APPLICATION OF SCFV PROTEIN, CAR GENE EXPRESSION VECTOR, CAR-T CELL AND APPLICATION THEREOF

Abstract

An application of SCFV protein in preparation of a CAR gene expression vector is disclosed. The protein sequence of the SCFV protein is shown in SEQ ID No. 1. The 1st˜21st amino acids are the extracellular signal peptide amino acid sequences. The 22nd˜31st amino acids are Flag amino acid sequences. And the remaining sequences are CXCR4 scfv sequences. An application of the gene sequence encoding the SCFV protein in preparation of a CAR gene expression vector is disclosed, and the gene sequence is shown in SEQ ID No.2. A CAR gene expression vector and a CAR-T cell are disclosed. The expression vector is plent-EF1a-Flag-CXCR4 CAR, and CAR-T cells are used in the preparation of a drug for a targeted therapy of tumor cells with high expression of CXCR4.

Claims

1. An application of SCFV protein in preparation of a CAR gene expression vector, wherein a protein sequence of the SCFV protein is shown in SEQ ID No.1; the 1st˜21st amino acids are extracellular signal peptide amino acid sequences; the 22nd˜31st amino acids are Flag amino acid sequences; and the remaining sequences are CXCR4 scfv sequences.

2. A CAR gene expression vector, wherein the expression vector is plent-EF1a-Flag-CXCR4 CAR; plent-EF1a is a lentivirus vector obtained by enzyme digestion of plent-EF1a-EGFP vector with AsisI and MIuI, and Flag-CXCR4 CAR is a recombinant plasmid obtained by enzyme digestion with AsisI and MIuI on the basis of the sequence shown in SEQ ID No.2; and a constructing process comprises: performing enzyme digestion on the vector containing CXCR4 CAR and the lentivirus vector plenti-EF1a-EGFP separately to obtain a digested product, ligating the digested product at 22° C. for 2 h to obtain a ligated product, purifying and recovering the ligated product to obtain the CAR gene expression vector.

3. A CAR-T cell, wherein the CAR-T cells are formed by infecting T cells with the plent-EF1a-Flag-CXCR4 CAR.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to explain the embodiments of the present disclosure or the technical solutions in the prior art more clearly, the following drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on the drawings disclosed without creative work.

[0020] FIG. 1 is a map of the CAR gene expression vector plent-EF1a-Flag-CXCR4 CAR of the present disclosure.

[0021] FIG. 2 is the gel electrophoresis diagram.

[0022] FIG. 3 is the diagram of the flow cytometry result.

[0023] FIG. 4 is the diagram of the flow cytometry result.

[0024] FIG. 5 is the diagram of the flow cytometry result.

[0025] FIG. 6 is the diagram of the flow cytometry result.

[0026] FIG. 7 is the diagram of the flow cytometry result.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Technical solutions of the present disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. Based on the embodiments of the disclosure, all other embodiments made by those skilled in the art without sparing any creative effort should fall within the protection scope of the disclosure.

Embodiment 1 Construction of plent-EF1a-Flag-CXCR4 CAR Plasmid

[0028] 1. Sequence Synthesis

[0029] CXCR4 ScFv sequence was synthesized by the whole gene. CD8a signal peptide and Flag sequence were added at the N-terminal And it was cloned into lentivirus expression plasmid plent-EF1a-EGFP by using Asisi and MluI enzyme digestion sites. The plasmid was Amp resistant.

[0030] The SCFV sequence is as follows:

[0031] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAAGATCTCTG AGACTGAGCTGCACAGCCTCCGGCTTTACTTTCACAGATAATTATATGTCTTGGGTGAGAC AGGCACCAGGCAAAGGCCTGGAGTGGGTGGGCTTTATCAGAAATAAGGCCAACGGATATA CTACTGAATATGCCGCCTCTGTGAAGGGAAGATTCACTATCTCTAGAGATGACTCTAAATCC ATAGCCTACCTCCAGATGAATAGCCTGAAAACTGAAGATACAGCAGTGTACTACTGTGCTA GGGATGTGGGCTCCAACTACTTCGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCA GCCAGCTGAAGTCCAGCGGCAGCGGCAGCGAGAGCAAGAGCACCGACATCGTGATGACC CAGTCTCCCTCCAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACAATGAGCTGCAAGTCT AGCCAGAGCCTGTTCAATAGCAGAACCAGGAAGAACTACCTGGCCTGGTACCAGCAGAA GCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCTCCGCCAGGGACAGCGGCGTGC CCGCCAGGTTCACCGGCAGCGGCAGCGAGACCTACTTTACCCTGACCATCTCCAGAGTGC AGGCCGAGGACCTGGCCGTGTACTACTGCATGCAGAGCTTCAACCTGAGAACCTTCGGCC AGGGCACCAAGGTGGAGATCAAGACCAGGACCGTGGCCGCCCCCAGCGTGTTTATCTTTC CCCCCAGCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTGAACAACT TCTACCCTAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAT AGCCAGGAGTCCGTGACCGAGCAGGACAGCAAGGACAGCACATACAGCCTGAGCTCCAC CCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCC ACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAATAGAGGCGAG, as shown in SEQ ID No.2.

[0032] SCFV amino acid sequence of human CXCR4 gene is as follows:

[0033] Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly ArgSer Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp AsnTyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val Gly Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gln Leu Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Asp Ser Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Ser Glu Thr Tyr Phe Thr Leu Thr Ile Ser Arg Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser Phe Asn Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu, as shown in SEQ ID NO.1.

[0034] 2. Enzyme Digestion and Ligation

[0035] The enzyme digestion is performed on the vector PUC57 containing CXCR4 CAR and lentivirus vector plent-EF1a-EGFP. The corresponding enzyme digestion system is shown in Table 1 and table 2:

TABLE-US-00001 TABLE 1 Composition of reaction solution Volume DNA fragment (0.1 μg/μL) 15 μL 10× Buffer 5 μL AsisI 1.5 μL MluI 1.5 μL ddH.sub.2O 27 μL Total 50 μL

TABLE-US-00002 TABLE 2 Composition of reaction solution Volume Vector (0.5 μg/μL) 2 μL 10× Buffer 5 μL AsisI 1.5 μL MluI 1.5 μL ddH2O 40 μL Total 50 μL

[0036] The enzyme digestion was performed at 37° C. for 1 h after the sample was prepared and mixed well. After the reaction, the size of the enzyme digestion target band was detected by 1% agarose gel electrophoresis, and the target fragments were recovered by gel recovery kit. Micro centrifugation was preformed after mixing evenly, and the ligation was performed at 22° C. for 2 h to obtain the CAR gene expression vector plent-EF1a-Flag-CXCR4 CAR. The mass spectrum is shown in FIG. 1.

[0037] 3. Transformation

[0038] The ligation product transformed the Escherichia coli DH5a competent cells and was spread into the corresponding resistant LB plate for screening.

[0039] The specific steps of the transformation are as follows.

[0040] (1) The prepared DH5a competent cells were taken out from −80° C. and put in an ice bath.

[0041] (2) After the DH5a competent cells were thawed, 1 μL of the ligation product was put into 20)(L of DH5a competent cells, fully mixed, and left in an ice bath for 30 minutes.

[0042] (3) The centrifuge tube was put into a 42° C. water bath for 40 seconds (do not shake the centrifuge tube during this period), and then quickly moved to an ice bath and stood for 2 minutes.

[0043] (4) 200)(L sterile LB medium (without antibiotics) was added into the centrifuge tube, mixed well, placed in a shaking table at 37° C., 200 rpm, and shaken for 1 hour. The purpose was to express the relevant resistance marker genes on the plasmid and resuscitate the bacteria.

[0044] (5) The above product was spread into the corresponding resistant solid medium plate.

[0045] (6) Culture was performed overnight in a 37° C. incubator, a single colony was selected for culture, and the plasmid was extracted for enzyme digestion identification, and the positive clone gel is shown in FIG. 2.

Embodiment 2 CXCR4 CAR Lentivirus Packaging

[0046] The information of instruments and consumables is shown in Table 3 and Table 4.

TABLE-US-00003 TABLE 3 Consumable Article No. Brand Cell culture plate 10 cm 704001 NEST Cell culture plate 15 cm 715001 NEST 10 ml Serological pipette 4488 Corning 50 ml Serological pipette 4490 Corning 100-1250 Sterile extended TF112-1000-Q QSP cartridge gun head with scale Cell cryopreservation tube 377267 Corning DMEM High-sugar medium C11965500BT GIBICO HEPES Solution 1M SH30237.01 HyClone Penicillin Streptomycin C0222 Beyotime Solution (100*) Fetal bovine serum 1122050 Gibico 0.25% Trypsin SH30042.01 Hyclone 10xPBS ST476 Beyotime DMSO D8372 SOLARBIO

TABLE-US-00004 TABLE 4 Specification Instrument and model Brand Biosafety cabinet BSC-1300IIA2 SUZHOU ANTAI AIRTECH CO., LTD (SAT) Inverted microscope CKX31SF OLYMPUS Carbon dioxide incubator MCO-15AC Sanyo Electric Co., Ltd Quantitative polymerase CFX96 BIO-RAD chain reaction instrument (QPCR instrument) Fluorescence microscope ELWD0.3 Nikon Ultra-low temperature DW-86L386 Qingdao Haier special storage box instrument Co., Ltd High speed refrigerated 1580R LAB GENE Co., Ltd centrifuge

[0047] 1. Virus Packaging Steps

[0048] Day1: 10 cmdish HEK293T cells with a degree of polymerization of 90% (about 6×10.sup.7 per plate) were transferred to 10 cmdish in the ratio of 1:3, and the cells reached a degree of polymerization of 90%-95% (about 6%×10.sup.7 per plate) on the next day. The medium was DMEM high sugar medium of GIBICO (containing 10% FBS).

[0049] Day2:

[0050] No fluid changed before transfection.

[0051] The transfection reagent was configured according to the proportion in Table 5. below.

TABLE-US-00005 Mix 1 Volume μL 1 DMEM (without FBS) 1000 μL 2 Target gene plasmid 10 μg 3 PMD2G 3 μg 4 PSPAX2 6 μg

[0052] Mix1 was mixed evenly and placed at room temperature for 5-10 min. 63 μl (1 μg/μl) PEI was added and mixed upside down evenly to obtain a mixture. Then the mixture was placed at room temperature for 5-10 min and added into 10 cm cell culture plate. (The cells reached a degree of polymerization of 90%, and too few cells would affect the transfection efficiency.)

[0053] Day3: the fluid was changed 6 hours after transfection.

[0054] Day5: the cell state was observed and taken photos after 72 h. The supernatant culture medium was collected, filtered with 0.45 μM membrane and removed the lysed HEK293T cells. The supernatant culture medium was added into the ultracentrifuge tube (355642) and centrifuged after balancing, at 25000 rpm, at 4° C. for 1.5 h. The supernatant was discarded, and 1 ml of virus preservation solution was used for redissolving, mixing and dissolving overnight.

[0055] Day6: the virus is collected for subpackaging, and the virus vector particle plent-EF1a-Flag-CXCR4 CAR was obtained.

[0056] 2. Titer Detection

[0057] The titer was detected by the integration number method of lentivirus infected cells. The activity unit of lentivirus (integration number method) of WZ Bioscience Inc. was IU/mL, that is, the number of virus particles with biological activity contained in each milliliter of virus solution. For example, the virus titer≥1×10E.sup.9 IU/mL means that there are at least 1×10.sup.9 lentivirus particles with biological activity in each milliliter of virus solution.

[0058] Viral infection of cells included the following steps.

[0059] (1) HEK293 cells were uniformly inoculated at 2.5×105 cells/well in a 24-well cell culture plate 6 hours before infection.

[0060] (2) The lentivirus was diluted in gradients, and three gradients were made, that is, each well (500 μL DMEM medium without penicillin, streptomycin and serum) contained 10 μL, 10 μL and 0.1 μL virus. After shaking and mixing, it was added to the 24-well plate inoculated with cells. Before adding the virus, the medium in the plate was aspirated.

[0061] (3) After 18-20 hours of infection, the medium in the culture plate was replaced with fresh DMEM complete medium.

[0062] (4) After 64-68 hours of infection, cells were collected and genomic DNA was extracted.

[0063] (5) For each detection, a group of lentiviruses with known TU with fluorescence was set as a control to check the detected value.

[0064] 3. Genomic DNA Extraction (Operating According to the Instructions of AxyGEN Genomic DNA Extraction Kit) and QPCR Detection

[0065] (1) With the gradient dilution of the detected lentivirus vector as the standard, QPCR was performed on the universal primers of the lentivirus vector to obtain the viral integrated copy number.

[0066] (2) With the gradient dilution of Actin plasmid as the standard, the genome copy number of the sample was detected by QPCR with Actin primers to obtain the genome copy number.

[0067] (3) SYBR Green Relatively Quantified the PCR Reaction System and Parameters

[0068] The amplification system was shown in Table 6.

TABLE-US-00006 TABLE 6 Template 2 μL 2× SYBR Green Mix 10 μL Upstream primer (10 μM) 0.4 μL Downstream primer (10 μM) 0.4 μL Replenishing water to 20 μL

[0069] 4. Calculation of Lentivirus IU (Integration Unit)

IU/ml=(C×N×D×1000)/V

[0070] C=average number of lentiviral integrated copies per genome

[0071] N=number of cells at infection (approximately 2.5×10.sup.5)

[0072] D=dilution multiple of virus

[0073] V=the volume of diluted virus added

[0074] The titer detection results are shown in Table 7.

TABLE-US-00007 Virus Titer Results Human CXCR4 gene CAR lentivirus 2.0 × 10E9 IU/mL pLent-EF1a-EGFP Control lentivirus 6 × 10E8 TU/mL

Embodiment 3 T Cell Identification CD3 Positive Rate Test

[0075] 1. Resuscitation of PBMC Cells

[0076] The culture medium was X-VIVO 15, CD3, CD28 antibody and IL-2 were added, and the culture density was controlled at 1-2*10∧6/mL.

[0077] 2. Test of CD3 Positive Rate of T Cells

[0078] The cells were cultured to D5 for flow cytometry, and CD3 and CD25 antibodies were stained.

[0079] 3. Flow Test Results

[0080] The flow cytometry results are shown in FIG. 3, showing that the proportion of CD3.sup.+ was 99.2% and the proportion of CD25+(T cell activation marker) was 98.1%, indicating that T cell activation and expansion were successful.

Embodiment 4 CAR-T Cell Preparation and CXCR4 CAR Positive Rate Test

[0081] 1. Resuscitation of T Cells

[0082] The culture medium was X-VIVO 15, and the culture density was controlled at 1-2*10∧6/ml.

[0083] 2. Infection of T cells with lentivirus particle plent-EF1a-Flag-CXCR4 CAR

[0084] 5*10∧5 cells, 500 μL culture medium, virus according to MOI 20 and 5 μL LVTE (lentiviral transduction enhancer) were added into each well of the 24-well plate, mixed gently, centrifuged at room temperature for 45 min at 800×g, and placed into the incubator for 5-6 h after centrifugation. And then 500 μL culture medium was added. 48 h after infection, 1.2*10∧6 cells were counted. The positive rate of CAR was identified by flow cytometry and flag antibodies were stained.

[0085] 3. Flow Cytometry Results

[0086] The flow cytometry results are shown in FIG. 4, which shows that the proportion of positive CAR-T cells infected with CXCR4 was 14.9%.

Embodiment 5 Identification of CXCR4 Expression in Lung Cancer Cells

[0087] 1. Human lung cancer cell A549 and two poorly differentiated and highly metastatic human lung adenocarcinoma cell lines, SK-LU-1 and PC-9, were selected to identify the CXCR4 expression in these three cells by flow cytometry.

[0088] 2. A549 cells were resuscitated in the medium of DMEM+10% FBS. After 2-5 days of culture, flow cytometry was performed and CXCR4 antibody was stained. The positive rate of CXCR4 in A549 cells was 5.5%, as shown in FIG. 5.

[0089] Note: at the same time, T cells were stained with CXCR4 antibody. It was found that T cells themselves also expressed CXCR4, and the positive rate reached 90%

[0090] 3. SK-LU-1 cells were resuscitated in the medium of MEM+10% FBS. After 2-5 days of culture, flow cytometry was performed and CXCR4 antibody was stained. The positive rate of CXCR4 in SK-LU-1 cells was 3.7%, and the flow cytometry diagram is shown in FIG. 6.

[0091] PC-9 cells were resuscitated in the medium of DMEM+10% FBS. After 2-5 days of culture, flow cytometry was performed and CXCR4 antibody was stained. The positive rate of CXCR4 in PC-9 cells was 2.1%, and the flow cytometry diagram is shown in FIG. 7.

[0092] Various embodiments in the present specification are described in a progressive manner, and the emphasizing description of each embodiment is different from the other embodiments. The same and similar parts of various embodiments can be referred to for each other.

[0093] The above description of the disclosed embodiments enables those skilled in the art to realize or use the present disclosure. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.