siRNA knocking down human PD-1 and recombinant expression CAR-T vector and their construction methods and applications

Abstract

Provided are a human PD-1 knockdown siRNA, a recombinant expression CAR-T vector, a preparation method thereof, and an application of the same. A PD-1 knockdown siRNA expression cassette and an siRNA expression product thereof can be applied to a CAR-T therapy of multiple myeloma (MM) for eliminating or alleviating a tumor immune escape mechanism, and in the suppression of an immune escape mechanism in a CAR-T therapy of a tumor, such as pancreatic cancer, brain glioma, and myeloma.

Claims

1. An shRNA for knocking down PD-1 in a CAR T cell, wherein the shRNA comprises complementary siRNAs selected from the group consisting of a. SEQ ID NO: 41 and SEQ ID NO: 42; b. SEQ ID NO: 43 and SEQ ID NO: 44; c. SEQ ID NO: 45 and SEQ ID NO: 46; d. SEQ ID NO: 47 and SEQ ID NO: 48, e. SEQ ID NO: 49 and SEQ ID NO: 50; f. SEQ ID NO: 51 and SEQ ID NO: 52; and g. SEQ ID NO: 53 and SEQ ID NO: 54.

2. The shRNA of claim 1, wherein, the shRNA is an essential material in a medicine for eliminating or relieving the immune escape mechanism of tumor.

3. A recombinant expression vector comprising the shRNA of claim 1.

4. The recombinant expression vector according to claim 3, wherein, the recombinant expression vector is a lentiviral expression vector, a retroviral expression vector, an adenovirus expression vector, an adeno-associated virus expression vector or a plasmid.

5. The recombinant expression vector according to claim 4, wherein, the recombinant expression vector is the lentiviral expression vector and the lentiviral expression vector comprises a prokaryotic replicon pUC Ori sequence as shown in SEQ ID NO: 2; and the recombinant expression vector comprises: an AmpR sequence with Ampicillin resistance gene as shown in SEQ ID NO: 1; a virus-replicon SV40 Ori sequence as shown in SEQ ID NO: 3; a lentivirus packaging cis element used for lentivirus packaging; a ZsGreen1 green fluorescent protein as shown in SEQ ID NO: 11; an IRES ribosome binding sequence as shown in SEQ ID NO: 12; a human EF1α promoter as shown in SEQ ID NO: 15; a gene encoding an anti-BCMA chimeric antigen receptor; an enhanced woodchuck hepatitis virus post-transcriptional regulatory element (eWPRE) as shown in SEQ ID NO: 13; and a human RNA polymerase III promoter hU6, as shown in SEQ ID NO: 14, operably linked to the shRNA of claim 1.

6. The recombinant expression vector according to claim 5, wherein, the lentiviral expression vector comprises: a lentivirus 5′ terminal LTR as shown in SEQ ID NO: 5, a lentivirus 3′ terminal self-inactivating LTR as shown in SEQ ID NO: 6, a Gag cis element as shown in SEQ ID NO: 7, an RRE cis element as shown in SEQ ID NO: 8, an env cis element as shown in SEQ ID NO: 9, and a cPPT cis element as shown in SEQ ID NO: 10.

7. The recombinant expression vector according to claim 5, wherein, the lentiviral expression vector comprises: a lentivirus 5′ terminal LTR as shown in SEQ ID NO: 5, a lentivirus 3′ terminal self-inactivating LTR as shown in SEQ ID NO: 6, a Gag cis element as shown in SEQ ID NO: 7, an RRE cis element as shown in SEQ ID NO: 8, an env cis element as shown in SEQ ID NO: 9, a cPPT cis element as shown in SEQ ID NO: 10, and an RSV promoter as shown in SEQ ID NO: 4.

8. The recombinant expression vector according to claim 5, wherein, the eWPRE has 6 enhanced nucleotide mutations at positions g.396G>A, g.397C>T, g.398T>C, g.399G>A, g.400A>T, and g.411A>T, as shown in SEQ ID NO: 13.

9. The recombinant expression vector according to claim 5, wherein, the anti-BCMA chimeric antigen receptor comprises: a serialized CD8 leader chimeric receptor signal peptide, as shown in SEQ ID NO: 16, a BCMA single-chain antibody light chain VL, as shown in SEQ ID NO: 17, an Optimal Linker C, as shown in SEQ ID NO: 18, a BCMA single-chain antibody heavy chain VH, as shown in SEQ ID NO; 19, a CD8 chimeric receptor hinge, as shown in SEQ ID NO: 20, a CD8 chimeric receptor transmembrane domain, as shown in SEQ ID NO: 21, a CD137 chimeric receptor inducible co-stimulator, as shown in SEQ ID NO: 22, and a TCR chimeric receptor T cell activation domain, as shown in SEQ ID NO: 23.

10. The recombinant expression vector according to claim 5, wherein, the anti-BCMA chimeric antigen receptor comprises: a serialized CD8 leader chimeric receptor signal peptide, as shown in SEQ ID NO: 16, a BCMA single-chain antibody light chain VL, as shown in SEQ ID NO: 17, an Optimal Linker C, as shown in SEQ ID NO: 18, a BCMA single-chain antibody heavy chain VH, as shown in SEQ ID NO: 19, a CD8 chimeric receptor hinge, as shown in SEQ ID NO: 20, a CD8 chimeric receptor transmembrane domain, as shown in SEQ ID NO: 21, a CD28 chimeric receptor inducible co-stimulator, as shown in SEQ ID NO: 24, a CDI 37 chimeric receptor inducible co-stimulator, as shown in SEQ ID NO: 22, and a TCR chimeric receptor T cell activation domain, as shown in SEQ ID NO: 23.

11. A method for constructing the recombinant expression vector of claim 3, the method comprising: (1) storing in a lentiviral skeleton plasmid pLenti-3G silencer the AmpR sequence with Ampicillin resistance gene of SEQ ID NO: 1, prokaryotic replicon pUC Ori sequence of SEQ ID NO: 2, virus-replicon SV40 Ori sequence of SEQ ID NO: 3, lentivirus packaging cis element used for lentivirus packaging, ZsGreen1 green fluorescent protein of SEQ ID NO: 11, IRES ribosome binding sequence of SEQ ID NO: 12, enhanced woodchuck hepatitis virus post-transcriptional regulatory element (eWPRE) of SEQ ID NO: 13, and human RNA polymerase III promoter hU6 of SEQ ID NO: 14; (2) combining the human EF1α promoter of SEQ ID NO: 15 and anti-BCMA chimeric antigen receptors; (3) cloning the shRNA of claim 1 into the recombinant lentiviral plasmid of step (2); (4) transfecting recombinant lentiviral plasmids pCAR19-1761 and pCAR19-1769 of step (3) with lentiviral packaging plasmids pPac-GP and pPac-R and membrane protein plasmid pEnv-G, respectively into HEK293T/17 cell, and collecting a supernatant containing recombinant lentiviral vectors; and (5) purifying recombinant lentivirus supernatant of step (4) by ion exchange modes of extraction, adsorption, and elution.

12. A pharmaceutical composition comprising the recombinant expression vector of claim 3.

13. A CAR T cell, wherein, the CAR T cell is a T lymphocyte modified by the shRNA of claim 1.

14. A pharmaceutical composition comprising the CAR T cell of claim 13.

15. A pharmaceutical composition comprising the recombinant expression vector of claim 4.

16. A pharmaceutical composition comprising the recombinant expression vector of claim 5.

17. A pharmaceutical composition comprising the recombinant expression vector of claim 6.

18. A pharmaceutical composition comprising the recombinant expression vector of claim 7.

19. A pharmaceutical composition comprising the recombinant expression vector of claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of PD-1/PD-L1 signaling pathway described in the invention;

(2) FIG. 2 is a schematic diagram of PD1siRNA delivery by lentivirus described in the invention;

(3) FIG. 3A is a structure diagram of the 3.sup.rd generation lentiviral vector adopted by the invention;

(4) FIG. 3B is a comparison between the 2.sup.nd generation and 3.sup.rd generation lentiviral vector structure;

(5) FIG. 4 is a flowchart of the construction of the recombinant lentiviral vector described in the invention; Among them, (A) is a structure diagram of lentiviral cytoskeleton plasmid pLenti-3G silencer; (B) is a structure diagram of pCARbcma-silencer plasmid; (C) is a schematic diagram of pCARbcma-1453-pCARbcma-1460 plasmid; (D) is a structure diagram of lentiviral packaging plasmid pPac-GP; (E) is a structure diagram of lentiviral packaging plasmid pPac-R; (F) is a structure diagram of membrane protein pEnv-G;

(6) FIG. 5A is the schematic diagram of the restriction enzyme digestion prediction of lentiviral cytoskeleton plasmid pLenti-3G silencer; of which lane 1 is 1 kb DNA ladder Marker: the bands from top to bottom 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; lane 2 is the Cla I+BamH I digestion prediction of pLenti-3G silencer: the bands from top to bottom are: 7381 bp, 23 bp;

(7) FIG. 5B is the enzymatic cleavage agarose gel electrophoregram of lentiviral cytoskeleton plasmid pLenti-3G silencer, and lane1 is the electrophoretic result of 1 kb DNA ladder Marker; lane2 is the enzyme-digested electrophoretic result of Cla I+BamH I;

(8) FIG. 6A is the schematic diagram of the restriction enzyme digestion prediction of recombinant lentiviral plasmid pCARbcma-silencer, of which lane 1 is 1 kb DNA ladder Marker: the bands from top to bottom 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; lane 2 is the Pvu I digestion prediction of pCARbcma-silencer: the bands from top to bottom are: 8898 bp, 896 bp, 249 bp;

(9) FIG. 6B the enzymatic cleavage agarose gel electrophoregram of recombinant lentiviral plasmid pCARbcma-silencer, and lane 1 is the result of electrophoresis of 1 kb DNA ladder Marker; lane 2 is the Pvu I enzyme-digested electrophoretic result of pCAR19-silencer;

(10) FIG. 7A shows the sequencing alignment result of pCARbcma-1453, the top line shows SEQ ID NO: 41, the bottom line shows SEQ ID NO: 65.

(11) FIG. 7B shows the sequencing alignment result of pCARbcma-1454, the top line shows SEQ ID NO: 43, the bottom line shows SEQ ID NO: 66;

(12) FIG. 7C shows the sequencing alignment result of pCARbcma-1455, the top line shows SEQ ID NO: 45, the bottom line shows SEQ ID NO: 67;

(13) FIG. 7D shows the sequencing alignment result of pCARbcma-1456, the top line shows SEQ ID NO: 47, the bottom line shows SEQ ID NO: 68;

(14) FIG. 7E shows the sequencing alignment result of pCARbcma-1457, the top line shows SEQ ID NO: 49, the bottom line shows SEQ ID NO: 69;

(15) FIG. 7F shows the sequencing alignment result of pCARbcma-1458, the top line shows SEQ ID NO: 51, the bottom line shows SEQ ID NO: 70;

(16) FIG. 7G shows the sequencing alignment result of pCARbcma-1459, the top line shows SEQ ID NO: 53, the bottom line shows SEQ ID NO: 71;

(17) FIG. 7H shows the sequencing alignment result of pCARbcma-1460, the top line shows SEQ ID NO: 55, the bottom line shows SEQ ID NO: 72;

(18) FIG. 8 is a flowchart of ion exchange chromatography for purification of recombinant lentiviral vectors;

(19) FIG. 9 shows the titer detection results of recombinant lentiviral vectors;

(20) FIG. 10 shows the detection results of mycoplasma in different purification methods of recombinant lentiviral vectors. lane 1 is DL2000 marker, and the bands from top to bottom are: 2 kb, 1 kb, 750 bp, 500 bp, 250 bp, 100 bp; lane 2 is a positive control; lane 3 is a negative control; lane 4 is PBS; lane 5 is water; lane 6 is lvCARbcma-1453; lane 7 is lvCARbcma-1454; lane 8 is lvCARbcma-1455; lane 9 is lvCARbcma-1456; lane 10 is lvCARbcma-1457; lane 11 is lvCARbcma-1458; lane 12 is lvCARbcma-1459; lane 13 is lvCARbcma-1460;

(21) FIG. 11 is a bar chart of relative expression level of mRNA;

(22) FIGS. 12A and 12B show a WB detection chart of CAR protein expression. In FIG. 12 A, lane 1 is PBMC blank cells; lane 2 is the control virus MOCK; lane 3 is lvCARbcma-1453; lane 4 is lvCARbcma-1454; lane 5 is lvCARbcma-1455; lane 6 is lvCARbcma-1456; lane 7 is lvCARbcma-1457; lane 8 is lvCARbcma-1458; lane 9 is lvCARbcma-1459; lane 10 is lvCARbcma-1460; FIG. 12 B are the beta-actin bands;

(23) FIG. 13 shows the mRNA transcriptional level of PD-1 upon RT-QPCR test and after target cells have been incubated with CAR-T cells transduced by PD-1 knock-down recombinant lentiviral vectors lvCARbcma-1453-lvCARbcma-1460 for 24 h;

(24) FIG. 14 shows the killing of target cells at 24 h after co-culture of different effector cells and target cells at a ratio of 10:1. E is the effector cell, and T is the target cell.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(25) The following embodiments are only to illustrate the invention not to limit its scope. An experimental method without specific conditions stated in an embodiment is generally in accordance with conventional conditions or conditions recommended by the manufacturer.

Embodiment 1 To Construct Recombinant Lentiviral Vector

(26) I. Material

(27) 1. Lentiviral cytoskeleton plasmid pLenti-3G silencer, lentiviral packaging plasmid pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T/17 cells, homologous recombinase, Oligo Annealing Buffer were provided by Shiao (Shanghai) Biotech Co., Ltd.;

(28) 2. Primers: Primers required for amplification of DNA fragments and target sites and designed in the principle of primer design were synthesized by a Shanghai-based company, including:

(29) TABLE-US-00001 EF1α-F: (SEQ ID NO: 25) 5′-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3′ EF1α-R: (SEQ ID NO: 26) 5′-TCACGACACCTGAAATGGAAGA-3′ CD8 leader-F: (SEQ ID NO: 27) 5′-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3′ CD8 leader-R: (SEQ ID NO: 28) 5′-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3′ VL-F: (SEQ ID NO: 29) 5′-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3′ VL-R: (SEQ ID NO: 30) 5′-ACGCTTGATCTCCAGTTTGGT-3′ OLC-VH-F: (SEQ ID NO: 31) 5′-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCG GGTGGCGGCGGATCTCAGGTGCAGCTGGTCCAGAG-3′ VH-R: (SEQ ID NO: 32) 5′-GCTGGACACGGTCACTAGTGTG-3′ CD8 Hinge-F: (SEQ ID NO: 33) 5′-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3′ CD8 Hinge-R: (SEQ ID NO: 34) 5′-GTAGATATCACAGGCGAAGTCCA-3′ CD8 Transmembrane-F: (SEQ ID NO: 35) 5′-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3′ CD8 Transmembrane-R: (SEQ ID NO: 36) 5′-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3′ CD137-F: (SEQ ID NO: 37) 5′-AAACGGGGCAGAAAGAAACTC-3′ CD137-R: (SEQ ID NO: 38) 5′-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3′ TCR-F: (SEQ ID NO: 39) 5′-AGAGTGAAGTTCAGCAGGAGCG-3′ TCR-R: (SEQ ID NO: 40) 5′-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3′ siRNA1453-F: (SEQ ID NO: 41) 5′-CCGGCTAAACTGGTACCGCATGAGCCTCGAGTCATGCGGTACCAGT TTAGCATTTTTTG-3′ siRNA1453-R: (SEQ ID NO: 42) 5′-AATTCAAAAAATGCTAAACTGGTACCGCATGACTCGAGGCTCATGC GGTACCAGTTTAG-3′ siRNA1454-F: (SEQ ID NO: 43) 5′-CCGGCATTGTCTTTCCTAGCGGAATCTCGAGTCCGCTAGGAAAGAC AATGGTTTTTTTG-3′ siRNA1454-R: (SEQ ID NO: 44) 5′-AATTCAAAAAAACCATTGTCTTTCCTAGCGGACTCGAGATTCCGCT AGGAAAGACAATG-3′ siRNA1455-F: (SEQ ID NO: 45) 5′-CCGGAGGCGCAGATCAAAGAGAGTTCTCGAGCTCTCTTTGATCTGC GCCTTGTTTTTTG-3′ siRNA1455-R: (SEQ ID NO: 46) 5′-AATTCAAAAAACAAGGCGCAGATCAAAGAGAGCTCGAGAACTCTC TTTGATCTGCGCCT-3′ siRNA1456-F: (SEQ ID NO: 47) 5′-CCGGCCCTGTGGTTCTATTATATTACTCGAGATATAATAGAACCAC AGGGAATTTTTTG-3′ siRNA1456-R: (SEQ ID NO: 48) 5′-AATTCAAAAAATTCCCTGTGGTTCTATTATATCTCGAGTAATATAA TAGAACCACAGGG-3′ siRNA1457-F: (SEQ ID NO: 49) 5′-CCGGGGAACCCATTCCTGAAATTATCTCGAGAATTTCAGGAATGGG TCCAATTTTTTG-3′ siRNA1457-R: (SEQ ID NO: 50) 5′-AATTCAAAAAATTGGAACCCATTCCTGAAATTCTCGAGATAATTTCA GGAATGGGTTCC-3′ siRNA1458-F: (SEQ ID NO: 51) 5′-CCGGCAGGCCTAGAGAAGTTTCAGGCTCGAGTGAAACTTCTCTAGG CCTGCATTTTTTG-3′ siRNA1458-R: (SEQ ID NO: 52) 5′-AATTCAAAAAATGCAGGCCTAGAGAAGTTTCACTCGAGCCTGAAA CTTCTCTAGGCCTG-3′ siRNA1459-F: (SEQ ID NO: 53) 5′-CCGGCAGGACTCATGTCTCAATGCCCTCGAGCATTGAGACATGAGT CCTGTGTTTTTTG-3′ siRNA1459-R: (SEQ ID NO: 54) 5′-AATTCAAAAAACACAGGACTCATGTCTCAATGCTCGAGGGCATTGA GACATGAGTCCTG-3′ siRNA1460-F: (SEQ ID NO: 55) 5′-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAG AATTTTTTG-3′ siRNA1460-R: (SEQ ID NO: 56) 5′-AATTCAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACG TTCGGAGAA-3′ PD-1-QPCR-F: (SEQ ID NO: 57) 5′-TGCAGCTTCTCCAACACAT-3′ PD-1-QPCR-R: (SEQ ID NO: 58) 5′-CTTGTCCGTCTGGTTGCT-3′ WPRE-QPCR-F: (SEQ ID NO: 59) 5′-CCTTTCCGGGACTTTCGCTTT-3′ WPRE-QPCR-R: (SEQ ID NO: 60) 5′-GCAGAATCCAGGTGGCAACA-3′ Actin-QPCR-F:  (SEQ ID NO: 61) 5′-CATGTACGTTGCTATCCAGGC-3′ Actin-QPCR-R:  (SEQ ID NO: 62) 5′-CTCCTTAATGTCACGCACGAT-3′ CAR-QPCR-F: (SEQ ID NO: 63) 5′-GACTTGTGGGGTCCTTCTCCT-3′ CAR-QPCR-R: (SEQ ID NO: 64) 5′-GCAGCTACAGCCATCTTCCTC-3′

(30) 3. The DNA sequences shown in SEQ ID NO: 15-SEQ ID NO: 64 were synthesized by Shanghai Generay Biotech Co., Ltd., and stored as oligonucleotide dry powder or plasmid;

(31) 4. Tool enzymes BspE I, EcoR I, BamH I, Pvu I, Cla I and T4 DNA ligases were purchased from NEB;

(32) 5. PrimerSTAR HS DNA Polymerase, RN were purchased from Takara;

(33) 6. 0.22 μm-0.8 μm PES filters were purchased from millipore;

(34) 7. The Plasmid Extraction Kit and Agarose Gel Recovery Kit were purchased from MN;

(35) 8. TOP 10 Competent Cell were purchased from tiangen;

(36) 9. NaCl, KCl, Na.sub.2HPO.sub.4.12H.sub.2O, KH.sub.2PO.sub.4, Trypsin, EDTA, CaCl.sub.2, NaOH, PEG6000 were purchased from Shanghai Sangon Biotech;

(37) 10. Opti-MEM, FBS, DMEM, 1640, Pen-Srep, Hepes were purchased from invitrogen;

(38) 11. Biotinylated protein L was purchased from GeneScript;

(39) 12. HRP-labeled secondary antibodies and DAB working fluid were purchased from ZSGB-BIO;

(40) 13. ECL+plus™ Western blotting system was purchased from Amersham;

(41) 14. DNeasy kit was purchased from Shanghai Generay Biotech Co., Ltd.;

(42) 15. Lymphocyte Separation Medium were purchased from Dakewe Biotech Co., Ltd.;

(43) 16. SA-HRP were purchased from Yeasen Biotech Co., Ltd.;

(44) 17. Mycoplasma Detection Kit, Endotoxin Detection Kit and BCMA-K562 Cell and BCMA-PDL1-K562 cell strains were purchased from Shiao (Shanghai) Biotech Co., Ltd.;

(45) 18. LDH Detection Kit were purchased from promega;

(46) II. Construction Method of Recombinant Lentiviral Vectors lvCARbcma-1453-lvCARbcma-1460 See FIG. 4. The construction method of the recombinant lentiviral vector described in the invention is as follows:

(47) 1. The human EF1α promoters, CD8 leader chimeric receptor signal peptide, BCMA single chain antibody light chain VL, Optimal Linker C, BCMA single chain antibody heavy chain VH, CD8 chimeric receptor hinge, CD8 transmembrane transmembrane domain chimeric receptor, the chimeric receptor co-stimulation factor—CD137, TCR and T cell activation domain chimeric receptor fragments were cloned into the lentiviral cytoskeleton plasmid pLenti-3G silencer to obtain recombinant lentiviral plasmid pCARbcma-silencer, and the siRNA fragments were connected into pCARbcma-silencer respectively to obtain IL-6 knock-down recombinant lentiviral plasmids pCARbcma-1453-pCARbcma-1460.

(48) (1) The lentiviral cytoskeleton plasmid pLenti-3G silencer was double digested with Cla I and BamH I restriction enzymes. The product was electrophoresed on a 1.5% agarose gel to confirm the 7381 bp fragment V1 (see FIG. 5), then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(49) TABLE-US-00002 TABLE 1 Procedures for the recovery of agarose gels 1. Sol Add the sol solution in a ratio of 200 μl NTI/100 mg gel, and place it in a 50° C. water bath for 5-10 minutes. 7. Bind to Centrifuge at 11,000 g for 30 seconds, and discard the filtrate. DNA 8. Wash Add 700 μl NT3, centrifuge at 11,000 g for 30 seconds, and membrane discard the filtrate 9. Wash Repeat the third step once membrane 10. Dry Centrifuge at 11,000 g for 1 minute, replace with a new collection tube, and leave it at room temperature for 1 minute. 11. Elute Add 15-30 μl NE, leave it at room temperature for 1 minute, DNA centrifuge at 11,000 g for 1 minute, and then collect the filtrate.

(50) (2) Use the primers EF1α-F and EF1α-R with the synthesized SEQ ID NO: 15 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 2 min)*35 cycle, 72° C. 10 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 1208 bp fragment a, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined-,

(51) TABLE-US-00003 TABLE 2 50 μl PCR reaction system Reagent Volume (μl) H.sub.2O 32.5 5 × Buffer (with Mg2+) 10 dNTP (2.5 mM each) 4 Primer1 (+)(10 μM) 1 Primer2 (−)(10 μM) 1 Template 1 PrimeSTAR 0.5

(52) (3) Use the primers CD8 leader-F and CD8 leader-R with the synthesized SEQ ID NO: 16 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 101 bp fragment b, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(53) (4) Use the primers VL-F and VL-R with the synthesized SEQ ID NO: 17 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 336 bp fragment c, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(54) (5) Use the primers OLC-VH-F and VH-R with the synthesized SEQ ID NO: 19 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 421 bp fragment d, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(55) (6) Use the primers CD8 Hinge-F and CD8 Hinge-R with the synthesized SEQ ID NO: 20 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 147 bp fragment e, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(56) (7) Use the primers CD8 Transmembrane-F and CD8 Transmembrane-R with the synthesized SEQ ID NO: 21 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 100 bp fragment f, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(57) (8) Use the primers CD137-F and CD137-R with the synthesized SEQ ID NO: 22 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 142 bp fragment g, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(58) (9) Use the primers TCR-F and TCR-R with the synthesized SEQ ID NO: 23 as a template, and apply the system in Table 2. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec)*35 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 335 bp fragment h, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(59) (10) Applying the system in Table 3, 1 μl each of DNA fragments b, c and d were taken as templates to add to Eppendorf tubes except for primers. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 60° C. 10 sec, 72° C. 40 sec)*6 cycle. To add primer CD8 leader-F/VH-R with the conditions as (98° C. 10 sec, 60° C. 10 sec, 72° C. 40 sec)*24 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 814 bp fragment i, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(60) TABLE-US-00004 TABLE 3 50 μl overlapping PCR reaction system Reagent Volume (μl) H.sub.2O 33.5-1* number of templates 5 × Buffer (with Mg2+) 10 dNTP (2.5 mM each) 4 Primer1 (+) (10 μM) 1 Primer2 (−) (10 μM) 1 Template 1* number of templates PrimeSTAR 0.5

(61) (11) Applying the system in Table 3, 1 μl each of DNA fragments e, f, g and h were taken as templates to add to Eppendorf tubes except for primers. PCR circulation condition was: 98° C. 3 min, (98° C. 10 sec, 60° C. 10 sec, 72° C. 40 sec)*6 cycle. To add primer CD8 Hinge-F/TCR-R with the conditions as (98° C. 10 sec, 60° C. 10 sec, 72° C. 40 sec)*24 cycle, 72° C. 5 min. The product was electrophoresed on a 1.5% agarose gel to confirm the 704 bp fragment j, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(62) (12) The DNA fragments V1, a, i, j were added to the Eppendorf tubes in a total volume of 5 μl with a molar ratio of 1:1:1:1. 15 μl of the homologous recombinase reaction solution was added to the tubes, and the mixtures were incubated at 42° C. for 30 minutes. Place them on ice for 2-3 minutes. Add the reaction solution to 50 μl of TOP10, gently rotate to mix the contents, place them on ice for 30 minutes, then put the tubes in the thermostatic water bath pre-warmed to 42° C. for 90 seconds, and quickly transfer the tubes in an ice bath. The cells were allowed to cool for 2-3 minutes. Add 900 μl of LB medium to each tube, then put the tubes to a 37° C. shaker and incubate for 1 hour to resuscitate the bacteria. Take 100 μl of transformant bacteria solution to apply to an Amp LB agar plate, invert the plate, and culture in a thermostatic incubator at 37° C. for 16 hours.

(63) The clones were picked for colony PCR identification, and the correct clones were identified as recombinant lentiviral plasmid pCARbcma-silencer. Enzyme digestion identification was performed for the correct clones (see FIG. 6);

(64) (13) The recombinant lentiviral plasmid pCARbcma-silencer was double digested with BspE I and EcoR I restriction enzymes. The product was electrophoresed on a 1.5% agarose gel to confirm the 10035 bp fragment V2, then such gel was recovered and placed in an Eppendorf tube. The corresponding fragments were recovered with Agarose Gel Recovery Kit of MN (see Table 1), and the purity and concentration of the product were determined;

(65) (14) The synthesized siRNA1453-F/R-siRNA1460-F/R were dissolved into 20 μM with oligo annealing buffer respectively, and each 30 μl of the corresponding F and R were mixed. The mixture of siRNA1453-F/R-siRNA1460-F&R was heated in a water bath at 95° C. for 5 minutes, and then the water bath was opened and allowed to cool to room temperature to form double-stranded oligonucleotide fragments. Take 1 μl for the ligation reaction (see Table 4), ligate at 4° C. for 16 h, and then place it on ice for 2-3 minutes. Add the reaction solution to 50 μl of TOP10, gently rotate to mix the contents, place them on ice for 30 minutes, then put the tubes in the thermostatic water bath pre-warmed to 42° C. for 90 seconds, and quickly transfer the tubes in an ice bath. The cells were allowed to cool for 2-3 minutes. Add 900 μl of LB medium to each tube, then put the tubes to a 37° C. shaker and incubate for 1 hour to resuscitate the bacteria. Take 100 μl of transformant bacteria solution to apply to an Amp LB agar plate, invert the plate, and culture in a thermostatic incubator at 37° C. for 16 hours.

(66) The clones were picked for colony PCR identification, and the correct clones were identified as PD-1 knock-down recombinant lentiviral plasmids pCARbcma-1453-pCARbcma-1460. The correct clones were sequenced and identified (see FIG. 7).

(67) TABLE-US-00005 TABLE 4 20 μl ligation reaction system Reagent Volume (μl) H.sub.2O 13 V2 3 10 × T4 DNA ligase Buffer 2 T4 DNA ligase 1 Annealed double-stranded 1 oligonucleotides

(68) 2. Packaging of Recombinant Lentiviral Vectors lvCARbcma-1453-lvCARbcma-1460

(69) (1) Complete medium: take out the pre-warmed fresh medium, add 10% FBS+5 ml Pen-Srep, and mix them upside down;

(70) (2) 1×PBS solution: weigh 8 g of NaCl, 0.2 g of KCl, 3.58 g of Na.sub.2HPO.sub.4.12H.sub.2O, 0.24 g of KH.sub.2PO.sub.4, and put them in a 1000 ml beaker, and add 900 ml of Milli-Q grade ultrapure water to dissolve. After completion of the dissolution, the volume was adjusted to 1000 ml using a 1000 ml measuring cylinder, and the mixture was sterilized by heat sterilization at 121° C. for 20 minutes; (3) 0.25% Trypsin solution: weigh 2.5 g of Trypsin, 0.19729 g EDTA, and put them in a 1000 ml beaker, and add 900 ml of 1×PBS solution to dissolve. After completion of the dissolution, the volume was adjusted to 1000 ml using a 1000 ml measuring cylinder, and the mixture was sterilized via 0.22 μM filter. It could be saved in the refrigerator at −20° C. for long-term use;

(71) (4) 0.5M CaCl.sub.2) solution: weigh 36.75 g of CaCl.sub.2, and dissolve it with 400 ml of Milli-Q grade ultrapure water; The volume was adjusted to 500 ml with Milli-Q grade ultrapure water, and mixed; The mixture was sterilized via 0.22 μM filter, and stored in 50 ml centrifuge tubes with about 45 ml in each tube at 4° C.

(72) (5) 2×HBS solution: weigh 4.09 g of NaCl, 0.269 g of Na.sub.2HPO.sub.4, 5.96 g of Hepes, and dissolve them with 400 ml Milli-Q grade ultrapure water; After calibrating the PH meter, the PH of the HBS solution was adjusted to 7.05 with 2M NaOH solution. It was about 3 ml of 2M NaOH to consume to adjust the PH of each bottle of HBS.

(73) (6) The frozen HEK293T/17 cells were removed from the liquid nitrogen container and repidly transferred to a 37° C. water bath for 1-2 minutes, and then put them on a super clean bench. Aseptically transfer all the liquid in the freezing tube to a 10 cm.sup.2 petri dish, and make up DMEM containing 10% FBS to 8 mL/10 cm.sup.2 dish, and observe the cells under microscope after 24 hours. Passage was performed with the degree of cell confluence greater than 80%;

(74) (7) HEK293T/17 cells with good cell status and no pollution were selected, and each 2-6 petri dishes were used as a group. After trypsinizing the cells, 4-12 ml of complete medium was pipetted with an electric pipette to add 2 ml to each digested dish to avoid drying the dish; All cells were isolated into single cell suspensions using a 1 ml pipette and transferred to medium bottles;

(75) (8) The remaining cells in the above 2-6 petri dishes were transferred to the medium bottles, and the petri dishes were rinsed with the medium again;

(76) (9) Close the cap of the medium bottles and turn them upside down for about 10 times to fully mixed the cell suspension. Transfer the cells to 8-24 10 cm.sup.2 petri dishes. The cell density of each dish shall be about 4×10.sup.6 cells/10 ml complete medium. In the case that the cell density was significantly different from the expected, the number of cells would be counted. Then the cells were inoculated according to the quantity of 4-10.sup.6 per dish;

(77) (10) Arrange each of the 6 petri dishes into a pile, and keep the fit between the upper and lower dishes. Shake the petri dishes left and right, back and forth several times to make cells fully spread out, and then put them into an incubator with 5% CO. The remaining cells were treated as the same;

(78) (11) Upon Checking the passage cells, the cells shall be at 70-80% confluence, with full contour, good attachment and even distribution in petri dishes;

(79) (12) For changing the solution, the medium was replaced with fresh complete medium with 9 ml per dish. The CO2 concentration of incubator was increased to 8%;

(80) (13) To prepare DNA/CaCl.sub.2 according to N+0.5. The amount of HEK293T/17 cell transfection plasmid per dish was used in the following ratios: recombinant lentiviral plasmid (20 μg), pPac-GP (15 μg), pPac-R (10 μg), pEnv-G (7.5 μg). Take a new 5 ml centrifuge tube, add 0.5M CaCl.sub.2: 0.25 ml, recombinant lentiviral plasmid 20 μg: pPac-GP 15 μg: pPac-R 10 μg: pEnv-G 7.5 μg, supplement ultrapure water to 0.5 ml, and cover the cap to mix them fully;

(81) (14) Take another 5 ml centrifuge tube and add 0.5 ml DNA/CaCl.sub.2 solution. Open a vortex mixer, hold the upper end of the 5 ml centrifuge tube with one hand, and make the bottom of the tube contact the oscillation chamber, so that the liquid could spread on the tube wall. Take a 1 ml pipette with anther hand to suck 0.5 mL 2×HBS solution, add it into the centrifuge tube slowly and control the flow velocity. It was advisable to complete the drip in half a minute. After 2-HBS was added, it should be oscillated for another 5 seconds, and then stop oscillating. It could be directly added into the cells that need transfection;

(82) (15) Take a dish of cells and drop 1 mL calcium transfection solution in the centrifuge tube in the dish to distribute the calcium transfection solution throughout the petri dish as much as possible;

(83) (16) After the calcium transfection solution was added, the petri dish was marked on the cover, and put back in another incubator with 5% CO2. Make sure that the petri dish was placed horizontally, and that there were no more than 6 petri dishes in each pile. These dishes were placed in the incubator with 5% CO2 for 6-8 h;

(84) (17) The CO.sub.2 concentration of the first incubator was adjusted at 5%;

(85) (18) The cells status was check 24 hours later. The cell confluence should be around 80-85% and in good condition. Aspirate the medium and replace 10 ml of fresh DMEM complete medium;

(86) (19) The transfection efficiency was observed 48 hours later. Most cells were still adherent. It could be seen that more than 95% of the cells would have green fluorescence. The supernatant of the same virus packaging was collected together, and 10 mL of fresh medium was added to the petri dish;

(87) (20) The same virus supernatant was collected again 72 hours later. The two collections were put together, and the petri dishes were discarded; the supernatant collected at this time contained the recombinant lentiviral vectors lvCARbcma-1453-lvCARbcma-1460.

Embodiment 2 Concentration and Detection of Recombinant Lentivirus Vector

(88) I. Purification of Recombinant Lentiviral Vectors by Ion Exchange Chromatography (see FIG. 8);

(89) (1) The collected supernatant was filtered through a 0.22 μm-0.8 μm PES filter using a Thermo vacuum pump to remove impurities;

(90) (2) 1.5M NaCl 250 mM Tris-HCl (PH6-8) was added to the supernatant at a ratio of 1:1 to 1:10

(91) (3) Two ion exchange columns were placed in series, and they were passed through sequentially by 4 ml 1M NaOH, 4 ml 1M NaCl, 5 ml 0.15M NaCl 25 mM Tris-HCl (pH 6-8) solution;

(92) (4) The solution obtained in step 2 was pumped into the ion exchange column with a peristaltic pump at a rate of 1-10 ml/min;

(93) (5) After all the supernatant was passed through the column, it was washed with 10 ml of 0.15M NaCl 25 mM Tris-HCl (pH 6-8) solution;

(94) (6) According to the sample size, 1-5 ml of 1.5M NaCl 25 mM Tris-HCl (pH 6-8) was used for elution and the eluate was collected;

(95) (7) The eluate was divided into tubes about 25 to 50 μl each, and stored in a refrigerator with −80° C. for long-term storage;

(96) II. Titre Determination;

(97) (1) 293T cells were inoculated with 24-well plates. The number of cells in each well was 5×104, and the volume of medium added was 500 ul. As the growth rate of different types of cells was different, the rate of cell fusion during viral infection was 40%-60%;

(98) (2) Three sterile EP tubes were prepared, and 90 μl fresh complete medium (high glucose DMEM+10% FBS) was added into each tube to inoculate the cells. 24 hours later, the cells in the two pores were taken and counted with a hemocytometer to determine the actual number of cells at the time of infection, denoted as N;

(99) (3) 10 μl of the virus stock to be determined was added to the first tube. After gently mixing, 10 μl of the virus stock was added to the second tube, and then sequentially operated until the last tube; 410 μl complete medium (high glucose DMEM+10% FBS) was added into each tube, and the final volume was 500 μl;

(100) (4) 20 hours after the infection, the cultural supernatant was removed and changed into 5001 complete medium (high glucose DMEM+10% FBS). The cells were continuously cultured for 48 hours in 5% C02;

(101) (5) After 72 hours, the fluorescence expression was observed. Under normal circumstances, the number of fluorescence cells decreased with the increase of dilution ratio. At the same time, photos were taken;

(102) (6) The cells were digested with 0.2 ml 0.25% trypsin-EDTA solution, and then they were placed at 37° C. for 1 minute. The whole cellular surface were purged with medium, and the cells were collected by centrifugation. Genomic DNA was extracted according to the instructions of DNeasy kit. 200 μl of eluent were added to each sample tube to remove DNA, and then they were quantified;

(103) (7) The DNA detection qPCRmix manifold I was prepared (QPCR primer sequences were SEQ ID NO: 59-SEQ ID NO: 60):

(104) TABLE-US-00006 2 × TaqMan Master Mix  25 μl × n Forward primer (100 pmol ml−1) 0.1 μl × n Reverse primer (100 pmol ml−1) 0.1 μl × n Probe (100 pmol ml−1) 0.1 μl × n H.sub.2O 19.7 μl × n  n = number of reactions. For example, the total n were 40. 1 ml of 2 × TaqMan Universal PCR Master Mix, 4 μl of forward primer, 4 μl of reverse primer, 4 μl of probe and 788 μl of H.sub.2O were mixed and Placed on ice after being shaken;

(105) (8) The reference DNA detection qPCRmix manifold II were prepared (QPCR primer sequences were SEQ ID NO: 59-SEQ ID NO: 60):

(106) TABLE-US-00007 2 × TaqMan Master Mix  25 μl × n 10 × RNaseP primer/probe mix 2.5 μl × n H.sub.2O 17.5 μl × n  n = number of reactions. For example, the total n were 40. 1 ml of 2 × TaqMan Universal PCR Master Mix, 100 μl pf 10 × RNaseP primer/probe mix and 700 μl of H.sub.2O were mixed and placed on ice after being shaken;

(107) (9) The PCR system was established on a pre-cooled 96-well PCR plate. Take 45 μl from each tube of manifold I to add to the wells of each row of A-D. Take 45 μl from each tube of manifold II to add to the wells of each row of E-G.

(108) (10) 5 μl of the standard plasmid and the genomic DNA from the samples to be tested were taken respectively to add to the A-D row, and each sample was repeated once. 1 well was left to add 5 μl of water as no-template control.

(109) (11) 5 μl of the genomic standards and the genomic DNA from the samples to be tested were taken respectively to add to the E-G row, and each sample was repeated once. 1 well was left to add 5 μl of water as no-template control.

(110) (12) The quantitative PCR instrument used was the ABI PRISM 7500 quantitative system. The cyclic conditions were set to: 50° C. 2 min, 95° C. 10 min, (95° C. 15 sec, 60° C. 1 min)×40 cycle.

(111) Data analysis: the copy number of lentiviral vectors integrated in the measured DNA samples was calibrated with the number of genomes to obtain the copy number of viruses integrated in each genome.

(112) The calculation formula of integration units per ml (IU ml.sup.−1) was as follows:
IU ml.sup.−1=(C×N×D×1000)/V Of C=the average virus copy number per genome which: integration N=number of cells at the time of infection (approximately 1×10.sup.−5) D=dilution of the viral vector V=the volume of diluted virus added

(113) (13) Titer results of recombinant lentiviral vector lvCAR19-1761-lvCAR19-1769 (see FIG. 9);

(114) III. Endotoxin Determination;

(115) (1) The working standard of endotoxin was 15 EU per dose;

(116) (2) Sensitivity of Tachypiens Amebocyte Lysate (TAL) λ=0.25 EU/ml, 0.5 ml/tube

(117) (3) Dilution of endotoxin standard: take one endotoxin standard, dilute it into 4× and 2× solution with BET water, seal with sealing film and vortex for 15 min; During dilution, each dilution step should be mixed on the vertex mixer for 30 s;

(118) (4) Adding: Several TAL were taken, each was dissolved in 0.5 ml of BET water, and then divided into several exdotoxin-free tubes (0.1 ml each tube). Two of them were negative control which were added 0.1 ml of BET water to each of them;

(119) Two tubes were positive control which were added 0.1 ml of endotoxin working standard solution with concentration of 2λ to each of them;

(120) Two tubes were positive control of sample which were added 0.1 ml sample solution contained 2× endotoxin standard (1 ml of 20× dilution of sample to be tested+1 ml of solution contained 4× endotoxin standard=2 ml of 40× dilution of sample contained 2λ endotoxin standard).

(121) 0.1 ml of sample was added to the sample tube. The dilution ratio was in accordance with the Table 5. They were placed in water bath (or incubator) at 37±1° C. for 60±1 min;

(122) TABLE-US-00008 TABLE 5 Dilution ratio of exdotoxin and corresponding endotoxin content Dilution Multiple Original Fluid 5 10 20 40 80 160 Corresponding 0.25 1.25 2.5 5 10 20 40 EU/ml Result

(123) (5) The endotoxin detection results of the recombinant lentiviral vector lvCAR19-1761-lvCAR19-1769 (as shown in Table 6) showed that the endotoxin content was between 0-2.5 EU/ml, which met the requirements;

(124) TABLE-US-00009 TABLE 6 Detection results of endotoxin Dilution Multiple Original Fluid 5 10 20 40 80 160 Corresponding 0.25 1.25 2.5 5 10 20 40 EU/ml lvCARbcma-1453 (+) (−) (−) (−) (−) (−) (−) lvCARbcma-1454 (+) (−) (−) (−) (−) (−) (−) lvCARbcma-1455 (+) (−) (−) (−) (−) (−) (−) lvCARbcma-1456 (+) (+) (−) (−) (−) (−) (−) lvCARbcma-1457 (+) (−) (−) (−) (−) (−) (−) lvCARbcma-1458 (+) (+) (−) (−) (−) (−) (−) lvCARbcma-1459 (+) (+) (−) (−) (−) (−) (−) lvCARbcma-1460 (+) (−) (−) (−) (−) (−) (−)

(125) IV. Determination and Comparison of Mycoplasma;

(126) (1) Cell samples were cultured in antibiotic-free medium three days before the experiment;

(127) (2) 1 ml of cell suspension was collected (the number of cells is greater than 1*10.sup.5) to place in a 1.5 ml centrifuge tube;

(128) (3) Centrifuge at 13000×g for 1 min, collect the precipitate, and discard the medium;

(129) (4) 500 μl of PBS was added. The mixture was blew and sucked with a pipette or vortex oscillated to resuspend the precipitate. Centrifuge at 13000×g for 5 min;

(130) (5) To repeat step 4 once;

(131) (6) 50 μl of Cell Lysis Buffer was added. The mixture was blew and sucked with a pipette. After fully mixed, it was incubated in the water at 55° C. for 20 min;

(132) (7) The sample was heated at 95° C. for 5 min;

(133) (8) After centrifugation at 13000×g for 5 min, 5 μl of supernatant was used as a template. 25 μl PCR reaction system was: 6.5 μl of ddH.sub.2O, 1 μl of Myco Mix, 12.5 μl of 2×Taq Plus Mix Master (Dye Plus), 5 μl of template; PCR circulation condition was: 95° C. 30 sec, (95° C. 30 sec, 56° C. 30 sec, 72° C. 30 sec)*30 cycle, 72° C. 5 min.

(134) (9) Detection results of mycoplasma (as shown in FIG. 10 and Table 7) showed that the recombinant lentiviral vectors lvCARbcma-1453-lvCARbcma-1460 did not contain mycoplasma.

(135) TABLE-US-00010 TABLE 7 Detection results of mycoplasma PCR Determination template PCR products description Positive 280 b and 150 bp Positive control were contained No band or only one Not positive band Negative 150 bp band Negative control No band or more Not negative than two bands Sample 280 and 150 bands Contaminated by were contained mycoplasma Only 280 band Severely contaminated by mycoplasma Only 150 bp band No mycoplasma contamination No band Too few cells or PRC reaction was inhibited

Embodiment 3 Functional Detection of Recombinant Lentivrial Vectors lvCARbcma-1453-lvCARbcma-1460

(136) I. Detection of Cellular Level Expression of CAR Gene:

(137) (1) After PBMC cells were infected with recombinant lentiviral vectors lvCARbcma-1453-lvCARbcma-1460 and control virus MOCK, the cells were collected. RT-PCR was used to detect the transcription level of CAR mRNA and verify the expression of CAR gene. If the transcription level of CAR mRNA increased, it indicated that the transcription level of CAR gene was successfully expressed;

(138) (2) After PBMC cells were infected with recombinant lentiviral vectors lvCARbcma-1453-lvCARbcma-1460 and control virus MOCK, the cells were collected. Western blot was used to detect the expression level of CAR protein and verify the expression of CAR gene. If the expression level of CAR protein increased, it indicated that the translation level of CAR gene was successfully expressed;

(139) (3) lvCARbcma-1453-lvCARbcma-1460 with MOI=15 and control virus MOCK were infected with cells respectively. After 48 h, total RNA and total protein of the cells in the 6-well plate were extracted for fluorescence quantitative PCR and western blot assay. Specific steps: four wells of the 6-well plate were coated. Relevant PBS and RN were added to each well and overnight at 4° C. After 12 hours, the virus was coated according to MOI=15, and placed the plate in an incubator at 37° C. for 5 h; Take out the 6-well plate, and discard viral supernatant. The plate was washed twice with PBS, coated with PBMC (isolated from human blood with lymphocyte separation solution) at 1*10.sup.6/well, and added 500 μl of medium (containing 10% serum, 20 U/ml IL-2, Polybrene 8 ug/ml). Then such plate was allowed to stand for 20 min, centrifuge at 1000 g for 20 min at 20° C., and culture for 48 h at 37° C.

(140) (4) The total RNA of PBMC cells in 6-well plate was extracted by Trizol method, and the cDNA was amplified by reverse transcription. QPCR primers (SEQ ID NO: 63-SEQ ID NO: 64) were used for fluorescence quantitative PCR assay (the reaction system was shown in Table 8) to verify the transcription of its mRNA with Action as its control group.

(141) TABLE-US-00011 TABLE 8 20 μl qPCR reaction system Reagent Volume (μl ) SYBR premix ex taq: 10 μl ROX Reverse Dye(50x) 0.4 μl Upstream primer (2.5 μM): 0.5 μl Downstream primer 0.5 μl (2.5 μM): cDNA 1.0 μl ddH.sub.2O 7.6 μl

(142) (5) Western Blot was used to separate the total protein extracted from PBMC by relative molecular weight by polyacrylamide gel electrophoresis. The protein was transferred to the PVDF membrane by wet transfer (4° C., 400 mA, 120 min). The PVDF membrane was blocked with a blocking solution (TBST solution containing 5% skim milk) for 1 h at room temperature. The blocking solution was diluted with Biotinylated protein L at 1:1000, and then incubated with the blocked PVDF membrane at room temperature and overnight at 4° C. The membrane was washed three times with TBST, 10 min each time. The blocking solution was diluted with the corresponding SA-HRP at 1:500, and then used to incubated PVDF membrane at room temperature for 2 h. The membrane was washed three times with TBST, 10 min each time. ECL+plus™ Western blotting system kit of Amersham was used for color development. X-ray optical development was used to obtain film showing bands.

(143) (6) RT-QPCR detection showed that the transcription level of CAR gene infected with PBMC by recombinant lentiviral vectors was significantly higher than that of control virus MOCK and blank cells (as shown in FIG. 11 and Table 9), indicating that the transcription level of CAR gene was successfully expressed.

(144) TABLE-US-00012 TABLE 9 Sample name Actin (CT) CAR (CT) −ΔCt −ΔΔCt 2.sup.−ΔΔCt lvCARbcma-1453 18.94781 27.9884 −9.04059 5.74813 53.74756 lvCARbcma-1454 19.63172 28.76517 −9.13345 5.65526 50.39696 lvCARbcma-1455 19.20012 23.48228 −9.28216 5.50655 45.46091 lvCARbcma-1456 18.34099 27.44321 −9.10222 5.6865 51.49997 lvCARbcma-1457 18.2057 27.45988 −9.25419 5.53453 45.35104 lvCARbcma-1458 18.31527 27.42907 −9.11379 5.67492 51.08833 lvCARbcma-1459 18.69971 27.8403 −9.1406 5.64812 50.14799 lvCARbcma-1460 19.56607 28.77406 −9.20799 5.58073 47.85924 MOCK 19.75225 34.55302 −14.8008 −0.01206 0.991675 Blank 19.72942 34.51814 −14.7887 0 1

(145) (7) Western Blot results showed that the expression level of CAR protein infected with PBMC with recombinant lentiviral vectors was significantly higher than that of control virus MOCK and blank cells (as shown in FIG. 12), indicating that the translation level of CAR gene was successfully expressed.

(146) II. Evaluation of PD-1 Knock-down Effect (PD-lmRNA Transcription Level)

(147) (1) BCMA-K562 cells and PBMC cells were cultured separately;

(148) (2) Four days before start of the experiment, the virus of lvCARbcma-1453-lvCARbcma-1460 with MOI=15 was infected with PBMC cells, and cultured for 72-96 h;

(149) (3) 4×10.sup.5 of target cells (CD19+K562) and 2.8×10.sup.6 of effector cells (lvCARbcma-1453-PBMC-lvCARbcma-1460-PBMC cells) were collected, centrifuged at 800 g for 6 min, and supernatant was discarded;

(150) (4) The target cells and effector cells were resuspended with 1 ml of 1×PBS solution respectively, centrifuged at 800 g for 6 min, and the supernatant was discarded;

(151) (5) Step 4 was repeated once;

(152) (6) Effector cells were resuspended with 700 μl medium (1640 medium+10% FBS), and target cells were resuspended with 2 ml medium (1640 medium+10% FBS).

(153) (7) The experimental ports were set with the ratio of effector cells to target cells of 10:1, and the Blank group was set;

(154) (8) Being plate centrifuged at 250×g for 5 min;

(155) (9) They were co-cultured in an incubator with 5% C02 at 37° C. for 24 hours. 100 μl of co-cultured supernatant was collected to detect the transcriptional level of PD-1 mRNA;

(156) (10) The total RNA of the mixed cells above was extracted by Trizol method, and the cDNA was amplified by reverse transcription. QPCR primers (SEQ ID NO: 57--SEQ ID NO: 58) were used for fluorescence quantitative PCR assay (the reaction system was shown in Table 6) to verify the transcription of its mRNA with Action as its control group.

(157) (11) RT-QPCR detection results showed that after a part of PBMC transduced by PD-1 knock-down recombinant lentiviral vectors were incubated with target cells, the mRNA content of PD-1 gene was significantly lower than that of control virus lvCARbcma-1460 (see FIG. 13 and Table 10), indicating PD1siRNA can knock down the transcriptional level of PD-1 gene, in which the knock-down effect of lvCARbcma-1453 was the best and reached over 70%. As Blank group is not activated by target cells, its PD-1 transcriptional level was not increased.

(158) TABLE-US-00013 TABLE 10 Sample name Actin (CT) PD-1 (CT) −ΔCt −ΔΔCt 2.sup.−ΔΔCt Blank 13.27986 35.211302 −21.9314 −7.01064 0.007755 lvCARbcma-1453 13.06856 30.328141 −17.2536 −2.33878 0.197677 lvCARbcma-1454 14.11226 30.347056 −16.2348 −1.314 0.402295 lvCARbcma-1455 13.23032 28.156707 −14.9284 −0.00759 0.994752 lvCARbcma-1456 14.93184 30.045561 −15.1137 −0.19293 0.874829 lvCARbcma-1457 14.74173 29.710368 −14.9686 −0.04784 0.967382 lvCARbcma-1458 14.89117 29.821291 −14.9301 −0.00933 0.993557 lvCARbcma-1459 13.08795 28.050568 −14.9626 −0.04182 0.971426 lvCARbcma-1460 14.17941 29.100206 −14.9208 0 1

(159) III. Effect Evaluation of Cell Killing Test

(160) (1) BCMA-PDL1-K562 cells and PBMC cells were cultured separately;

(161) (2) Four days before start of the experiment, the viruses of lvCARbcma-1453-lvCARbcma-1460 with MOI=15 was infected with PBMC cells, and cultured for 72-96 h;

(162) (3) 4×10.sup.5 of target cells (BCMA-PDL1-K562) and 2.8×10.sup.6 of effector cells (CART cells) were collected, centrifuged at 800 g for 6 min, and supernatant was discarded; (4) The target cells and effector cells were resuspended with 1 ml of 1×PBS solution respectively, centrifuged at 800 g for 6 min, and the supernatant was discarded;

(163) (5) Step 3 was repeated once;

(164) (6) Effector cells were resuspended with 700 μl medium (1640 medium+10% FBS), and target cells were resuspended with 2 ml medium (1640 medium+10% FBS);

(165) (7) The experimental ports were set with the ratio of effector cells to target cells of 1:1, 5:1, 10:1, and the control group was set with 3 multiple wells each group;

(166) (8) Being plate centrifuged at 250×g for 5 min;

(167) (9) They were co-cultured in an incubator with 5% CO2 at 37° C. for 24 hours;

(168) (10) Being plate centrifuged at 250×g for 5 min;

(169) (11) 50 μl of supernatant taken from each well was added into a new 96-well plate with 50 μl of substrate solution each well (light protection operation);

(170) (12) They were incubated in the dark for 25 min;

(171) (13) 50 μl of TMAH was added to each well;

(172) (14) 490 nm absorbance was detected by enzyme-labeled instrument;

(173) (15) Average of the three multiple wells was taken: The average of median background absorbance was subtracted from the absorbance of all experimental ports, target cell wells and effector cell wells; The average of control absorbance of volume correction was subtracted from the maximum absorbance of target cells.

(174) (16) The corrected values obtained in step 15 were taken into the following formula to calculate the percentage of cytotoxicity produced by each ratio of effector cells to target cells. Results as shown in FIG. 14, the killing efficiency of PD-1 knock-down recombinant lentiviral vector transduced PBMC cells was significantly higher than that of PBMC blank cells at the ratio of effector cells to target cells 10:1, in which in killing efficiency on target cells, lvCARbcma-1453-PBMC was the highest and more than 40%, the next was lvCARbcma-1454-PBMC, and the third was lvCARbcma-1455-PBMC-lvCARbcma-1458-PBMC, which was roughly same as lvCARbcma-1460-PBMC in killing efficiency on target cells; by comparing the results in FIG. 13, it can be seen that as there is PD-L1 in target cells, which transmits inhibiting signals and inhibits the immunological competence of T cells after binding to PD-1, resulting in the significant reduction in the killing efficiency of T cells on target cells. When its expression level is greatly decreased, PD-1 can effectively interfere with the activation of PD-1/PDL-1 signaling pathway, so that T cells can normally kill target cells. In the future, lvCARbcma-1453 vector and T cells transduced by it can be used clinically to the inhibit the expression level of PD-1 in CAR-T cells and reinforce the killing effect of CAR-T cells on tumor in vivo to inhibit immune escape.
Killing efficiency=(experimental ports−effector cell wells−target cell wells)/(target cell maximum well−target wells)×100%.

(175) The better embodiments of the invention have been specified above, but the invention is not limited to the said embodiments. Technical personnel familiar with the field, without violating the spirit of the invention, make a variety of equivalent variations or substitutions, which are all included in the scope of the claim of this application.