CN111533808B - Chimeric antigen receptor modified T cell capable of autocrine TLR4 scFv and targeting cMet and application thereof - Google Patents

Abstract

The invention provides a chimeric antigen receptor modified T cell capable of autocrine TLR4 scFv and targeting cMet and application thereof; the T cell of the invention contains a coding sequence of a chimeric antigen receptor recognizing cMet and a coding sequence of a TLR4 scFv antibody with a secretory signal peptide; the T cell modified by the chimeric antigen receptor can specifically target cMet high-expression tumor cells, activate the T cells, autocrine TLR4 scFv antibody, weaken the immunosuppressive action of a tumor microenvironment, reduce the expression of Treg cells and promote CD8⁺Killing effect of T cells on tumor.

Description

Chimeric antigen receptor modified T cell capable of autocrine TLR4 scFv and targeting cMet and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a T lymphocyte modified by a specific chimeric antigen receptor with an autocrine antibody function, a preparation method and application thereof.

Background

With the development of tumor immunology theory and clinical technology, in recent years, chimeric antigen receptor T-cell immunotherapy (CAR-T), which is a representative tumor immune cell therapy, has attracted extensive attention and is expected to be a major breakthrough in immunotherapy, CAR-T cells are a major breakthrough in immunotherapy by constructing a specific chimeric antigen receptor vector, combining the high affinity of antibodies with the killing effect of T cells, and after combining genetically modified antibodies with corresponding tumor antigens, activating T cells in a major histocompatibility complex non-restricted Manner (MHC), thereby exerting an anti-tumor effect. Typically CD 3). Since the intracellular part contains only the CD3 zeta domain, although it can induce activation of T cells and initial cytotoxic response, it has short survival time and poor effect; the second generation CAR-T cells fused the intracellular part of costimulatory molecules such as CD28, CD137 upstream of the CD3 zeta domain, with a significantly enhanced effect compared to the first generation; the third generation of CAR-T cells simultaneously fuses two costimulatory molecules to the CD3 zeta domain to form scFv-CD28-CD134-CD3 zeta or scFv-CD28-CD137-CD3 zeta structure, thereby providing long-term T cell expansion signal and prolonging survival time of CAR-T cells in vivo.

CAR-T cell therapy currently achieves better efficacy in the treatment of hematological disorders, but is still under investigation in the treatment of solid tumors. One of the important reasons for poor tumor immunotherapy effect is the existence of tumor tissue immunosuppressive microenvironment, that is, the tumor tissue can change and maintain the conditions of self survival and development by using the tumor microenvironment, so as to achieve the purpose of avoiding immune regulation and further promote the occurrence and development of tumors. CAR-T alone against tumor antigens is therefore poorly therapeutic and susceptible to relapse. c-Met is a high affinity receptor of Hepatocyte Growth Factor (HGF), and is abnormally highly expressed, mutated or activity-changed in tumor tissues of liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer and the like, and is lowly expressed or not expressed in normal tissues. The c-Met is highly expressed in liver cancer tissues and is mostly expressed in low and medium liver cancers, the expression of the c-Met gene is related to the size of liver cancer tumors, the stage of TNM, lymph node metastasis, distant metastasis and the like, and the high expression of the c-Met gene indicates poor prognosis of liver cancer patients. Because c-Met is the cross point of a plurality of tumor signal channels, the c-Met is taken as a target to block the HGF/c-Met signal channel which is abnormally activated in the hepatoma cells, so that the tumor cells have a series of changes such as morphological change, slow proliferation, reduced tumorigenicity, reduced invasive ability and the like. Toll-like receptor 4 (Toll-like receptor)l-like receptor 4, TLR4) is one member of the Toll-like receptor family, and is a class of pattern recognition receptors. Current research proves that TLR4 can be activated by Lipopolysaccharide (LPS) and heat shock protein 70 (HSP 70), so that downstream signal pathways such as NF-kappa B, PI3K/Akt and EGFR are activated, secretion of various cytokines such as PGE2, VEGF, IL-6 and IL-8 is promoted, inflammatory cell infiltration at a damaged part is induced, and finally tumor occurrence is accelerated. Research has shown that TLR4 is also expressed in CD4⁺CD25⁺FoxP3⁺The Treg cell surface, the signaling pathway of which can directly influence the number and function of Treg cells. After TLR4 is activated by LPS (lipopolysaccharide) endotoxin, CD4 can be enhanced by 10 times⁺CD25⁺FoxP3⁺Suppressive potency of Treg cells. Meanwhile, activation of TLR4 can promote release of some inflammatory factors, and indirectly promote CD4 by up-regulating expression of co-stimulatory molecules such as CD80 and the like⁺CD25⁺FoxP3⁺Proliferation of Treg cells.

At present, no research report is available on a chimeric antigen receptor and a related modified T cell immunotherapy which take c-Met as a target point and simultaneously integrate an autocrine anti-TLR 4 antibody with a neutralizing effect.

Disclosure of Invention

Based on the above, the invention aims to provide the specific chimeric antigen receptor modified T lymphocyte capable of effectively regulating the tumor microenvironment, and the specific chimeric antigen receptor modified T lymphocyte obtained by the invention can specifically recognize and kill the tumor cells expressing c-Met genes, and can autocrine out a TLR4 scfv antibody to inhibit the proliferation of Treg cells, thereby enhancing the killing function of the tumor cells.

The second object of the present invention is to provide a lentiviral vector comprising a chimeric antigen receptor capable of secreting TLR4 scFv and targeting cMet.

It is a further object of the present invention to provide an immune cell expressing a chimeric antigen receptor with autocrine TLR4 scFv prepared using the lentiviral vector comprising a chimeric antigen receptor capable of secreting TLR4 scFv targeting cMet.

The fourth purpose of the invention is to provide the application of the chimeric antigen receptor with autocrine TLR4 scFv in the anti-tumor immune cell therapy.

The invention realizes the purpose by the following technical scheme:

the T lymphocyte surface expresses specific chimeric antigen receptor, wherein the tumor specific antigen or tumor associated antigen comprises: AFP, BCMA, B7H4, CD52, CD56, CD80, CDK4/m, CEA, CT, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, Epcam, G250, GAGE, GnTV, HAGE, HER2, HPVE7, HSP70, hTERT, iCE, IGF1R, IL2R, IL5, LAGE, LDLR/FUT, MAGE, MART1, MART2, Mesothelin, MUCl, PRAME, PSMA, RAGE, SAGE, TGFf3, TPI/m, VEGF, WTL, and the like; in the preferred T lymphocyte modified by the specific secretory chimeric antigen receptor, the selected tumor specific antigen or tumor associated antigen is c-Met.

The invention provides a chimeric antigen receptor (CsT CAR) capable of autocrine TLR4 scFv and targeting cMet, which is composed of a CD8 alpha chimeric receptor signal peptide, a cMet single-chain antibody light chain variable region, a Linker region, a cMet single-chain antibody heavy chain variable region, a human CD8 alpha transmembrane region, a CD137 chimeric receptor co-stimulatory factor, an immune receptor tyrosine activation motif CD3 zeta, a T2A self-cleavage peptide segment, a VH3-3 antibody signal peptide, a TLR4 antibody heavy chain variable region, a Linker region and a TLR4 antibody light chain variable region which are connected in series.

Preferably, the amino acid sequence of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown in SEQ ID No. 1.

Preferably, the coding nucleotide sequence of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown in SEQ ID No. 2.

Preferably, the amino acid sequence of the CD8 α chimeric receptor signal peptide of the chimeric antigen receptor that autocrine TLR4 scFv and target cMet is shown in SEQ ID No. 3;

preferably, the coding nucleotide sequence of the CD8 alpha chimeric receptor signal peptide capable of autocrine TLR4 scFv and targeting the chimeric antigen receptor of cMet is shown as SEQ ID NO. 11;

preferably, the amino acid sequence of the cMet scFv that autocrine TLR4 scFv and target the chimeric antigen receptor of cMet is shown in SEQ ID No. 4;

preferably, the nucleotide sequence encoding the cMet scFv autocrine for TLR4 scFv and targeting the chimeric antigen receptor of cMet is shown in SEQ ID NO. 12;

preferably, the amino acid sequence of the human CD8 a transmembrane region of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown in SEQ ID No. 5;

preferably, the encoding nucleotide sequence of the human CD8 alpha transmembrane region of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown as SEQ ID No. 13;

preferably, the amino acid sequence of the CD137 chimeric receptor co-stimulatory factor autocrine to TLR4 scFv and targeting the chimeric antigen receptor of cMet is shown as SEQ ID NO. 6;

preferably, the coding nucleotide sequence of the CD137 chimeric receptor co-stimulatory factor capable of autocrine TLR4 scFv and targeting the chimeric antigen receptor of cMet is shown as SEQ ID NO. 14;

preferably, the amino acid sequence of the immunoreceptor tyrosine activation motif CD3 ζ of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown in SEQ ID No. 7;

preferably, the nucleotide sequence encoding the immunoreceptor tyrosine activation motif CD3 ζ autocrine to TLR4 scFv and targeting cMet's chimeric antigen receptor is shown in SEQ ID No. 15;

preferably, the amino acid sequence of the T2A self-cleavage peptide fragment of the chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet is shown as SEQ ID NO. 8;

preferably, the coding nucleotide sequence of the T2A self-cleavage peptide fragment of the chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet is shown as SEQ ID NO. 16;

preferably, the amino acid sequence of the VH3-3 antibody signal peptide of the chimeric antigen receptor autocrine to TLR4 scFv and targeting cMet is shown as SEQ ID No. 9;

preferably, the encoding nucleotide sequence of the VH3-3 antibody signal peptide of the chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet is shown as SEQ ID NO. 17;

preferably, the amino acid sequence of the TLR4 scFv autocrine to TLR4 scFv and targeting the chimeric antigen receptor of cMet is shown in SEQ ID NO. 10;

preferably, the nucleotide sequence encoding the TLR4 scFv autocrine to TLR4 scFv and targeting the cMet chimeric antigen receptor is shown in SEQ ID NO. 18;

preferably, the expression in the recombinant plasmid taking the pCDH-CMV-MCS-EF1a-CopGFP lentiviral vector as the lentiviral expression vector contains the expression amino acid SEQ ID NO.1 and the coding gene SEQ ID NO. 2.

Preferably, a composition comprising:

(1) a vector comprising an expression cassette for the chimeric antigen receptor of claim for integrating said expression cassette into the genome of a host cell; and

(2) a vector comprising the expression cassette for the TLR4 scFv antibody of any one of the above claims for integrating the expression cassette into the genome of a host cell.

Preferably, the specific chimeric antigen receptor-modified T lymphocytes are prepared by the following steps:

(1) obtaining a nucleic acid sequence of a chimeric antigen receptor which can specifically autocrine TLR4 scFv and target cMet;

(2) respectively cloning a nucleic acid sequence of a chimeric antigen receptor which can specifically autocrine TLR4 scFv and targets cMet into a lentiviral expression vector to obtain a cMet sTLR4 CAR expression plasmid;

(3) transfecting the expression plasmid into GP2-293T cells; packaging to obtain virus particles, and centrifugally concentrating to obtain a lentivirus concentrated solution;

(4) infecting T lymphocytes with the lentivirus concentrate to obtain chimeric antigen receptor-modified T lymphocytes autocrine for TLR4 scFv and targeted to cMet.

The application of the T lymphocyte modified by the specific chimeric antigen receptor in preparing antitumor drugs.

Preferably, the cancer is a cancer whose cancer cell surface abnormally expresses cMet.

Preferably, the cancer is selected from: liver cancer, lung cancer, colon cancer, carcinoma of large intestine, breast cancer, ovarian cancer, and pancreatic cancer.

Preferably, the tumor is a tumor positive for cMet expression.

The method of the invention has the following advantages:

the T lymphocyte modified by the specific secretory chimeric antigen receptor (named CsT CAR-T for short) can specifically recognize and kill tumor cells highly expressing c-Met antigen, and can secrete TLR4 scfv in an autocrine manner, so that the immunosuppressive effect of a tumor microenvironment is weakened, the expression of Treg cells is reduced, and CD8 is promoted⁺Killing effect of T cells on tumor. And the secretory type can overcome the tumor immune evasion problem caused by immunosuppressive signals of the existing CAR-T cell therapy, and can better kill tumor cells.

Description of the drawings:

FIG. 1 is a schematic structural diagram of the CAR of example 1;

FIG. 2 is a nucleic acid electrophoretogram of the CAR plasmid in example 2;

FIG. 3 is a verification plot of the expression of the CAR plasmid in example 3;

FIG. 4 is a post-expression flow cytometric map of the CAR plasmid in example 3;

FIG. 5 is a titer test chart of the CAR virus in example 5;

FIG. 6 is a graph of CAR-T cell infection efficiency in example 6;

FIG. 7 is a phenotypic assay of CAR-T cells in example 6;

FIG. 8 is the killing effect of CAR-T cells on different target cells in example 7;

FIG. 9 is the killing effect of different doses of CAR-T cells on tumor cells differentially expressing cMet in example 7;

FIG. 10 shows the secretion of IL-2 and IFN- γ by CAR-T cells on different target cells in example 8;

FIG. 11 is the effect of CsT CAR on Trag cell proliferation in example 9;

FIG. 12 is the detection of the change in Trag cell expression during the killing of CAR-T cells in example 9;

FIG. 13 is a measurement of the expression of FoxP3, CD86, MyD88 in T cells by CsT CAR in example 9;

FIG. 14 is the effect of proliferation of the CsT CAR-bearing mice in example 10;

fig. 15 is tumor re-detection in tumor-bearing mice after CsT CAR treatment in example 10.

Detailed Description

The present invention will be described in detail with reference to the following detailed description and accompanying drawings. The following embodiments are illustrative of the present invention and are not intended to limit the scope of the present invention. Various changes or modifications may be made by those skilled in the art without departing from the spirit of the invention, and equivalents may be made within the scope of the claims appended hereto.

Example 1: synthesis of CAR-expressing genes

The bispecific chimeric antigen receptor provided by the invention is formed by connecting a CD8 alpha signal peptide, c-Met scFv, a CD8 transmembrane region, a CD137 intracellular signal region, a CD3 zeta intracellular signal region, a T2A self-cleavage sequence, an antibody secretion signal peptide VH3 and TLR4 scFv in series, and the structure of the bispecific chimeric antigen receptor is shown in figure 1; the gene SEQUENCEs of the anti-c-Met and TLR4 single-chain antibodies (anti-c-Met scFv and TLR4 scFv) are from the monoclonal antibody SEQUENCE constructed in the method, and are subjected to codon optimization to ensure that the monoclonal antibody SEQUENCE is more suitable for expression of T cells under the condition of no change of an encoding amino acid SEQUENCE, and the information of each gene SEQUENCE is shown as SEQUENCE LISTING (SEQ ID NO. 1-18).

Example 2: construction of CAR plasmid

The CAR structures in the embodiment 1 are spliced by adopting overlap PCR (polymerase chain reaction), so that CAR is obtained_c-MetSecretory CAR1_c-Met/TLR4And a control CAR_CD19（Unrelated CAR）。

Xba I restriction site is added to the 5 ' end, Not I restriction site is added to the 3 ' end, and EcoR I restriction site is added to the 5 ' end of T2A 5. The vector pCDH-CMV-MCS-EF1a-CopGFP is digested with Xba I/Not I and then separately digested with In-fusion PCRCAR_c-MetFragment, secreted CAR_{c-Met t/TLR4}Fragment and control CAR_CD19The fragment was ligated with the pCDH-CMV-MCS-EF1a-CopGFP vector (see FIG. 2).

Transforming the ligation product into E.coli (DH5 alpha) competence, picking out single clone, culturing, extracting plasmid and sequencing to obtain pCDH-CAR_c-MetpCDH-secretory CAR_{c-Met t/TLR4}And pCDH-CAR_CD19A plasmid.

Example 3: characterization of secretory specific CAR binding Capacity and exogenous CD3 zeta expression

X-293T cells in logarithmic growth phase were collected at 1D before transfection, and the X-293T cells were seeded in 10cm cell culture dishes at an inoculum size of 10⁷The cells were cultured in DMEM medium containing 10% FBS, cultured in a 5% CO2 cell culture box at 37 ℃ and then transfected by observing the cell density to 60-80% under a microscope.

Separately mixing pCDH-CAR_c-MetpCDH-secretory CAR_{c-Met t/TLR4}And pCDH-CAR_CD19The plasmid was transfected into X-293T cells.

After transfection of 1D and 2D, fluorescence microscopy was used to observe the expression of GFP fluorescence after transfection, and after 2D transfection, X-293T cells were collected for extraction of total cell protein. The Western Blot method was used to detect exogenous CD3 ζ expression. As shown in figure 3, each CAR plasmid had exogenous CD3 ζ expression. The CAR molecules were all effective in activating T cells, which were normally differentiated as shown in figure 4.

Example 4: endotoxin removal bulk extraction of lentivirus expression plasmids and packaging plasmids

Mixing pCDH-CAR_c-MetpCDH-secretory CAR_{c-Met t/TLR4}And pCDH-CAR_CD19The plasmid and the strain for packaging the plasmid are cultured in LB culture solution in large quantity, and the plasmid is extracted in large quantity by an alkaline lysis method for transfection.

1) Adding the bacterial liquid into 100ml of LB culture solution containing ampicillin according to the ratio of 1:1000, and shaking in a constant-temperature incubator at 37 ℃ at 220rpm for 12-16 h;

2) measuring absorbance of A600 with a small amount of bacterial liquid, and stopping shaking until the absorbance reaches about 0.4;

3) adding 2.5ml of balance liquid BL into the adsorption column CP6, centrifuging at 8000rpm for 2min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube;

4) centrifuging the bacterial liquid at 4 ℃ and 8000rpm for 3min, sucking the supernatant as much as possible, and collecting bacterial precipitates;

5) adding 8ml of solution P1 (ensuring that RNaseA has been added), thoroughly suspending the bacterial cell pellet;

6) adding 8ml of solution P2, immediately and gently turning upside down for 6-8 times to fully crack the thallus, and standing at room temperature for 5 min;

7) adding 8ml of solution P4, immediately and gently turning upside down for 6-8 times, mixing well until the solution appears white dispersed flocculent precipitate, standing at room temperature for 10min, and centrifuging at 8000rpm for 10 min; separating the white precipitate to the bottom of the tube, carefully pouring the whole solution into a filter CS1, slowly pushing a push handle to filter, and collecting the filtrate in a clean 50ml centrifuge tube;

8) adding isopropanol with the volume of 0.3 time of that of the filtrate into the filtrate, turning the filtrate upside down and uniformly mixing the filtrate, and transferring the mixture into adsorption column CP 6;

9) centrifuging at room temperature of 8000rpm for 2min, removing waste liquid in the collecting tube, and replacing the adsorption column CP6 in the collecting tube;

10) adding 10ml of rinsing solution PW (please check whether absolute ethyl alcohol is added or not) into the adsorption column CP6, centrifuging at 8000rpm for 2min, discarding the waste liquid in the collection tube, and replacing the adsorption column into the collection tube;

11) repeating the operation step 10;

12) adding 3ml of anhydrous ethanol into the adsorption column CP6, centrifuging at room temperature of 8000rpm for 2min, and pouring off waste liquid;

13) putting the adsorption column CP6 back into the collection tube again, centrifuging at 8000rpm for 5min, and removing residual rinsing liquid in the adsorption column;

14) placing the adsorption column CP6 in a new 50ml collecting tube in sterile ultra-clean environment, suspending and dripping 2ml elution buffer TB in the middle part of the phase adsorption membrane, standing at room temperature for 5min, centrifuging at 8000rpm for 2min, placing all the eluent in a 50ml centrifuge tube into 2 clean 1.5ml centrifuge tubes, and preserving at-20 ℃ for later use;

15) 2 mul of DNA solution is taken to measure the plasmid DNA concentration and A260/A280 by an ultraviolet spectrophotometry method, and the quality of the plasmid is analyzed by agarose gel electrophoresis at the same time.

Example 5: packaging and detection of CAR lentiviruses

Cell treatment: trypsinized 24h before transfection, the 4 th-12 th GP2-293T cells in logarithmic growth phase were collected, the cells were transferred to a 10cm dish, grown in 10ml DMEM medium containing 10% FBS and 1% PS and placed at 37 ℃ in 5% CO ₂Culturing for 24h in an incubator, and performing transfection when the confluence rate reaches 60-70%;

and (3) configuring a transfection system:

1) to a 1.5ml sterile EP tube, 50. mu.l of serum-free Opti-MEM medium was added, followed by 10. mu.g of plasmid (pCDH-CAR)_c-MetpCDH-secretory CAR_{c-Met t/TLR4}、pCDH- CAR_CD19Mixing plasmid or pCDH-no-load plasmid, VSVG, r-8.91, 4: 3, mixing, and standing at room temperature for 5 min; adding 50 μ l of serum-free Opti-MEM culture medium and 30 μ l of PEI (PEI: total DNA (μ g) ═ 2: 1) as transfection reagent into another 1.5 sterilized centrifuge tube, mixing well, standing at room temperature for 5min, mixing the solution in the two tubes, and standing at room temperature for 20 min;

2) carefully sucking out the culture solution in the original culture dish, and adding 8ml of serum-free Opti-MEM culture medium;

3) adding the plasmid-PEI mixed solution into a 10cm culture dish, and carefully and uniformly mixing;

4) placing the culture dish in 5% CO at 37 deg.C₂Culturing for 12-16h, adding 2ml FBS, and continuing culturing;

5) observing Red fluorescence expression conditions of 293T cells after transfection 24h under a fluorescence microscope, collecting cell culture supernatants 48h and 2h after transfection respectively, centrifuging at room temperature of 3000rpm for 15min, and collecting the supernatants;

6) viral supernatants were filtered through 0.45 μm filters to obtain pCDH-null and pCDH-CAR virus stocks, respectively.

Concentration of lentivirus and determination of viral titer

Collecting cells and culture supernatant, concentrating virus with PEG8000, adding 1/4 volume of PEG8000/NaCl solution (25% PEG8000+ 4.4% NaCl) into the virus solution, mixing, and standing at 4 deg.C for 2 hr;

centrifuge at 4500rpm for 40 min at 4 ℃. Abandoning the supernatant, and taking 1: dissolving lentivirus precipitate in DMEM 100, subpackaging and storing at-80 deg.C to obtain lentivirus concentrate.

The virus titer was determined. Pre-seeding 293 cells into 96-well plates, 10³Pore, 5% CO at 37 ℃₂Culturing in a cell culture box for 24 hours;

dissolving the virus, preparing from 10^-2To 10^-710-fold dilution of the virus sample;

removing cell culture fluid, adding cell culture fluid containing different virus amount, adding polybrene (polybrene) at a ratio of 1:1000, and standing at 37 deg.C with 5% CO₂Culturing in a cell culture box for 48 hours;

the number of stained cells was counted by fluorescence microscopy and monitored for viral titer, see FIG. 5.

Diluting the virus to 10⁷TU/ml, stored at-80 ℃.

Example 6: isolated culture of T cells and CAR lentivirus transduction

And (3) carrying out Ficoll density gradient centrifugation on lymphocyte separation liquid to obtain the human PBMC cells.

PBMC cells were plated in 24-well plates pre-coated with CD3/CD28 antibody at 37 ℃ in 5% CO₂The culture was carried out for 24 hours.

After an additional 48 hours of culture with IL-2, the lentivirus concentrate was infected with T cells at MOI = 5. Expression of GFP on the surface of T cells was detected by flow cytometry 5 days after infection (see figure 6). Flow cytometry detection of CD4 in CAR-T cells⁺T cell, CD8⁺T cell ratio. The results are shown (FIG. 7).

Example 7: CAR-T cell killing activity in vitro

Implanting liver cancer cell strains MHCC97, HepG2, BEL7402, 7721, SK-HEP-1, QGY7701, human malignant melanoma cell A375 and liver cell LO2 into 96-well plate, and measuring 2 × 10 by LDH release method⁴And each hole is provided with 3 multiple holes, and after the cells adhere to the wall, the effective target ratio is 10: 1. CsT CAR-T cells, cMet CAR-T cells, Unrelated CAR-T cells, TLR4 scFv, and activated cultured T cells were added at 5:1 and 2:1, respectively. After a total of 18 hours of culture,the 96-well plate was centrifuged at 1500rpm for 5 minutes, and the supernatant was collected and tested for CAR-T cell specific killing by LDH release (see fig. 8).

Preparation of HepG2 c-Met stably knockdown cell line HepG2 using specific shRNA^-/-HepG2-Luc stable cell line was prepared using Luc lentivirus.

Detection of different pairs of Effector cells HepG2 and HepG2 according to the scheme that the ratio of E to T is 10:1^-/-The killing effect of (1). Supernatants were collected and tested for CAR-T cell specific killing using LDH release (see figure 9).

The CytoTox 96 nonradioactive cytotoxicity method is used for detecting the killing activity of the CAR-T cells on tumor cells: the kit was purchased from Promega corporation and was operated according to the instructions.

1) Preparing target cells: cells in logarithmic growth phase were collected by centrifugation at 800rpm for 5min, and the cell concentration was adjusted to 2X 10 with 5% FBS-containing RPMI1640⁶/ml；

2) Preparing effector cells: one week post-infection CAR-T cells were harvested by centrifugation at 1000rpm for 10min, and the cell concentration was adjusted to 2X 10 with 5% FBS in RPMI1640⁶/ml。

3) The following control and experimental groups (triplicate wells) were set in a U-bottom 96-well plate:

4) according to different effects: target proportion effector cells and target cells were added, and the volume was made up to 200. mu.l/well with 5% FBS in RPMI 1640;

5) centrifuging 96-well plate 200g for 4min, culturing at 37 deg.C with 5% CO2 and saturated humidity incubator for 7h, adding 10 μ l cell lysate (10X) to the maximum release hole of target cell 45min in advance, centrifuging plate 200g for 4 min;

6) measuring LDH activity;

7) calculation of CTL Activity: the average absorbance (A490) was calculated for each group (3 duplicate wells), and the percentage of CTL lysis target cells was calculated according to the procedure.

Example 8: CAR-T cell IL-2 and IFN-gamma secretion determination

The secretion levels of cytokines IFN-. gamma.and IL-2 were measured by ELISA after the CAR-T cells were co-cultured with hepatoma cells (see FIG. 10). ELISA kits (purchased from R & D) were used according to the kit instructions.

1) Taking out supernatant from C, D, E group culture for 36h from-20 deg.C, and thawing at room temperature;

2) taking the concentrated washing liquid, the diluent, the standard substance and the sealing plate strip out of the refrigerator, and pre-warming at room temperature;

3) diluting the concentrated washing liquid and the diluent to working concentration by using double distilled water respectively;

4) preparing a standard product: adding 500ml diluent into lyophilized standard, dissolving naturally for at least 20min, mixing, and diluting to 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.6 and 0 pg/ml;

5) adding the sample and standard substances with different concentrations into corresponding holes at a concentration of 100 μ l/hole, sealing the reaction holes with sealing plate gummed paper, and incubating at 37 deg.C for 2 h;

6) washing the plate for 5 times, and drying the liquid in the plate;

7) balancing the corresponding antibody to room temperature, adding 100 mu l/hole, sealing the reaction hole by using sealing plate adhesive paper, and incubating for 1h at 37 ℃;

8) washing the plate for 5 times, and drying the liquid in the plate;

9) the chromogenic substrate is balanced to the room temperature, 100 mu l of chromogenic substrate is added into each hole, and the chromogenic substrate is incubated for 30min at 37 ℃ in a dark place;

10) balancing the stop solution to room temperature, adding 50 μ l/hole of the stop solution, uniformly mixing, and measuring absorbance values with a microplate reader at a wavelength of 450nm (detection wavelength) and a wavelength of 570nm (correction wavelength) within 30 min;

11) a4 parameter (4-PL) linear standard curve is drawn, and the concentration of the sample is checked by the OD value of the sample.

Example 9: CAR-T cell Pair CD4⁺CD25⁺Foxp3⁺Effect of Treg cells

Peripheral blood isolation from selected donors PBMC cells obtained using magnetic bead sorting to obtain Na-meive CD4⁺T cells are subjected to in vitro induction culture by adopting TGF-beta, and the expression levels of CD4, CD25 and FoxP3 are detected by FACS.

The CsT CAR-T cells, cMet CAR-T cells, Unrelated CAR-T cells, activated T cells were transferred to the lower layer of the Transwell chamber and the induced cultured Treg cells were transferred to the upper layer of the Transwell chamber.

And collecting cell culture supernatant after 48 hours for detection. And (3) adding LPS into the obtained and cultured DCs cells to induce the activation of TLR4, and detecting the change of cell phenotype after adding culture supernatants of each group of effector cells.

The result shows that the specific binding rate of the TLR4 scFv to macrophages marked by FTIC is the highest, 17.54%, which is much higher than that of cMet CAR-T cell group, unalated CAR-T cell group, LPS group and blank group.

After the CART cell culture supernatant and the Tregs are incubated for 2h, the cells are stimulated by LPS for 4h, 8h and 12h respectively.

Extracting cell RNA, detecting the mRNA expression of inflammatory factors TNF-alpha, IL-6, IL-1 and IFN-beta in the cells by using Q-PCR, and observing the inhibition effect of TLR4 scFv on the mRNA expression of the inflammatory factors and whether the inhibition effect changes with time.

In the LPS group, after LPS stimulates macrophages, QPCR detects that the mRNA expression levels of inflammatory factors TNF-alpha, IL-6, IL-1 and IFN-beta are obviously increased compared with that of a blank control group, while compared with the LPS group, the expression level of the inflammatory factor mRNA is obviously reduced by CsT CAR-T cell supernatant + the LPS group which is pre-incubated by CsT CAR-T cell culture supernatant, which indicates that TLR4 scFv can inhibit the expression of the inflammatory factor mRNA after LPS stimulates the macrophages, is strongest in 4 hours of LPS stimulating cells, and has about 50% inhibition rate on four inflammatory factors. Over time, this inhibitory effect gradually diminishes.

The nucleic acid and protein detection of the Treg cells extracted from the experimental group shows that the expression of the Foxp3 gene is obviously inhibited, the expression of the CD86 and MyD88 genes is up-regulated, and the gene expression of the Treg cells is obviously abnormal (figure 13).

The effect of TLR4 scFv on T cell differentiation was examined by co-culturing the CART cell culture supernatants of each experimental group into PBMCs, and flow cytometry results showed that Treg cell expression was down-regulated by 2.94% in the CsT CAR-T cell group, 4.71% in the cMet CAR-T cell group, and 7.08% in the unlated CAR-T cell group (fig. 12).

Human peripheral blood PBMC cells are obtained, human-derived macrophages are separated and cultured, the culture supernatant of the Treg cells treated by the treatment group is added into the DCs cells, and the activation condition of the DCs cells is detected to indirectly reflect the difference condition of secreted protein expression after the Treg cells are influenced by CAR-T cells and influence on a TLR4-LPS channel (figure 11).

The results show that in the experimental group of PBLC + DSCs + LPS + CsT CAR-T, the expression of Treg cells is down-regulated, the expression of CD4+ T cells is up-regulated, and the expression of Treg cells/CD 4+ T cells is down-regulated.

Thus, the CsT CAR-T cells can autocrine TLR4 scFv; the TLR4 scFv has good neutralizing activity, can influence T cell differentiation and promote CD4⁺T cell CD8⁺And (4) the proliferation of the T cells and the proliferation of the Treg cells are inhibited. Under the influence of TLR4 scFv, the expression of Foxp3 gene in Treg cells is obviously inhibited, the expression of CD86 and MyD88 is up-regulated, and the expression of inflammatory related factors TNF-alpha, IL-1, IFN-beta and IL-1 is influenced.

Example 10: killing effect of CAR-T cells on tumor-bearing mice

After the nude mice are injected with HepG2-Luc cells subcutaneously for 2 weeks, the tumor volume reaches 200-300 mm³And carrying out animal experiments.

Mice were randomly divided into four groups, and injected with CsT CAR-T cells, cMet CAR-T cells, unlated CAR-T cells, activated T cells, respectively, on days 0, 3, and 6 post-tumorigenesis.

In vivo imaging was performed on days 0, 3, 6, 9, 12 post injection. The CsT CAR-T cells and the cMet CAR-T cells can obviously inhibit the growth of HepG2 transplantation tumor, and the CsT CAR-T cells have more obvious tumor inhibition effect than the cMet CAR-T cells (figure 14) (P < 0.05).

Tumor bearing mice were sacrificed 15 days after treatment and tumor body load weight was examined.

The tumor load of the mice treated by the CsT CAR-T cells is lowest, the Luciferase fluorescence of the tumor cells can not be detected in 3 of 6 mice, the tumor of the mice treated by the cMet CAR-T cells is obviously faded, but the Luciferase fluorescence can still be detected by in vivo imaging of part of individuals; in contrast, in mice treated with unregulated CAR-T cells and activated T cells, the tumor continued to grow (FIG. 15).

The research results indicate that both the CsT CAR-T cell and the cMet CAR-T cell can inhibit the growth of liver cancer cells, the inhibition effect of the CsT CAR-T cell is superior to that of the cMet CAR-T cell, the inflammation of tumor tissues after treatment is reduced, and infiltrating T cells are increased.

The invention has been described in detail with respect to the general description and the specific examples, but it will be apparent to those skilled in the art that modifications or improvements may be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Nanjing university of medical science

<120> chimeric antigen receptor modified T cell capable of autocrine TLR4 scFv and targeting cMet and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 771

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Asp Asn Trp Gly Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

145 150 155 160

Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly Asp Arg Val

165 170 175

Thr Ile Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

195 200 205

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

225 230 235 240

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro His Thr Phe Gly

245 250 255

Gln Gly Thr Lys Leu Glu Ile Lys Phe Trp Val Leu Val Val Val Gly

260 265 270

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

275 280 285

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

290 295 300

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

305 310 315 320

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu

485 490 495

Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu

500 505 510

Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Gln Ile

515 520 525

Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg

530 535 540

Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu

545 550 555 560

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr

565 570 575

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

580 585 590

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

595 600 605

Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro Trp Thr

610 615 620

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

625 630 635 640

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

645 650 655

Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu

660 665 670

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met

675 680 685

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val

690 695 700

Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly

705 710 715 720

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

725 730 735

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

740 745 750

Asp Pro Gly Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

755 760 765

Val Ser Ser

770

<210> 2

<211> 2313

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc agctggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 120

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 180

gctccaggca aggggctgga gtgggtggca gttatatggt atgatggaag taataaatac 240

tatgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 300

tatctgcaaa tgaacagcct gagagccgag gacacggctg tgtattactg tgcgagagat 360

aactggggat ttgactactg gggccagggc accctggtca ccgtctcctc tggtggtggt 420

ggttctggtg gtggtggttc tggcggcggc ggctccggtg gtggtggatc cgatatccag 480

atgacccagt ctccatcctt actctctgca tctacaggag acagagtcac catcagttgt 540

cgggcaagtc agagcattag cagctattta aattggtatc agcagaaacc agggaaagcc 600

cctaagctcc tgatctatgc tgcatccagt ttgcaaagtg gggtcccatc aaggttcagt 660

ggcagtggat ctgggacaga tttcactctc accatcagca gtctgcaacc tgaagatttt 720

gcaacttact actgtcaaca gagttacagt acccctcaca cttttggcca ggggaccaag 780

ctggagatca aattttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 840

ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 900

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 960

tatgccccac cacgcgactt cgcagcctat cgctccaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg aagcggagag ggcagaggaa gtcttctaac atgcggtgac 1500

gtggaggaga atcccggccc tatggagttt gggctgagct ggctttttct tgtggctatt 1560

ttaaaaggtg tccagtgcca gattgtgctc actcagtctc cagcaatcat gtctgcatct 1620

ctaggggaac gggtcaccat gacctgcact gccagctcaa gtgtaagttc cagttacttg 1680

cactggtacc agcagaagcc aggatcctcc cccaaactct ggatttatag cacatccaac 1740

ctggcttctg gagtcccagc tcgcttcagt ggcagtgggt ctgggacctc ttactctctc 1800

acaatcagca gcatggaggc tgaagatgct gccacttatt actgccacca gtatcatcgt 1860

tccccgtgga cgttcggtgg agggaccaaa ctggaaatca aaggtggtgg tggttctggt 1920

ggtggtggtt ctggcggcgg cggctccggt ggtggtggat cccaggtgca gctggtggaa 1980

tcggggggag gcgtggtcca gcctgggagg tccctgagac tctcctgtgc agcgtctgga 2040

ttcaccttca gtagctatgg catgcactgg gtccgccagg ctccaggcaa ggggctggag 2100

tgggtggcag ttatatggta tgatggaagt aataaatact atgcagactc cgtgaagggc 2160

cgattcacca tctccagaga caattccaag aacacgctgt atctgcaaat gaacagcctg 2220

agagccgagg acacggctgt gtattactgt gcgagagatc cgggctgggg atttgactac 2280

tggggccagg gaactctggt cactgtctct tca 2313

<210> 3

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 4

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asn Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

130 135 140

Ser Leu Leu Ser Ala Ser Thr Gly Asp Arg Val Thr Ile Ser Cys Arg

145 150 155 160

Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser

180 185 190

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Ser Tyr Ser Thr Pro His Thr Phe Gly Gln Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys

<210> 5

<211> 68

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210> 6

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 7

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 8

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 9

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 10

<211> 245

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

130 135 140

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

145 150 155 160

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

165 170 175

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Asp Pro Gly Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 11

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 12

<211> 729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagataac 300

tggggatttg actactgggg ccagggcacc ctggtcaccg tctcctctgg tggtggtggt 360

tctggtggtg gtggttctgg cggcggcggc tccggtggtg gtggatccga tatccagatg 420

acccagtctc catccttact ctctgcatct acaggagaca gagtcaccat cagttgtcgg 480

gcaagtcaga gcattagcag ctatttaaat tggtatcagc agaaaccagg gaaagcccct 540

aagctcctga tctatgctgc atccagtttg caaagtgggg tcccatcaag gttcagtggc 600

agtggatctg ggacagattt cactctcacc atcagcagtc tgcaacctga agattttgca 660

acttactact gtcaacagag ttacagtacc cctcacactt ttggccaggg gaccaagctg 720

gagatcaaa 729

<210> 13

<211> 204

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210> 14

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 15

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 16

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggaagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60

cct 63

<210> 17

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgc 57

<210> 18

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cagattgtgc tcactcagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60

atgacctgca ctgccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120

ccaggatcct cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240

gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccgtg gacgttcggt 300

ggagggacca aactggaaat caaaggtggt ggtggttctg gtggtggtgg ttctggcggc 360

ggcggctccg gtggtggtgg atcccaggtg cagctggtgg aatcgggggg aggcgtggtc 420

cagcctggga ggtccctgag actctcctgt gcagcgtctg gattcacctt cagtagctat 480

ggcatgcact gggtccgcca ggctccaggc aaggggctgg agtgggtggc agttatatgg 540

tatgatggaa gtaataaata ctatgcagac tccgtgaagg gccgattcac catctccaga 600

gacaattcca agaacacgct gtatctgcaa atgaacagcc tgagagccga ggacacggct 660

gtgtattact gtgcgagaga tccgggctgg ggatttgact actggggcca gggaactctg 720

gtcactgtct cttca 735

Claims (8)

1. a chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet, is characterized in that: chimeric antigen receptor is composed of CD8α chimeric receptor signal peptide, cMet single-chain antibody light chain variable region, Linker district , cMet single chain antibody heavy chain variable region, human CD8α transmembrane region, CD137 chimeric receptor costimulator, immunoreceptor tyrosine activation motif CD3ζ, T2A self-cleaving peptide, VH3-3 antibody signal peptide , TLR4 antibody heavy chain variable region, Linker region and TLR4 antibody light chain variable region are formed in series; the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1. 2. A gene encoding the chimeric antigen receptor of claim 1. 3. The gene according to claim 2, characterized in that: the nucleotide sequence of the gene is shown in SEQ ID NO.2; wherein the coding nucleotide sequence of the CD8α chimeric receptor signal peptide is shown in SEQ ID NO.11 ; The coding nucleotide sequence of cMet scFv is shown in SEQ ID NO.12; The coding nucleotide sequence of human CD8α transmembrane region is shown in SEQ ID NO.13; The coding nucleoside of CD137 chimeric receptor costimulatory factor The acid sequence is shown in SEQ ID NO.14; the coding nucleotide sequence of the immunoreceptor tyrosine activation motif CD3ζ is shown in SEQ ID NO.15; the coding nucleotide sequence of the T2A self-cleaving peptide segment is shown in SEQ ID NO.15 ID NO. 16; the coding nucleotide sequence of the VH3-3 antibody signal peptide is shown in SEQ ID NO. 17; the coding nucleotide sequence of TLR4 scFv is shown in SEQ ID NO. 18. 4 . A lentiviral expression vector, characterized in that: the vector carries the nucleic acid sequence encoding the chimeric antigen receptor of claim 1 . 5 . The lentiviral expression vector according to claim 4 , wherein the lentiviral vector recombinant plasmid inserted into the chimeric antigen receptor is pCDH-CMV-MCS-EF1a-CopGFP. 6 . 6. a composition, it is characterised in that the composition contains the carrier of the expression cassette of the chimeric antigen receptor of claim 1, the carrier is used to integrate the expression cassette into the genome of the host cell . 7. A method for modifying T lymphocytes using the chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet according to claim 1, characterized in that, comprising the steps: (1) Obtain the nucleic acid sequence of a chimeric antigen receptor that can specifically autocrine TLR4 scFv and target cMet; (2) Recombining the nucleic acid sequence of the chimeric antigen receptor specific for autocrine TLR4 scFv and targeting cMet into a lentiviral expression vector to obtain a cMet sTLR4 CAR expression plasmid; (3) The expression plasmid was transfected into GP2-293T cells; the virus particles were obtained by packaging, and the lentivirus concentrate was obtained after centrifugation and concentration; (4) Infecting T lymphocytes with the lentivirus concentrate to obtain chimeric antigen receptor-modified T lymphocytes that can autocrine TLR4 scFv and target cMet. 8 . The application of a T lymphocyte modified with the chimeric antigen receptor capable of autocrine TLR4 scFv and targeting cMet according to claim 7 in the preparation of an antitumor drug. 9 .

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