WO2018103503A1 - 一种封闭pdl1的用于抑制免疫逃脱的car-t转基因载体及其构建方法和应用 - Google Patents

一种封闭pdl1的用于抑制免疫逃脱的car-t转基因载体及其构建方法和应用 Download PDF

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WO2018103503A1
WO2018103503A1 PCT/CN2017/110656 CN2017110656W WO2018103503A1 WO 2018103503 A1 WO2018103503 A1 WO 2018103503A1 CN 2017110656 W CN2017110656 W CN 2017110656W WO 2018103503 A1 WO2018103503 A1 WO 2018103503A1
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seq
set forth
lentiviral
car
vector
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PCT/CN2017/110656
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French (fr)
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祁伟
俞磊
康立清
余宙
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上海优卡迪生物医药科技有限公司
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Priority to US16/464,682 priority Critical patent/US10736920B2/en
Priority to EP17879118.2A priority patent/EP3550024A4/en
Priority to KR1020197017093A priority patent/KR102157197B1/ko
Priority to JP2019530074A priority patent/JP6736109B2/ja
Publication of WO2018103503A1 publication Critical patent/WO2018103503A1/zh

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Definitions

  • the invention belongs to the field of medical biology, and in particular relates to a vector, in particular to a CAR-T transgenic vector for blocking immune escaping which blocks PDL1. Furthermore, the invention relates to a method and application of the construction of the vector.
  • tumor immunotherapy The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's attack on tumor cells (highly specific cell lysis).
  • Burnet and Thomas proposed the theory of "immuno-monitoring", which believed that the mutant tumor cells that often appear in the body can be cleared by the immune system and lay a theoretical foundation for tumor immunotherapy [Burnet FM.Immunological aspects of malignant disease.Lancet, 1967; 1:171-4].
  • various tumor immunotherapy including cytokine therapy, monoclonal antibody therapy, adoptive immunotherapy, vaccine therapy, and the like are successively applied to the clinic.
  • CAR-T In 2013, a more advanced tumor immunotherapy---CAR-T therapy was successfully used in clinical practice and showed unprecedented clinical efficacy.
  • CAR-T the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy.
  • CAR-T therapy is the most clinically advanced Novartis CLT019.
  • CLT019 is used to treat patients with relapsed and refractory acute lymphoblastic leukemia.
  • the progression-free survival rate of tumors is 67% for six months, and the longest response time is more than two years. .
  • CAR-T therapy is effective, it encounters many difficulties in the treatment of solid tumors.
  • One of the most important reasons is the PD1/PDL1 inhibitory immune checkpoint (as shown in Figure 1A), which combines the transmission of inhibition signals. It inhibits the immune activity of T cells, plays an important role in immune tolerance, and is also an important reason for the escape of tumor cells.
  • PD-1 (also known as CD279) is an immunosuppressive receptor, a type I transmembrane protein belonging to the CD28 family member, and a programmed cell death molecule-1 receptor in 1992 by Ishida et al [Ishida Y, Agata Y, Shibahara K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death [J].
  • the human PD-1 gene is located on chromosome 2q37.35 and encodes a transmembrane glycoprotein of approximately 55 kD.
  • PD-1 is widely expressed on the surface of activated T cells, B cells, monocytes and dendritic cells.
  • PD-1 is structurally 30% homologous to CTLA-4, and two tyrosines are present in the intracellular region.
  • Residues which are involved in an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N-terminus and an immunoreceptor tyrosine-dependent translation motif at the C-terminus (immunoreceptor tyrosin-based switch) Motif, ITSM);
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITSM immunoreceptor tyrosine-based switch
  • the extracellular domain is composed of an IgV-like domain, contains multiple glycosylation sites and is heavily glycosylated, which binds to the ligand and functions to inhibit T cell activation.
  • PD-L1 is overexpressed in most cancer tissues, including NSCLC, melanoma, breast cancer, glioma, lymphoma, leukemia and various urological tumors, digestive tract tumors, germline tumors, etc.
  • NSCLC nuclear-derived neurotrophic factor
  • melanoma breast cancer
  • glioma lymphoma
  • leukemia various urological tumors
  • digestive tract tumors germline tumors, etc.
  • J J Leukoc Biol, 2013, 94(1): 25-39.
  • PD-L1 is highly expressed and can pass Over-inhibition of RAS and PI3K/AKT signaling pathways, which in turn regulates cell cycle checkpoint protein and cell proliferation-associated protein expression, ultimately leading to inhibition of T cell proliferation [11].
  • One of the technical problems to be solved by the present invention is to provide a CAR-T transgenic vector for blocking immune escaping which blocks PDL1.
  • it saves costs and eliminates the expensive cost of purchasing antibody drugs.
  • the problem of inefficient delivery of the scFv gene in vivo is avoided.
  • the PDL1scFv gene transduced by lentivirus can effectively utilize the intracellular protein translation system and express the corresponding PDL1scFv in a large amount. After the circulation of the body fluid, a good PDL1 blocking effect can be achieved without affecting the therapeutic effect of CAR-T treatment.
  • the second technical problem to be solved by the present invention is to provide a method for constructing the carrier.
  • the third technical problem to be solved by the present invention is to provide an application of the carrier.
  • a CAR-T transgenic vector for blocking immune escaping comprising PDL1
  • PDL1 comprising:
  • An ampicillin resistance gene AmpR sequence for large-scale amplification of a strain of interest as shown in SEQ ID NO.
  • Prokaryotic replicon pUC Ori sequence for plasmid replication as shown in SEQ ID NO. 2;
  • a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells as set forth in SEQ ID NO.
  • a human EF1 ⁇ promoter for eukaryotic transcription of a chimeric antigen receptor gene as set forth in SEQ ID NO.
  • a humanized single-chain antibody of human PDL1 is PDL1scFv1 as shown in SEQ ID NO. 21, or PDL1scFv2 as shown in SEQ ID NO. 22, or as SEQ ID NO PDL1scFv3 shown in .23;
  • An IL6 signal peptide as set forth in SEQ ID NO.
  • a chimeric antigen receptor for a second generation CAR or a third generation CAR that is used to form a collection recognition, delivery, and initiation.
  • the humanized single-chain antibody of human PDL1 is PDL1 scFv1 as shown in SEQ ID NO.
  • the lentiviral packaging cis element can be a second generation lentiviral vector or a third generation lentiviral vector, preferably a third generation lentiviral vector.
  • the second generation lentiviral vector comprises: a lentiviral 5terminal LTR as set forth in SEQ ID NO. 5, a lentiviral 3terminal Self-Inactivating LTR as set forth in SEQ ID NO. 6, as shown in SEQ ID NO. Gag cis element, RRE cis element as shown in SEQ ID NO. 8, env cis element as shown in SEQ ID NO. A cPPT cis element as shown in SEQ ID NO.
  • the third generation lentiviral vector comprises: a lentiviral 5terminal LTR as set forth in SEQ ID NO. 5, a lentiviral 3terminal Self-Inactivating LTR as set forth in SEQ ID NO. 6, as shown in SEQ ID NO. a Gag cis element, an RRE cis element as set forth in SEQ ID NO. 8, an env cis element as set forth in SEQ ID NO. 9, a cPPT cis element as set forth in SEQ ID NO. 10, and a SEQ ID NO.
  • the chimeric antigen receptor for the second generation CAR of the composition recognition, delivery and initiation comprises: the CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. BCMA single-chain antibody light chain VL as shown in SEQ ID NO. 14, Optimal Linker C as shown in SEQ ID NO. 15, BCMA single-chain antibody heavy chain VH as shown in SEQ ID NO. 16, as SEQ CD8 Hinge chimeric receptor hinge shown by ID NO. 17 , CD8 Transmembrane chimeric receptor transmembrane region as shown in SEQ ID NO. 18, co-stimulation of CD137 chimeric receptor as shown in SEQ ID NO.
  • the chimeric antigen receptor for the three generations of CARs constituting the set recognition, delivery, and initiation comprises: SEQ ID NO.
  • the eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element has a 6-nucleotide enhancing mutation, specifically: g.396G>A, g.397C>T, g.398T >C, g.399G>A, g.400A>T, g.411A>T.
  • a method for constructing a CAR-T transgenic vector for inhibiting immune escape by blocking PDL1 as described above comprising the steps of:
  • a ampicillin-resistant gene AmpR sequence as shown in SEQ ID NO. 1 a prokaryotic replicon pUC Ori sequence as shown in SEQ ID NO. 2, a viral replicon as shown in SEQ ID NO. a SV40Ori sequence, a lentiviral packaging cis element for lentiviral packaging, an eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element as set forth in SEQ ID NO. 11 is stored on a lentiviral backbone plasmid;
  • the obtained recombinant lentiviral plasmids pCARmm-PDL1scFv1, pCARmm-PDL1scFv2, or pCARmm-PDL1scFv3 were co-transfected into HEK293T/17 cells with the lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein granule pEnv-G, respectively.
  • the packaged recombinant recombinant lentiviral vector is released into the cell culture supernatant, and the supernatant of the recombinant lentiviral vector contained is collected;
  • the obtained recombinant lentiviral supernatant was purified by column filtration using suction filtration, adsorption and elution to obtain a recombinant lentiviral vector.
  • step (3) the entire CAR gene expression is initiated by the human EF1 ⁇ promoter; the CAR protein is localized on the surface of the cell membrane, recognizes the BCMA antigen, stimulates T cell proliferation and cytokine secretion, and activates the downstream signaling pathway. Expression; when the scfv region binds to the BCMA antigen, the signal is transmitted to the cell through the chimeric receptor, thereby producing T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic protein, delayed cell death, and lysis of target cells.
  • the filtration step is to control the supernatant volume from 200 ml to 2000 ml, and the control vacuum degree is from -0.5 MPA to -0.9 MPA to prevent carrier loss due to plugging.
  • the adsorption step is to control the pH of the solution to be 6-8, to prevent the change of pH from causing the carrier to be deactivated;
  • the elution step is to control the ionic strength of the eluent from 0.5 M to 1.0 M to prevent the change of the ionic strength. Causes incomplete elution or inactivation of the carrier.
  • the present invention has the following beneficial effects:
  • the vector backbone used in the present invention is a third generation lentiviral vector (as shown in FIG. 2A) (a CAR-T transgenic vector based on replication-defective recombinant lentivirus and its application filed on March 17, 2016
  • the construction method and application "disclosed in the invention patent", 3'SIN LTR removes the U3 region, eliminates the possibility of lentiviral vector self-replication, greatly improves safety; increases cPPT and WPRE components, improves transduction efficiency and The efficiency of expression of the transgene; the use of the RSV promoter ensures the sustained and efficient transcription of the core RNA during lentiviral vector packaging; using the human EF1 ⁇ promoter, the CAR gene can be continuously expressed in the human body for a long time.
  • the third generation lentiviral backbone plasmid used in the present invention (disclosed as a "CAR-T transgenic vector based on replication-defective recombinant lentivirus and its construction method and application” patent application filed on March 17, 2016 Using the eWPRE element, compared with the conventional WPRE, it can enhance the polyadenylation of the primary transcription product, increase the intracellular mRNA content, and enhance the expression efficiency of the transgene.
  • a lentiviral vector column used in the present invention (as shown in FIG. 7) (a CAR-T transgenic vector based on replication-defective recombinant lentivirus and its construction method and application filed on March 17, 2016 Application "disclosed in the invention patent", different from the commonly used ultracentrifugation or high-speed centrifugation, semi-automatic operation, avoiding the cumbersome and error of manual operation, the recovered lentiviral vector endotoxin, mycoplasma, host DNA residue and other indicators Fully meet clinical standards.
  • the recombinant lentiviral vector system of the present invention (published in the invention patent of "a replication-defective recombinant lentivirus-based CAR-T transgenic vector and its construction method and application", filed on March 17, 2016,
  • the 3'SIN LTR removed the U3 region, eliminating the possibility of lentiviral vector self-replication, greatly improving safety; increasing cPPT and eWPRE elements, improving transduction efficiency and transgene expression Efficiency; using the RSV promoter ensures sustained and efficient transcription of the core RNA during lentiviral vector packaging; using the human's own EF1 ⁇ promoter, the CAR gene can be expressed continuously in humans for a long time.
  • the Linker design of the scfv segment used in the present invention (disclosed in the invention patent of "a CAR-T transgenic vector based on replication-defective recombinant lentivirus and its construction method and application” filed on March 17, 2016)
  • the invention adopts a single-chain antibody blocking technology for PDL1, and a single chain antibody fragment (scFv) is a short peptide of 15-20 amino acids which is passed from the antibody heavy chain variable region and the light chain variable region. (linker) connected.
  • scFv can retain its affinity for antigen well, and has the characteristics of small molecular weight, strong penetrability and weak antigenicity.
  • the human PDL1 blocking single-chain antibody designed by the invention can effectively overexpress and secrete into the extracellular space in T cells, can effectively block the binding of PD1 and PDL1, and block the transmission inhibition signal of PD1/PDL1 signaling pathway.
  • QPCR detection can effectively increase the transcription level of IL2, TNF ⁇ and IFN ⁇ mRNA in T cells, and relieve the inhibition of T cell activation-related genes, and block PD1/PDL1 signaling pathway in vivo in the future. To achieve the effect of inhibiting immune escape and improve the efficacy of CAR-T cell therapy against solid tumors.
  • the scFv fragment and the Fc fragment of the antibody used in the present invention are all humanized, and can effectively reduce the production of human anti-mouse antibodies (HAMA) in vivo, and improve the half-life and effect of scFv.
  • HAMA human anti-mouse antibodies
  • the present invention employs the mode of action of PDL1 scFv (as shown in Figure 1B). First of all, saving costs and eliminating the need to buy anti- The expensive cost of body medicine. Secondly, the problem of inefficient delivery of the scFv gene in vivo is avoided. Third, the PDL1scFv gene transduced by lentivirus can effectively utilize the intracellular protein translation system, and express the corresponding PDL1scFv in a large amount, and achieve a good PDL1 blocking effect through body fluid circulation.
  • the present invention screens a series of bioinformatics information such as the gene sequence and amino acid sequence of the PDL1 antibody, predicts the variable regions of the heavy and light chains of the PDL1scFv, and analyzes the secondary structure and physicochemical properties of the PDL1scFv combination.
  • the affinity constant of PDL1scFv was determined by soluble expression and indirect ELISA. Three scFvs were selected to detect the cell function level. PDL1scFv1 was finally selected as the best choice and could enter the clinical research stage in the future.
  • the recombinant lentiviral vector backbone of the present invention can be loaded with different therapeutic genes and widely used in the field of adoptive cell therapy, and the PDL1 scFv gene is used to block PDL1, thereby inhibiting the immunoregulatory signaling pathway, thereby inhibiting tumor immune escape.
  • the lentiviral vector of the invention can realize the expression of BCMA chimeric antigen receptor on human T lymphocytes, guide and activate the killing effect of T lymphocytes on BCMA positive cells, and is clinically used for treating multiple myeloma (MM).
  • MM multiple myeloma
  • the expression of cF1 in the human T lymphocytes which effectively blocks PDL1 and blocks the immunoregulatory signaling pathway, can be used clinically to inhibit tumor immune escape and improve CAR- The efficacy of T cell immunotherapy.
  • the recombinant lentiviral vector of the present invention can provide a reliable transgenic guarantee for CAR-T treatment of multiple myeloma (MM), and can block the immune escape mechanism of the tumor and improve the therapeutic effect of the CAR-T cell therapy.
  • MM multiple myeloma
  • the medical cost incurred by the patient is greatly reduced, the technical problems in the field are solved, and the unexpected technical effects are achieved.
  • the PDL1 single-chain antibody expression cassette and the gene expression product thereof of the invention can be applied not only to the immune escape mechanism for eliminating or alleviating tumors in CAR-T treatment of multiple myeloma (MM), but also for inhibiting CAR -T
  • the immune escape mechanism when treating tumors of the type such as pancreatic cancer, glioma, myeloma and the like.
  • FIG. 1A is a schematic diagram of a PD1/PDL1 signal path of the present invention.
  • FIG. 1B is a schematic view showing the mode of action of the PDL1 scFv of the present invention
  • FIG. 2 is a schematic diagram showing the structure of a lentiviral vector of the present invention
  • FIG. 2A is a schematic diagram showing the structure of a third generation lentiviral vector used in the present invention
  • FIG. 2B is a schematic diagram showing a comparison of the structures of the second generation and third generation lentiviral vectors
  • FIG. 3 is a flow chart showing the construction of the recombinant lentiviral vector of the present invention in Example 1 of the present invention.
  • A is a schematic diagram of the structure of the lentiviral skeleton plasmid pLenti-3G Basic2
  • B is a schematic diagram of the structure of the pCARmm-Basic2 plasmid
  • C is a diagram of pCARmm-PDL1scFv1, pCARmm-PDL1scFv2, pCARmm-PDL1scFv3, pCARmm Schematic diagram of the -scFv0 plasmid
  • D is a schematic diagram of the structure of the lentiviral packaging plasmid pPac-GP
  • E is a schematic diagram of the structure of the lentiviral packaging plasmid pPac-R
  • F is the structure of the membrane protein pEnv-G schematic diagram
  • Figure 4 is a diagram showing the restriction enzyme digestion and enzymatic cleavage agarose gel electrophoresis of the lentiviral backbone plasmid pLenti-3G Basic2 in Example 1 of the present invention
  • Figure 4A is a schematic diagram of the restriction enzyme digestion of the lentiviral backbone plasmid pLenti-3G Basic2, wherein Lane 1 is the Cla I+BamH I restriction of pLenti-3G Basic2, the bands are: 5854bp from top to bottom; lane 2 is 1kb DNA ladder Marker prediction, the bands are from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp; FIG.
  • 4B is an enzyme-cut agarose gel electrophoresis pattern of the lentiviral backbone plasmid pLenti-3G Basic2, wherein Lane 1 is the result of Cla I+BamH I digestion of pLenti-3G Basic2; lane 2 is the result of electrophoresis of 1 kb DNA ladder Marker;
  • Figure 5 is a diagram showing the restriction enzyme digestion and enzymatic cleavage agarose gel electrophoresis of the recombinant lentiviral plasmid pCARmm-Basic2 in Example 1 of the present invention
  • Figure 5A is a restriction diagram of the recombinant lentiviral plasmid pCARmm-Basic2, wherein lane 1 It is a 1 kb DNA ladder Marker, and the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp; lane 2 is pCARmm -Basic2 Xba I+Xho I digestion prediction, the bands from top to bottom are: 6839 bp, 1692 bp;
  • Figure 5B is the recombinant
  • Figure 6 is a diagram showing the recombinant lentiviral plasmids pCARmm-PDL1scFv1 and pCARmm-PDL1scFv2 in Example 1 of the present invention. And pCARmm-PDL1scFv3, pCARmm-scFv0 digestion prediction and enzymatic cleavage agarose gel electrophoresis map;
  • Figure 6A is the enzymatic cleavage prediction map of pCARmm-PDL1scFv1, wherein lane1 is 1kb DNA ladder Marker, the strips are from top to bottom They are: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp; lane2 is the BsrG I restriction of pCARmm-PD
  • Figure 6B is the electrophoresis pattern of pCARmm-PDL1scFv1, wherein lane1 is the electrophoresis result of 1 kb DNA ladder Marker, and lane2 is the result of BsrG I digestion of pCARmm-PDL1scFv1;
  • 6C is a restriction map of pCARmm-PDL1scFv2, wherein lane1 is a 1 kb DNA ladder marker, and the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5.
  • lane2 is the Sal I restriction of pCARmm-PDL1scFv2, the bands are: 8484 bp, 1588 bp, 818 bp from top to bottom;
  • Figure 6D is the enzymatically modified agarose gel of pCARmm-PDL1scFv2 Electropherogram, where lane1 is a 1 kb DNA ladder Marker
  • Lane2 is the result of Sal I digestion of pCARmm-PDL1scFv2;
  • Figure 6E is the restriction map of pCARmm-PDL1scFv3, wherein lane1 is 1kb DNA ladder Marker, and the bands are from top to bottom: 10kb, 8kb, 6kb , 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500
  • Figure 6F is an electrophoresis map of pCARmm-PDL1scFv3, wherein lane1 is the electrophoresis result of 1 kb DNA ladder Marker, and lane2 is the result of Pvu II digestion of pCARmm-PDL1scFv3;
  • 6G is a restriction map of pCARmm-scFv0, wherein lane1 is a 1 kb DNA ladder marker, and the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5.
  • lane2 is the Kpn I restriction of pCARmm-scFv0, the bands are: 7028 bp, 3626 bp, 895 bp, 347 bp from top to bottom;
  • Figure 6H is the enzymatically cut agarose of pCARmm-scFv0 Gel electrophoresis map, where lane1 is 1 kb DNA ladder Marker Electrophoresis results, lane2 is the result of Kpn I digestion of pCARmm-scFv0.
  • Example 7 is a flow chart of purifying a recombinant lentiviral vector by ion exchange chromatography in Example 2 of the present invention.
  • Figure 8 is a schematic diagram showing the results of titer detection of the recombinant lentiviral vector in Example 2 of the present invention.
  • lane1 is DL2000marker, and the strips from top to bottom are from top to bottom: 2 kb, 1 kb, 750 bp, 500bp, 250bp, 100bp;
  • lane2 is a positive control;
  • lane3 is a negative control;
  • lane4 is PBS;
  • lane5 is water;
  • lane6 is lvCARmm-PDL1scFv1;
  • lane7 is lvCARmm-PDL1scFv2;
  • lane8 is lvCARmm-PDL1scFv3;
  • lane9 is lvCARmm-scFv0;
  • Figure 10 is a bar graph showing the relative expression levels of mRNA in Example 3 of the present invention.
  • Figure 10A is a schematic diagram of RT-QPCR results, indicating that the CAR gene is efficiently transcribed in PBMC cells
  • Figure 10B is a schematic diagram of RT-QPCR results, indicating scFv gene Efficient transcription in PBMC cells
  • Figure 11 is a WB detection diagram of CAR protein expression in Example 3 of the present invention. The results indicate that CAR protein is highly expressed in PBMC cells.
  • M is the protein Marker
  • lane1 is the PBMC empty cell
  • lane2 is the control virus MOCK.
  • Lane3 is lvCARmm-PDL1scFv1
  • lane4 is lvCARmm-PDL1scFv2
  • lane5 is lvCARmm-PDL1scFv3
  • lane6 is lvCARmm-scFv0
  • Figure 11B is beta-actin internal reference strip;
  • Figure 12 is a graph showing the results of ELISA detection of scFv protein expression in Example 3 of the present invention, and the results indicate that scFv protein is highly expressed in PBMC cells;
  • Figure 13 is a diagram showing the PBMC transduced by the recombinant lentiviral vector in Example 3 of the present invention, co-cultured under different target-target conditions, and the killing condition of the target cells was detected after 24 hours;
  • Figure 14 is a schematic diagram showing changes in the transcription level of PD1 mRNA at 24 h under different conditions of co-culture of different effector cells with target cells in Example 3 of the present invention
  • FIG. 15 is a schematic diagram showing changes in transcript levels of cytokines at 24 h under different co-culture conditions of different effector cells in the present invention; wherein, FIG. 15A represents RT-QPCR results, and IL2 gene is present in each experimental group PBMC. Intracellular mRNA transcription levels; Figure 15B represents RT-QPCR results, mRNA transcription levels of TNF ⁇ genes in PBMC cells of each experimental group; Figure 15C represents RT-QPCR results, mRNA transcription levels of IFN ⁇ genes in PBMC cells of each experimental group.
  • Lentiviral skeleton plasmid pLenti-3G Basic2, lentiviral packaging plasmid pPac-GP, pPac-R and membrane protein granule pEnv-G, HEK293T/17 cells, homologous recombinase by Shiyi (Shanghai) Biomedical Technology Co., Ltd. provide;
  • Primers designed to amplify DNA fragments and target sites according to primer design principles. The primers were synthesized by Shanghai Biotech Co., Ltd., specifically:
  • EF1 ⁇ -F 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO. 29)
  • CD8 leader-F 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO. 31)
  • CD8 leader-R 5'-GGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO. 32)
  • VL-F 5'-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3' (SEQ ID NO. 33)
  • VL-R 5'-ACGCTTGATCTCCAGTTTGGT-3' (SEQ ID NO. 34)
  • OLC-VH-F 5'-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCAGGTGCAGCTGGTCCAGAG-3' (SEQ ID NO. 35)
  • VH-R 5'-GCTGGACACGGTCACTAGTGTG-3' (SEQ ID NO. 36)
  • CD8 Hinge-F 5'-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3' (SEQ ID NO. 37)
  • CD8 Hinge-R 5'-GTAGATATCACAGGCGAAGTCCA-3' (SEQ ID NO. 38)
  • CD8 Transmembrane-F 5'-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3' (SEQ ID NO. 39)
  • CD8 Transmembrane-R 5'-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3' (SEQ ID NO. 40)
  • CD137-F 5'-AAACGGGGCAGAAAGAAACTC-3' (SEQ ID NO. 41)
  • CD137-R 5'-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3' (SEQ ID NO. 42)
  • TCR-F 5'-AGAGTGAAGTTCAGCAGGAGCG-3' (SEQ ID NO. 43)
  • TCR-R 5'-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3' (SEQ ID NO. 44)
  • IRES-F 5'-GCCCTGCCCCCTCGCTAAGCCCCTCTCCCTCCCC-3' (SEQ ID NO. 45)
  • IRES-R 5'-CCAGGGAGAAGGCAACTGGACCGAAGGCGCTTGTGGAGAAGGAGTTCATGGTGGCATTATCATCGTGTTTTTCAAAGGA-3' (SEQ ID NO. 46)
  • PDL1s1-F 5'-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGATATTGTGCTGACCCAGAG-3' (SEQ ID NO. 47)
  • PDL1s1-R 5'-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3' (SEQ ID NO. 48)
  • PDL1s2-F 5'-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGATATTCAGATGACCCAGAGC-3' (SEQ ID NO. 49)
  • PDL1s2-R 5'-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3' (SEQ ID NO. 50)
  • PDL1s3-F 5'-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG CCCCAGATATTGTGCTGACCCAGAGC-3' (SEQ ID NO. 51)
  • PDL1s3-R 5'-GCAGCTTTTCGGTTCCGCGCTCGCGGTCACCAGGGT-3' (SEQ ID NO. 52)
  • s0-F 5'-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCATTGTTCTGGATTCCTGCTTCCA-3' (SEQ ID NO. 53)
  • s0-R 5'-GCAGCTTTTCGGTTCTGCAGAGACAGAGACCAGAGT-3' (SEQ ID NO. 54)
  • Fc-F 5'-GAACCGAAAAGCTGCGATAAAAC-3' (SEQ ID NO. 55)
  • Fc-R 5'-CTAGCAATCTAGAGGTTATTTGCCCGGGCTCAGGCTCA-3' (SEQ ID NO. 56)
  • WPRE-QPCR-F 5'-CCTTTCCGGGACTTTCGCTTT-3' (SEQ ID NO. 57)
  • WPRE-QPCR-R 5'-GCAGAATCCAGGTGGCAACA-3' (SEQ ID NO. 58)
  • Actin-QPCR-F 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO. 59)
  • CAR-QPCR-F 5'-GACTTGTGGGGTCCTTCTCCT-3' (SEQ ID NO. 61)
  • PD1-QPCR-F 5'-TGCAGCTTCTCCAACACAT-3' (SEQ ID NO. 63)
  • PD1-QPCR-R 5'-CTTGTCCGTCTGGTTGCT-3' (SEQ ID NO. 64)
  • IL2-QPCR-F 5'-CACCAGGATGCTCACATTTAAGT-3' (SEQ ID NO. 65)
  • IL2-QPCR-R 5'-GTCCCTGGGTCTTAAGTGAAAGT-3' (SEQ ID NO. 66)
  • Fc-QPCR-F 5'-GACATTGGAAATGTGAACATGT-3' (SEQ ID NO. 67)
  • Fc-QPCR-R 5'-CACAGCTGGGGTTTGGTGA-3' (SEQ ID NO. 68)
  • TNF ⁇ -QPCR-F 5'-TCTCTAATCAGCCCTCTG-3' (SEQ ID NO. 69)
  • TNF ⁇ -QPCR-R 5'-GGGTTTGCTACAACATGG-3' (SEQ ID NO. 70)
  • IFN ⁇ -QPCR-F 5'-GACTAATTATTCGGTAACTGA-3' (SEQ ID NO. 71)
  • IFN ⁇ -QPCR-R 5'-GATGCTCTTCGACCTCGAAACA-3' (SEQ ID NO. 72)
  • the DNA sequence shown in SEQ ID NO. 15 to SEQ ID NO. 72 is synthesized by Shanghai Jierui Bioengineering Co., Ltd. and stored as an oligonucleotide dry powder or a plasmid;
  • Tool enzymes Xba I, Xho I, Pvu II, Sal I, BsrG I, BamH I, Kpn I, Cla I, T4 DNA ligase were purchased from NEB;
  • the plasmid extraction kit and the agarose gel recovery kit were purchased from MN Company;
  • Competent cells TOP10 were purchased from tiangen;
  • Opti-MEM FBS, DMEM, 1640, Pen-Srep, Hepes, purchased from Invitrogen;
  • Biotinylated protein L proteinG-HRP was purchased from GeneScript;
  • ECL+plusTM Western blotting system was purchased from Amersham;
  • DNeasy kit was purchased from Shanghai Jierui Company;
  • Lymphocyte separation solution was purchased from Shenzhen Dakco as the company;
  • phycoerythrin (PE)-conjugated streptavidin was purchased from BD Bioscience;
  • SA-HRP, TMB substrate solution, and ELISA reaction stop solution were purchased from Shanghai Shengsheng Company;
  • Mycoplasma test kit endotoxin test kit, BCMA-K562 cells, BCMA-PDL1-K562 cells were purchased from Shiyi (Shanghai) Co., Ltd.;
  • the LDH test kit was purchased from Promega.
  • the construction method of the recombinant lentiviral vector of the present invention is as follows:
  • the fragments were ligated into pCARmm-Basic2, respectively, to obtain IL-6R blocking recombinant lentiviral plasmids pCARmm-PDL1scFv1, pCARmm-PDL1scFv2, pCARmm-PDL1scFv3, and control pCARmm-scFv0.
  • the lentiviral backbone plasmid pLenti-3G Basic2 was digested with Cla I and BamH I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 5854 bp fragment V1 (as shown in Fig. 4). ), and the tapping recovery was placed in an Eppendorf tube, and the corresponding fragment was recovered using MN's agarose gel recovery kit (see Table 1), and the purity and concentration of the product were determined;
  • sol The sol solution was added in a ratio of 200 ⁇ l NTI/100 mg gel, and placed in a water bath at 50 ° C for 5-10 minutes. 2, combined with DNA Centrifuge at 11,000 g for 30 seconds and discard the filtrate. 3, wash the film 700 ⁇ l of NT3 was added and centrifuged at 11,000 g for 30 seconds, and the filtrate was discarded. 4, wash the film Repeat the third step once 5, dry Centrifuge at 11000g for 1 minute, replace with a new collection tube and leave it at room temperature for 1 minute. 6, eluting DNA 15-30 ⁇ l of NE was added, and the mixture was allowed to stand at room temperature for 1 minute, centrifuged at 11,000 g for 1 minute, and the filtrate was collected.
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C) 30 sec) * 35 cycles, 72 ° C for 5 min.
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 336 bp fragment c was confirmed, and the gel was collected and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C 30 sec) * 35 cycles, 72 ° C 5 min.
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 421 bp fragment d was confirmed, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 704 bp fragment j was confirmed, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the clones were picked for colony PCR identification, and the correct clone was identified as recombinant lentiviral plasmid pCARmm-Basic2, and the correct clone was identified by enzyme digestion (see Figure 5).
  • the recombinant lentiviral plasmid pCARmm-Basic2 was digested with Sal I and Nhe I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment of VARI of 8491 bp, and the tapping recovery was carried out in Eppendorf. In the tube, the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the purity and concentration of the product were determined;
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C) 2min) *35cycle, 72 ° C for 10 min.
  • the product was electrophoresed on a 1.5% agarose gel to confirm the 729 bp fragment o, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered using MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the clones were picked for colony PCR identification, and the correct clones were identified as recombinant lentiviral plasmids pCARmm-PDL1scFv1, pCARmm-PDL1scFv2, pCARmm-PDL1scFv3 and control pCARmm-scFv0, and the correct clones were identified by restriction enzyme digestion (see Figure 6); , packaging of recombinant lentiviral vector lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm-scFv0.
  • Trypsin solution Weigh Trypsin 2.5g, EDTA 0.19729g in 1000ml beaker, add 900ml 1XPBS to dissolve, dissolve it, use 1000ml measuring cylinder to make up to 1000ml, 0.22 ⁇ M filter sterilization, long-term use can be saved to -20 ° C refrigerator;
  • a DNA/CaCl 2 solution was prepared in accordance with N + 0.5.
  • the amount of HEK293T/17 cell transfection plasmid per dish was used in the following ratios: recombinant lentiviral plasmid (20 ⁇ g), pPac-GP (15 ⁇ g), pPac-R (10 ⁇ g), pEnv-G (7.5 ⁇ g).
  • the same virus supernatant was collected again, and the two collected viruses could be put together and the culture dish discarded; the supernatant collected at this time contained the recombinant lentiviral vector lvCARmm-PDL1scFv1, lvCARmm -PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm-scFv0.
  • the eluate is divided into 25 to 50 ⁇ l tubes, frozen and stored in a -80 ° C refrigerator for long-term storage;
  • a 24-well plate was used to inoculate 293T cells.
  • the cell volume per well is 5 ⁇ 10 4
  • the volume of the added medium is 500 ul
  • the growth rate of different kinds of cells is different
  • the cell fusion rate when the virus is infected is 40%-60%;
  • the cells were digested with 0.2 ml of 0.25% trypsin-EDTA solution and allowed to stand at 37 ° C for 1 minute. The entire cell surface was washed with a medium, and the cells were collected by centrifugation. Genomic DNA was extracted according to the instructions of the DNeasy kit. 200 ⁇ l of eluate was added to each sample tube to wash the DNA and quantify;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 4 ⁇ l of forward primer, 4 ⁇ l of reverse primer, 4 ⁇ l of probe and 788 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 100 ⁇ l of 10 ⁇ RNaseP primer/probe mix and 700 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the quantitative PCR instrument used was the ABI PRISM 7500 quantitative system.
  • the cycle conditions were set to: 50 ° C for 2 minutes, 95 ° C for 10 minutes, then 95 ° C for 15 seconds, 60 ° C for 1 minute of 40 cycles.
  • N number of cells at the time of infection (approximately 1 ⁇ 10 5 )
  • V volume of diluted virus added
  • the endotoxin working standard is 15EU/piece;
  • Step 4 is repeated once;
  • PCR reaction system was: ddH20 6.5 ⁇ l, Myco Mix 1 ⁇ l, 2 ⁇ Taq Plus Mix Master (Dye Plus) 12.5 ⁇ l, template 5 ⁇ l; PCR cycle conditions were: 95 ° C 30 sec, (95 ° C 30 sec, 56 ° C 30 sec, 72 ° C 30 sec) * 30 cycle, 72 ° C 5 min.
  • Example 3 Functional detection of recombinant lentiviral vectors lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm-scFv0.
  • Recombinant lentiviral vectors lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm-scFv0 and control virus Mock were infected with PBMC cells, and the collected cells were detected by RT-PCR for mRNA and scFv mRNA transcription levels.
  • the expression of the CAR gene and the scFv gene if the mRNA level of the CAR gene and the scFv gene are increased, indicates that the transcription levels of the CAR gene and the scFv gene are successfully expressed;
  • the cell separation solution was isolated from human blood), and 500 ul of medium (containing 10% serum, 20 U/ml IL-2, Polybrene 8 ug/ml) was added. The cells were allowed to stand for 20 min, centrifuged at 1000 g for 20 min at 20 ° C, and cultured at 37 ° C for 48 h.
  • the total protein extracted from PBMC was separated by relative molecular mass by polyacrylamide gel electrophoresis.
  • the protein was transferred to a PVDF membrane using a wet transfer (4 ° C, 400 mA, 120 min).
  • the PVDF membrane was blocked with a blocking solution (containing 5% skim milk in TBST solution) for 1 h at room temperature, and the blocking solution was diluted 1:1000 with Biotinylated protein L, and then incubated with the blocked PVDF membrane at room temperature for 4 ° C overnight.
  • the membrane was washed 3 times with TBST for 10 min each time.
  • the blocking solution was diluted 1:500 with the corresponding SA-HRP.
  • the PVDF membrane was incubated for 2 h at room temperature, and the membrane was washed 3 times with TBST for 10 min each time. Color development was performed using an Amersham ECL+plusTM Western blotting system kit. X-ray development obtained a film showing the strip.
  • Enzyme Linked ImmunoSorbent Assay by coating 1:2, 1:5, 1:10 diluted cell culture supernatant into 96-well plates, and setting a negative control, positive control And blank wells, overnight at 4 °C. After washing 3 times the next day, 0.1 ml of fresh 1:10000 diluted proteinG-HRP was added to the well, incubated at 37 ° C for 30-60 minutes, washed, and washed with pure water for the last time. 0.1 ml of the TMB substrate solution added to each reaction well was incubated at 37 ° C for 10 to 30 minutes. 0.05 ml of an ELISA reaction stop solution was added to each reaction well. The OD value of each well was measured at 405 nm on a microplate reader.
  • RT-QPCR analysis showed that the transcription level of CAR gene and scFv gene after PBMC infection by recombinant lentiviral vector was significantly higher than that of empty cells (as shown in Figure 10), indicating the transcription level expression of CAR gene and scFv gene. success.
  • step 15 The corrected values obtained in step 15 were taken to the following formula to calculate the percentage of cytotoxicity produced by each of the target ratios.
  • the results are shown in Figure 13.
  • the killing efficiency of PBMC cells transduced with lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm-scFv0 recombinant lentiviral vector was significantly higher than that of PBMC empty cells and control viruses at several target ratios. , indicating that the expression of the scFv gene has little effect on the function of the CAR gene.
  • Killing efficiency (experimental well - effector cell hole - target cell well) / (target cell maximum pore - target cell well) X100%
  • PDL1 blocking effect evaluation (PD1, IL2, TNF ⁇ , IFN ⁇ mRNA transcription level).
  • RT-QPCR test results show that lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2 After lvCARmm-PDL1scFv3, lvCARmm-scFv0 transduced PBMC and incubated with target cells, the mRNA level of PD1 gene was significantly higher than that of Mock group and empty cell group, lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3, lvCARmm- There was no significant difference in the mRNA level of the PD1 gene between the four groups of scFv0 (as shown in Figure 14), indicating that the expression level of PD1 also increased synchronously after T cells were activated.
  • the transcript levels of IL2, TNF ⁇ and IFN ⁇ mRNA were significantly increased in the lvCARmm-PDL1scFv1, lvCARmm-PDL1scFv2, lvCARmm-PDL1scFv3 groups compared with the control virus lvCARmm-scFv0 group, among which lvCARmm-
  • the transcript mRNA levels of IL2, TNF ⁇ and IFN ⁇ genes in PDL1scFv1 group were the most elevated (as shown in Figure 15).
  • lvCARmm-PDL1scFv1 can Effectively block the PD1/PDL1 signaling pathway and release the inhibitory effect on T cell activation-related genes.
  • the PD1/PDL1 signaling pathway can be blocked in vivo to inhibit the immune escape and improve the treatment of CAR-T cells against solid tumors. Efficacy.

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Abstract

提供了一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体，包括：含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；原核复制子pUC Ori序列，如SEQ ID NO.2所示；病毒复制子SV40 Ori序列如SEQ ID NO.3所示；eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.11所示；人EF1α启动子，如SEQ ID NO.12所示；用于慢病毒包装的慢病毒包装顺式元件；人PDL1的人源化单链抗体PDL1scFv1，如SEQ ID NO.21所示、PDL1scFv2，如SEQ ID NO.22所示、或PDL1scFv3，如SEQ ID NO.23所示；IRES核糖体结合序列，如SEQ ID NO.25所示；IL6信号肽，如SEQ ID NO.26所示；人源抗体Fc段，如SEQ ID NO.27所示；以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体。此外，还提供了该载体的构建方法及其在制备抑制免疫逃脱的药物中的应用。

Description

一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体及其构建方法和应用 技术领域
本发明属于医学生物领域，具体涉及一种载体，尤其涉及一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体。此外，本发明还涉及该载体的构建方法和应用。
背景技术
肿瘤免疫治疗的理论基础是免疫系统具有识别肿瘤相关抗原、调控机体攻击肿瘤细胞(高度特异性的细胞溶解)的能力。1950年代，Burnet和Thomas提出了“免疫监视”理论，认为机体中经常会出现的突变的肿瘤细胞可被免疫系统所识别而清除，为肿瘤免疫治疗奠定了理论基础[Burnet FM.Immunological aspects of malignant disease.Lancet,1967；1:1171-4]。随后，各种肿瘤免疫疗法包括细胞因子疗法、单克隆抗体疗法、过继免疫疗法、疫苗疗法等相继应用于临床。
2013年一种更先进的肿瘤免疫疗法---CAR-T疗法成功用于临床，并表现了前所未有的临床疗效。CAR-T，全称是Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法。CAR-T疗法在临床上最领先的有诺华的CLT019，采用CLT019治疗复发难治急性淋巴细胞白血病患者，六个月的肿瘤无进展生存率达到67％，其中最长的应答时间达到两年多。总部位于中国上海的上海优卡迪生物医药科技有限公司与医院合作，共治疗复发难治急性淋巴细胞白血病患者36例，其中完全24例，缓解比例达到66.6％。这是抗癌研究的颠覆性突破。CAR-T细胞疗法可能是最有可能治愈癌症的手段之一，并被《Science》杂志评为2013年度十大科技突破之首。
CAR-T疗法虽然效果显著，但是在治疗实体肿瘤的过程中遇到很多困难，其中一个很重要的原因就是PD1/PDL1抑制性免疫检查点(如图1A所示)，它们结合传递抑制信号，抑制了T细胞的免疫活性，在免疫耐受中发挥了重要作用，同时也是促使肿瘤细胞逃逸的重要原因。
PD-1(又称CD279)是一种免疫抑制性受体，属于CD28家族成员的Ⅰ型跨膜蛋白，程序性细胞死亡分子-1受体1992年由Ishida等[Ishida Y,Agata Y,Shibahara K,et al.Induced expression of PD-1,a novel member of the immunoglobulin gene superfamily,upon programmed cell death〔J〕.EMBO J,1992,11(11):3887-3895.]采用消减杂交方法于凋亡的T细胞杂交瘤中得到并命名。人PD-1基因位于2q37.35染色体上，编码一个约55kD的跨膜糖蛋白。PD-1在激活的T细胞、B细胞、单核细胞和树突状细胞表面广泛表达，PD-1结构上与CTLA-4有30％的同源性，胞内区存在两个酪氨酸残基，分别参与构成了N端的一个免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif，ITIM)和C端的一个免疫受体酪氨酸依赖的转换基序(immunoreceptor tyrosin-based switch motif，ITSM)；胞外区则是由一个IgV样结构域组成，含有多个糖基化位点并被重度糖基化，该结构域可以与配体结合，从而发挥抑制T细胞活化的功能[李瑛,焦顺昌等.PD-1/PD-L1信号通路在肿瘤免疫逃逸中的作用及临床意义[J].Acad J Chin PLA Med Sch,Jul 2015,36(7).]。
PD-L1在多数癌症组织中过量表达，包括NSCLC、黑色素瘤、乳腺癌、胶质瘤、淋巴瘤、白血病及各种泌尿系肿瘤、消化道肿瘤、生殖系肿瘤等[Intlekofer AM,Thompson CB.At the bench:preclinical rationale for CTLA-4and PD-1blockade as cancer immunotherapy[J].J Leukoc Biol,2013,94(1):25-39.].Parsa在鼠和人的肿瘤细胞中，发现T细胞异常分泌的IFN-γ，IFN-γ可以诱导肿瘤细胞上的PD-L1高表达[Ding H,Wu X,Wu J,et al.Delivering PD-1inhibitory signal concomitant with blocking ICOS co-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice[J].Clin Immunol,2006,118(2/3):258-267.]。PD-L1高表达，可以通 过抑制RAS及PI3K/AKT信号通路，进而调控细胞周期检查点蛋白和细胞增殖相关蛋白表达，最终导致T细胞增殖的抑制[11]。Dong等体外实验和小鼠模型还发现，PD-1/PD-L1信号通路的激活可以诱导特异性CTL调亡，使CTL的细胞毒杀伤效应敏感性下降，促使肿瘤细胞发生免疫逃逸[Dong H,Strome SE,Salomao DR,et al.Tumor-associated B7-H1promotes T-cell apoptosis:a potential mechanism of immune evasion[J].Nat Med,2002,8(8):793-800.]。
目前临床上主要是是使用商品化的PD1单抗，作为免疫检查点抑制剂，来抑制肿瘤细胞的免疫逃脱。2014年9月3日，百时美施贵宝公司抗PD-1药物Opdivo(Nivolumab)在日本正式上市，在日本国内此药物目前仍然只限于黑色素瘤患者。药物被设定为每100mg，729849日元(约合人民币43000元)的价格。体重每1kg就需要2mg的使用量，因此体重50kg的人则需要100mg的使用量。并且，体重每增加10kg用药剂量需要增加20mg(150200日元，约合人民币8778元)，每3周为一个疗程。价格相当昂贵，普通家庭难以承受。
因此，如何采用低成本的方法来抑制肿瘤细胞免疫逃脱的发生，不影响CAR-T治疗的疗效，成为CAR-T治疗的一个技术难题。
发明内容
本发明要解决的技术问题之一是提供一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体。首先，节约了成本，省去了购买抗体药物的昂贵费用。其次，避免了scFv基因的在体递送效率低的问题。第三，通过慢病毒转导的PDL1scFv基因，能够有效利用细胞内的蛋白质翻译体系，大量表达出相应的PDL1scFv，经过体液循环，达到良好的PDL1封闭效果，不影响CAR-T治疗的疗效。
本发明要解决的技术问题之二是提供该载体的构建方法。
本发明要解决的技术问题之三是提供该载体的应用。
为解决上述技术问题，本发明采用如下技术方案：
在本发明的一方面，提供一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体，包括：
用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；
用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；
用于增强真核细胞内的复制的病毒复制子SV40Ori序列，如SEQ ID NO.3所示；
用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.11所示；
用于嵌合抗原受体基因的真核转录的人EF1α启动子，如SEQ ID NO.12所示；
用于慢病毒包装的慢病毒包装顺式元件；
人PDL1的人源化单链抗体，所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1、或者如SEQ ID NO.22所示的PDL1scFv2、或者如SEQ ID NO.23所示的PDL1scFv3；
用于共同转录表达蛋白质的IRES核糖体结合序列，如SEQ ID NO.25所示；
IL6信号肽，如SEQ ID NO.26所示；
人源抗体Fc段，如SEQ ID NO.27所示；
以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体。
作为本发明优选的技术方案，所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1。
所述慢病毒包装顺式元件可以采用第二代慢病毒载体或者第三代慢病毒载体，优选第三代慢病毒载体。所述第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元 件、如SEQ ID NO.10所示的cPPT顺式元件。所述第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
作为本发明优选的技术方案，所述用于组成集识别、传递、启动于一体的二代CAR的嵌合抗原受体包括：如SEQ ID NO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域；所述用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括：如SEQ ID NO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.28所示的CD28嵌合受体共刺激因子。
作为本发明优选的技术方案，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
在本发明的第二方面，提供一种上述封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体的构建方法，包括以下步骤：
(1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上；
(2)将如SEQ ID NO.12所示的人EF1α启动子、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体组合成二代CAR或三代CAR设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到二代CAR或三代CAR设计的重组慢病毒质粒；
(3)将人PDL1的人源化单链抗体PDL1scFv1、PDL1scFv2、或PDL1scFv3，IRES核糖体结合序列、IL6信号肽以及人源抗体Fc段分别克隆至重组慢病毒质粒中，得到PDL1阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3；
(4)将得到的重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
(5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化，分别得到重组慢病毒载体。
作为本发明优选的技术方案，步骤(3)中，由人EF1α启动子启动整个CAR基因表达；CAR蛋白定位于细胞膜表面，识别BCMA抗原，刺激T细胞增殖和细胞因子分泌，激活下游信号通路的表达；当scfv区域与BCMA抗原结合时，信号通过嵌合受体传递至细胞内，从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞一系列生物学效应；由IRES核糖体结合序列共表达PDL1scFv与Fc的融合蛋白，并在IL6 信号肽的引导下分泌到细胞外，通过与PDL1的结合，阻断PD1与PDL1的结合，从而阻断PD1/PDL1的信号通路，达到抑制免疫逃脱的效果。
作为本发明优选的技术方案，步骤(5)中，所述抽滤步骤要控制上清体积在200ml～2000ml，控制真空度在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤要控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤要控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
在本发明的第三方面，提供上述载体在制备抑制免疫逃脱的药物中的应用。
与现有技术相比，本发明具有如下有益效果：
本发明所采用的载体骨架为第三代慢病毒载体(如图2A所示)(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)，3’SIN LTR去除了U3区域，消除了慢病毒载体自我复制的可能性，大大提高了安全性；增加了cPPT和WPRE元件，提高了转导效率和转基因的表达效率；采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录；采用人自身的EF1α启动子，使CAR基因能够在人体内长时间持续表达。
本发明所采用的第三代慢病毒骨架质粒(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)采用eWPRE元件，与常规WPRE相比，能够增强初级转录产物的多聚腺苷化，增加细胞内mRNA的含量，增强转基因的表达效率。
本发明所采用的慢病毒载体柱纯化方式(如图7所示)(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)，不同于通常采用的超速离心或者高速离心的方式，半自动化操作，避免人工操作的繁琐和失误，所回收的慢病毒载体在内毒素、支原体、宿主DNA残留等指标上完全达到临床标准。
本发明所述的重组慢病毒载体系统(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)，为第三代慢病毒载体，3’SIN LTR去除了U3区域，消除了慢病毒载体自我复制的可能性，大大提高了安全性；增加了cPPT和eWPRE元件，提高了转导效率和转基因的表达效率；采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录；采用人自身的EF1α启动子，使CAR基因能够在人体内长时间持续表达。
本发明采用的scfv段的Linker设计(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)，能够显著提高细胞因子的分泌、CAR-T细胞的体外杀伤作用以及临床治疗效果。
本发明所采用的是针对PDL1的单链抗体封闭技术，单链抗体(single chain antibody fragment，scFv)，是由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成。scFv能较好地保留其对抗原的亲和活性,并具有分子量小、穿透力强和抗原性弱等特点。
本发明所采用的人PDL1阻断单链抗体设计，能够有效在T细胞中过表达并分泌至胞外，能够有效阻断PD1与PDL1结合，阻断PD1/PDL1信号通路的传递抑制信号，在T细胞杀伤实验中，经过QPCR检测，能有效提高T细胞中IL2、TNFα、IFNγ的mRNA的转录水平，解除对T细胞活化相关基因的抑制作用，将来可以在体内阻断PD1/PDL1的信号通路，达到抑制免疫逃脱的效果，提高CAR-T细胞治疗针对实体肿瘤的疗效。
本发明中采用的scFv片段和抗体Fc段，均经过人源化改造，能有有效减少体内人抗鼠抗体(Human anti-mouse antibodies，HAMA)的产生，提高scFv的半衰期和作用效果。
本发明采用PDL1scFv的作用方式(如图1B所示)。首先，节约了成本，省去了购买抗 体药物的昂贵费用。其次，避免了scFv基因的在体递送效率低的问题。第三，通过慢病毒转导的PDL1scFv基因，能够有效利用细胞内的蛋白质翻译体系，大量表达出相应的PDL1scFv，经过体液循环，达到良好的PDL1封闭效果。本发明筛选了一系列PDL1抗体的基因序列、氨基酸序列等生物信息学方面的信息，预测了PDL1scFv的重链及轻链的可变区，分析了PDL1scFv组合的二级结构及其物理化学性质，通过可溶性表达和间接ELISA法测定了PDL1scFv的亲和力常数，从中优选出3条scFv进行细胞功能水平的检测，最终确定了PDL1scFv1作为最佳选择，未来可进入临床研究阶段。本发明的重组慢病毒载体骨架可以搭载不同的治疗性基因并广泛的用于过继性细胞治疗领域，搭载PDL1scFv基因用于封闭PDL1，抑制免疫负调节信号通路，从而抑制肿瘤的免疫逃脱。本发明的慢病毒载体可以实现在人T淋巴细胞上表达BCMA嵌合抗原受体，引导并激活T淋巴细胞对BCMA阳性细胞的杀伤作用，在临床上用于治疗多发性骨髓瘤(MM)。在人T淋巴细胞内表达细胞程式死亡配体1(Programmed cell death 1ligand 1，PDL1)的scFv，有效封闭PDL1，阻断免疫负调节信号通路，临床上可用于抑制肿瘤的免疫逃脱，提高CAR-T细胞免疫治疗的疗效。
可见，本发明所述的重组慢病毒载体在给多发性骨髓瘤(MM)的CAR-T治疗提供可靠转基因保障的同时，可以阻断肿瘤的免疫逃脱机制，提高CAR-T细胞治疗的疗效，大大降低患者承担的医疗成本，解决了本领域的技术难题，且达到了预料不到的技术效果。
本发明所述的PDL1单链抗体表达框及其基因表达产物，不仅可以应用于CAR-T治疗多发性骨髓瘤(MM)中用于消除或减轻肿瘤的免疫逃脱机制，还可应用于抑制CAR-T治疗诸如胰腺癌、脑胶质瘤、骨髓瘤等等类型肿瘤时的免疫逃脱机制。
附图说明
图1A是本发明的PD1/PDL1信号通路示意图。
图1B是本发明的PDL1scFv的作用方式示意图；
图2是本发明的慢病毒载体结构示意图；其中图2A是本发明采用的第三代慢病毒载体结构示意图，图2B是第二代和第三代慢病毒载体结构比较示意图；
图3为本发明实施例1中构建本发明所述的重组慢病毒载体的构建流程图。其中，(A)图是慢病毒骨架质粒pLenti-3G Basic2的结构示意图；(B)图是pCARmm-Basic2质粒的结构示意图；(C)图是pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3、pCARmm-scFv0质粒的示意图；(D)图是慢病毒包装质粒pPac-GP的结构示意图；(E)图是慢病毒包装质粒pPac-R的结构示意图；(F)图是膜蛋白pEnv-G的结构示意图；
图4是本发明实施例1中慢病毒骨架质粒pLenti-3G Basic2的酶切预测及酶切琼脂糖凝胶电泳图；图4A是慢病毒骨架质粒pLenti-3G Basic2的酶切预测示意图，其中，lane 1是pLenti-3G Basic2的Cla I+BamH I酶切预测，条带从上到下依次为：5854bp；lane 2是1kb DNA ladder Marker预测，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图4B是慢病毒骨架质粒pLenti-3G Basic2的酶切琼脂糖凝胶电泳图，其中，lane 1是pLenti-3G Basic2的Cla I+BamH I酶切电泳结果；lane 2是1kb DNA ladder Marker的电泳结果；
图5是本发明实施例1中重组慢病毒质粒pCARmm-Basic2的酶切预测及酶切琼脂糖凝胶电泳图；图5A是重组慢病毒质粒pCARmm-Basic2的酶切预测图，其中，lane 1是1kb DNA ladder Marker，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane 2是pCARmm-Basic2的Xba I+Xho I酶切预测，条带从上到下依次为：6839bp、1692bp；图5B是重组慢病毒质粒pCARmm-Basic2的酶切琼脂糖凝胶电泳图，其中，lane1是1kb DNA ladder Marker的电泳结果；lane2是pCARmm-Basic2的Xba I+Xho I酶切电泳结果；
图6是本发明实施例1中重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2 和pCARmm-PDL1scFv3、pCARmm-scFv0的酶切预测及酶切琼脂糖凝胶电泳图；图6A是pCARmm-PDL1scFv1的酶切预测图，其中，lane1是1kb DNA ladder Marker，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pCARmm-PDL1scFv1的BsrG I酶切预测，条带从上到下依次为：9592bp、1298bp；图6B是pCARmm-PDL1scFv1的酶切琼脂糖凝胶电泳图，其中，lane1是1kb DNA ladder Marker的电泳结果，lane2是pCARmm-PDL1scFv1的BsrG I酶切电泳结果；图6C是pCARmm-PDL1scFv2的酶切预测图，其中，lane1是1kb DNA ladder Marker，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pCARmm-PDL1scFv2的Sal I酶切预测，条带从上到下依次为：8484bp、1588bp、818bp；图6D是pCARmm-PDL1scFv2的酶切琼脂糖凝胶电泳图，其中lane1是1kb DNA ladder Marker的电泳结果；lane2是pCARmm-PDL1scFv2的Sal I酶切电泳结果；图6E是pCARmm-PDL1scFv3的酶切预测图，其中，lane1是1kb DNA ladder Marker，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pCARmm-PDL1scFv3的Pvu II酶切预测：条带从上到下依次为：3354bp、2364bp、1920bp、1460bp、823bp、733bp；图6F是pCARmm-PDL1scFv3的酶切琼脂糖凝胶电泳图，其中lane1是1kb DNA ladder Marker的电泳结果，lane2是pCARmm-PDL1scFv3的Pvu II酶切电泳结果；图6G是pCARmm-scFv0的酶切预测图，其中，lane1是1kb DNA ladder Marker，条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pCARmm-scFv0的Kpn I酶切预测，条带从上到下依次为：7028bp、3626bp、895bp、347bp；图6H是pCARmm-scFv0的酶切琼脂糖凝胶电泳图，其中，lane1是1kb DNA ladder Marker的电泳结果，lane2是pCARmm-scFv0的Kpn I酶切电泳结果。
图7是本发明实施例2中离子交换色谱法纯化重组慢病毒载体的流程图；
图8是本发明实施例2中重组慢病毒载体的滴度检测结果示意图；
图9是本发明实施例2中重组慢病毒载体的不同纯化方式的支原体检测结果示意图，其中，lane1为DL2000marker，从上到下条带条带从上到下依次为：2kb、1kb、750bp、500bp、250bp、100bp；lane2为阳性对照；lane3为阴性对照；lane4为PBS；lane5为水；lane6为lvCARmm-PDL1scFv1；lane7为lvCARmm-PDL1scFv2；lane8为lvCARmm-PDL1scFv3；lane9为lvCARmm-scFv0；
图10是本发明实施例3中mRNA相对表达量的柱状图；其中，图10A是RT-QPCR结果示意图，表明CAR基因在PBMC细胞内高效转录；图10B是RT-QPCR结果示意图，表明scFv基因在PBMC细胞内高效转录；
图11是本发明实施例3中CAR蛋白表达量的WB检测图，结果表明CAR蛋白在PBMC细胞内高效表达，图11A中，M为蛋白Marker，lane1为PBMC空细胞，lane2为对照病毒MOCK，lane3为lvCARmm-PDL1scFv1，lane4为lvCARmm-PDL1scFv2，lane5为lvCARmm-PDL1scFv3，lane6为lvCARmm-scFv0；图11B是beta-actin内参条带；
图12是本发明实施例3中scFv蛋白表达量的ELISA检测结果图，结果表明scFv蛋白在PBMC细胞中高效表达；
图13是本发明实施例3中重组慢病毒载体转导的PBMC，在不同效靶比条件下共培养，24h后检测对靶细胞的杀伤情况示意图；
图14是本发明实施例3中不同效应细胞分别与靶细胞共培养条件下，24h PD1mRNA转录水平的变化情况示意图；
图15是本发明实施例3中不同效应细胞分别与靶细胞共培养条件下，24h细胞因子转录水平的变化情况示意图；其中，图15A代表RT-QPCR结果，IL2基因在各实验组PBMC 细胞内mRNA转录水平；图15B代表RT-QPCR结果，TNFα基因在各实验组PBMC细胞内mRNA转录水平；图15C代表RT-QPCR结果，IFNγ基因在各实验组PBMC细胞内mRNA转录水平。
具体实施方式
下面结合具体实施例进一步阐述此发明。应理解的是，在此描述的特定实施方式通过举例的方式来表示，并不作为对本发明的限制。在不偏离本发明范围的情况下，本发明的主要特征可以用于各种实施方式。
实施例1构建重组慢病毒载体
一、材料
1、慢病毒骨架质粒pLenti-3G Basic2，慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G，HEK293T/17细胞、同源重组酶由世翱(上海)生物医药科技有限公司提供；
2、引物：根据引物设计原则设计扩增DNA片段和靶位点所需的引物，该引物由上海生物公司合成，具体为：
EF1α-F：5’-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3’(SEQ ID NO.29)
EF1α-R：5’-TCACGACACCTGAAATGGAAGA-3’(SEQ ID NO.30)
CD8 leader-F：5’-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3’(SEQ ID NO.31)
CD8 leader-R：5’-GGTCATCTGGATGTCCGGCCTGGCGGCGTG-3’(SEQ ID NO.32)
VL-F：5’-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3’(SEQ ID NO.33)
VL-R：5’-ACGCTTGATCTCCAGTTTGGT-3’(SEQ ID NO.34)
OLC-VH-F：5’-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCAGGTGCAGCTGGTCCAGAG-3’(SEQ ID NO.35)
VH-R：5’-GCTGGACACGGTCACTAGTGTG-3’(SEQ ID NO.36)
CD8 Hinge-F：5’-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3’(SEQ ID NO.37)
CD8 Hinge-R：5’-GTAGATATCACAGGCGAAGTCCA-3’(SEQ ID NO.38)
CD8 Transmembrane-F：5’-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3’(SEQ ID NO.39)
CD8 Transmembrane-R：5’-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3’(SEQ ID NO.40)
CD137-F：5’-AAACGGGGCAGAAAGAAACTC-3’(SEQ ID NO.41)
CD137-R：5’-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3’(SEQ ID NO.42)
TCR-F：5’-AGAGTGAAGTTCAGCAGGAGCG-3’(SEQ ID NO.43)
TCR-R：5’-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3’(SEQ ID NO.44)
IRES-F：5’-GCCCTGCCCCCTCGCTAAGCCCCTCTCCCTCCCC-3’(SEQ ID NO.45)
IRES-R：5’-CCAGGGAGAAGGCAACTGGACCGAAGGCGCTTGTGGAGAAGGAGTTCATGGTGGCATTATCATCGTGTTTTTCAAAGGA-3’(SEQ ID NO.46)
PDL1s1-F：5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGATATTGTGCTGACCCAGAG-3’(SEQ ID NO.47)
PDL1s1-R：5’-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3’(SEQ ID NO.48)
PDL1s2-F：5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGATATTCAGATGACCCAGAGC-3’(SEQ ID NO.49)
PDL1s2-R：5’-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3’(SEQ ID NO.50)
PDL1s3-F：5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG CCCCAGATATTGTGCTGACCCAGAGC-3’(SEQ ID NO.51)
PDL1s3-R：5’-GCAGCTTTTCGGTTCCGCGCTCGCGGTCACCAGGGT-3’(SEQ ID NO.52)
s0-F：5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCATTGTTCTGGATTCCTGCTTCCA-3’(SEQ ID NO.53)
s0-R：5’-GCAGCTTTTCGGTTCTGCAGAGACAGAGACCAGAGT-3’(SEQ ID NO.54)
Fc-F：5’-GAACCGAAAAGCTGCGATAAAAC-3’(SEQ ID NO.55)
Fc-R：5’-CTAGCAATCTAGAGGTTATTTGCCCGGGCTCAGGCTCA-3’(SEQ ID NO.56)
WPRE-QPCR-F：5’-CCTTTCCGGGACTTTCGCTTT-3’(SEQ ID NO.57)
WPRE-QPCR-R：5’-GCAGAATCCAGGTGGCAACA-3’(SEQ ID NO.58)
Actin-QPCR-F：5’-CATGTACGTTGCTATCCAGGC-3’(SEQ ID NO.59)
Actin-QPCR-R：5’-CTCCTTAATGTCACGCACGAT-3’(SEQ ID NO.60)
CAR-QPCR-F：5’-GACTTGTGGGGTCCTTCTCCT-3’(SEQ ID NO.61)
CAR-QPCR-R：5’-GCAGCTACAGCCATCTTCCTC-3’(SEQ ID NO.62)
PD1-QPCR-F：5’-TGCAGCTTCTCCAACACAT-3’(SEQ ID NO.63)
PD1-QPCR-R：5’-CTTGTCCGTCTGGTTGCT-3’(SEQ ID NO.64)
IL2-QPCR-F：5’-CACCAGGATGCTCACATTTAAGT-3’(SEQ ID NO.65)
IL2-QPCR-R：5’-GTCCCTGGGTCTTAAGTGAAAGT-3’(SEQ ID NO.66)
Fc-QPCR-F：5’-GACATTGGAAATGTGAACATGT-3’(SEQ ID NO.67)
Fc-QPCR-R：5’-CACAGCTGGGGTTTGGTGA-3’(SEQ ID NO.68)
TNFα-QPCR-F：5’-TCTCTAATCAGCCCTCTG-3’(SEQ ID NO.69)
TNFα-QPCR-R：5’-GGGTTTGCTACAACATGG-3’(SEQ ID NO.70)
IFNγ-QPCR-F：5’-GACTAATTATTCGGTAACTGA-3’(SEQ ID NO.71)
IFNγ-QPCR-R：5’-GATGCTCTTCGACCTCGAAACA-3’(SEQ ID NO.72)
3、SEQ ID NO.15～SEQ ID NO.72所示的DNA序列由上海捷瑞生物工程有限公司合成，并以寡核苷酸干粉或者质粒形式保存；
4、工具酶Xba I、Xho I、Pvu II、Sal I、BsrG I、BamH I、Kpn I、Cla I、T4 DNA连接酶均购自NEB公司；
5、高保真酶PrimeSTAR、RN购自Takara公司；
6、0.22μm-0.8μm PES滤器购自millipore公司；
7、质粒抽提试剂盒、琼脂糖凝胶回收试剂盒均购自MN公司；
8、感受态细胞TOP10购自tiangen公司；
9、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2、NaOH、PEG6000均购自上海生工；
10、Opti-MEM、FBS、DMEM、1640、Pen-Srep、Hepes、购自invitrogen公司；
11、Biotinylated protein L、proteinG-HRP购自GeneScript公司；
12、辣根过氧化物酶标记的二抗、DAB工作液均购自北京中杉金桥；
13、ECL+plusTM Western blotting system购自Amersham公司；
14、DNeasy试剂盒购自上海捷瑞公司；
15、淋巴细胞分离液购自深圳达科为公司；
16、phycoerythrin(PE)-conjugated streptavidin购自BD Bioscience公司；
17、SA-HRP、TMB底物溶液、ELISA反应终止液购自上海翊圣公司；
18、支原体检测试剂盒、内毒素检测试剂盒、BCMA-K562细胞、BCMA-PDL1-K562细胞购自世翱(上海)公司；
19、LDH检测试剂盒购自promega公司。
二、重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的构建方法。
参见图3，本发明所述重组慢病毒载体的构建方法如下：
1、将人EF1α启动子、CD8 leader嵌合受体信号肽、BCMA单链抗体轻链VL、Optimal Linker C、BCMA单链抗体重链VH、CD8 Hinge嵌合受体铰链、CD8 Transmembrane嵌合受体跨膜区、CD137嵌合受体共刺激因子、TCR嵌合受体T细胞激活域片段克隆至慢病毒骨架质粒pLenti-3G Basic2，得到重组慢病毒质粒pCARmm-Basic2，再将scFv片段与Fc片段分别连接到pCARmm-Basic2中，得到IL-6R阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3以及对照pCARmm-scFv0。
(1)将慢病毒骨架质粒pLenti-3G Basic2使用Cla I和BamH I限制性内切酶进行双酶切，产物经过1.5％的琼脂糖凝胶电泳，确认5854bp的片段V1(如图4所示)，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
1、溶胶 按200μl NTI/100mg gel比例加入溶胶液，50℃水浴放置5-10分钟。
2、结合DNA 11000g离心30秒，弃去滤液。
3、洗膜 加入700μl NT3，11000g离心30秒，弃去滤液。
4、洗膜 重复第三步一次
5、晾干 11000g离心1分钟，换新的收集管，室温放置1分钟。
6、洗脱DNA 加入15-30μl NE，室温放置1分钟，11000g离心1分钟，收集滤液。
表1 琼脂糖凝胶回收步骤
(2)用引物EF1α-F和EF1α-R以合成的SEQ ID NO.12为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认1208bp的片段a，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
试剂 体积(μl)
H2O 32.5
5×Buffer(with Mg2+) 10
dNTP(各2.5mM) 4
Primer1(+)(10μM) 1
Primer2(－)(10μM) 1
Template 1
PrimeSTAR 0.5
表2 50μl PCR反应体系
(3)用引物CD8 leader-F和CD8 leader-R以合成的SEQ ID NO.13为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认101bp的片段b，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(4)用引物VL-F和VL-R以合成的SEQ ID NO.14为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认336bp的片段c，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(5)用引物OLC-VH-F和VH-R以合成的SEQ ID NO.16为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。 产物经过1.5％的琼脂糖凝胶电泳，确认421bp的片段d，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(6)用引物CD8 Hinge-F和CD8 Hinge-R以合成的SEQ ID NO.17为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认147bp的片段e，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(7)用引物CD8 Transmembrane-F和CD8 Transmembrane-R以合成的SEQ ID NO.18为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认100bp的片段f，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(8)用引物CD137-F和CD137-R以合成的SEQ ID NO.19为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认142bp的片段g，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(9)用引物TCR-F和TCR-R以合成的SEQ ID NO.20为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认355bp的片段h，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(10)将DNA片段b、c、d各1μl作为模板，使用表3中的体系，除引物外加入Eppendorf管内，PCR循环条件为：98℃3min，(98℃10sec，60℃10sec，72℃30sec)*6cycle，加入引物CD8 leader-F/VH-R,(98℃10sec，60℃10sec，72℃40sec)*24cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认814bp的片段i，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
试剂 体积(μl)
H2O 33.5-1*模板数
5×Buffer(with Mg2+) 10
dNTP(各2.5mM) 4
Primer1(+)(10μM) 1
Primer2(－)(10μM) 1
Template 1*模板数
PrimeSTAR 0.5
表3 50μl重叠PCR反应体系
(11)将DNA片段e、f、g、h各1μl作为模板，使用表3中的体系，除引物外加入Eppendorf管内，PCR循环条件为：98℃3min，(98℃10sec，60℃10sec，72℃30sec)*6cycle，加入引物CD8 Hinge-F/TCR-R,(98℃10sec，60℃10sec，72℃30sec)*24cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认704bp的片段j，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(12)将DNA片段V1、a、i、j以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内，加入同源重组酶反应液15μl，混匀后在42℃孵育30分钟，转移至冰上放置2-3分钟，将反应液加入50μl TOP10中，轻轻旋转以混匀内容物，在冰中放置30分钟，将管放到预加温到42℃的恒温水浴锅中热激90秒，快速将管转移到冰浴中，使细胞冷却2-3分钟，每管加900μl LB培养液，然后将管转移到37℃摇床上，温育1小时使细菌复苏，取100μl的转化菌液 涂布于Amp LB琼脂平板上，倒置平皿，于恒温培养箱中37℃培养，16小时。
挑取克隆进行菌落PCR鉴定，鉴定正确的克隆即为重组慢病毒质粒pCARmm-Basic2，对正确的克隆进行酶切鉴定(见图5)；
(13)将重组慢病毒质粒pCARmm-Basic2使用Sal I和Nhe I限制性内切酶进行双酶切，产物经过1.5％的琼脂糖凝胶电泳，确认8491bp的片段V2，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(14)用引物IRES-F和IRES-R以合成的SEQ ID NO.25为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认605bp的片段k，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(15)用引物PDL1s1-F和PDL1s1-R以合成的SEQ ID NO.21为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认754bp的片段l，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(16)用引物PDL1s2-F和PDL1s2-R以合成的SEQ ID NO.22为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认777bp的片段m，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(17)用引物PDL1s3-F和PDL1s3-R以合成的SEQ ID NO.23为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认774bp的片段n，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(18)用引物s0-F和s0-R以合成的SEQ ID NO.24为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认729bp的片段o，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(19)用引物Fc-F和Fc-R以合成的SEQ ID NO.27为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认726bp的片段p，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(20)将DNA片段(V2、k、l、p)、(V2、k、m、p)、(V2、k、n、p)、(V2、k、o、p)分别以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内，加入同源重组酶反应液15μl，混匀后在42℃孵育30分钟，转移至冰上放置2-3分钟，将反应液加入50μl TOP10中，轻轻旋转以混匀内容物，在冰中放置30分钟，将管放到预加温到42℃的恒温水浴锅中热激90秒，快速将管转移到冰浴中，使细胞冷却2-3分钟，每管加900μl LB培养液，然后将管转移到37℃摇床上，温育1小时使细菌复苏，取100μl的转化菌液涂布于Amp LB琼脂平板上，倒置平皿，于恒温培养箱中37℃培养，16小时。挑取克隆进行菌落PCR鉴定，鉴定正确的克隆即为重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3以及对照pCARmm-scFv0，对正确的克隆进行酶切鉴定(见图6)；2、重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的包装。
(1)完全培养基：取出预热好的新鲜培养基，加入10％FBS+5ml Pen-Srep，上下颠倒混匀即可；
(2)1XPBS溶液：称量NaCl 8g，KCl 0.2，Na2HPO4.12H2O 3.58g，KH2PO4 0.24g 置于1000ml烧杯中，加入900ml Milli-Q grade超纯水溶解，溶解完成后，使用1000ml量筒定容至1000ml，121℃高温湿热灭菌20min；
(3)0.25％Trypsin溶液:称量Trypsin 2.5g，EDTA 0.19729g置于1000ml烧杯中，加入900ml1XPBS溶解，溶解完成后，使用1000ml量筒定容至1000ml，0.22μM过滤除菌，长期使用可保存至-20℃冰箱；
(4)0.5M CaCl2溶液：称量36.75g CaCl2用400ml Milli-Q grade超纯水溶解；用Milli-Q grade超纯水将总体积定容至500ml，混匀；0.22μm过滤除菌，分装保存到50ml离心管中，每管45ml左右，4℃保存。
(5)2XHBS溶液：称量4.09g NaCl，0.269g Na2HPO4，5.96g Hepes，用400ml Milli-Q grade超纯水溶解；校准pH仪后，用2M NaOH溶液将HBS溶液的pH调到7.05。调整每瓶HBS的PH消耗2M NaOH为3ml左右；
(6)从液氮罐中取出冻存的HEK293T/17细胞，迅速转移到37℃水浴中，1～2min后转移到超净台中，无菌操作将冻存管中的液体全部转移至10cm2培养皿中，补足含10％FBS的DMEM至8mL/10cm2dish，24h后显微镜观察细胞，细胞汇合的程度大于80％进行传代；
(7)选择细胞状态良好、无污染的HEK293T/17细胞，每2-6个培养皿为一组，将细胞胰酶消化后，用电动移液器吸取4-12ml完全培养基，向每个消化后的培养皿中加2ml，避免培养皿变干；使用1ml移液器将所有细胞吹打成单细胞悬液，转移到培养基瓶中；
(8)将上述2-6个培养皿中的剩余细胞转移到培养基瓶中，并用培养基再冲洗一便培养皿；
(9)盖紧培养基瓶盖，上下颠倒10次左右充分混匀细胞悬液，将细胞传到8-24个10cm2培养皿中，每皿的细胞密度应当约4×106个/10ml完全培养基左右。如果细胞密度和预期的相差较大，则需要对细胞进行计数，然后按照4×106个/皿的量接种；
(10)每6个培养皿整理为一摞，注意保持上下皿之间的配合。将培养皿左右，前后晃动数次，使细胞充分铺开，然后放入5％CO2培养箱。剩余细胞做同样处理；
(11)检查所传代细胞，细胞汇合度应当为70-80％，轮廓饱满，贴壁良好，在细胞培养皿中均匀分布；
(12)为细胞换液，将培养基替换为新鲜完全培养基,每皿9ml，并将培养箱的CO2浓度设定值提高到8％；
(13)按照N+0.5配DNA/CaCl2溶液。每皿HEK293T/17细胞转染质粒量按照下列比例使用：重组慢病毒质粒(20μg),pPac-GP(15μg)，pPac-R(10μg),pEnv-G(7.5μg)。取一个新的5ml离心管，加入0.5M CaCl2：0.25ml，重组慢病毒质粒20μg：pPac-GP 15μg：pPac-R 10μg：pEnv-G 7.5μg，补充超纯水至0.5ml盖上盖子，充分混匀；
(14)另取一支5ml离心管，加入0.5ml DNA/CaCl2溶液。打开涡旋振荡器，一只手拿住5ml离心管的上端，使管底接触振荡头，使液体在管壁上散开流动，另一只手拿一把1mL移液枪，吸取0.5mL 2×HBS溶液，缓慢滴加进入离心管，控制流速，以半分钟滴完为宜。2×HBS加入后，继续振荡5秒钟，停止振荡，可直接加入需要转染的细胞中；
(15)取一皿细胞，将离心管中的1mL钙转液滴加进去，尽可能使钙转试剂分布到整个培养皿中；
(16)钙转液加入后，在皿盖上做好标记，将培养皿放还到另一个5％CO2培养箱中。确保培养皿水平放置，每摞培养皿不要超过6个。在5％CO2培养箱中放置(6–8h)；
(17)将第一个培养箱的CO2浓度设定值调回到5％；
(18)24小时后，检查细胞状态。细胞汇合度应当为80–85％左右，状态良好。将培养基吸走，更换10ml新鲜的DMEM完全培养基；
(19)48小时后，观察转染效率。绝大多数细胞仍然是贴壁的。可以看到超过95％细胞都会带有绿色荧光。将同一个病毒包装上清液收集到一起，并向培养皿中继续添加10mL新鲜 培养基；
(20)72小时后，再次将同一个病毒上清液收集到一起，两次收集的病毒可以放在一起，丢弃培养皿；此时收集的上清里包含了重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0。
实施例2重组慢病毒载体的浓缩及检测
一、离子交换色谱法纯化重组慢病毒载体(如图7所示)；
(1)将收集的上清液使用Thermo真空泵，经0.22μm-0.8μm的PES滤器抽滤，除去杂质；
(2)按1:1～1:10的比例往上清中加入1.5M NaCl 250mM Tris-HCl(pH 6-8)；
(3)将2个离子交换柱串联放置，用4ml 1M NaOH、4ml 1M NaCl、5ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液依次过柱；
(4)将步骤2中获得的溶液通过蠕动泵以1-10ml/min的速度给离子交换柱上样；
(5)全部上清液过柱后，使用10ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液清洗一遍；
(6)根据上样量使用1-5ml 1.5M NaCl 25mM Tris-HCl(pH 6-8)进行洗脱，收集洗脱液；
(7)将洗脱液分成25到50μl一管，冻存到-80℃冰箱，进行长期保存；
二、滴度测定；
(1)取24孔板接种293T细胞。每孔细胞为5×104个，所加培养基体积为500ul,不同种类的细胞生长速度有所差异，进行病毒感染时的细胞融合率为40％-60％；
(2)准备3个无菌EP管，在每个管中加入90ul的新鲜完全培养基(高糖DMEM+10％FBS)接种细胞24小时后，取两个孔的细胞用血球计数板计数，确定感染时细胞的实际数目，记为N；
(3)取待测定的病毒原液10ul加入到第一个管中，轻轻混匀后，取10ul加入到第二个管中，然后依次操作直到最后一管；在每管中加入410ul完全培养基(高糖DMEM+10％FBS),终体积为500ul；
(4)感染开始后20小时，除去培养上清，更换为500μl完全培养基(高糖DMEM+10％FBS)，5％CO2继续培养48小时；
(5)72小时后，观察荧光表达情况，正常情况下，荧光细胞数随稀释倍数增加而相应减少,并拍照；
(6)用0.2ml 0.25％胰酶-EDTA溶液消化细胞，在37℃放置1分钟。用培养基吹洗整个细胞面，离心收集细胞。按照DNeasy试剂盒的说明抽提基因组DNA。每个样品管中加入200μl洗脱液洗下DNA并定量；
(7)准备目的DNA检测qPCRmix总管Ⅰ(QPCR引物序列为SEQ ID NO.57---SEQ ID NO.58)：
Figure PCTCN2017110656-appb-000001
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，4μl forward primer，4μl reverse primer，4μl probe和788μl H2O混和。震荡后放在冰上；
(8)准备内参DNA检测qPCRmix管Ⅱ(QPCR引物序列为SEQ ID NO.59---SEQ ID NO.60)：
2×TaqMan Master Mix                   25μl×n
10×RNaseP primer/probe mix            2.5μl×n
H2O                                    17.5μl×n
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，100μl 10×RNaseP primer/probe mix和700μl H2O混和。震荡后放在冰上；
(9)在预冷的96孔PCR板上完成PCR体系建立。从总管Ⅰ中各取45μl加入到A-D各行的孔中，从总管Ⅱ中各取45μl加入到E-G各行的孔中。
(10)分别取5μl质粒标准品和待测样品基因组DNA加入到A-D行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(11)分别取5μl基因组标准品和待测样品基因组DNA加入到E-G行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(12)所使用定量PCR仪为ABI PRISM 7500定量系统。循环条件设定为：50℃2分钟，95℃10分钟，然后是95℃15秒，60℃1分钟的40个循环。
数据分析：测得的DNA样品中整合的慢病毒载体拷贝数用基因组数加以标定，得到每基因组整合的病毒拷贝数。
滴度(integration units per ml，IU ml-1)的计算公式如下：
IU ml-1＝(C×N×D×1000)/V
其中：C＝平均每基因组整合的病毒拷贝数
N＝感染时细胞的数目(约为1×105)
D＝病毒载体的稀释倍数
V＝加入的稀释病毒的体积数
(13)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的滴度结果(如图8所示)；
三、内毒素测定；
(1)、内毒素工作标准品为15EU/支；
(2)、鲎试剂灵敏度λ＝0.25EU/ml,0.5ml/管
(3)、内毒素标准品稀释：取内毒素标准品一支，分别用BET水按比例稀释成4λ和2λ的溶解，封口膜封口，震荡溶解15min；稀释时每稀释一步均应在漩涡混合器上混匀30s；
(4)、加样：取鲎试剂若干支，每支加入BET水0.5ml溶解，分装至若干支无内毒素试管中，每管0.1ml。其中2支为阴性对照管，加入BET水0.1ml；
2支为阳性对照管，加入2λ浓度的内毒素工作标准品溶液0.1ml；
2支为样品阳性对照管，加入0.1ml含2λ内毒素标准品的样品溶液(稀释20倍的待测样品1ml+4λ的内毒素标准品溶液1ml＝2ml含2λ内毒素标准品的稀释40倍样品)。
样品管中加入0.1ml样品，稀释比例见表5，37±1℃水浴(或培养箱)保温60±1min；
Figure PCTCN2017110656-appb-000002
表5内毒素稀释比例及对应内毒素含量
(5)、重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的内毒素检测结果(如表6所示)，内毒素含量在0～2.5EU/ml之间，符合要求；
稀释倍数 原液 5 10 20 40 80 160
对应EU/ml 0.25 1.25 2.5 5 10 20 40
lvCARmm-PDLlscFv1 (+) (-) (-) (-) (-) (-) (-)
lvCARmm-PDLlscFv2 (+) (-) (-) (-) (-) (-) (-)
lvCARmm-PDLlscFv3 (+) (-) (-) (-) (-) (-) (-)
lcCARmm-scFv0 (+) (-) (-) (-) (-) (-) (-)
表6重组慢病毒载体的内毒素检测结果
四、支原体测定及比较；
(1)在实验前三日，细胞样品用无抗生素培养基进行培养；
(2)收集1ml细胞悬浮液(细胞数大于1*105)，置于1.5ml离心管中；
(3)13000×g离心1min，收集沉淀，弃去培养基；
(4)加入500ul PBS用枪头吹吸或涡旋振荡，重悬沉淀。13000×g离心5min；
(5)步骤4重复一次；
(6)加入50μl Cell Lysis Buffer，用枪头吹吸，充分混匀后,在55℃水浴中孵育20min；
(7)将样品置于95℃中加热5min；
(8)13000×g离心5min后，取5μl上清作为模板，25μlPCR反应体系为：ddH20 6.5μl、Myco Mix 1μl、2x Taq Plus Mix Master(Dye Plus)12.5μl、模板5μl；PCR循环条件为：95℃30sec，(95℃30sec，56℃30sec，72℃30sec)*30cycle，72℃5min。
(9)支原体检测结果显示(如图9所示)，重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0均不含支原体。
实施例3重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的功能检测。
一、CAR基因的细胞水平表达检测：
(1)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后，收集细胞采用RT-PCR进行CAR基因和scFv基因mRNA转录水平的检测，验证CAR基因和scFv基因的表达，如果CAR基因和scFv基因mRNA转录水平增高，则说明CAR基因和scFv基因的转录水平表达成功；
(2)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后，收集细胞采用western blot进行CAR蛋白表达水平的检测，验证CAR基因的表达，如果CAR蛋白表达水平增高，则说明CAR基因的翻译水平表达成功；
(3)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后，收集细胞培养上清采用ELISA进行scFv蛋白表达水平的检测，验证scFv基因的表达，如果scFv蛋白表达水平增高，则说明scFv基因的翻译水平表达成功；
(4)分别将MOI＝15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染细胞，48h后提取6孔板中 细胞的总RNA和总蛋白分别进行荧光定量PCR实验和免疫印迹实验。具体步骤：包被6孔板的四个孔，每个孔加入相应的PBS和RN，4℃过夜。12小时后按MOI＝15包被病毒，37℃培养箱放置5h；取出的6孔板，弃掉病毒上清，用PBS洗两遍，按1*106/孔，包被PBMC(用淋巴细胞分离液从人血中分离)，加入500ul培养基(含10％血清、20U/ml IL-2、Polybrene8ug/ml)。静置20min，1000g 20℃离心30min，37℃培养48h。
(5)Trizol法提取6孔板中PBMC细胞的总RNA，逆转录扩增cDNA,用CAR基因QPCR引物(序列为SEQ ID NO.61---SEQ ID NO.62)和scFv基因QPCR引物(序列为SEQ ID NO.67---SEQ ID NO.68)进行荧光定量PCR实验，反应体系见表7，以内参Actin为对照组，验证其mRNA的转录情况。
试剂 体积(μl)
SYBR premix ex taq: 10μl
ROX Reverse Dye(50x) 0.4μl
上游引物(2.5μM)： 0.5μl
下游引物(2.5μM)： 0.5μl
cDNA 1.0μl
ddH2O 7.6μl
表7 20μl qPCR反应体系
(6)蛋白免疫印迹(Western Blot)通过聚丙烯酰胺凝胶电泳将从PBMC中提取的总蛋白质按相对分子质量分离。采用湿转(4℃，400mA，120min)，将蛋白转移到PVDF膜上。用封闭液(含5％脱脂牛奶的TBST溶液)室温封闭PVDF膜1h，封闭液1:1000稀释Biotinylated protein L，然后与封闭好的PVDF膜室温孵育4℃过夜。TBST洗膜3次，每次10min。封闭液1:500稀释相应的SA-HRP，室温下孵育PVDF膜2h，TBST洗膜3次，每次10min。采用Amersham公司ECL+plusTM Western blotting system试剂盒进行显色。X光显影获得显示条带的胶片。
(7)酶联免疫吸附测定(Enzyme Linked ImmunoSorbent Assay,ELISA)通过将1:2、1:5、1:10稀释的细胞培养上清包被至96孔板中，同时设置阴性对照、阳性对照和空白孔，4℃过夜。次日洗涤3次，于反应孔中，加入新鲜1:10000稀释的proteinG-HRP 0.1ml，37℃孵育30-60分钟，洗涤，最后一遍用纯水洗涤。于各反应孔中加入的TMB底物溶液0.1ml，37℃孵育10～30分钟。于各反应孔中加入ELISA反应终止液0.05ml。在酶标仪上，于405nm处，测各孔OD值。
(8)RT-QPCR检测显示，重组慢病毒载体感染PBMC后的CAR基因和scFv基因的转录水平比空细胞有明显升高(如图10所示)，说明CAR基因和scFv基因的转录水平表达成功。
(9)蛋白免疫印迹(Western Blot)的结果表明，重组慢病毒载体感染PBMC后CAR蛋白的表达水平比对照病毒MOCK和空细胞有明显升高(如图11所示)，说明CAR基因的翻译水平表达成功。
(10)酶联免疫吸附测定(ELISA)的结果表明，重组慢病毒载体感染PBMC后scFv蛋白的表达水平比对照病毒MOCK和空细胞有明显升高(如图12所示)，说明scFv基因的翻译水平表达成功。
二、细胞杀伤实验效果评估。
(1)分别培养BCMA-K562细胞和PBMC细胞；
(2)实验开始前4天，分别用MOI＝15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的病毒感染PBMC细胞，培养72-96h后可安排开始实验；
(3)收集靶细胞(BCMA-K562)4x105cells和效应细胞(lvCARmm-PDL1scFv1、 lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0分别转导的PBMC细胞)2.8x106cells，800g，6min离心，弃上清；
(4)用1ml 1xPBS溶液分别重悬靶细胞和效应细胞，800g，6min离心，弃上清；
(5)重复步骤3一次；
(6)用700ul培养基(1640培养基+10％FBS)重悬效应细胞，用2ml培养基(1640培养基+10％FBS)重悬靶细胞；
(7)设置效靶比为1:1、5:1、10:1的实验孔，并设置对照组，每组3个复孔；
(8)250xg，5min平板离心；
(9)37℃5％CO2培养箱中培养24小时；
(10)250xg，5min平板离心；
(11)取每个孔的50ul上清到新96孔板中，并且每孔加入50ul底物溶液(避光操作)；
(12)避光孵育25分钟；
(13)每孔加入50ul终止液；
(14)酶标仪检测490nm吸光度；
(15)将3个复孔取平均值；将所有实验孔、靶细胞孔和效应细胞孔的吸光值减去培养基背景吸光值的均值；将靶细胞最大值的吸光值减去体积校正对照吸光值的均值。
(16)将步骤15中获得的经过校正的值带入下面公式，计算每个效靶比所产生的细胞毒性百分比。结果如图13所示，lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0重组慢病毒载体转导的PBMC细胞在几个效靶比条件下杀伤效率明显高于PBMC空细胞和对照病毒，说明scFv基因的表达对CAR基因的功能影响较小。
杀伤效率＝(实验孔-效应细胞孔-靶细胞孔)/(靶细胞最大孔-靶细胞孔)X100％
三、PDL1阻断效果评估(PD1、IL2、TNFα、IFNγmRNA转录水平)。
(1)分别培养BCMA-PDL1-K562细胞和PBMC细胞；
(2)实验开始前4天，分别用MOI＝15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的病毒感染PBMC细胞，培养72-96h后可安排开始实验；
(3)收集靶细胞(BCMA-PDL1-K562)4x105cells和效应细胞(lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0分别转导的PBMC细胞)2.8x106cells，800g，6min离心，弃上清；
(4)用1ml 1xPBS溶液分别重悬靶细胞和效应细胞，800g，6min离心，弃上清；
(5)重复步骤4一次；
(6)用700ul培养基(1640培养基+10％FBS)重悬效应细胞，用2ml培养基(1640培养基+10％FBS)重悬靶细胞；
(7)设置效靶比为10:1的实验孔，并设置对照组；
(8)250xg，5min平板离心；
(9)37℃5％CO2培养箱中共培养24小时，1000xg，2min平板离心，收集细胞抽总mRNA，反转cDNA，检测PD1、IL2、TNFα、IFNγmRNA转录水平；
(10)用PD1基因QPCR引物(序列为SEQ ID NO.63---SEQ ID NO.64)，IL2基因QPCR引物(序列为SEQ ID NO.65---SEQ ID NO.66)，TNFα基因QPCR引物(序列为SEQ ID NO.69---SEQ ID NO.70)，IFNγ基因QPCR引物(序列为SEQ ID NO.71---SEQ ID NO.72)，进行荧光定量PCR实验，反应体系见表6，以内参Actin为对照组，验证其mRNA的转录情况。
(11)RT-QPCR检测结果显示，lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、 lvCARmm-PDL1scFv3、lvCARmm-scFv0转导的PBMC并与靶细胞孵育后，PD1基因的mRNA水平与Mock组和空细胞组相比明显升高，lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0四组之间的PD1基因的mRNA水平则没有太大差别(如图14所示)，说明T细胞被激活后，PD1的表达水平也同步上升。而其后通过检测IL2、TNFα、IFNγmRNA转录水平发现，lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3三组与对照病毒lvCARmm-scFv0组相比IL2、TNFα、IFNγmRNA转录水平明显升高，其中lvCARmm-PDL1scFv1组的IL2、TNFα、IFNγ基因的转录mRNA水平升高最为明显(如图15所示)，IL2、TNFα、IFNγ基因的表达水平越高说明T细胞越处于活化状态，说明lvCARmm-PDL1scFv1组能有效阻断PD1/PDL1信号通路，解除对T细胞活化相关基因的抑制作用，将来可以在体内阻断PD1/PDL1的信号通路，达到抑制免疫逃脱的效果，提高CAR-T细胞治疗针对实体肿瘤的疗效。
Figure PCTCN2017110656-appb-000003
Figure PCTCN2017110656-appb-000004
Figure PCTCN2017110656-appb-000005
Figure PCTCN2017110656-appb-000006
Figure PCTCN2017110656-appb-000007
Figure PCTCN2017110656-appb-000008
Figure PCTCN2017110656-appb-000009
Figure PCTCN2017110656-appb-000010
Figure PCTCN2017110656-appb-000011
Figure PCTCN2017110656-appb-000012
Figure PCTCN2017110656-appb-000013
Figure PCTCN2017110656-appb-000014
Figure PCTCN2017110656-appb-000015
Figure PCTCN2017110656-appb-000016
Figure PCTCN2017110656-appb-000017
Figure PCTCN2017110656-appb-000018
Figure PCTCN2017110656-appb-000019
Figure PCTCN2017110656-appb-000020
Figure PCTCN2017110656-appb-000021

Claims (10)

  1. 一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体，其特征在于，包括：
    用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；
    用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；
    用于增强真核细胞内的复制的病毒复制子SV40 Ori序列，如SEQ ID NO.3所示；
    用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.11所示；
    用于嵌合抗原受体基因的真核转录的人EF1α启动子，如SEQ ID NO.12所示；
    用于慢病毒包装的慢病毒包装顺式元件；
    人PDL1的人源化单链抗体，所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1、或者如SEQ ID NO.22所示的PDL1scFv2、或者如SEQ ID NO.23所示的PDL1scFv3；
    用于共同转录表达蛋白质的IRES核糖体结合序列，如SEQ ID NO.25所示；
    IL6信号肽，如SEQ ID NO.26所示；
    人源抗体Fc段，如SEQ ID NO.27所示；
    以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体。
  2. 如权利要求1所述的载体，其特征在于，所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1。
  3. 如权利要求1所述的载体，其特征在于，所述慢病毒包装顺式元件采用第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。
  4. 如权利要求1所述的载体，其特征在于，所述慢病毒包装顺式元件采用第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
  5. 如权利要求1所述的载体，其特征在于，所述用于组成集识别、传递、启动于一体的二代CAR的嵌合抗原受体包括：如SEQ ID NO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域；
    所述用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括：如SEQ ID NO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.28所示的CD28嵌合受体共刺激因子。
  6. 如权利要求1-5任一项所述的载体，其特征在于，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
  7. 一种如权利要求1-6任一项所述的封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体的构建方法，其特征在于，包括以下步骤：
    (1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40 Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上；
    (2)将如SEQ ID NO.12所示的人EF1α启动子、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体组合成二代CAR或三代CAR设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到二代CAR或三代CAR设计的重组慢病毒质粒；
    (3)将人PDL1的人源化单链抗体PDL1scFv1、PDL1scFv2、或PDL1scFv3，IRES核糖体结合序列、IL6信号肽以及人源抗体Fc段分别克隆至重组慢病毒质粒中，得到PDL1阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3；
    (4)将得到的重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
    (5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化，分别得到重组慢病毒载体。
  8. 如权利要求7所述的方法，其特征在于，步骤(3)中，由人EF1α启动子启动整个CAR基因表达；CAR蛋白定位于细胞膜表面，识别BCMA抗原，刺激T细胞增殖和细胞因子分泌，激活下游信号通路的表达；当scfv区域与BCMA抗原结合时，信号通过嵌合受体传递至细胞内，从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞一系列生物学效应；由IRES核糖体结合序列共表达PDL1scFv与Fc的融合蛋白，并在IL6信号肽的引导下分泌到细胞外，通过与PDL1的结合，阻断PD1与PDL1的结合，从而阻断PD1/PDL1的信号通路，达到抑制免疫逃脱的效果。
  9. 如权利要求7所述的方法，其特征在于，步骤(5)中，所述抽滤步骤要控制上清体积在200ml～2000ml，控制真空度在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤要控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤要控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
  10. 如权利要求1-6任一项所述的载体在制备抑制免疫逃脱的药物中的应用。
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