to ensure the stability and reliability of clinical trials.
  • the transfection efficiency was detected by transfecting different immune cells from human peripheral blood.
  • the Fab fragment against ScFv was used as the objective basis for the positive transfection efficiency of flow cytometry.
  • the detection efficiency of transfection efficiency can also be carried out by real-time quantitative PCR.
  • the immune cells such as CAR-T cells transfected with the virus were mixed with the CD19-positive cell line in a certain ratio and co-incubated, and the in vitro killing assay was performed by flow cytometry at 0h, 24h and 48h, and the incubation time was compared. Cell killing effect was compared to control virus.
  • CD3/CD28 immunomagnetic beads were added for stimulation activation and Virus transfection, continue to give CD3/CD28 immunomagnetic beads, Interleukin7 and Interleukin15 for in vitro expansion, generally 7 to 8 days can be amplified more than 100 times, after reaching the appropriate number of cells, washed back into the human body.
  • the number of human CAR-T positive cells returned is 1 ⁇ 10 4 /Kg to 5 ⁇ 10 7 /Kg, and is adjusted according to the internal load, preferably 2 ⁇ 10 5 /Kg to 5 ⁇ 10 7 /Kg.
  • the double-targeted chimeric antigen receptor vector of the invention is prepared for CD19-positive leukemia and lymphoma virus, and the transfection efficiency is generally more than 20% after in vitro transfection, and most of them are between 40% and 70%, as attached.
  • a to c are transfection efficiencies of other lentiviral vector packages
  • d to e are lentiviral transfection efficiencies packaged using the patented technology
  • f is an untransfected control.
  • the cells were co-incubated with the test group; the virus obtained by the vector preparation of the irrelevant sequence was used as a control group.
  • the results of the killing effect of the two groups of cells on the target cells are shown in Fig. 5.
  • 5a-5c are the results of in vitro cell killing experiments on the 0th day, the 1st day, and the 2nd day of the test group; FIG. 5d- FIG.
  • 5f are the 0th day, the 1st day, and the 2nd day of the control group, respectively.
  • the virus-transfected CAR-T cells prepared by the CD19 dual-targeting chimeric antigen receptor vector of the present scheme greatly enhanced the killing effect on the target cells after being incubated at 24h and 48h.
  • Test Example 1 A virus-transfected CAR-T cell prepared by this CD19 double-targeting chimeric antigen receptor vector in a patient with relapsed CD19-positive acute lymphoblastic leukemia, the results are shown in Fig. 6.
  • 6A-6C is the result of detection of residual disease before infusion of CD19 dual-targeted specific CAR-T cells
  • 6D-6F is the result of the 8th day after infusion of CD19 dual-targeted specific CAR-T cells
  • MRD decreased from 33.1% to less than 0.01%, indicating that on the 8th day after CAR-T treatment, bone marrow MRD reached molecular remission (MRD ⁇ 0.01%).
  • Test Example 2 A virus-transfected CAR-T cell prepared by CD19 dual-targeting chimeric antigen receptor vector in a patient with CD19-positive acute lymphoblastic leukemia who failed to induce relapse after secondary recurrence, and the results are shown in FIG.
  • 7A-7C is the result of detection of residual disease in CD19 dual-targeted specific CAR-T cells prior to infusion, with an MRD of 11.7%
  • 7D-7F is after infusion of CD19 dual-targeted specific CAR-T cells
  • Test Example 3 The results of detection of virus-transfected CAR-T cells prepared from CD19-positive lymphoblastic lymphoma/leukemia patients with bone marrow relapse via the CD19 dual-targeting chimeric antigen receptor vector are shown in FIG.
  • Test Example 4 In vitro expansion of CAR-T cells It was found that, as shown in Fig. 9, a lentivirus prepared by the double-targeting chimeric antigen receptor vector of the present application and a lentivirus of another company were transfected into human T lymphocytes in vitro. The growth after the cells is very different. After transfection of other companies, CAR-T cells expanded about 14-fold in 7 days, while the CAR-T cells transfected with the vector of the present application amplified 160-fold in 7 days of rare, in vitro expansion. The speed is greatly improved. Since these patients are at the end of the disease, the shortening of the in vitro culture amplification time is very important for the safety of the treatment.
  • Test Example 5 The virus-transfected CART cells prepared by the present invention were compared with CART cells from other companies for in vitro killing experiments, and the experimental results are shown in FIG. The results showed that the in vitro killing activity of the CART cells (Double-CAR-CD19) prepared by the dual-targeted genetic engineering vector of the present invention was significantly enhanced as compared with the CD19-CAR killing effect of other companies, and the results are shown in FIG. 10A is a comparison of the effects of infusion for 0 hours, and FIG. 10B is a comparison of effects of infusion for 16 hours.
  • Test Example 6 The in vitro killing effect of the present application was compared with the technique in the patent CN103483452A, and the present application focused on the problem of the effective target ratio.
  • the effective target ratio is effector cells (CAR-T cells): target cells (tumor cells), we can obtain 99.9% killing effect from 1:10 to 1:20, and the patent CN103483452A is done from 1 to 50: 1, the resulting killing effect is not up to the level of this application.
  • CAR-T cells effector cells
  • target cells tumor cells
  • Test Example 7 We found in in vitro studies that a suitable ratio of CD4 to CD8 cells in transfected positive CAR-T cells is one of the key factors for better cytotoxicity. In vitro experiments revealed that when CD4:CD8 When the ratio is between 40% and 60%, high-efficiency killing of target cells can be obtained. Because patients receive chemotherapy drugs, the proportion of CD4:CD8 cells often deviates from the above range, and the clinical efficacy is greatly affected. The corresponding test results are shown in Figure 12.
  • Test Example 8 High transfection efficiency, high-efficiency amplification and appropriate ratio of CAR-T cells are the basis for efficient killing of target cells in vivo and obtaining clinical efficacy in vivo.
  • the clinically-incorporated patients are treated with the patented CAR-T cell.
  • All 29 patients with refractory and recurrent CD19-positive hematological malignancies were treated with morphological changes in the remaining 27 patients from 8 days to 1 month after CD19-CAR-T treatment. Complete remission and molecular remission, the latter by flow cytometry to detect minimal residual disease (MRD) ⁇ 0.01%.
  • MRD minimal residual disease

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Abstract

提供了一种嵌合抗原受体基因工程载体、修饰的免疫细胞及其应用，包括慢病毒载体骨架、连接在慢病毒载体骨架上的调控元件和嵌合抗原受体基因序列，所述的调控元件包括WPRE元件、cPPT元件和RRE元件；所述的嵌合抗原受体基因序列的上游插入有EF1A启动子；所述的嵌合抗原受体基因序列包括独立表达的共刺激信号通路序列和效应器信号通路序列；或者，所述的嵌合抗原受体基因序列包括融合表达的共刺激信号通路序列和效应器信号通路序列。

Description

一种嵌合抗原受体基因工程载体、修饰的免疫细胞及其应用 技术领域
本发明属于细胞治疗领域，具体地说，涉及一种嵌合抗原受体基因工程载体、修饰的免疫细胞及其应用。该基因工程载体经病毒包装后可以稳定转染自体或者异体免疫细胞，通过双靶向识别达到特异性杀伤体内具有靶分子的目的细胞，从而起到临床治疗作用。
背景技术
嵌合抗原受体-Chimeric antigen receptor，简写为CAR。其中基于T细胞的嵌合抗原受体细胞治疗简称CAR-T治疗，基于NK细胞的嵌合抗原受体细胞治疗简称CAR-NK治疗，也有基于γ/δT细胞的嵌合抗原受体细胞治疗和T细胞受体αβ链敲除的通用型CART。不管采用哪种细胞，均是利用相应免疫细胞对靶细胞的强大的杀伤能力，达到清除体内肿瘤和某些类型细胞的作用。
自国际上首例复发的急性淋巴细胞白血病患者接受CAR-T治疗到现在，对于复发难治B系急性淋巴细胞白血病和淋巴瘤的疗效取得了显著的临床疗效，并得到了业界公认。CAR-T治疗技术经历了不断的更新，从最早的一代CAR-T鲜有临床疗效，到二代CAR-T取得显著疗效，后者是在一代CAR-T的基础上增加了共刺激分子，如4-1BB或CD28。三代CAR-T是在二代CAR-T的基础上增加了两种共刺激分子，但一般认为三代CAR-T不比二代CAR-T具有优势，因此目前主流的还是基于二代CAR-T技术进行的嵌合抗原受体治疗。
二代CAR-T技术主要是针对肿瘤细胞膜表面抗原，将可识别该抗原的抗体分子中的轻链和重链可变区，通过连接肽进行串联，形成单链亲和抗体结构，然后与铰链区、跨膜区、共刺激分子和效应器分子进行病毒制备后在免疫细胞中融合表达，使得相应的免疫细胞具有特异性杀伤靶细胞的作用。目前基于CD19、CD20、CD30、CD33、GD2、BCMA、EGFR VIII、Mesothelin、CD138、CD38等的CAR-T载体构建均是采用相同的融合表达策略。当前，基于嵌合抗原受体细胞治疗技术对于CD19阳性淋巴细胞白血病和淋巴瘤取得了非常显著的疗效。联合PD-1抗体和CAR-T细胞治疗是一个不错的选择，目前有很多临床实验正在进行中。
然而，CAR-T细胞应用于癌症治疗，在显示出疗效的同时，伴随有毒性和风险，甚至导致病人死亡。主要面临的问题有两个，首先，细胞因子释放综合症(Cytokines release syndrome，CRS)是最显著的毒性，为头号安全风险。细胞因子释放综合症是基于T细胞的激活，是T细胞激活活性的一个反应，所以副作用是与CAR-T的治疗机制正相关的临床反应。高度增殖的T细胞能引起CRS，表现为高热和肌痛，不稳定的低血压和呼吸衰竭。一般而言，CRS风暴的严重程度与患者接受CAR-T治疗前体内的肿瘤细胞负荷有很大的相关性，但不排除少数患者在低负荷的情况下也会产生比较严重的CRS。其次，少数患者出现急性脑水肿，我们推测是由于患者基因组中转录翻译成为神经元细胞膜上某个蛋白的编码序列存在多态性(Single nucleotide polymorphisms,SNP)或者突变(Single nucleotide variant,SNV)，使得其抗原表位在结构上与CD19抗原表位结构上趋同，从而导致CAR-T细胞对CD19阳性白血病细胞产生杀伤的同时，也产生了针对自身神经元细胞的细胞毒作用(脱靶效应)，并且我们觉得应该称为CAR-T相关急性坏死性脑炎(CAR-T related acute necrotic encephalitis,CANE)更为合适。综上，目前一代、二代及三代的嵌合抗原受体仍然存在治疗副作用多、特异性差等问题。有鉴于此特提出本发明。
发明内容
本发明要解决的技术问题在于克服现有技术的不足，提供一种嵌合抗原受体基因工程载 体、修饰的免疫细胞及其应用。本发明的嵌合抗原受体基因工程载体安全性好，转染效率高，转染的免疫细胞能够快速扩增，杀伤效果大大提高。另外，本发明较单一靶点的嵌合抗原受体免疫细胞对靶细胞的识别更加精准，防止单一靶点的脱靶效应。
为解决上述技术问题，本发明采用技术方案的基本构思是：
本发明的第一目的是提供一种嵌合抗原受体基因工程载体，包括慢病毒载体骨架、连接在慢病毒载体骨架上的调控元件和嵌合抗原受体基因序列，所述的调控元件包括WPRE元件、cPPT元件和RRE元件。
本申请将Gag(编码病毒结构蛋白)、Pol(编码病毒所需酶类蛋白)、Env(编码病毒膜蛋白)和Rev(编码一个可作用于Rev反应元件，即RRE的蛋白，调节病毒mRNA的细胞核外转运)等慢病毒所需元件放到三种不同的载体中表达，形成三质粒系统，大大降低了病毒重组的概率，可以确保人体使用的安全性。通过对入组患者的检测，最长患者治疗后22个月，体内未检测到慢病毒分子存在。
本申请的慢病毒载体骨架可以为常用的慢病毒载体，例如HIV-1来源的病毒载体。WPRE元件有助于提高病毒mRNA分子的稳定性，cPPT元件有助于病毒向细胞核内转移，从而更容易进行基因组的整合，提高稳定转染效率，而RRE元件有助于mRNA分子的核外转运，从而使得包装获得的病毒滴度高，病毒活力强，这是提高转染效率的关键。由于只有病毒转染阳性的细胞才具有特异性杀伤肿瘤细胞的作用，提高转染效率可以大大降低培养细胞的体系，节约前期生产成本。
进一步的方案，所述的嵌合抗原受体基因序列的上游插入有哺乳类组成型启动子，优选EF1A启动子。本申请的载体优选采用EF1A启动子启动外源基因的表达，EF1A启动子为人延长因子1α来源的哺乳动物组成型表达启动子，使得外源基因表达十分稳定，并且不受细胞类型的影响，可广泛用于T细胞、NK细胞、巨噬细胞等不同细胞类型。相对于CMV、SV40等启动子，除不受细胞类型影响外，更为重要的是外源蛋白表达稳定，不会过高的表达，从而对细胞生长影响小，这是CAR阳性T淋巴细胞在体外高速扩增的基础。
进一步的方案，所述的嵌合抗原受体基因序列包括独立表达的共刺激信号通路序列和效应器信号通路序列；或者，所述的嵌合抗原受体基因序列包括融合表达的共刺激信号通路序列和效应器信号通路序列。
独立表达的共刺激信号通路序列和效应器信号通路序列之间连接有自剪切序列；共刺激信号通路序列和效应器信号通路序列分别独立跨膜表达，且在同时与靶位点结合、被激活时产生效应。本申请载体上的共刺激信号通路序列和效应器信号通路序列，在表达后为两条独立的跨膜的信号通路融合蛋白，一条为共刺激信号通路，一条为效应器信号通路；当两条信号通路均被激活时，嵌合抗原受体基因工程载体所修饰的免疫细胞才能够被激活，对靶细胞产生杀伤作用。当只有一条通路被激活时，免疫细胞不能够被激活，也不能产生杀伤作用。因此，与单一靶点的嵌合抗原受体免疫细胞对靶细胞的识别相比，这种方式更加精确；同时双靶向的联合识别作用也可以更好的防止单一靶点的脱靶，增强对靶细胞的特异性和杀伤力。
所述的独立表达的共刺激信号通路序列和效应器信号通路序列，包含但不限于仅由两个独立的信号通路序列组成，可以由一个或一个以上的共刺激信号通路序列和一个或一个以上的效应器信号通路序列组成，便于实现多个信号通路的协同作用。
有研究发现CD19-CAR-T治疗患者中部分患者出现急性脑水肿等严重危及生命的副反应，我们分析可能是患者神经元细胞中某种膜蛋白分子发生了突变，从而结构上与CD19抗 体识别的抗原表位相近，造成了脱靶效应。而采用本申请的针对CD19抗原不同的抗原表位(至少两个抗原表位)的ScFv抗体，可以在很大程度上避免类似严重不良事件发生。
本申请提供的嵌合抗原受体基因工程载体策略，也可以也包括采用IRES和/或双启动子载体和/或多启动子载体等构建的嵌合抗原受体载体。
进一步的方案，所述的共刺激信号通路序列按照转录方向包括依次连接的第一引导肽序列、第一抗体序列和共刺激分子序列，所述的效应器信号通路序列按照转录方向包括依次连接的第二引导肽序列、第二抗体序列和效应器分子序列；所述的共刺激分子序列与第二引导肽序列之间，或者效应器分子序列与第一引导肽序列连接有自剪切序列。
本发明中提到的“第一引导肽、第一抗体序列和共刺激分子序列”和“第二引导肽序第二抗体序列和效应器分子序列”应理解为是为了对应于不同信号通路而做的简单描述，这里的共刺激信号通路和效应器信号通路可以由两种、三种或者更多种独立表达的信号通路组成。
所述的独立表达的共刺激信号通路序列，不仅限于激活效应器细胞的信号通路序列，也包含抑制效应器细胞的信号通路序列。
第一引导肽序列和第二引导肽序列表达相同或不同的引导肽，能够分别引导共刺激信号通路序列表达的蛋白、效应器信号通路序列表达的蛋白至细胞膜；第一抗体和第二抗体位于细胞膜表面。当靶细胞表面存在与两个抗体分别作用的分子时，本申请的两个抗体分别与其结合，导致抗体结构发生改变，产生的信号可以激活其下游融合的蛋白。第一抗体产生的信号激活其下游的共刺激分子，第二抗体产生的信号激活其下游的效应器分子；共刺激分子和效应器分子均被激活后，导致ZAP70激活，这样免疫细胞，也就是CART细胞就激活了，且产生各种细胞因子，能够对靶细胞产生快速、特异性高的杀伤作用。
进一步的方案，所述的第一抗体序列或第二抗体序列包括但不限于：与靶细胞的细胞膜表面分子相互作用的分子序列、与靶细胞内部递呈到细胞膜表面的胞内抗原或胚胎期表达的抗原相互作用的分子序列、与靶细胞内部递呈到细胞膜表面的肿瘤新抗原相互作用的分子序列。抗体可以与靶细胞的细胞膜表面原有的分子序列发生相互的结合作用包括：与靶细胞的细胞膜表面抗原作用，或者与非抗原蛋白作用。另外，靶细胞内表达的蛋白经降解等过程而递呈(包含但不限于MHC递呈途径)到细胞膜表面的蛋白片段或短肽所形成的特异性嵌合抗原受体靶点，抗体也可以与此类靶点结合而被激活。本申请中的双靶向是指该嵌合抗原受体基因工程表达载体上同时插入了两种针对靶细胞表面的两种相同或者不同的分子(抗原)所对应的ScFv抗体序列。优选的，第一抗体序列或第二抗体序列均为ScFv抗体序列，选自：与靶细胞的细胞膜表面抗原相互作用的分子序列。
进一步的方案，第一抗体序列或第二抗体序列选自：CD1、CD2、CD3、CD4、CD5、CD7、CD8、CD9、CD10、CD11a、CD11b、CD13、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD28、CD30、CD33、CD34、CD36、CD37、CD38、CD40、CD41、CD42、CD43、CD44、CD45、CD56、CD58、CD64、CD66c、CD70、CD71、CD73、CD74、CD80、CD81、CD83、CD86、CD90、CD94、CD97、CD99、CD102、CD117、CD123、CD133、CD134、CD137、CD138、CD200、GD2、EGFR VIII、GD3、NG2、CA125、CA153、CA199、CA242、CA724、HLA-DR、BDCA4、TCRαβ、TCRγδ、A33、CEA、CEACAM6、CS1、EGFR、ERBB2、FGF19、HER3、IL3Ra、NCAM、NKG2A、BCMA、NTBA、PD-1、PDL-1、PSMA、PSGL1、ROR1、VEGF、HER2、NY-ESO、GPC3、PSCA、PDL1、EPCAM、c-MET、MESO、AFP、FRa、EGFRvIII、IL13Ra2、GBM、LMP1、CD171、FGFR4、CSPG4中的至少一种。
进一步的方案，第一抗体序列和第二抗体序列的组合选自CD1、CD2、CD3、CD4、CD5、CD7、CD8、CD9、CD10、CD11a、CD11b、CD13、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD28、CD30、CD33、CD34、CD36、CD37、CD38、CD40、CD41、CD42、CD43、CD44、CD45、CD56、CD58、CD64、CD66c、CD70、CD71、CD73、CD74、CD80、CD81、CD83、CD86、CD90、CD94、CD97、CD99、CD102、CD117、CD123、CD133、CD134、CD137、CD138、CD200、GD2、EGFR VIII、GD3、NG2、CA125、CA153、CA199、CA242、CA724、HLA-DR、BDCA4、TCRαβ、TCγδ、A33、CEA、CEACAM6、CS1、EGFR、ERBB2、FGF19、HER3、IL3Ra、NCAM、NKG2A、BCMA、NTBA、PD-1、PDL-1、PSMA、PSGL1、ROR1、VEGF、HER2、NY-ESO、GPC3、PSCA、PDL1、EPCAM、c-MET、MESO、AFP、FRa、EGFRvIII、IL13Ra2、GBM、LMP1、CD171、FGFR4、CSPG4等上述抗原对应的抗体中任意两个相同抗体或者不同抗体间的组合，
优选的，包含但不限于：CD19/CD19、CD19/CD20、CD19/CD123、CD19/CD66c、CD19/CD58、CD19/CD56、CD19/CD13、CD19/CD33、CD19/CD44、CD19/CD73、CD19/CD86、CD19/CD86、CD19/CD99、CD19/CD24、CD19/CD200、CD19/CD97、CD19/BDCA4、CD19/CD133、CD19/CD15、CD19/NG2、CD19/sIgM、CD20/CD20、CD20/CD20、CD20/CD123、CD20/CD66c、CD20/CD58、CD20/CD56、CD20/CD13、CD20/CD33、CD20/CD44、CD20/CD73、CD20/CD86、CD20/CD86、CD20/CD99、CD20/CD24、CD20/CD200、CD20/CD97、CD20/BDCA4、CD20/CD133、CD20/CD15、CD20/NG2、CD20/sIgM、CD22/CD22、CD22/CD22、CD22/CD123、CD22/CD66c、CD22/CD58、CD22/CD56、CD22/CD13、CD22/CD33、CD22/CD44、CD22/CD73、CD22/CD86、CD22/CD86、CD22/CD99、CD22/CD24、CD22/CD220、CD22/CD97、CD22/BDCA4、CD22/CD133、CD22/CD15、CD22/NG2、CD22/sIgM、CD33/CD117、CD33/CD34、CD56/CD81、CD7/CD99、CD3/CD99、CD138/BCMA、CD33/CD123、CD33\CD133等。上述组合可以单一，或者联合使用，从而可减少由于靶点丢失造成的治疗失败。针对B系急性淋巴细胞白血病和B系淋巴瘤，优选地，采用CD19/CD19的组合；针对AML，优选地，采用CD33/CD117或CD33/CD34组合；针对多发性骨髓瘤，优选地，采用BCMA/BCMA、CD138/CD138、CD138/BCMA组合；针对神经母细胞瘤，优选地，采用GD2/CD2或CD56/CD81组合；针对T系急性淋巴细胞白血病和T系淋巴瘤，优选地，采用CD7/CD99、CD3/CD99、CD7/CD34或CD3/CD34组合。针对肝癌，优选的，采用EPCAM/AFP或EPCAM/GPC3组合；针对乳腺癌，优选的，采用EPCAM/HER2或EPCAM/MESO组合；针对前列腺癌，优选的，采用EPCAM/PSMA组合；针对结直肠癌，优选的，采用EGFR/HER2或EGFR/CEA组合。
上述方案中提到的不同ScFv抗体组合，是根据急性白血病细胞等肿瘤细胞独特的免疫表型特征设计的，上述组合中CD19和CD22基本上是所有B系白血病细胞都表达的细胞膜抗原，而CD20是所有B系淋巴瘤淋巴瘤均表达的细胞膜抗原，同时联合相同的抗体序列或者其它抗体序列，可以做到特异性杀伤肿瘤细胞的作用，同时对正常的B淋巴细胞没有影响。我们进行了双靶向嵌合抗原受体的临床实验，11例入组患者均在CAR-T细胞输注后8～28天获得分子缓解(MRD<0.01％)，从而证明了上述基于双靶向嵌合抗原受体治疗的有效性。
或者，所述的第一抗体序列和第二抗体序列还包括针对靶细胞内表达的蛋白经降解等过程而递呈到细胞膜表面的蛋白片段或短肽所形成的特异性前核抗原受体靶点设计的抗体序列。具体是指细胞内一些肿瘤源性基因突变蛋白产物或异常表达蛋白产物经蛋白酶体或其它机制降解后，经MHC分子或其它分子递呈到细胞膜表面，可以作为免疫细胞识别的靶点。针对这些递呈的肿瘤细胞突变蛋白产物或异常表达蛋白产物设计相应的抗体序列，经组装成 嵌合抗原受体基因工程载体和病毒，通过转染免疫细胞并表达，可以识别特定的肿瘤细胞从而产生靶向杀伤作用。
优选的，突变蛋白产物或异常表达蛋白产物包括但不限于ABL1、ALK、ASXL1、ATM、BCOR、BCORL1、BRAF、CALR、CBL、CEBPA、CSF3R、CSMD1、CUX1、DNMT3A、EP300、ETNK1、ETV6、EZH2、FLT3、GATA1、GATA2、GNAS、IDH1、IDH2、IKZF1、JAK1、JAK2、JAK3、KIT、KMT2A、KMT2C、KMT2D、KRAS、MPL、NF1、NOTCH1、NPM1、NRAS、PDGFRA、PHF6、PRPF40B、PRPF8、PTEN、PTPN11、RAD21、ROBO1、ROBO2、RUNX1、SETBP1、SF1、SF3A1、SH2B3、SMC1A、SMC3、SRSF2、STAG2、SUZ12、TET2、TP53、U2AF1、U2AF2、WT1、ZRSR2等基因的突变；包括但不限于AML1-ETO、EWS-ETV1、NPM1-RARα、SCAF11-PDGFRA、AML1-MDS1/EVI1、EWS-ETV4、NUMA1-RARα、SDC4-ROS1、AML1-MTG16、EWSR1-ZNF384、NUP214-ABL1、SEC31A-ALK、ATF7IP-JAK2、EZR-ROS1、NUP98-HoxA9/11/13、SET-CAN、AXL-MBIP、FAM46C-MYC、NUP98-HoxC11、SFPQ-ABL1、BCOR-RARα、FGFR2-CIT、NUP98-HoxD13、SIL-TAL1、BCR-ABL1、FGFR2-WARS、NUP98-PMX1、SLC34A2-ROS1、BMP6-MYC、FGFR3-TACC3、OFD1-JAK2、SNX2-ABL1、CARS-ALK、FIG-ROS1、P2RY8-CRLF2、SPECC1-PDGFRB、CBFβ-MYH11、FIP1L1-PDGFRA、PAG1-ABL2、SQSTM1-ALK、CCDC6-PDGFRB、FIP1L1-RARα、PAX5-ASXL1、SSBP2-CSF1R、CCDC6-ROS1、FOXJ2-MEF2D、PAX5-AUTS2、SSBP2-JAK2、CCND1-MYC、FOXO3-MYC、PAX5-CBFA2T3、SSBP2-PDGFRB、CCND3-MYC、FOXP1-ABL1、PAX5-ESRRB、STAT5b-RARα、CD74-ROS1、FUS-CHOP、PAX5-JAK2、STRN3-JAK2、CLTC-ALK、FUS-FEV、PAX5-KIF3B、STRN-ALK、CREBBP-ZNF384、HBA1-CD74、PAX5-MLLT3、STRN-PDGFRA、DEK-CAN、HLXB9-ETV6、PAX5-RNF38、SYNRG-ZNF384、DGKH-ZFAND3、IQGAP2-TSLP、PAX5-SP2、TAF15-ZNF384、E2A-HLF、JAK2-SNX29、PAX5-TMEM14B、TCF3-ZNF384、E2A-PBX1、KDELR2-ROS1、PAX5-ZNF521、TERF2-JAK2、EBF1-JAK2、KIF5B-ALK、PCM1-JAK2、TFG-ALK、EBF1-PDGFRB、KIF5B-PDGFRA、PLEKHA6-NTRK3、TLS-ERG、EML4-ALK、KIF5B-RET、PLZF-RARα、TNIP1-PDGFRB、EP300-ZNF384、KLC1-ALK、PML-RARα、TPM3-ALK、ETV6-ABL、LRIG3-ROS1、PPF1BP1-JAK2、TPM3-ROS1、ETV6-ABL1、MEF2D-BCL9、PPFIBP1-ALK、TPM4-ALK、ETV6-ABL2、MEF2D-DAZAP1、PRKAR1A-RARα、TPR-JAK2、ETV6-JAK2、MEF2D-HNRNPUL1、PTK2B-KDM6A、TPR-MET、ETV6-NIPBL、MEF2D-JAK2、PTK2B-STAG2、TTL-ETV6、ETV6-NTRK3、MEF2D-SS18、PTPN3-ALK、TXNDC5-MYC、ETV6-PDGFRA、MLL rearrangement、PTPRZ1-MET、TYK2-MYB、ETV6-PDGFRB、MN1-ETV6、RANBP2-ABL1、UQCRH-EWS、ETV6-RUNX1、MN1-TEL、RB1-MYC、XBP1-MYC、ETV6-STL、MSN-ALK、RBM15-MKL1、ZC3HAV1-ABL2、EWS-ATF1、MYH9-ALK、RCSD1-ABL1、ZEB2-PDGFRB、EWS-CHN、MYH9-IL2RB、RCSD1-ABL2、ZMIZ1-ABL1、EWS-CHOP、NCOR1-LYN、RET-KTN1、ZMYM2-FGFR1、EWS-CREB1、NPM1-ALK、RET-RAB6IP2、ZNF384-RNF180、EWS-ERG、NPM1-MLF1、RNF213-ALK、ZSG-UQCRH等融合基因产物；包含但不限于IG和TCR重排产生的蛋白产物等。
进一步，共刺激信号通路序列包括按照转录方向依次连接的第一引导肽序列、第一抗体轻链VL序列、第一抗体连接肽序列、第一抗体重链VH序列、铰链区序列、跨膜区序列和共刺激分子序列；效应器信号通路序列包括按照转录方向依次连接的第二引导肽序列、第二抗体轻链VL序列、第二抗体连接肽序列、第二抗体重链VH序列、铰链区序列、跨膜区序列以及效应器分子序列；所述的共刺激分子序列与第二引导肽序列之间连接有自剪切序列。
进一步的方案，所述的共刺激分子序列包括激活性共刺激分子序列，所述激活性共刺激分子序列选自4-1BB、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1、ICOS序列中的至少一种；
优选的，共刺激分子序列为4-1BB序列。
进一步的方案，所述的共刺激分子序列包括抑制性共刺激分子序列，所述的抑制性共刺激分子序列包括PD1、CTLA4的效应器分子序列；
优选的，包括PI3K、Akt、TCRζ、ZAP70、PKC序列；
优选的，抑制性共刺激分子序列包括PI3K、Akt序列。
所述的共刺激分子序列选自4-1BB、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS序列中的至少一种；优选的，共刺激分子序列为4-1BB序列。
所述的效应器分子序列选自CD3ζ。
自剪切序列选自P2A、T2A、F2A、E2A、BmCPV2A、BmIFV2A序列中的至少一种。优选的，自剪切序列为P2A序列。
进一步的方案，第一抗体序列或第二抗体序列选自SEQ ID NO:11～50所示的核苷酸序列；优选的，针对CD19阳性淋巴细胞白血病和淋巴瘤，选择SEQ ID NO:11～40中的序列；优选的，针对神经母细胞瘤，选择SEQ ID NO:41～50中的序列。也可以使用相同的上述序列用于急性白血病和淋巴瘤B系恶性肿瘤的细胞免疫治疗。
进一步的，第一引导肽序列或者第一引导肽序列包括但不限于：CD1、CD2、CD3、CD4、CD5、CD7、CD8、CD9、CD10、CD11a、CD11b、CD13、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD36、CD37、CD38、CD40、CD41、CD42、CD43、CD44、CD45、CD56、CD58、CD66c、CD70、CD73、CD74、CD80、CD81、CD86、CD94、CD97、CD99、CD102、CD123、CD133、CD134、CD137、CD138、CD200、EGFR、GD3、NG2、CA125、A33、CEA、CEACAM6、CS1、EGFR、ERBB2、FGF19、HER3、IL3Ra、NCAM、NKG2A、BCMA、NTBA、PD-1、PDL-1、PSMA、PSGL1、ROR1、VEGF等膜蛋白引导肽序列。优选的，引导肽序列选自SEQ ID NO:1～10。
连接肽序列是指将VL和VH串联起来的氨基酸序列，优选的序列为SEQ ID NO:51所示的核苷酸序列。铰链区序列是指介导激活信号从ScFv抗体传递到跨膜区的氨基酸序列，优选的序列为SEQ ID NO:52所示的核苷酸序列。跨膜区序列是指介导激活信号从铰链区跨膜传递到胞内区的氨基酸序列，优选的序列为SEQ ID NO:53所示的核苷酸序列。共刺激分子包含但不限于4-1BB、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS等分子的序列，优选序列为SEQ ID NO:54所示的核苷酸序列。自剪切序列是指包含但不限于P2A、T2A、F2A、E2A、BmCPV2A、BmIFV2A序列，优选地，选自SEQ ID NO:55～58所示的核苷酸序列。效应器分子序列是指包含但不限于CD3ζ序列，优选SEQ ID NO:59所示的核苷酸序列。
本发明的第二目的是提供一种采用如上所述的嵌合抗原受体基因工程载体制备的病毒，嵌合抗原受体基因工程载体与包装质粒转染于包装细胞系中，获得相应病毒；优选的，所述的病毒选自慢病毒、逆转录病毒、腺病毒、腺相关病毒等。
本发明的第三目的是提供一种嵌合抗原受体基因工程载体修饰的免疫细胞，所述的免疫细胞转染了如上所述的嵌合抗原受体基因工程载体或如上所述的病毒；
优选的，免疫细胞包括T细胞、NK细胞、单核巨噬细胞；所述的T细胞包括未经改 造的T细胞、改造的T细胞、自体T细胞、异体T细胞；所述的NK细胞包括未经改造的NK细胞、改造的NK细胞、自体NK细胞、异体NK细胞；优选的，T细胞选自CD4阳性T淋巴细胞、CD8阳性T淋巴细胞、CD4和CD8双阳性T淋巴细胞中的至少一种；
优选的，嵌合抗原受体基因工程载体修饰的免疫细胞包括CD4阳性T淋巴细胞和CD8阳性T淋巴细胞，且CD4阳性T淋巴细胞的数量占总细胞数量的40％～60％。
本发明的第四目的是提供如上所述的嵌合抗原受体基因工程载体，或者如上所述的病毒，或者如上所述的免疫细胞在制备治疗肿瘤疾病或免疫系统疾病的药物中的应用；
优选的，治疗肿瘤疾病的药物针对的疾病包括：急性淋巴细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、慢性粒细胞白血病、毛细胞白血病、肥大细胞白血病、浆细胞白血病、骨髓瘤、骨髓增生性疾病、幼年型粒单核细胞型白血病、混合系白血病、各种淋巴瘤和淋巴母细胞瘤、何杰金氏病、神经母细胞瘤、肺母细胞瘤、胰母细胞瘤、肾母细胞瘤、原始神经外胚层肿瘤、肾癌、膀胱癌、生殖腺肿瘤、横纹肌肉瘤、滑膜肉瘤、骨肿瘤、骨肉瘤、尤文肉瘤、软组织肉瘤、黑色素瘤、各种小圆细胞瘤、肾、输尿管、膀胱、尿道肿瘤、肾上腺皮质肿瘤、肾上腺神经母细胞瘤、视网膜瘤、星形细胞瘤、脑胶质瘤、室管膜瘤、软黄囊瘤、畸胎瘤、髓母细胞瘤、小细胞和非小细胞肺癌和支气管肿瘤、朗格罕氏细胞淋巴组织细胞增生症、嗜酸细胞增多综合征、嗜酸细胞肿瘤、各种类型皮肤癌、各种纤维瘤和纤维肉瘤、乳腺癌、口腔癌、鼻咽癌、颅咽管瘤、唇癌、涎腺肿瘤、食管癌、胃癌、小肠肿瘤、结直肠癌、胰腺癌、肝癌、胆管癌、心脏肿瘤、阴道肿瘤、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸肿瘤、多发性内分泌瘤综合征、垂体瘤、胸腺瘤、粘液瘤；
优选的，治疗免疫系统疾病的药物针对的疾病包括：自身免疫性疾病、病毒感染性疾病、细菌性或真菌性感染疾病；其中，自身免疫性疾病包括类风湿性关节炎、慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、口眼干燥综合征、强直性脊柱炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病。细菌性或真菌性感染疾病包括葡萄球菌、链球菌、变形杆菌、绿脓杆菌痢疾杆菌、百日咳杆菌、放线菌、破伤风杆菌、产气荚膜杆菌、伤寒杆菌、霍乱弧菌、脑膜炎双球菌、炭疽杆菌、白喉杆菌、肺炎球菌、肺炎杆菌、肠球菌、不动杆菌、流血嗜血杆菌、大肠杆菌、军团菌、芽孢菌、厌氧菌感染；各种念珠菌、曲霉菌、毛霉菌、隐球菌、马内菲青霉菌、孢子丝菌、着色芽生菌感染；立克次氏体、螺旋体、支原体、衣原体、各种原虫感染。
采用上述技术方案后，本发明与现有技术相比具有以下有益效果：
1、本发明通过改造嵌合抗原受体基因工程载体，插入调控元件、启动子、优化碱基序列等使载体的安全性好，转染效率高，转染的免疫细胞能够快速扩增，杀伤效果大大提高。
2、本发明通过采用双靶向的嵌合抗原受体基因工程载体作为CAR-T治疗的的载体，相应的载体经病毒包装后可以进行自体或异体免疫细胞(T细胞和/或NK细胞和/或γ/δT细胞)的转染和扩增，用以细胞免疫治疗。双靶向嵌合抗原受体可以针对细胞膜表面的一种或者几种抗原设计，相同抗原可以为相同或者不同的ScFv序列。嵌合抗原受体与靶细胞膜表面抗原结合后，经过信号转导，可以实现共刺激信号分子和效应器信号分子的同时且单独激活，从而激活修饰的免疫细胞发挥抗肿瘤等的作用。因此，与单一靶点的嵌合抗原受体免疫细胞对靶细胞的识别相比，这种方式更加精确；同时双靶向的联合识别作用也可以更好的防止单一靶点的脱靶，增强对靶细胞的特异性和杀伤力。另外，能够降低与正常细胞表面类似的或者异变分子的结合作用，减少了其攻击正常细胞的几率，提高了使用的安全性。
3、本申请的嵌合抗原受体序列中，跨膜区序列与共刺激信号分子或者效应器信号分子之间没有linker，能够缩短信号通路，提高信号转导效率，从而使得T细胞等免疫细胞的激活效率提高，对于提高CART细胞的体外杀伤效果非常重要。
下面结合附图对本发明的具体实施方式作进一步详细的描述。
附图说明
附图作为本发明的一部分，用来提供对本发明的进一步的理解，本发明的示意性实施例及其说明用于解释本发明，但不构成对本发明的不当限定。在附图中：
图1是本发明的嵌合抗原基因工程载体的结构示意图；
图2是独立表达的共刺激分子信号通路和效应器分子信号通路的核心序列；
图3是本发明双靶向嵌合抗原受体结构图；
图4是本申请的载体与其他公司慢病毒载体的转染效率的比较结果；其中，a～c为其它公司慢病毒载体包装的转染效率，d～e为采用本专利载体包装的慢病毒转染效率，f为未转染的对照。
图5是本发明CD19双靶向特异性CAR的T淋巴细胞体外细胞杀伤实验；其中，图5a-图5c分别为试验组第0天、第1天以及第2天的体外细胞杀伤实验结果；图5d-图5f分别为对照组第0天、第1天以及第2天的体外细胞杀伤实验结果；
图6是本发明一例难治性急性淋巴细胞白血病CD19双靶向特异性CAR-T输注前后微量残留病检测结果；6A-6C是CD19双靶向特异性CAR-T细胞输注前残留病的检测结果；6D-6F是输注CD19双靶向特异性CAR-T细胞后第8天的检测结果；
图7是一例二次复发急性淋巴细胞白血病CD19双靶向特异性CAR-T输注前后微量残留病检测结果；
图8是一例移植后复发急性淋巴细胞白血病CD19双靶向特异性CAR-T输注前后微量残留病检测结果；
图9是CD19双靶向特异性CAR-T细胞体外增殖实验与其他公司的比较结果；其中，起始细胞的数目为1×10 7；
图10是本申请的Double-CAR-CD19与Novartis CD19-CAR杀伤效果对比(E/T＝1:20)；图10A是0h，图10B是16h；
图11是本申请双靶向载体其他慢病毒载体公司转染细胞体外杀伤效果比较；
图12是CD4与CD8的合适比例与细胞毒作用的关系；
图13是入组患者预计2年的无事件生存率(Event-free survival,EFS)；
图14是入组患者预计2年的总生存率(Overall survival,OS)。
需要说明的是，这些附图和文字描述并不旨在以任何方式限制本发明的构思范围，而是通过参考特定实施例为本领域技术人员说明本发明的概念。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述，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本实施例以嵌合抗原受体基因工程载体包含一个共刺激信号通路序列和一个效应器信号通路序列为例进行详细阐述，还可以包括多个共刺激信号通路序列和多个效应器信号通路序列。
1)首先，构建嵌合抗原受体基因工程载体。通过基因合成的方法合成相应的序列，并测序验证。选择合适的酶切位点将上述序列插入到腺病毒、逆转录病毒或慢病毒载体中合适的位置，并经测序验证。如图1和图2所示，嵌合抗原受体基因工程载体包括：
RRE序列插入到载体的多克隆位点的上游，WPRE序列和cPPT序列插入到多克隆位点的下游，EF1A启动子插入到RRE序列与多克隆位点的上游之间，用于启动插入多克隆位点的嵌合抗原受体基因。对嵌合抗原受体结构蛋白上游的序列进行优化，优化后序列如SEQ ID NO60所示，使得外源蛋白表达稳定且持续，对于提高CART细胞的体外杀伤效果非常重要。
引导肽序列构建：膜蛋白引导肽序列，优选引导肽序列见SEQ ID NO:1～10。
与靶细胞上A或B靶点对应的第一抗体和第二抗体的序列，也就是ScFv抗体轻链VL序列和重链VH序列构建：针对急性白血病和淋巴瘤，我们选择SEQ ID NO:11～40中的序列，可以选择相同的或者不同的两个序列用于这些疾病的细胞免疫治疗。以CD19为靶点治疗CD19阳性白血病和淋巴瘤的的ScFv抗体轻链VL序列和重链VH序列为例，可以选择SEQ ID NO:11～14，19～40中的序列，可以选择相同的基于CD19的上述序列用于B系恶性肿瘤的细胞免疫治疗。针对神经母细胞瘤，我们选择SEQ ID NO:19～20，41～50中的序列。
连接肽序列构建：指将VL和VH串联起来的氨基酸序列，优选的序列见SEQ ID NO:51中提供的序列。铰链区序列构建：指介导激活信号从ScFv传递到跨膜区的氨基酸序列，优选的序列见SEQ ID NO:52中提供的序列。跨膜区序列构建：是指介导激活信号从铰链区跨膜传递到胞内区的氨基酸序列，优选的序列见SEQ ID NO:53中提供的序列。共刺激分子序列构建：是指包含但不限于4-1BB、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS等分子的序列。优选地，4-1BB的序列见SEQ ID NO:54中提供的序列。自剪切序列构建：指包含但不限于P2A、T2A、F2A、E2A、BmCPV2A、BmIFV2A等，优选地，P2A序列见SEQ ID NO:55～58中提供的序列。效应器分子序列：是指包含但不限于CD3ζ序列，相应的序列见SEQ ID NO:59中提供的序列。
序列组装：除了一般的表达载体组件，以CD19为靶点的嵌合抗原受体基因工程载体序列组成包含但不限于下述结构：CD8引导肽—CD19ScFv—铰链区—CD8跨膜区—4-1BB—P2A—CD8引导肽—CD19ScFv—铰链区—CD8跨膜区—CD3zeta-TGA。本申请的跨膜区序列与共刺激信号分子或者效应器信号分子之间没有linker，能够缩短信号通路，提高信号转导效率，从而使得T细胞等免疫细胞的激活效率提高，对于提高CART细胞的体外杀伤效果非常重要。
通过设计合适的酶切位点，将上述序列进行酶切后与采用相同酶切的载体进行连接，连接产物转化DH5α或stbl3感受态大肠杆菌，阳性克隆接种后过夜，细菌抽提质粒后酶切鉴定和送测序。测序结果完全正确的载体即为本实施例的嵌合抗原受体基因工程载体。共刺激信号通路序列和效应器信号通路序列分别表达后的双靶向嵌合抗原受体的结构如图3所示。
其次，通过将上述载体和病毒包装质粒进行合适比例混合，转染293T细胞72h和96h后收集培养上清并进行病毒浓缩。再次，通过实时定量PCR方法，结合病毒的不同浓度梯度转染293细胞进行病毒滴度的确定。再次，通过收集人外周血不同的免疫细胞进行上述病毒的转染，确保转染效率在20％以上，从中找到合适的MOI值用于临床治疗。需要指出的 是，每批病毒均需要进行上述操作，确保临床试验的疗效稳定可靠。
再次，通过转染人外周血不同的免疫细胞进行转染效率的检测，对CD19为靶点的CAR-T而言，采用针对ScFv的Fab片段作为流式细胞检测阳性转染效率的客观依据。也可以采用实时定量PCR的方法进行转染效率的检测方案。
再次，通过将病毒转染的CAR-T细胞等免疫细胞与CD19阳性细胞株进行一定比例混合和共孵育，在0h、24h和48h后通过流式细胞技术进行体外杀伤检测，比较不同孵育时间的细胞杀伤效果并与对照病毒进行比较。
再次，通过采集外周血0.5～2ml/Kg，磁珠分选获得CD3阳性T细胞或者CD56获得NK细胞，或者TCRγ/δ分选获得γ/δT细胞，加入CD3/CD28免疫磁珠进行刺激激活和病毒转染，继续给予CD3/CD28免疫磁珠、Interleukin7和Interleukin15进行体外扩增，一般7～8天可以扩增100倍以上，达到合适细胞数量后经洗涤回输人体。回输人体CAR-T阳性细胞数量在1×10 4/Kg～5×10 7/Kg，并根据体内负荷进行调整，优选地在2×10 5/Kg～5×10 7/Kg。
由于病毒转染阳性的细胞才具有特异性杀伤肿瘤细胞的作用，提高转染效率可以大大降低培养细胞的体系，节约前期生产成本。同时转染阴性的细胞在培养时也会被CD3/CD28免疫磁珠激活，与病毒转染阳性的细胞争夺营养，且由于转染阴性的细胞没有产生额外表达外源蛋白的压力，培养时间长后会导致转染体系中病毒转染阳性细胞比例的进一步降低。本发明的双靶向嵌合抗原受体载体制备的针对CD19阳性白血病和淋巴瘤病毒，经体外转染后一般转染效率在20％以上，绝大部分在40～70％之间，如附图4所示，图4中，a～c为其它慢病毒载体包装的转染效率，d～e为采用本项专利技术载体包装的慢病毒转染效率，f为未转染的对照。
本发明的双靶向嵌合抗原受体载体制备的针对CD19阳性白血病和淋巴瘤病毒，经体外转染后按照CAR-T细胞：CD19阳性白血病细胞株＝1:10～1:20的比例混合和共孵育，作为试验组；同时采用无关序列制备载体包装获得的病毒作为对照组。两组细胞对靶细胞的杀伤作用的结果如图5所示。其中，图5a-图5c分别为试验组第0天、第1天以及第2天的体外细胞杀伤实验结果；图5d-图5f分别为对照组第0天、第1天以及第2天的体外细胞杀伤实验结果。可以看出，与对照组相比，本方案的CD19双靶向嵌合抗原受体载体制备的病毒转染的CAR-T细胞在24h和48h共孵育后，对靶细胞的杀伤作用大大增强。
试验例1：一例复发的CD19阳性急性淋巴细胞白血病患者经此CD19双靶向嵌合抗原受体载体制备的病毒转染的CAR-T细胞，结果如附图6所示。图6中，6A-6C是CD19双靶向特异性CAR-T细胞输注前残留病的检测结果；6D-6F是输注CD19双靶向特异性CAR-T细胞后第8天的检测结果，MRD由33.1％降到低于0.01％的水平，说明其在CAR-T治疗后第8天，骨髓MRD达到分子缓解(MRD<0.01％)。
试验例2：一例二次复发后再诱导失败的CD19阳性急性淋巴细胞白血病患者经此CD19双靶向嵌合抗原受体载体制备的病毒转染的CAR-T细胞，结果如附图7所示.图7中，7A-7C是CD19双靶向特异性CAR-T细胞输注前残留病的检测结果，MRD为11.7％；7D-7F是输注CD19双靶向特异性CAR-T细胞后第8天的检测结果。说明在CAR-T治疗后第8天，骨髓MRD达到分子缓解(MRD<0.01％)。
试验例3：一例骨髓复发的CD19阳性淋巴母细胞性淋巴瘤/白血病患者经此CD19双靶向嵌合抗原受体载体制备的病毒转染的CAR-T细胞后检测结果如图8所示。图中，如8A-8C所示，没有输注CAR-T细胞前，骨髓中有大量的肿瘤细胞，MRD＝95.6％，而其在CAR-T治疗后第8天，骨髓中仅有少量肿瘤细胞(MRD＝0.012％)，如8D-8F所示；CAR-T治疗后第28天，骨髓MRD达到分子缓解(MRD<0.01％)，如8G-8I所示，说明治疗的过程快速且有效。
试验例4：CAR-T细胞的体外扩增实验发现，如图9所示，采用本申请双靶向嵌合抗原受体载体制备的慢病毒、其他公司的慢病毒在体外转染人T淋巴细胞后的生长大为不同。其它公司的病毒转染后CAR-T细胞在7天的时间内扩增了14倍左右，而本申请载体转染的CAR-T细胞在7天的少见内扩增了160倍，体外扩增速度大大提高。由于这些患者均处于疾病终末期，体外培养扩增时间的缩短对于治疗安全非常重要。
试验例5：将本发明制备的病毒转染的CART细胞与来自其它公司的CART细胞进行了体外杀伤实验的比较，实验结果如图10所示。结果表明，与其它公司的CD19-CAR杀伤效果对比，本发明的双靶向基因工程载体制备获得的CART细胞(Double-CAR-CD19)的体外杀伤活性明显增强，其结果如图10所示。其中，图10A为输注0小时效果的比较，图10B为输注16小时的效果比较。
试验例6：将本申请的体外杀伤作用与专利CN103483452A中的技术进行了比较，本申请着重提出效靶比的问题。效靶比是效应器细胞(CAR-T细胞)：靶细胞(肿瘤细胞)，我们在1:10～1:20的时候可以获得99.9％的杀伤效果，专利CN103483452A中做的是1～50:1，得到的杀伤效果也达不到本申请的水平。如图11所示。说明：采用E:T不同比例组合进行体外杀伤实验，在E:T＝1:20时，可以将99.9％的靶细胞杀死，而专利CN103483452A的CAR-T在E:T＝50:1时尚达不到上述杀伤效果，值得注意的是，由于T细胞与靶细胞存在非特异性杀伤，在E:T＝1:1或更低时，专利CN103483452A的CAR-T细胞与对照无显著差异。
试验例7：我们在体外研究中发现，转染阳性的CAR-T细胞中CD4与CD8细胞的合适比例是取得更好细胞毒作用的关键因素之一，通过体外实验我们发现，当CD4:CD8比例介于40％～60％之间时，可以获得对靶细胞的高效杀伤，而由于患者接受化疗药物等，其CD4:CD8细胞比例常常偏离上述范围，对其临床疗效的发挥影响巨大。相应的检测结果见图12所示。
试验例8：CAR-T细胞获得高的转染效率、高效扩增和合适比例是造成体内高效杀伤靶细胞和获得体内临床疗效的基础，采用本专利技术的CAR-T细胞治疗临床入组患者，所有入组29例难治、复发的CD19阳性血液系统恶性肿瘤患者，除两例患者未检测MRD外，其余27例患者均在CD19-CAR-T治疗后8天～1个月获得形态学完全缓解和分子缓解，后者经过流式细胞技术检测微量残留病(MRD)均<0.01％。29例患者中2例早期死于严重的中枢神经系统细胞因子风暴，5例患者复发，其中2例患者经二次CAR-T治疗后获得再次形态和分子缓解.经过近两年的临床观察，入组患者预计两年的无事件生存率为62.1％，总生存率达到72.7％，结果如图13和图14所示。
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，任何熟悉本专利的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。

Claims (15)

  1. 一种嵌合抗原受体基因工程载体，其特征在于，包括慢病毒载体骨架、连接在慢病毒载体骨架上的调控元件和嵌合抗原受体基因序列，所述的调控元件包括WPRE元件、cPPT元件和RRE元件。
  2. 根据权利要求1所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的嵌合抗原受体基因序列的上游插入有哺乳类组成型启动子，优选EF1A启动子。
  3. 根据权利要求1或2所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的嵌合抗原受体基因序列包括独立表达的共刺激信号通路序列和效应器信号通路序列；
    或者，所述的嵌合抗原受体基因序列包括融合表达的共刺激信号通路序列和效应器信号通路序列。
  4. 根据权利要求3所述的一种嵌合抗原受体基因工程载体，其特征在于，独立表达的共刺激信号通路序列和效应器信号通路序列中，共刺激信号通路序列按照转录方向包括依次连接的第一引导肽序列、第一抗体序列和共刺激分子序列，所述的效应器信号通路序列按照转录方向包括依次连接的第二引导肽序列、第二抗体序列和效应器分子序列；
    所述的共刺激分子序列与第二引导肽序列之间，或者效应器分子序列与第一引导肽序列连接有自剪切序列。
  5. 根据权利要求4所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的第一抗体序列或第二抗体序列选自：与靶细胞的细胞膜表面分子相互作用的分子序列、与靶细胞内部递呈到其细胞膜表面的特异分子相互作用的分子序列；
    优选的，所述的第一抗体序列或第二抗体序列选自：CD1、CD2、CD3、CD4、CD5、CD7、CD8、CD9、CD10、CD11a、CD11b、CD13、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD28、CD30、CD33、CD34、CD36、CD37、CD38、CD40、CD41、CD42、CD43、CD44、CD45、CD56、CD58、CD64、CD66c、CD70、CD71、CD73、CD74、CD80、CD81、CD83、CD86、CD90、CD94、CD97、CD99、CD102、CD117、CD123、CD133、CD134、CD137、CD138、CD200、GD2、EGFR VIII、GD3、NG2、CA125、CA153、CA199、CA242、CA724、HLA-DR、BDCA4、TCRαβ、TCRγδ、A33、CEA、CEACAM6、CS1、EGFR、ERBB2、FGF19、HER3、IL3Ra、NCAM、NKG2A、BCMA、NTBA、PD-1、PDL-1、PSMA、PSGL1、ROR1、VEGF、HER2、NY-ESO、GPC3、PSCA、PDL1、EPCAM、c-MET、MESO、AFP、FRa、EGFRvIII、IL13Ra2、GBM、LMP1、CD171、FGFR4、CSPG4中的至少一种；
    优选的，第一抗体序列或第二抗体序列包括SEQ ID NO:11～50所示的核苷酸序列；
    优选的，针对急性白血病和淋巴瘤，包括SEQ ID NO:11～40中的序列；
    优选的，针对神经母细胞瘤，包括SEQ ID NO:41～50中的序列。
  6. 根据权利要求5所述的一种嵌合抗原受体基因工程载体，其特征在于，第一抗体序列和第二抗体序列的组合选自CD1、CD2、CD3、CD4、CD5、CD7、CD8、CD9、CD10、CD11a、CD11b、CD13、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD27、CD28、CD30、CD33、CD34、CD36、CD37、CD38、CD40、CD41、CD42、CD43、CD44、CD45、CD56、CD58、CD64、CD66c、CD70、CD71、CD73、CD74、CD80、CD81、CD83、CD86、CD90、CD94、CD97、CD99、CD102、CD117、CD123、CD133、CD134、CD137、CD138、CD200、GD2、EGFR VIII、GD3、NG2、CA125、CA153、CA199、CA242、CA724、HLA-DR、BDCA4、TCRαβ、TCγδ、A33、CEA、CEACAM6、CS1、EGFR、ERBB2、FGF19、HER3、IL3Ra、NCAM、NKG2A、BCMA、NTBA、PD-1、PDL-1、PSMA、PSGL1、ROR1、VEGF、HER2、NY-ESO、GPC3、PSCA、PDL1、EPCAM、c-MET、MESO、AFP、FRa、EGFRvIII、IL13Ra2、GBM、LMP1、CD171、FGFR4、CSPG4抗原对应的抗体中任意两个相同抗体或者不同抗体间的组合；
    优选的，包括：CD19/CD19、CD19/CD20、CD19/CD123、CD19/CD66c、CD19/CD58、CD19/CD56、CD19/CD13、CD19/CD33、CD19/CD44、CD19/CD73、CD19/CD86、CD19/CD86、CD19/CD99、CD19/CD24、CD19/CD200、CD19/CD97、CD19/BDCA4、CD19/CD133、CD19/CD15、CD19/NG2、CD19/sIgM、CD20/CD20、CD20/CD20、CD20/CD123、CD20/CD66c、CD20/CD58、CD20/CD56、CD20/CD13、CD20/CD33、CD20/CD44、 CD20/CD73、CD20/CD86、CD20/CD86、CD20/CD99、CD20/CD24、CD20/CD200、CD20/CD97、CD20/BDCA4、CD20/CD133、CD20/CD15、CD20/NG2、CD20/sIgM、CD22/CD22、CD22/CD22、CD22/CD123、CD22/CD66c、CD22/CD58、CD22/CD56、CD22/CD13、CD22/CD33、CD22/CD44、CD22/CD73、CD22/CD86、CD22/CD86、CD22/CD99、CD22/CD24、CD22/CD220、CD22/CD97、CD22/BDCA4、CD22/CD133、CD22/CD15、CD22/NG2、CD22/sIgM、CD33/CD117、CD33/CD34、CD56/CD81、CD7/CD99、CD3/CD99、CD138/BCMA、CD33/CD123、CD33\CD133。
  7. 根据权利要求4-6任意一项所述的一种嵌合抗原受体基因工程载体，其特征在于，共刺激信号通路序列包括按照转录方向依次连接的第一引导肽序列、第一抗体轻链VL序列、第一抗体连接肽序列、第一抗体重链VH序列、铰链区序列、跨膜区序列以及共刺激分子序列；
    效应器信号通路序列包括按照转录方向依次连接的第二引导肽序列、第二抗体轻链VL序列、第二抗体连接肽序列、第二抗体重链VH序列、铰链区序列、跨膜区序列以及效应器分子序列；
    所述的共刺激分子序列与第二引导肽序列之间连接有自剪切序列。
  8. 根据权利要求4-7任意一项所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的共刺激分子序列包括激活性共刺激分子序列，所述激活性共刺激分子序列选自4-1BB、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1、ICOS序列中的至少一种；
    优选的，共刺激分子序列为4-1BB序列。
  9. 根据权利要求4-7任一所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的共刺激分子序列包括抑制性共刺激分子序列，所述的抑制性共刺激分子序列包括PD1、CTLA4的效应器分子序列；
    优选的，包括PI3K、Akt、TCRζ、ZAP70、PKC序列；
    优选的，抑制性共刺激分子序列包括PI3K、Akt序列。
  10. 根据权利要求4-9任意一项所述的一种嵌合抗原受体基因工程载体，其特征在于，所述的效应器分子序列包括CD3ζ序列。
  11. 根据权利要求4-10任意一项所述的一种嵌合抗原受体基因工程载体，其特征在于，自剪切序列选自P2A、T2A、F2A、E2A、BmCPV2A、BmIFV2A序列中的至少一种；优选的，自剪切序列为P2A序列。
  12. 一种嵌合抗原受体基因工程载体修饰的免疫细胞，其特征在于，所述的免疫细胞转染了如权利要求1-11任意一项所述的嵌合抗原受体基因工程载体；
    优选的，免疫细胞包括T细胞、NK细胞、单核巨噬细胞；所述的T细胞包括未经改造的T细胞、改造的T细胞、自体T细胞、异体T细胞；所述的NK细胞包括未经改造的NK细胞、改造的NK细胞、自体NK细胞、异体NK细胞；
    优选的，T细胞选自CD4阳性T淋巴细胞、CD8阳性T淋巴细胞、CD4和CD8双阳性T淋巴细胞中的至少一种；
    优选的，嵌合抗原受体基因工程载体修饰的免疫细胞包括CD4阳性T淋巴细胞和CD8阳性T淋巴细胞，且CD4阳性T淋巴细胞的数量占总细胞数量的40％～60％。
  13. 如权利要求1-11任意一项所述的嵌合抗原受体基因工程载体或者如权利要求12所述的免疫细胞在制备治疗肿瘤疾病或免疫系统疾病的药物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，治疗肿瘤疾病的药物针对的疾病包括：急性淋巴细胞白血病、急性髓系白血病、慢性淋巴细胞白血病、慢性粒细胞白血病、毛细胞白血病、肥大细胞白血病、浆细胞白血病、骨髓瘤、骨髓增生性疾病、幼年型粒单核细胞型白血病、慢性粒单核细胞白血病、混合系白血病、各种淋巴瘤和淋巴母细胞瘤、何杰金氏病、神经母细胞瘤、肺母细胞瘤、胰母细胞瘤、肾母细胞瘤、原始神经外胚层肿瘤、肾癌、膀胱癌、生殖腺肿瘤、横纹肌肉瘤、滑膜肉瘤、骨肿瘤、骨肉瘤、尤文肉瘤、软组织肉瘤、黑色素瘤、各种小圆细胞瘤、肾、输尿管、膀胱、尿道肿瘤、肾上腺皮质肿瘤、肾上腺神经母细胞瘤、视网膜瘤、星形细胞瘤、脑胶质瘤、室管膜瘤、软黄囊瘤、畸胎瘤、髓母细胞瘤、小细 胞和非小细胞肺癌和支气管肿瘤、朗格罕氏细胞淋巴组织细胞增生症、嗜酸细胞增多综合征、嗜酸细胞肿瘤、各种类型皮肤癌、各种纤维瘤和纤维肉瘤、乳腺癌、口腔癌、鼻咽癌、颅咽管瘤、唇癌、涎腺肿瘤、食管癌、胃癌、小肠肿瘤、结直肠癌、胰腺癌、肝癌、胆管癌、心脏肿瘤、阴道肿瘤、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸肿瘤、多发性内分泌瘤综合征、垂体瘤、胸腺瘤、粘液瘤。
  15. 根据权利要求13所述的应用，其特征在于，治疗免疫系统疾病的药物针对的疾病包括：自身免疫性疾病、病毒感染性疾病、细菌性或真菌性感染疾病；
    优选的，自身免疫性疾病包括类风湿性关节炎、慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、口眼干燥综合征、强直性脊柱炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病；
    优选的，细菌性或真菌性感染疾病包括葡萄球菌、链球菌、变形杆菌、绿脓杆菌痢疾杆菌、百日咳杆菌、放线菌、破伤风杆菌、产气荚膜杆菌、伤寒杆菌、霍乱弧菌、脑膜炎双球菌、炭疽杆菌、白喉杆菌、肺炎球菌、肺炎杆菌、肠球菌、不动杆菌、流血嗜血杆菌、大肠杆菌、军团菌、芽孢菌、厌氧菌感染；各种念珠菌、曲霉菌、毛霉菌、隐球菌、马内菲青霉菌、孢子丝菌、着色芽生菌感染；立克次氏体、螺旋体、支原体、衣原体、各种原虫感染。
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