CN105753991B

CN105753991B - Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof

Info

Publication number: CN105753991B
Application number: CN201510843720.9A
Authority: CN
Inventors: 姚永超; 戚何妹; 邓萃兰; 郭文中; 黄烁洲; 刘立宝; 贺倩倩; 秦莉; 陈小平
Original assignee: Guangzhou Cas Lamvac Biotech Co Ltd
Current assignee: Zhongke Lanhua Guangzhou Biomedical Technology Co ltd
Priority date: 2015-11-26
Filing date: 2015-11-26
Publication date: 2019-12-27
Anticipated expiration: 2035-11-26
Also published as: CN105753991A

Abstract

The invention relates to the field of tumor cellular immunotherapy, in particular to a chimeric antigen receptor for resisting placental chondroitin sulfate (pl-CS) and application thereof, wherein the chimeric antigen receptor mainly comprises an antigen recognition region, a hinge region, a transmembrane region and an intracellular region which are connected in series for resisting the pl-CS; wherein the antigen recognition region of the anti-pl-CS is any one of plasmodium protein VAR2CSA, a partial peptide segment of the plasmodium protein VAR2CSA or a pl-CS antibody; the partial peptide segment of the plasmodium protein VAR2CSA is the peptide segment of the plasmodium protein VAR2CSA with the amino acid number more than 500. The T cell expressing the chimeric antigen receptor can kill tumor cells to the maximum extent, has small damage degree to normal tissues, can break through a tumor immunosuppressive microenvironment, has a better curative effect on solid tumors, and can almost treat various tumors.

Description

Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof

Technical Field

The invention relates to the field of tumor cell immunotherapy, in particular to a placenta-like chondroitin sulfate-resistant chimeric antigen receptor and application thereof, and specifically relates to a construction method of a tumor-specific target pl-CS-based chimeric antigen receptor T (CAR-T) cell technology and application thereof in anti-tumor therapy.

Background

Chimeric antigen receptor T cell (CAR-T) technology is a novel cell therapy currently undergoing large-scale clinical trials. Has significant efficacy in the treatment of acute leukemia and non-hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment modalities. The basic technology is that T cells are modified to recognize tumor antigens in an HLA-independent mode through a genetic engineering means, so that the CAR-T cells have stronger capacity of recognizing and killing tumor cells compared with natural T cells. The core component of CAR-T is CAR, which typically comprises a tumor associated antigen binding region, typically a monoclonal antibody scFV fragment derived from a tumor associated antigen; an extracellular hinge region; a transmembrane region and an intracellular stimulatory signal region.

The key to the success of CAR-T technology is the selection of tumor antigens, i.e. targets. The most desirable antigens are those that are highly expressed on the surface of tumor cells and are not expressed on normal cells. Only by selecting such an antigen, the CAR-T cells can be specifically bound and kill tumor cells, while normal cells are not bound and normal tissues are not damaged. However, there are not many antigens that are ideal, and most antigens are highly expressed or overexpressed in tumor tissues and poorly expressed in normal tissues. At present, CAR-T has far less effect than leukemia in treating solid tumors, and one reason for this is that it does not find a very desirable target in every solid tumor.

Another reason why CAR-T technology is not effective for treating solid tumors is the presence of a tumor immunosuppressive microenvironment. The tumor immunosuppressive microenvironment is an internal environment formed in the process of generating and developing tumors and is composed of tumor cells, interstitial cells, immunoregulatory cells, capillaries, tissue fluid, cytokines, extracellular matrix and the like. Due to the existence of immune microenvironment, the immune killer cells cannot effectively reach the tumor cell parts or cannot effectively kill the tumor cells after reaching the target parts. For the CAR-T cell to effectively treat the solid tumor, the CAR-T cell needs to break through the tumor immune microenvironment limitation and enter the solid tumor to kill the tumor cell.

CN 104788573A discloses a chimeric antigen receptor hCD19scFv-CD8 alpha-CD 28-CD3 zeta and application thereof, wherein the chimeric antigen receptor is formed by connecting an anti-human CD19 monoclonal antibody HI19a light chain and heavy chain variable region (hCD19scFv), a human CD8 alpha hinge region, a human CD28 transmembrane region and intracellular region, and a human CD3 zeta intracellular region in series. The chimeric antigen receptor is used for modifying T lymphocytes, and the modified T cells (CAR-T cells) can be used for treating surface CD19 positive tumors. The chimeric antigen receptor of the patent is only directed at surface CD19 positive tumors, and the recognition effect is poor, so that a proper tumor target is screened, and meanwhile, the tumor microenvironment can be broken through, and the chimeric antigen receptor is the key for treating solid tumors by using the CAR-T technology.

Chondroitin Sulfate (CS) is a class of glycosaminoglycans that is covalently linked to proteins to form proteoglycans. Chondroitin sulfate is widely distributed on the extracellular matrix and cell surface of animal tissues, and sugar chains consisting of alternating glucuronate and N-acetylgalactosamine (also called N-acetylgalactosamine) disaccharide units are linked to serine residues of core protein by a sugar chain-like domain. Chondroitin sulfate is present in all organisms from nematodes to humans, except plants, and performs a number of important physiological functions. Although the backbone structure of the polysaccharide is not complex, it exhibits a high degree of heterogeneity in terms of degree of sulfation, sulfate groups and distribution of the two differences into the isomeric uronic acids within the chain. The fine structure of chondroitin sulfate determines the specificity of function and the interaction with various protein molecules.

The placenta contains a special chondroitin sulfate, the glycosylation pattern of the chondroitin sulfate is different from that of the conventional chondroitin sulfate, a protein named as VAR2CSA exists in the plasmodium, the protein can be specifically combined with the chondroitin sulfate in the placenta, and placenta-like chondroitin sulfate (pl-CS) exists on the surface of tumor cells, so the pl-CS is the most ideal target point for treating tumors by the CAR-T technology.

Disclosure of Invention

Aiming at the situations that the target selection in the current CAR-T technology for treating the tumor is not ideal and the tumor microenvironment influences the treatment effect of the CAR-T technology, the invention provides the placenta chondroitin sulfate-resistant chimeric antigen receptor and the application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a chimeric antigen receptor against placental-like chondroitin sulfate, which mainly comprises an antigen recognition region, a hinge region, a transmembrane region and an intracellular region which are connected in series and are resistant to pl-CS;

wherein the antigen recognition region of the anti-pl-CS is any one of plasmodium protein VAR2CSA, a partial peptide segment of the plasmodium protein VAR2CSA or a pl-CS antibody;

the partial peptide segment of the plasmodium protein VAR2CSA is the peptide segment of the plasmodium protein VAR2CSA with the amino acid number more than 500.

In the present invention, the partial peptide of the plasmodium protein VAR2CSA refers to a peptide of the plasmodium protein VAR2CSA with an amino acid number greater than 500, and may be any 500 amino acids or more than 500 amino acids of the plasmodium protein VAR2 CSA. In the invention, the plasmodium protein VAR2CSA, the partial peptide fragments ID1-ID2a of the plasmodium protein VAR2CSA and the pl-CS antibody can be specifically combined with pl-CS, wherein the pl-CS exists on the surfaces of almost all types of tumor cells, but does not exist on the surfaces of normal cells; selecting pl-CS as a specific target of the tumor, taking the VAR2CSA as a recognition structure domain of the CAR to recognize the pl-CS of the tumor, and enabling the CAR-T cells to specifically recognize the tumor cells in a mode that the VAR2CSA recognizes the pl-CS, so that the CAR-T cells can be more easily targeted to a tumor microenvironment and enter the interior of tumor tissues without damaging normal cells and tissues; in addition, pl-CS is also a component of tumor extracellular matrix, is a material basis for forming a tumor microenvironment, and is selected as a target, so that the CAR-T cells can break through the tumor immunosuppressive microenvironment.

Preferably, the plasmodium protein VAR2CSA is a VAR2CSA protein of plasmodium pf 3D7 and/or a VAR2CSA protein of plasmodium pf FCR 3.

Preferably, the amino acid sequence of the VAR2CSA protein of the plasmodium pf 3D7 is SEQ ID NO.3, and the nucleic acid sequence is SEQ ID NO. 4.

Preferably, the amino acid sequence of the VAR2CSA protein of the plasmodium pf FCR3 is SEQ ID NO.5, and the nucleic acid sequence is SEQ ID NO. 6.

Preferably, the partial peptide segment of the plasmodium protein VAR2CSA is ID1-ID2a, the amino acid sequence of the ID1-ID2a is SEQ ID NO.7, and the nucleic acid sequence is SEQ ID NO. 8.

In the invention, the ID1-ID2a is the partial peptide segment, and the partial peptide segment is smaller in length and is the segment with the best effect.

Preferably, the hinge region is a human CD8 a hinge region.

Preferably, the amino acid sequence of the human CD8 a hinge region is SEQ ID No.9 and the nucleic acid sequence is SEQ ID No. 10.

Preferably, the transmembrane region is the human CD28 transmembrane region.

Preferably, the amino acid sequence of the transmembrane region of the human CD28 is SEQ ID NO.11, and the nucleic acid sequence is SEQ ID NO. 12.

As a preferred embodiment, the intracellular domain is any one or a combination of at least two of the human CD3 ζ, human CD28, and human 4-1BB intracellular domains, such as CD3 ζ, human CD28, human 4-1BB intracellular domain, tandem of human CD28 and human CD3 ζ intracellular domains, tandem of human 4-1BB intracellular domain and human CD3 ζ intracellular domain, tandem of human CD3 ζ and human 4-1BB intracellular domain, tandem of human CD28 intracellular domain, tandem of human 4-1BB intracellular domain and human CD3 ζ intracellular domain, preferably CD3 ζ intracellular domain, tandem of human CD28 and human CD3 intracellular domain, tandem of human 4-1BB intracellular domain and human CD3 intracellular domain, or tandem of human CD28, human BB 4-1BB intracellular domain and human CD3 intracellular domain.

Preferably, the amino acid sequence of the intracellular domain of human CD3 ζ is SEQ ID No.13 and the nucleic acid sequence is SEQ ID No. 14.

Preferably, the amino acid sequence of the tandem of the intracellular region of human CD28 and the intracellular region of human CD3 ζ is SEQ ID NO.15 and the nucleic acid sequence is SEQ ID NO. 16.

Preferably, the amino acid sequence of the tandem of the human 4-1BB intracellular region and the human CD3 zeta intracellular region is SEQ ID NO.17 and the nucleic acid sequence is SEQ ID NO. 18.

Preferably, the amino acid sequence of the tandem of the intracellular region of human CD28, the intracellular region of human 4-1BB and the intracellular region of human CD3 zeta is SEQ ID NO.19 and the nucleic acid sequence is SEQ ID NO. 20.

Preferably, the amino terminus of the chimeric antigen receptor comprises a human CD8 α signal peptide.

Preferably, the amino acid sequence of the human CD8 alpha signal peptide is SEQ ID NO.21, and the nucleic acid sequence is SEQ ID NO. 22.

Preferably, the chimeric antigen receptor comprises a human CD8 α signal peptide, VAR2CSA, a human CD8 α hinge region, a human CD28 transmembrane region and intracellular region, a human 4-1BB intracellular region, and a human CD3 zeta intracellular region in tandem.

Preferably, the amino acid sequence of the chimeric antigen receptor is SEQ ID NO.1, and the nucleic acid sequence is SEQ ID NO. 2.

Preferably, the chimeric antigen receptor is expressed by transfection of its encoded nucleic acid sequence into a T cell.

Preferably, the transfection is by any one or a combination of at least two of a viral vector, a eukaryotic expression plasmid or an mRNA sequence.

Preferably, the viral vector is any one of a lentiviral vector, an adenoviral vector or a retroviral vector or a combination of at least two thereof.

In a second aspect, the present invention provides a composition comprising a chimeric antigen receptor according to the first aspect.

In a third aspect, the present invention provides the chimeric antigen receptor according to the first aspect and/or the composition according to the second aspect for preparing chimeric antigen receptor T cells and their use in drugs for treating tumors.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the chimeric antigen receptor, through the mode that the PL-CS is recognized by the VAR2CSA, the CAR-T cells can specifically recognize tumor cells, can be more easily targeted to a tumor microenvironment and enter the interior of a tumor tissue, can specifically recognize the tumor cells, and has better targeting property on the tumor microenvironment;

(2) the T cell expressing the chimeric antigen receptor can kill tumor cells to the maximum extent, has small damage degree to normal tissues, can break through a tumor immunosuppressive microenvironment, has a better curative effect on solid tumors, and can almost treat various tumors.

Drawings

FIG. 1 is a schematic diagram of a pI-CS targeted CAR lentiviral expression vector;

FIG. 2(A) is a graph showing the expression of Green Fluorescence (GFP) of T cells after 96h of lentivirus infection under a fluorescence microscope, and FIG. 2(B) is a graph showing the GFP positivity of T cells after 96h of lentivirus infection by a flow cytometer;

FIG. 3(A) is a comparison of IL-2 concentration in the supernatants of the experimental, negative control and wild-type T cell groups, FIG. 3(B) is a comparison of IFN-. gamma.concentration in the supernatants of the experimental, negative control and wild-type T cell groups, and FIG. 3(C) is a comparison of TNF-. alpha.concentration in the supernatants of the experimental, negative control and wild-type T cell groups;

fig. 4(a) is a comparison of cytotoxicity of the experimental group, the negative control group and the wild type T cell group to the lung cancer cell line a549 in the case of different effective target ratios, fig. 4(B) is a comparison of cytotoxicity of the experimental group, the negative control group and the wild type T cell group to the liver cancer cell line Huh-7 in the case of different effective target ratios, fig. 4(C) is a comparison of cytotoxicity of the experimental group, the negative control group and the wild type T cell group to the human osteosarcoma cell line MG63 in the case of different effective target ratios, and fig. 4(D) is a comparison of cytotoxicity of the experimental group, the negative control group and the wild type T cell group to the human colon cancer cell line Colo205 in the case of different effective target ratios.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

Example 1: determination of PL-CS-Targeted CAR Gene sequences

The plasmodium VAR2CSA gene, the human CD8 α signal peptide gene, the human CD8 α hinge region gene, the human CD28 transmembrane and intracellular region gene, the human 4-1BB intracellular region gene and the human CD3 ζ intracellular region gene sequences were searched from GenBank databases and the literature. The codon optimization is carried out on the protein, so that the protein is more suitable for human cell expression under the condition of unchanging the coding amino acid sequence.

The SEQUENCE information of each gene is shown in SEQUENCE LISTING (SEQUENCE LISTING SEQ ID NO. 1-22).

Example 2: construction of Lentiviral expression vector pLHlentiCAR004

The gene sequences are connected in sequence according to a human CD8 alpha signal peptide gene, VAR2CSA, a human CD8 alpha hinge region gene, a human CD28 transmembrane region and intracellular region gene, a human 4-1BB intracellular region gene and a human CD3 zeta intracellular region gene sequence to form a chimeric antigen receptor, the nucleic acid sequence of the antigen receptor is shown as SEQ ID NO.2, a proper enzyme cutting site is introduced, artificial whole gene synthesis is carried out, and the chimeric antigen receptor is cloned into a lentivirus expression vector to obtain pLHlentCAR 004 plasmid. The plasmid is schematically shown in FIG. 1.

The recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd for sequencing, and the sequencing results were compared with the sequences to be synthesized to confirm that the sequences were completely correct.

Example 3: mass extraction of lentiviral expression vectors and packaging vectors

The plasmid pLHlentiCAR004, psPAX2 and pMD2.G packaged plasmid stbl3 escherichia coli are cultured in LB culture solution in large quantity, and plasmids are extracted in large quantity by an alkaline lysis method for transfection preparation, and the specific process comprises the following steps:

1) respectively inoculating stbl3 escherichia coli strains with pLHlentiCAR004, psPAX2 and pMD2.G into a culture bottle containing 500mL of LB/antibiotic culture solution, and performing shake culture at 37 ℃ for 12-16 hours;

2) centrifuging at 3000 × g for 10min, collecting thallus, pouring out culture medium, and lightly beating with absorbent paper to completely absorb residual liquid. The collected thallus adopts Maxure Plasmid Maxi Kit of Guangzhou Meiji biological technology limited company to extract plasmids, and the specific steps of extracting the plasmids are as follows:

(1) adding 20mL of buffer P1/RNase A into the bacteria, and carrying out vortex resuspension on the bacteria (ensuring that the bacteria are completely resuspended, and the resuspended solution has no cell mass); adding 20mL Buffer P2 into the heavy suspension, reversing and mixing evenly for 10-15 times, and standing for 1-2min (mixing gently, taking care that the operation time cannot exceed 4 min);

(2) adding 20mL Buffer E3 into the lysate, immediately reversing and mixing for 15-20 times, wherein the mixing process needs to be gentle and sufficient, and the standard for sufficient neutralization is as follows: the solution became non-viscous and the precipitate was dispersed to appear white; centrifuging at 3000 × g for 10min, transferring the supernatant to a new centrifuge tube, adding 1/3 times of Buffer E4 into the supernatant, and mixing by vortex;

(3) sleeving a MaxPure Micro Column in a 50mL centrifuge tube, transferring 15mL of mixed solution to a Column, and centrifuging for 10min at 3000 × g; pouring off the effluent, putting the column back into the collection tube, continuously transferring the remaining solution into the column, and centrifuging for 10 minutes at 3000 × g; the effluent was decanted, the column was mounted back on the collection tube, 20mL Buffer E5 was added to the column, and 3000 Xg was centrifuged for 10 min; pouring out the filtrate and sleeving the column into the collecting pipe;

(4) 20mL of Buffer PW2 (diluted with absolute ethanol) was added to the column and centrifuged at 3000 × g for 10 min; the filtrate was decanted and the beads were mounted back in the collection tube, 20mL Buffer PW2 (diluted with absolute ethanol) was added to the column, and centrifuged at 3000 × g for 10 min; the filtrate was decanted and the column was mounted back on the collection tube and centrifuged at 4000 × g for 10 min;

(5) sleeving the column in a sterilized 50mL centrifuge tube, adding 30-200mL sterilized water to the center of the membrane of the column, standing for 2 minutes, centrifuging for 5min at 10000 × g, and collecting the separation solution;

(6) further extracting to remove endotoxin, and adjusting the plasmid concentration to be between 0.1g and 0.6mg/mL by using sterilized water;

(7) adding 0.1 times of Buffer ER1 and 0.1 times of Buffer ER2 into the plasmid solution, and reversing and uniformly mixing; standing on ice for 20min, and mixing by reversing for several times; centrifuging at room temperature at 10000 × g speed for 3min after water bath at 42 deg.C for 5 min;

(8) gently taking out the sample, finding that a layer of red solution is formed at the bottom, carefully transferring the supernatant to a centrifugal tube, adding 0.7 times of isopropanol into the supernatant, and turning and uniformly mixing for multiple times; centrifugation was carried out at 10000 × g for 10min at room temperature, the supernatant was decanted off, and 1-fold volume of 70% ethanol was added to the precipitate. Uniformly mixing for 15-30s in a vortex manner; centrifuging at 10000 × g for 3min at room temperature, and carefully pouring off the supernatant; centrifuging at 10000 × g for 1min at room temperature to collect droplets on the tube wall, drying in air for 10min without sucking precipitate;

(9) adding appropriate amount of sterile water into the plasmid, vortexing for 10-20s, mixing, standing at room temperature for 30min to dissolve the plasmid, and mixing repeatedly;

the plasmid concentration was measured on a Nanodrop UV spectrophotometer and the plasmid was stored at-20 ℃.

Example 4: packaging, concentration and titration of lentiviruses

1) Packaging of lentiviruses

(1) Cell preplating: at 24h prior to transfection, confluent 293T cells were plated, preferably to 70% confluence the next day (500 ten thousand plates in 75mm dishes).

(2) The next day: transfection

(a) Observing the growth of the cells in the pre-plated plate, sucking out the culture medium when the confluency of the cells is about 90%, and adding fresh pre-heated complete culture medium (15 mL in a 75mm culture dish);

(b) PEI, plasmid, Opti-MEM I Reduced Serum Medium and the like required by transfection are placed at room temperature for balancing before transfection;

(c) preparing a plasmid mixed solution: the packaging is carried out on 10 dishes of 293T cells, 500 mu L of mixed liquor is prepared for each dish of cells, and therefore the total volume of the mixed liquor is required to be prepared to be 5 mL; adding three plasmids according to the dosage of each plasmid, wherein the total amount of the three plasmids is 420 mu g (pLHlentCAR 004: psPAX2: pMD2. G: 3:2:1), adding Opti-MEM I Reduced Serum Medium to adjust the volume to 2.5mL, blowing and uniformly mixing, and standing at room temperature for 10 min; then 1.25mL each of PEI and Opti-MEM I Reduced Serum Medium are taken, mixed uniformly and placed at room temperature for 10min, and finally the two solutions are mixed, and placed at room temperature for 10min after being mixed uniformly, thus obtaining mixed solution for transfection;

(d) the mixed solution is added into a culture dish drop by drop, and each dish is 500 mu L;

(3) after 6h of transfection, each dish of culture medium needs to be changed into 17mL of UltraCLUTURE serum-free culture medium;

(4) third to fifth days, virus collection: transfecting for 48-72 h, respectively collecting culture supernatants, and temporarily storing the collected virus supernatants at 4 ℃.

2) Lentiviral concentration

The collected culture supernatant was filtered through a 0.45um membrane, centrifuged at 4 ℃ for 2 hours using a SW-32Ti rotor 25000rpm (corresponding to a centrifugal force of 106750g), and after removing the supernatant, it was resuspended in 200uL of PBS and stored at-80 ℃.

3) Titration of lentiviruses

(1) Pre-plating 293T cells in 6-well plates, 3X 10⁵cells/well，37℃5％CO₂Incubating overnight (about 18-20h) in an incubator;

(2) preparing polybrene mother liquor: sterilizing pure water, filtering with 0.22 μm filter head at 8mg/mL, subpackaging, and storing at-20 deg.C for use;

(3) preparing a DMEM medium containing polybrene: 10% FBS, 1% double antibody, 8. mu.g/mL polybrene (one thousandth of the volume of the medium);

(4) thawing the virus: taking out the virus frozen stock solution from-80 ℃, melting at room temperature, putting on ice after melting, and taking in cells for later use;

(5) and (3) virus dilution: diluting the virus stock solution 10 times with 5% FBS DMEM complete medium containing polybrene to obtain 10^-1-10^-4The virus diluent is uniformly mixed before the virus liquid is sucked each time for further dilution;

(6) old medium was discarded and 1mL of virus-free medium was added to the first well as a negative control. Adding 1mL of corresponding virus diluent into each of the other wells, and culturing at 37 ℃ for 18-20 h;

(7) the virus-containing medium was removed the next morning and replaced with 2mL of 5% FBS DMEM medium without polybrene; observing the cells under a microscope to express GFP;

(8) the medium was aspirated and the cells were then digested with pancreatin; blowing the cells into single cells, adding a proper amount of complete culture medium to stop pancreatin reaction, centrifuging for 3min at 300g, and collecting the cells;

(9) pouring out the supernatant, re-suspending with a proper amount of precooled PBS, centrifuging for 3min at 300g, collecting cells, and repeating once again;

(10) finally, resuspend with 1mL of pre-cooled PBS, place on ice, and use for flow analysis.

(11) Selecting holes with GFP positive rate of 1% -20%, calculating virus titer of each hole, and finally taking an average value;

calculating the formula: titer { (F × Cn)/V } × DF, unit TU/mL;

wherein, F: GFP positive cell rate; cn: number of cells plated per well; v: volume of added virus diluent; DF: dilution factor of virus

The virus titer was calculated as: 1.6X 10⁸-1×10¹⁰In the meantime.

Example 5: isolation, culture and infection of T cells

1) Isolation of PBMC cells

(1) Blood sampling is 50mL, 800g is centrifuged for 10min (acceleration is set to be 3, and deceleration is set to be 4);

(2) taking a leucocyte layer, and diluting the leucocyte layer to 8mL by using 2% FBS;

(3) sucking 4mL LymphoPrep, adding the LymphoPrep into a 15mL centrifuge tube, carefully adding diluted blood onto the layering solution along the tube wall, and keeping the interface between the two clear;

(4) centrifuging at 800g for 20min (acceleration set to 6, deceleration set to 1);

(5) gently sucking off-white mononuclear cells by using a Pasteur pipette, adding the off-white mononuclear cells into another centrifugal tube containing 10mLRF-10, and uniformly mixing;

(6) centrifuging at 500g for 5min, and discarding the supernatant;

(7) 10mL of RF-10 was added to resuspend the cells, trypan blue was counted, centrifuged at 350g for 10min, and the supernatant was discarded.

2) Magnetic bead sorting of CD4+ T (CD8+ T) cells

(1) Vortex and mix the magnetic beads evenly, take 25 μ L magnetic beads in the test tube, add 1mL Buffer 1 and mix evenly, put the test tube on the magnetic shelf for 1min, remove the supernatant. Add 25. mu.L of Bffer1 to resuspend the magnetic beads for use;

(2) the PBMC are resuspended by Buffer 1 to the density of 107/mL;

(3) adding 25 mu L of washed magnetic beads into 1mL of PBMC, incubating for 20min at 2-8 ℃, and placing on a shaking table for inclined rotation;

(4) placing the test tube on a magnet for 2min, and collecting the supernatant;

(5) removing the test tube, adding 1mL of Buffer 1, blowing, uniformly mixing, placing on a magnetic bead for 2min, collecting the supernatant, and repeating once;

(6) add 100 u L Buffer 2 heavy suspension cells, add 10 u L DETCHaBEAD, at room temperature and incubation for 45min to make cells from the magnetic beads release;

(7) placing the test tube on a magnetic frame for 1min, transferring the cell-containing supernatant into a new test tube, adding 500 μ L Buffer 2 to wash the magnetic beads for 2-3 times, and collecting the supernatant;

(8) 4mL of Buffer 2 is added, 350g of the mixture is centrifuged for 5min, the supernatant is removed, and the cells are resuspended by the Buffer 2;

(9) supernatants collected during sorting of CD4+ T cells CD8+ T cells were collected as per kit procedures.

3) Culture of T cells

(1) Centrifuging 350g of CD4+ T (CD8+ T) cells subjected to magnetic bead sorting for 10 min;

(2) RF-10 resuspension counting;

(3) according to the following steps of 1:1 in a ratio of 5X 10 in addition to CD4+ T and CD8+ T cell culture plates⁵Per mL;

(4) adding CD3/CD28 antibody magnetic beads into a special culture medium for the T cells, wherein the amount of the added magnetic beads is 1:1 of the amount of the added magnetic beads to the cells;

(5) adding rhIL-2 to make the final concentration be 10 ng/mL;

(6) the cells were counted 2-3 times per week and proliferation was recorded.

4) Lentiviral transfection of T cells.

(1) Centrifuging 350g of CD4+ T (CD8+ T) cells subjected to magnetic bead sorting for 10min, and performing suspension counting by adding complete culture medium;

(2) according to the following steps of 1:1, adding CD4+ T cells and CD8+ T cells to a 96-well plate, and culturing the cells at a cell density of 5 multiplied by 10⁵Per mLThe number of cells per well was 1X 10⁵A plurality of;

(3) washing the magnetic beads, and adding the magnetic beads in a ratio of 1:1 to the cells into a culture dish;

(4) adding rhIL-2 to a final concentration of 10. mu.g/L;

(5) after 24h of stimulation, infecting cells, adding polybrene to make the final concentration 6 mug/mL, and mixing evenly;

(6) changing the liquid after 16-24 h;

(7) after 3 days, a fluorescent microscope photograph was taken.

FIG. 2(A) shows the fluorescence microscope result chart and FIG. 2(B) shows the flow-based infection detection efficiency, which indicates that CAR-T cells can be efficiently produced with an infection efficiency of about 50% by flow-based detection.

Example 6: CAR-T cell in vitro cytokine release and detection

(1) CAR-T cells 1.5X 10/well in 48-well plates⁵1.5X 10 cells/well of Huh7 cells⁵Co-culturing for 24h, adding 500 μ L RPMI-1640 medium (without serum and rhIL-2);

(2) sample collection and storage: centrifuging to collect supernatant, and directly starting experiment or subpackaging and storing at-20 deg.C (100 μ L per tube);

(3) preparation of reagents: before use, all reagents are taken out and returned to room temperature, and the Wash Buffer concentrated solution is diluted to 500mL by deionized water; substrate color development liquid, mixing A and B in equal volume, and using up 200 mu L of mixed liquid in each hole within 15 min; removing 500 mu L of stock solution of a cytokine standard product, and diluting 6 groups in gradient concentration;

(4) adding the sample and the standard substance diluted by concentration gradient into an ELISA pore plate coated with the antibody, wherein each pore is 100 mu L, removing bubbles, incubating for 2h (each group of samples is provided with three compound pores);

(5) removing liquid in the holes, adding 300 mu LWB into each hole for washing, washing for three times, and finally sucking up WB for the last time;

(6) adding 200 mu L of corresponding detection antibody, and incubating for 2h at room temperature;

(7) removing liquid in the holes, adding 300 mu LWB into each hole for washing, washing for three times, and finally sucking up WB for the last time;

(8) adding 200. mu.L substrate solution, and incubating at room temperature for 20min (in the dark);

(9) adding 50 μ L of stop solution, changing the solution from blue to yellow, and if the solution is green or has not changed color, tapping to mix well;

(10) detecting the absorbance at 450nm within 30 min;

(11) the concentration of each cytokine was counted according to a standard curve,

results as shown in figures 3(a), 3(B) and 3(C), it can be seen that CAR-T cells co-cultured with tumor cells released a large amount of cytokines, indicating that CAR-T cells generated a strong immune response to tumor cells.

Example 7: CAR-T cells killing tumor cells in vitro

1) Determination of optimal cell plating concentration

(1) Each tumor cell was collected, washed with an assay medium, and adjusted to a cell concentration of 1X 10⁵Per mL;

(2) adding 100 μ L of medium for analysis into 96-well plate, and spreading;

(3) add 100. mu.L of cell suspension to a 96-well plate, dilute it sequentially, and aspirate 100. mu.L of cell suspension with the final concentration gradient. Setting six multiple holes, wherein three holes are a High control group, three holes are a Low control group, and three holes are used as a Background control medium (range, 500-;

(4) incubating in an incubator for 18 h;

(5) adding 5 μ L cell lysis solution into each hole of HC group, incubating for 15min, and vibrating to accelerate cell lysis;

(6) adding 100 mu L of the prepared reaction solution into each well, and incubating for 30min at 15-25 ℃ (taking care that the 96-well plate is protected from light);

(7) adding 50 mu L of stop solution into each hole, and shaking for 10 s;

(8) measuring the absorbance at 490 nm;

(9) the data was analyzed to determine the optimal cell concentration (maximum difference between HC and LC).

2) Cell-mediated toxicity assay

(1) Collecting the CAR-T cells, washing the cells with assay media;

(2) determining the number of CAR-T cells plated in 96-well plates from the different effective target ratios and the optimal concentration of the results in 1);

(3) the volume of each well of the corresponding cell suspension plated in a 96-well plate was adjusted to 50. mu.L;

(4) collecting each tumor cell, adjusting the cell concentration to be 2 times of the optimal concentration, and adding 50 mu L of cell suspension into the T cells;

(5) setting different control groups;

(6) incubating in a 37-degree incubator for 18 h;

(7) adding 5 μ L cell lysis solution into each well, incubating for 15min, and vibrating to accelerate cell lysis;

(8) adding 100 mu L of the prepared reaction solution into each well, and incubating for 30min at 15-25 ℃ (taking care that the 96-well plate is protected from light);

(9) adding 50 mu L of stop solution into each hole, and shaking for 10 s;

(10) measuring the absorbance at 490 nm;

(11) the percentage of cytotoxicity was calculated for each sample.

As shown in FIGS. 4(A) -4(D), it can be seen that CAR-T cells have toxicity to tumor cells and can kill tumor cells efficiently.

In conclusion, the T cell expressing the chimeric antigen receptor can kill tumor cells to the maximum extent, has little damage to normal tissues, can break through a tumor immunosuppressive microenvironment, has better curative effect on solid tumors and can almost treat various tumors.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A chimeric antigen receptor for resisting placenta-like chondroitin sulfate is characterized by mainly comprising an antigen recognition region, a hinge region, a transmembrane region and an intracellular region which are connected in series and used for resisting pl-CS;

wherein the antigen recognition region of the anti-pl-CS is a partial peptide segment of plasmodium protein VAR2 CSA;

the partial peptide segment of the plasmodium protein VAR2CSA is a peptide segment of the plasmodium protein VAR2CSA, wherein the number of amino acids in the peptide segment is more than 500;

the partial peptide segment of the plasmodium protein VAR2CSA is ID1-ID2a, the amino acid sequence of the ID1-ID2a is SEQ ID NO.7, and the nucleic acid sequence is SEQ ID NO. 8;

the hinge region is a human CD8 alpha hinge region, and the amino acid sequence of the human CD8 alpha hinge region is SEQ ID NO. 9;

the transmembrane region is a human CD28 transmembrane region, and the amino acid sequence of the human CD28 transmembrane region is SEQ ID NO. 11;

the intracellular domain is any one of or a combination of at least two of human CD3 zeta intracellular domain, human CD28 intracellular domain or human 4-1BB intracellular domain.

2. The chimeric antigen receptor according to claim 1, wherein the nucleic acid sequence of the human CD8 a hinge region is SEQ ID No. 10.

3. The chimeric antigen receptor according to claim 1, wherein the nucleic acid sequence of the human CD28 transmembrane region is SEQ ID No. 12.

4. The chimeric antigen receptor according to claim 1, wherein the intracellular domain is CD3 ζ intracellular domain, human CD28 intracellular domain in tandem with human CD3 ζ intracellular domain, human 4-1BB intracellular domain in tandem with human CD3 ζ intracellular domain, or human CD28 intracellular domain, human 4-1BB intracellular domain in tandem with human CD3 ζ intracellular domain.

5. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the intracellular domain of human CD3 ζ is SEQ ID No.13 and the nucleic acid sequence is SEQ ID No. 14.

6. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the tandem of the intracellular domain of human CD28 and the intracellular domain of human CD3 ζ is SEQ ID No.15 and the nucleic acid sequence is SEQ ID No. 16.

7. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the tandem of the human 4-1BB intracellular domain and the human CD3 zeta intracellular domain is SEQ ID No.17 and the nucleic acid sequence is SEQ ID No. 18.

8. The chimeric antigen receptor according to claim 4, wherein the amino acid sequence of the human CD28 intracellular domain, the human 4-1BB intracellular domain and the human CD3 zeta intracellular domain in tandem is SEQ ID No.19 and the nucleic acid sequence is SEQ ID No. 20.

9. The chimeric antigen receptor according to claim 1, wherein the amino terminus of said chimeric antigen receptor further comprises a human CD8 α signal peptide.

10. The chimeric antigen receptor according to claim 9, wherein the amino acid sequence of the human CD8 a signal peptide is SEQ ID No.21 and the nucleic acid sequence is SEQ ID No. 22.

11. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor is composed of a human CD8 a signal peptide, VAR2CSA, human CD8 a hinge region, human CD28 transmembrane and intracellular regions, human 4-1BB intracellular region, and human CD3 ζ intracellular region in tandem.

12. The chimeric antigen receptor according to claim 11, wherein the amino acid sequence of the chimeric antigen receptor is SEQ ID No.1 and the nucleic acid sequence is SEQ ID No. 2.

13. The chimeric antigen receptor according to claim 1, wherein said chimeric antigen receptor is expressed by transfection into a T cell with its encoded nucleic acid sequence.

14. The chimeric antigen receptor according to claim 13, wherein the transfection is by any one or a combination of at least two of a viral vector, a eukaryotic expression plasmid, or an mRNA sequence.

15. The chimeric antigen receptor according to claim 14, wherein the viral vector is any one of a lentiviral vector, an adenoviral vector or a retroviral vector or a combination of at least two thereof.

16. A composition comprising the chimeric antigen receptor of any one of claims 1-15.

17. Use of the chimeric antigen receptor of any one of claims 1-15 and/or the composition of claim 16 for the preparation of chimeric antigen receptor T cells and their use in medicaments for the treatment of tumors.