CN114957484A

CN114957484A - CAR vector targeting solid tumor cell B7-H3 protein, CAR-T cell and construction method and application thereof

Info

Publication number: CN114957484A
Application number: CN202210469541.3A
Authority: CN
Inventors: 田晓丽; 张谦; 刘国迪; 李德华; 张林松; 顾章杰
Original assignee: Shanghai Yihao Medical Technology Co ltd
Current assignee: Shanghai Yihao Biotechnology Co ltd
Priority date: 2022-04-30
Filing date: 2022-04-30
Publication date: 2022-08-30

Abstract

The invention provides a CAR vector targeting a solid tumor cell B7-H3 protein, a CAR-T cell and a construction method and application thereof, wherein the CAR vector comprises a chimeric antigen receptor and a vector, the chimeric antigen receptor has a structure of SP-B7-H3scFv-TM-CD28-4-1BB-CD3 zeta, a B7-H3 single-chain antibody comprises a heavy chain variable region VH, a light chain variable region VL and a flexible Linker, and the flexible Linker is used for connecting the heavy chain variable region VH and the light chain variable region VL. The CAR-T cell is combined with a CAR vector on a T lymphocyte of the CAR-T cell, has high transduction efficiency, can target a solid tumor cell B7-H3 protein, can rapidly recognize and kill the tumor cell in a short time at low target ratio, and avoids the problem of immune escape caused by the down-regulation of tumor cell MHC expression.

Description

CAR vector targeting solid tumor cell B7-H3 protein, CAR-T cell and construction method and application thereof

Technical Field

The invention belongs to the field of biological medicines, relates to an immune cell technology, and particularly relates to a CAR vector targeting a solid tumor cell B7-H3 protein, a CAR-T cell, and a construction method and application thereof.

Background

Among the current global cancer incidence rates, 90% of the current global cancer incidence rates are solid tumors, such as melanoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer and other cancers, the treatment methods of the current solid tumors include surgical treatment, chemotherapy, radiotherapy, immunotherapy, and combination therapy of multiple treatment methods, and with the continuous development of basic scientific research in recent years, immunotherapy is becoming an important adjuvant treatment method for solid tumors due to its unique curative effect and multiple cancer adaptability.

Given the complexity of the immune system, tumor immunotherapy encompasses a variety of mechanisms of action and drug forms, such as: LAK cells, DC, and CIK cells have been used as immunotherapeutic means for tumor, but the cells lack the ability to specifically recognize Tumor Associated Antigens (TAA) and kill specific tumor target cells, and the expression of Major Histocompatibility Complex (MHC) on the surface of target cells is reduced during tumor immunoediting process to form immune escape (i.e., tumor cells successfully avoid T cell attack), so that LAK cells, DC, CIK cells, etc. are gradually eliminated.

For another example: chimeric Antigen Receptor (CAR) -T cell therapy is artificially over-expressed on the surface of a T cell through a genetic engineering technology, has a great breakthrough in the aspect of treatment of malignant tumors in the blood system, can recognize single-chain antibody variable region gene fragments of specific tumor surface antigens, so that the T cell can recognize the specific antigens and further kill target cells expressing the antigens, and because CAR-T cells adopt a mode of recognizing the antigens by antibodies and are not limited by MHC (major histocompatibility complex), a plurality of researchers try to apply CAR-T technology to the treatment of solid tumors, but because of the specificity of the solid tumors, the curative effect of CAR-T immunotherapy on the treatment of the solid tumors is far from that of the treatment of the blood malignant tumors, so how to avoid the occurrence of tumor immune escape and enhance the killing of T lymphocytes on the tumor cells so as to improve the curative effect of the CAR-T immunotherapy on the solid tumors, still remains a challenge to CAR-T therapy.

With the development of basic scientific research, the B7-H3 protein cloned from a cDNA library derived from human Dendritic Cells (DCs) was studied to consist of 316 amino acids, which has a signal peptide at the amino terminus comprising an extracellular immunoglobulin-like variable region (IgV), a constant region (IgC), a transmembrane region, and a 45 amino acid cytoplasmic region, and which has some homology in amino acid sequence to other members of the B7 family in the extracellular region. The research shows that the B7-H3 protein is expressed in organs such as liver, lung, bladder, testis, prostate, breast, placenta and lymphoid tissue, and the B7-H3 protein is closely related to the generation and development of tumors in the organs and is widely and excessively expressed in solid tumors. The B7-H3 protein can be used as a site for targeted therapy of the solid tumor, and provides a brand-new therapeutic idea for the therapy of the solid tumor, but the current research on CAR-T of a B7-H3 protein target of a solid tumor cell is less, and how to construct a specific CAR structure is the key for the success of the therapeutic method.

Disclosure of Invention

In order to enable the CAR-T cell targeting the B7-H3 protein to recognize the solid tumor cell positively expressed by the B7-H3 and enhance the killing capability of the T lymphocyte so as to improve the anti-tumor cell effect of CAR-T immunotherapy, the invention discloses a CAR vector targeting the solid tumor cell B7-H3 protein, a CAR-T cell and a construction method and application thereof.

The technical scheme for realizing the purpose of the invention is as follows:

in a first aspect, the invention provides a CAR vector comprising a chimeric antigen receptor and a vector linked to the chimeric antigen receptor, wherein the chimeric antigen receptor comprises a leader peptide SP, a B7-H3 single chain antibody, a transmembrane region TM, a costimulatory domain CD28-4-1BB, and a signaling domain CD3 zeta.

The B7-H3 single-chain antibody comprises a heavy chain variable region VH, a light chain variable region VL and a flexible Linker, wherein the flexible Linker is used for connecting the heavy chain variable region VH and the light chain variable region VL.

In one embodiment of the CAR vector, the nucleotide sequence of the VH of the heavy chain variable region is shown in any one of SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequence of the VL of the light chain variable region is shown in any one of SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7.

According to the invention, through designing the CAR vector, firstly, the B7-H3 single-chain antibody of the CAR vector is designed, and the high expression of the B7-H3 protein in solid tumor tissues is realized, so that the CAR vector is used as a specific structure designed by a target spot, not only can be targeted to combine with solid tumor cells, but also can endow T cells with new antigen specificity, and can effectively avoid an immune escape mechanism of tumor cells with down-regulated MHC expression; secondly, the co-stimulation domain is designed, and the CD28 and the 4-1BB are jointly applied as the co-stimulation domain CD28-4-1BB of the CAR vector, so that the CAR-T cells formed by the method have high transduction efficiency, and the aim of quickly killing tumor cells in a short time by using low-efficiency target ratio can be fulfilled.

In one embodiment of the CAR vector, the nucleotide sequence of the above leader peptide SP is set forth in SEQ ID No. 1; the nucleotide sequence of the transmembrane region TM is shown in SEQ ID NO. 8; in the co-stimulatory domain CD28-4-1BB, the nucleotide sequence of CD28 is shown in SEQ ID NO.9, and the nucleotide sequence of 4-1BB is shown in SEQ ID NO. 10; the nucleotide sequence of the signaling domain CD3 ζ is shown in SEQ ID No. 11.

Further, the amino acid sequence of the flexible Linker is GGGGSGGGGSGGS.

In another embodiment of the CAR vector, the vector is a lentiviral vector.

Further, the lentiviral vector was pCDH-CMV-MCS-EF 1-Puro.

In a modified embodiment of the CAR vector, the method for preparing the CAR vector for B7-H3 expression protein targeting solid tumor cells comprises the following steps:

storing a gene encoding a chimeric antigen receptor on a PUC19 plasmid;

carrying out enzyme digestion on the PUC19 plasmid with the chimeric antigen receptor preserved therein, and separating to obtain a target fragment containing the chimeric antigen receptor;

carrying out enzyme digestion on the vector, and separating to obtain an enzyme digestion vector;

connecting the target fragment containing the chimeric antigen receptor with an enzyme digestion vector, converting and extracting to obtain a recombinant plasmid, namely the CAR vector.

Further, the enzyme digestion can be carried out by adopting a single enzyme digestion method or double enzyme digestion, in order to improve the enzyme digestion efficiency, the invention preferentially adopts double enzyme digestion to carry out enzyme digestion on the PUC19 plasmid and the vector which store the chimeric antigen receptor, and the enzyme digestion sites are EcoRI and NotI.

Furthermore, when the target fragment containing the chimeric antigen receptor is connected with the enzyme digestion vector, the molar ratio of the target fragment to the enzyme digestion vector is preferably selected to be 5: 1.

In a second aspect, the invention provides a CAR-T cell for targeting a B7-H3 expression protein of a solid tumor cell, comprising a T lymphocyte having bound thereto a CAR vector as described in the first aspect.

The CAR-T cell designed by the invention is formed by combining the T lymphocyte and the CAR vector designed by the first aspect, can recognize and kill solid tumor cells in a targeting manner, and avoids the problem of immune escape caused by the down-regulation of tumor cell MHC expression.

In a third aspect, the present invention provides a method for constructing a CAR-T cell, the CAR-T cell of the second aspect targeting a B7-H3 expression protein of a solid tumor cell is constructed, comprising the steps of:

packaging the CAR vector into a lentivirus;

centrifuging and concentrating the lentivirus to obtain a lentivirus suspension;

infecting T lymphocytes with the lentivirus suspension to obtain CAR-T cells.

In one embodiment of the method for constructing the CAR-T cell, the above-described lentivirus packaging method is: the PSPAX2 plasmid, the pMD2G plasmid and the CAR vector are used as a three-plasmid packaging system, the 293T cell is used as a packaging cell, and the CAR vector is packaged into lentivirus.

Furthermore, in the three-plasmid packaging system, the molar ratio of the PSPAX2 plasmid, the pMD2G plasmid and the CAR vector is preferably 27:3: 20.

In one embodiment of the method for constructing the CAR-T cell, the T lymphocyte is isolated from PBMC (peripheral blood mononuclear cell).

In one example of a method for constructing CAR-T cells, after CAR-T cells are obtained, it is also necessary to perform antibody binding antigen detection on the CAR-T cells, i.e., infection positive screening of the CAR-T cells using B7-H3 protein using a flow cytometry method.

In a fourth aspect, the invention provides an application of a CAR-T cell, wherein the CAR-T cell is used for targeting B7-H3 expression protein of a solid tumor cell, and the solid tumor cell comprises any one of melanoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, osteosarcoma, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma and glioma.

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

1. according to the invention, the B7-H3 protein is highly expressed in solid tumor tissues, so that the B7-H3 single-chain antibody with a specific structure designed by taking the protein as a target can be targeted to combine with solid tumor cells, when the CAR vector is combined with T cells, the T cells can be endowed with new antigen specificity, and an immune escape mechanism of tumor cell MHC (major histocompatibility complex) expression down regulation can be effectively avoided.

2. The co-stimulation domain of the CAR vector is designed, and the CD28 and the 4-1BB are jointly applied as the co-stimulation domain of the CAR vector, namely the CD28-4-1BB, so that the CAR-T cells formed by the method have high transduction efficiency, and the aim of quickly killing tumor cells in a short time by using a low-efficiency target ratio can be fulfilled.

3. The CAR-T cell designed by the invention can target B7-H3 protein of various solid tumor cells (such as melanoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, osteosarcoma, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma, glioma and the like), and avoids the problem of immune escape caused by the downregulation of tumor cell MHC expression.

Drawings

In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings used in the description of the embodiment will be briefly introduced below. It should be apparent that the drawings in the following description are only for illustrating the embodiments of the present invention or technical solutions in the prior art more clearly, and that other drawings can be obtained by those skilled in the art without any inventive work.

FIG. 1 is a schematic diagram of the structure of the chimeric antigen receptor of the CAR vector in example 1;

FIG. 2 is a schematic diagram of a lentiviral vector of pCDH-CMV-MCS-EF1-Puro structure of the CAR vector in example 1;

FIG. 3 is a flow chart of the preparation of the CAR vector in example 2;

FIG. 4 is a flowchart of the method of constructing B7-H3 protein-expressing CAR-T cells targeting solid tumor cells in example 4;

FIG. 5 is a schematic diagram showing the high expression of B7-H3 in one embodiment on the solid tumor cell lines gastric carcinoma HGC27, osteosarcoma HOS, ovarian carcinoma SKOV3 and colorectal carcinoma HCT 116;

FIG. 6 is a graph of the results of a positive rate test of CAR-T cells in particular embodiments;

FIG. 7 is a graph showing the effect of CAR-T cells on the killing effect of the solid tumor cell lines ovarian cancer SKOV3 and osteosarcoma HOS on the RTCA monitoring platform in accordance with an embodiment;

FIG. 8 is a graph showing the effect of CAR-T cells on killing solid tumor cell lines ovarian cancer SKOV3 and osteosarcoma HOS;

FIG. 9 is a graph of the effect of B7-H3 CAR-T cells on the pharmacodynamic effects of osteosarcoma PDX animal model in a specific embodiment.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

In the description of the present embodiments, it is to be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.

Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicit to a number of indicated technical features. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the invention, the meaning of "a plurality" is two or more unless otherwise specified.

Example 1:

this example provides a CAR vector comprising a chimeric antigen receptor comprising leader peptide SP, B7-H3 single chain antibody, transmembrane region TM, costimulatory domain CD28-4-1BB, and signaling domain CD3 ζ, and a vector linked to the chimeric antigen receptor. The B7-H3 single-chain antibody comprises a heavy chain variable region VH, a light chain variable region VL and a flexible Linker, wherein the flexible Linker is used for connecting the heavy chain variable region VH and the light chain variable region VL, namely as shown in figure 1, the structural composition of the chimeric antigen receptor is SP-B7-H3scFv-TM-CD28-4-1BB-CD3 zeta.

In one embodiment of the B7-H3 single chain antibody of the CAR vector, the VH heavy chain variable region has a nucleotide sequence shown in any one of SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the VL light chain variable region has a nucleotide sequence shown in any one of SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO. 7; the flexible Linker can select the existing general amino acid sequence and also can select the amino acid sequence consisting of GGGGSGGGGSGGS. For example, the B7-H3 single-chain antibody can be composed of the nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.5 and a flexible Linker; can also be composed of nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.7 and flexible Linker, and can also be formed by other combination modes. The B7-H3 single-chain antibody is used for expressing the antibody Anti-B7-H3 of the tumor-associated antigen B7-H3 protein on the surface of the solid tumor cell, and the Anti-B7-H3 is responsible for recognizing the B7-H3 protein highly expressed in the solid tumor cell, so that the targeting property of the CAR-T cell can be greatly increased, and the tumor immune escape is reduced

In one embodiment of the chimeric antigen receptor of the CAR vector, the leader peptide SP described above can be expressed to direct the transmembrane transfer of a newly synthesized protein, the nucleotide sequence of which is shown in SEQ ID No. 1; the transmembrane region TM is used to link the extracellular antigen-binding domain and the intracellular signal domain, anchoring the CAR structure to the T cell membrane, and its nucleotide sequence is shown in SEQ ID NO. 8; the co-stimulation structure domain CD28-4-1BB is used for transducing proliferation signals, inducing cytokine production and stimulating T cell activation, and the nucleotide sequence of CD28 is shown in SEQ ID NO.9, and the nucleotide sequence of 4-1BB is shown in SEQ ID NO. 10; the signal transduction domain CD3 ζ is one that, when the extracellular region binds to the target antigen, transmits a TCR-like signal intracellularly to activate T cells, and has a nucleotide sequence shown in SEQ ID NO. 11. The chimeric antigen receptor can endow T cells with stronger proliferation and durable vitality, so that the T cells can show stronger tumor cell killing capacity

In one example of a vector for the CAR vector, the vector is a lentiviral vector, preferably selected from pCDH-CMV-MCS-EF1-Puro, see FIG. 2. The analysis of the lentiviral vector and the related documents shows that the lentiviral vector with pCDH-CMV-MCS-EF1-Puro structure can be inserted into a target Gene fragment containing a chimeric antigen receptor after double enzyme digestion by EcoRI and NotI in the lentiviral vector by Snap Gene software, wherein the expression vector comprises: a CMV promoter, Multiple Cloning Site (MCS), WPRE element, SV40 polyA sequence, hybrid RSV/5' LTR, genetic element, SV40 origin, CMV promoter (i.e. mammalian cell specific promoter), which has strong driving ability; a Multiple Cloning Site (MCS) comprising a plurality of restriction sites (restriction sites) at which a foreign gene is inserted; WPRE element can raise polyA tailing efficiency of mRNA and improve the expression efficiency of transferred gene; the SV40 polyA sequence can effectively terminate transcription and add polyA tail to transcribed mRNA; hybrid RSV/5' LTR containing regulatory elements such as promoters and enhancers to allow high level expression of full length viral transcripts in 293T cells; genetic elements (cPPT, gag, env, LTRs) for packaging, transduction and stable integration of viral expression constructs into the genomic DNA of a host; SV40 origin, which is used for stable propagation of plasmids in packaging cells. The lentivirus vector can be used as the most effective vector for expressing a target gene segment containing a chimeric antigen receptor in almost all mammalian cells including non-dividing cells and dividing cells, has large capacity of carrying exogenous gene segments and high transfection efficiency, and can achieve satisfactory transfection effect on T cells.

In the embodiment, by designing the CAR vector, the B7-H3 single-chain antibody is highly expressed in solid tumor tissues according to B7-H3 protein, so that the antibody is used as a specific structure designed by a target spot, can be combined with solid tumor cells in a targeting manner, can endow T cells with new antigen specificity, and can effectively avoid an immune escape mechanism of tumor cells under MHC expression; the co-stimulation domain CD28-4-1BB is the co-stimulation domain CD28-4-1BB which uses CD28 and 4-1BB together as a CAR vector, and compared with the prior art which only uses CD28 or 4-1BB as a co-stimulation domain, the co-stimulation domain CD28-4-1BB has the advantages that the CAR-T cell transduction efficiency can be improved, and the purpose that tumor cells can be rapidly killed in a short time by using an inefficient target ratio can be achieved.

Example 2:

this example provides a method of making the CAR vector of example 1 for targeting B7-H3 expression protein of a solid tumor cell.

As shown in fig. 3, the preparation method of the CAR vector includes the following steps:

s1, the gene encoding the chimeric antigen receptor was stored on the PUC19 plasmid.

In this step, the chimeric antigen receptor with the structure SP-B7-H3scFv-TM-CD28-4-1BB-CD3 ζ was stored in plasmid form on the PUC19 plasmid.

S2, carrying out enzyme digestion on the PUC19 plasmid with the stored chimeric antigen receptor, and separating to obtain a target fragment containing the chimeric antigen receptor.

In this step, the chimeric antigen receptor stored on PUC19 plasmid was isolated by enzymatic digestion to form the target fragment when CAR vector was prepared. Specifically, the enzyme digestion can be carried out by adopting a single enzyme digestion method or double enzyme digestion, in order to improve the enzyme digestion efficiency, the invention preferentially adopts double enzyme digestion to carry out enzyme digestion on the PUC19 plasmid and the vector which store the chimeric antigen receptor, and the sites of the enzyme digestion are EcoRI and NotI.

And S3, carrying out enzyme digestion on the vector, and separating to obtain the enzyme digestion vector.

In the step, the vector is a lentiviral vector with a structure of pCDH-CMV-MCS-EF1-Puro, and the lentiviral vector needs to be subjected to enzyme digestion so as to form an enzyme digestion vector which can be connected with the target fragment containing the chimeric antigen receptor in the step S2. Specifically, in order to ensure the ligation of the enzyme-cleaved vector and the target fragment containing the chimeric antigen receptor in step S4, the enzyme-cleaved method of the vector is the same as that of the PUC19 plasmid having the chimeric antigen receptor stored therein in step S2.

And S4, connecting the target fragment containing the chimeric antigen receptor with a restriction enzyme vector, converting, and extracting to obtain a recombinant plasmid, namely the CAR vector.

In the step, when the target fragment containing the chimeric antigen receptor is connected with the enzyme digestion carrier, the molar ratio of the target fragment to the enzyme digestion carrier is preferably selected to be 5: 1.

Example 3:

this example provides a CAR-T cell, a B7-H3 expression protein for targeting solid tumor cells, including T lymphocytes, with the CAR vector described in examples 1 and 2 bound to the T lymphocytes.

The CAR-T cell designed by the invention is formed by combining a T lymphocyte and the CAR vector designed by the first aspect, can recognize and kill solid tumor cells in a targeting manner, and avoids the problem of immune escape caused by the downregulation of tumor cell MHC expression.

Example 4:

this example provides the method of construction of CAR-T cells of example 3, i.e. CAR-T cells expressing protein B7-H3 targeted to solid tumor cells are constructed.

As shown in fig. 4, a method of constructing B7-H3 protein expressing CAR-T cells targeted to solid tumor cells, comprising the steps of:

s5, packaging the CAR vector into lentivirus.

In this step, the lentivirus packaging method is as follows: the PSPAX2 plasmid, the pMD2G plasmid and the CAR vector are used as a three-plasmid packaging system, the 293T cell is used as a packaging cell, and the CAR vector is packaged into lentivirus. Furthermore, in the three-plasmid packaging system, the molar ratio of the PSPAX2 plasmid, the pMD2G plasmid and the CAR vector is preferably 27:3: 20.

S6, centrifuging and concentrating the lentivirus to obtain a lentivirus suspension;

s7, infecting the T lymphocyte with the lentivirus suspension to obtain the CAR-T cell.

In this step, T lymphocytes are isolated from PBMC (peripheral blood mononuclear cells).

In one embodiment of the CAR-T cell construction method, after obtaining the CAR-T cell, the method further comprises step S8, detecting the antibody binding antigen of the CAR-T cell, i.e. performing infection positive screening on the CAR-T cell by using B7-H3 protein by using a flow cytometry method.

The CAR-T cell constructed in the embodiment is used for targeting B7-H3 expression protein of a solid tumor cell, wherein the solid tumor cell comprises any one of melanoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, osteosarcoma, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma and glioma.

The preparation method of the CAR vector targeting the solid tumor cell B7-H3 protein, the construction method of the CAR-T cell and the application thereof are described in detail below.

1. Experimental materials:

1.1 Lentiviral vector pCDH-CMV-MCS-EF1-Puro, lentiviral packaging plasmid pMD2G, vector plasmid PSPAX2 from SBI; the structure of the lentivirus expression plasmid pCDH-CMV-MCS-EF1-Puro is shown in FIG. 1.

1.2. The chimeric antigen receptor (SP-B7-H3 scFv-TM-CD28-4-1BB-CD3 zeta) was stored as PUC19 plasmid;

1.3. restriction endonucleases EcoRI, NotI, which were used for double digestion of the gene encoding the chimeric antigen receptor on the PUC19 plasmid and the lentiviral vector with the structure pCDH-CMV-MCS-EF1-Puro were purchased from NEB.

T4 DNA ligase, Free H2O was purchased from Takara;

1.5. competent cells were purchased from Trans;

1.6. the gel recovery kit and the plasmid small-extraction kit are purchased from Tiangen Biotechnology limited company;

1.7.293T cells, SKOV3 cells were purchased from the cell bank of Chinese academy of sciences;

FBS, DMEM, 1640 medium, PBS, Opti-MEM, lipofectamine 2000 from Gibco;

CD3 monoclonal antibody, CD28 monoclonal antibody, CH38 protein and IL-2 were purchased from Shanghai offshore protein science and technology Co., Ltd;

1.10 Multiskan GO enzyme-labeling instrument + uDrop ultramicro plate, flow cytometer from ThermoFisher;

HE120 horizontal electrophoresis tank, Tanon gel imager from Tanon;

1.12. the water-proof constant-temperature incubator and the constant-temperature culture shaking table are purchased from Shanghai-Hengshi Co., Ltd;

Bio-Rad Mini-PROTECTAN Tetra Cell Mini electrophoresis System purchased from Bio-Rad;

1.14. olympus microscope purchased from olympus;

1.15. the inoculating loop and the coating rod are purchased from self-cleaning special biological filtration corporation;

1.16. syringes, 0.45 μm filters, petri dishes of various sizes, culture flasks, multi-well plates, and centrifuge tubes of various sizes were purchased from Corning.

2. Preparation of CAR vector targeting solid tumor cells and specifically highly expressing protein B7-H3

2.1, obtaining a lentiviral vector with a structure of pCDH-CMV-MCS-EF1-Puro and a PUC19 plasmid with a preserved chimeric antigen receptor.

2.2 respectively carrying out enzyme digestion, connection and transformation on a lentiviral vector with the structure of pCDH-CMV-MCS-EF1-Puro and a PUC19 plasmid with a preserved chimeric antigen receptor: EcoRI and NotI double digestion is respectively carried out on a lentiviral vector of pCDH-CMV-MCS-EF1-Puro, a PUC19 plasmid preserved with a chimeric antigen receptor and a PUC19 plasmid preserved with the chimeric antigen receptor, the digestion products are respectively subjected to agarose gel electrophoresis, and the results are observed and recorded by a gel imaging instrument. And (3) recovering the target band by using an agarose gel recovery kit, connecting the target fragment containing the chimeric antigen receptor with the enzyme digestion vector at a molar ratio of 5:1, transforming to competent cells, dripping the transformed competent cells on a preheated solid culture medium, marking, and incubating overnight at 37 ℃.

2.3 enzyme digestion verification and sequencing are carried out on the CAR vector: picking part of colonies in 5mL LB liquid medium (AMP resistance), enrichment culturing in a constant temperature shaking table, extracting plasmids, taking out 500 μ L of colonies before plasmid extraction, preserving in 1.5mL Ep tubes for strains, and storing at-80 ℃; verifying the extracted product by EcoRI and NotI double enzyme digestion, sequencing 1 mu g of plasmid with correct band, and discarding the plasmid with abnormal band and the remained bacterial liquid; and extracting the plasmid with the correct sequencing result, and discarding the plasmid with the wrong sequencing result and the bacterial liquid thereof.

3. Construction of specific high-expression protein B7-H3 CAR-T cell targeting solid tumor cell

3.1 packaging the CAR vector prepared in 2 into lentivirus: and (3) carrying out lentivirus packaging by adopting a three-plasmid packaging system, wherein three plasmids respectively comprise a CAR vector, a pMD2G plasmid and a PSPAX2 plasmid, and packaging by using 293T cells as packaging cells.

The specific implementation steps are as follows:

3.1.1 plating within 24h before staining, generally selecting cells with passage times within 3 generations, adjusting the cell density according to the cell growth density and state, and plating 293T cells with the growth density reaching 80%;

3.1.2 when the growth density reaches 60-90%, the cell state is good, and then the virus packaging can be carried out;

3.1.3 Virus packaging was performed according to the ratio of PSPAX2 plasmid, pMD2G plasmid, CAR vector (i.e.recombinant plasmid) 27:3:20 (i.e.3. mu.g of CAR vector, 0.5. mu.g of pMD2G plasmid, 4. mu.g of PSPAX2 plasmid), using a 6cm dish as an example.

3.1.4 transfection reagent selection Lipofectamine 2000 (4 degrees C storage), adding the amount of 2 u L/g plasmid;

3.1.5 mixing the plasmid mixture in 3.1.3 and the transfection reagent mixture in 3.1.4 in a tube, standing at room temperature for 20min, adding into the liquid-changing cells, and continuing culturing;

3.1.6 collecting culture supernatant after 48h and 72h respectively, and filtering through a 0.45 mu m filter membrane;

3.1.7 concentrating the collected virus liquid by PEG8000 concentration method, determining virus titer, and storing at-80 deg.C.

3.2 Lentiviral infection of T lymphocytes

3.2.1 obtaining T lymphocytes: about 6mL of fresh peripheral blood was collected with a heparin-containing vacuum blood collection tube, and PBMCs were obtained: adding PBS with the same volume to 3.2.1 at room temperature, slightly blowing, beating, uniformly mixing and diluting; take 50mL centrifuge tubeSucking 6mL of Ficoll (lymphocyte separation fluid) into a centrifugal tube (the volume ratio of the Ficoll to the blood before dilution is 1: 1), inclining the centrifugal tube at 45 degrees, and slowly adding the diluted blood to the Ficoll along the tube wall at a position about 1cm above the Ficoll liquid level; centrifuging: centrifuging at 18-20 deg.C at 2000rpm for 30min, and separating into four layers from tube bottom to liquid surface, including erythrocyte and granulocyte layer, layered liquid layer, mononuclear cell layer, and plasma layer; and (3) recovering: directly inserting a pipette into the cloud layer (or sucking the upper plasma layer first), slightly sucking out the cloud layer, and putting into a new centrifuge tube; washing: adding PBS to a volume of less than 3 times the volume of PBMC (peripheral blood mononuclear cells), at 18-20 deg.C, 2000rpm, for 10min twice; cell counting: the supernatant was discarded, 1mL of lymphocyte culture medium was added, and the mixture was blown and homogenized to prepare a PBMC cell suspension. Counting with a blood cell counting plate: a drop of PBMC suspension was mixed with a drop of 2% Trypan blue stain and applied to a blood cell counting plate, and the total number of cells in 4 grids was counted under a microscope. Cell number/mL =4 total large square lattice cells/4 × 10 ⁴ X2 (dilution factor).

3.2.2 activation of T cells and lentivirus infection:

3.2.2.1 preparation before experiment: preparing Anti-CD3 monoclonal antibody solution: dissolving 50 mu g of CD3 monoclonal antibody in 5mL PBS solution to prepare solution with the concentration of 10 mu g/mL, subpackaging according to 400 mu L of each EP tube after dissolving, and storing in a refrigerator at-80 ℃; (175. mu.L of antibody solution was added to each 24-well plate at this concentration); preparing Anti-CD28 monoclonal antibody solution: dissolving 50 mu g of CD28 monoclonal antibody in 5mL PBS solution to prepare solution with the concentration of 10 mu g/mL, subpackaging according to 400 mu L of each EP tube after dissolving, and storing in a refrigerator at-80 ℃; (175. mu.L of antibody solution was added to each 24-well plate at this concentration); preparing a CH-38 protein solution: dissolving 500 mu g of CH38 protein in 10mL of PBS solution to prepare 50 mu g/mL solution, subpackaging according to 400 mu L of each EP tube after dissolving, and storing in a refrigerator at-80 ℃; (175. mu.L of antibody solution was added to each 24-well plate at this concentration); IL-2 factor preparation: IL-2 protein solid at 1X 10 ⁷ U/mg, 500. mu.L of PBS solution is added to 50. mu.g of IL-2 protein to prepare 10 ³ The concentration of U/microliter is prepared, then 32 microliter is added into each EP for split charging, and the mixture is stored in a refrigerator at minus 80 ℃; shower nozzlePreparation of a culture medium of the blast cells: takara-551 h3 lymphocyte culture medium every 50mL was added with 30. mu.L of the dispensed IL-2 solution, 0.5mL of the double antibody, 250. mu.L of the autologous serum.

3.2.2.2 Experimental flow:

day 0: coating with 24-hole plate: a Corning 24-well plate was taken, and 175. mu.L of a CD3 monoclonal antibody solution, 175. mu.L of a CD28 monoclonal antibody solution, and 175. mu.L of a CH-38 protein solution were added to each of 2 wells, for example, 2 wells. After adding, lightly shaking and uniformly mixing, sealing the pore plate by using a sealing film, and putting the pore plate into a refrigerator at 4 ℃ for one night; cell recovery: taking PBMC cells in liquid nitrogen, and recovering;

day 1: cleaning a coating plate: taking out a 24-hole plate coated yesterday, discarding the supernatant, washing for 2 times by PBS, and adding PBS for later use;

PBMC (plated nucleic acid) plating: PBMC cells were collected, counted and the concentration finally adjusted to 0.7X 10 ⁶ cells/mL, 400. mu.L of cell suspension per well, i.e., 2.8X 10 per well ⁵ Cells;

viral infection: infecting with MOI =30, preparing 1mL of virus culture medium suspension, adding into a 24-well plate, centrifuging for 30min at 1000g, and adjusting the temperature of a centrifuge to 32 ℃;

day1-Day 2: observing the state of the cells;

day 3: all cells in a 24-well plate were transferred to 25cm with 10mL of medium added ² Observing the state of the cells in the culture flask;

day4-Day 7: observing the cell state and the cell number, and if the cells begin to obviously expand and the cell density of a local area is high, adding 10mL of culture medium;

day 8: at this time 25cm ² The cells in the flask were already full and transferred to 75cm with 20mL of medium added ² Continuing culturing in a culture bottle;

day9-Day 10: observing the state of the cells when the cells are at 75cm ² And when the culture bottle is in a full state, stopping continuously growing, enriching cells, calculating an amplification ratio, detecting cell typing by flow detection, and performing subsequent experiments such as cell killing detection or cell cryopreservation.

Identification and detection of CAR-T cells

4.1 flow cytometry detection of Positive expression rates of CAR Structure

4.1.1 test cells for positive rate of CD3, CD4, CD8, CCR7 and CD45 RA: the obtained NC group cells (not infected by virus) and sample group cells (infected by virus) are washed gently for 2 times by PBS +2% BSA, and the centrifugation condition is 1500rpm and 3 min; adding 1000 mu L of PBS +2% BSA into an NC tube, subpackaging into 5 tubes, namely NC, NC-CD3, NC-CD4, NC-CD8 and NC-CD3/4/8, adding 200 mu L of PBS into a sample tube, marking as sample-CD 3/4/8, adding 5 mu L/20 mu L of CD3/4/8 antibody, and mildly and uniformly mixing; incubating at room temperature in dark for 30min, then, 1500rpm for 3min, and discarding waste liquid; adding 200 μ L PBS +2% BSA, gently mixing and re-suspending, 1500rpm/3min, and discarding the waste liquid; adding 100 mu L PBS +2% BSA, gently mixing, uniformly mixing and re-suspending, and then carrying out on-machine detection;

4.1.2 detection of positive expression rate of CAR structure: gently washing the obtained NC group cells (not subjected to virus infection) and sample group cells (subjected to virus infection) with PBS +2% BSA for 2 times at 1500rpm/6min, and discarding the waste liquid; adding 200 mu L PBS into an NC tube, and resuspending; adding 100 mu L PBS into the sample tube, resuspending, adding 100 mu L primary antibody working solution (3 mu g/mL), and mixing; incubating at room temperature for 1h at 1500rpm/3min, and discarding waste liquid; adding 200 mu LPBS, gently mixing, re-suspending at 1500rpm/3min, and discarding waste liquid; adding 200 mu L PBS into an NC tube, and resuspending; adding 200 mu L PBS into the sample tube, resuspending, adding 5 mu L secondary antibody working solution, and mixing uniformly; incubating at room temperature in a dark place for 1h, removing waste liquor at 1500rpm/3min, mildly washing for 3 times by using PBS and 2% BSA, centrifuging at 1500rpm for 3min, and removing waste liquor; add 100. mu.L PBS, mix gently, resuspend, and test on the machine (FIGS. 5 and 6).

4.2 killing of solid tumor cells by CAR-T cells assay: taking solid tumor cell ovarian cancer SKOV3 and osteosarcoma HOS as examples, digesting, preparing into cell suspension, blowing, mixing well, and counting cells; diluting the cell suspension to 5X 10 ⁴ cells/mL, placed on ice for use; taking out the RTCA detection plate, and adding 50 mu L of culture medium; selecting a test program of the test in an RTCA detector program; placing the RTCA detection board into a detector, observing whether the Messege item in the program is normal, and starting an experimental program after the Messege item is normal; taking out the detection plate after the program 1 is operated, and adding tumor cells into corresponding holesAdding 100 mu L of cell suspension, and uniformly mixing each tube of cell suspension before adding; after the cell suspension is added, placing the detection plate in an incubator and standing for 30min to enable cells to naturally settle; after 30min, putting the detection plate into a detector, and operating the program 2; observing a cell growth curve after 24h, and preparing to add effector T cells when the cells are in a logarithmic growth phase; taking out the effector T cells from the culture flask, centrifuging, cleaning, counting, and preparing effector group cell concentrations according to different effector target ratios; the program was suspended, the test plate was removed, 50. mu.L of effector cells were added to the corresponding position, and the plate was returned to the tester, and the program was continued for daily observation.

4.3 ELISA detection of cytokine secretion: by ddH ₂ Diluting 10 times of coating solution buffer by O, preparing 250 times of coating protein according to the proportion, for example, adding 8 mu L of 250 times of coating protein into 2mL of coating solution buffer; adding the coating solution prepared in the step 1) into a Corning 9018 Elisa high-affinity 96-well plate by 100 mu L/well, sealing and putting the plate into a refrigerator at 4 ℃ for overnight; the coated 96-well plates were washed 3 times with PBST (0.05% Tween 20); by ddH ₂ Preparing 5 times of confining liquid by O, adding 200 mu L of confining liquid per hole, confining for 1h at room temperature, adding 1 times of confining liquid according to the requirements of bottled standard products for preparation, diluting for 7 times, and diluting a sample (taking supernate of CAR-T cells in an RTCA detection experiment after killing SKOV3 and HOS for 24 h) for 5 times; PBST washes the plate after closing 5 times, add the sample solution after the standard substance and dilution, incubate 2h or 4 duC overnight at room temperature; PBST cleaning for 4 times; diluting 250 × detection antibody with 1 × blocking solution, adding 100 μ L/well, and incubating at room temperature for 1h; PBST is washed for 4 times, diluted with 1 × blocking solution to 250 × HRP, added into 100 μ L/well, and incubated at room temperature for 30min, PBST is washed for 5 times, added with 1 × TMB reagent 100 μ L per well, and incubated at room temperature for 15 min; adding 50 mu L/hole stop solution to stop color development; detecting OD value with a microplate reader at 450 nm.

The effect of the CAR-T cells described above on targeting the B7-H3 expression protein of solid tumor cells is illustrated by the following example:

example 1: the B7-H3 positive rate of the solid tumor cell line was detected by flow cytometry using humanized B7-H3 antibody, and B7-H3 protein is highly expressed in solid tumor cells as shown in FIG. 5.

Example 2: the positive rate of the CAR-T cells is detected by using B7-H3 protein through flow cytometry, and the positive rate of the CAR-T cells can reach more than 60 percent as shown in figure 6.

Example 3: the killing effect of CAR-T cells on solid tumor cells was tested on the RTCA monitoring platform: ovarian cancer SKOV3 cells and osteosarcoma HOS cells are killed by the CAR-T cells, real-time monitoring is carried out by an RTCA monitoring platform, and the CAR-T cells effectively kill ovarian cancer SKOV3 cells and osteosarcoma HOS cells under the condition of different effective target ratios as shown in figure 7.

Example 4: test CAR-T cells killing solid tumor cells: after killing ovarian cancer SKOV3 cells and osteosarcoma HOS cells for 24h by using CAR-T cells, ELISA of TNF-alpha and IFN-gamma factors is detected, and as shown in figure 8, in the experiment of ovarian cancer SKOV3 cells, the equivalent target ratio is 5:1 and 2.5: 1, the cytokine TNF-alpha and IFN-gamma release of the CAR-T cell group is higher than that of the NC-T control group; in the experiment on osteosarcoma HOS cells, when the effective target ratio is 1:1 and 2.5: 1, the cytokine TNF-alpha and IFN-gamma release was higher in the CAR-T cell group than in the NC-T control group.

Example 8: detecting the drug effect of the CAR-T cells on an osteosarcoma PDX animal model: referring to fig. 9, an osteosarcoma PDX animal model was constructed and a control group was set, CAR-T cells at different doses were injected into the osteosarcoma PDX animal model through tail vein, and tumor volumes in the animal model and the animal model were measured at regular time to evaluate the drug effect of CAR-T cells on the osteosarcoma PDX animal model.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Shanghai Yi Hao medical science and technology Limited

<120> CAR vector targeting solid tumor cell B7-H3 protein, CAR-T cell and construction method and application thereof

<130> 2021-07-09

<160> 11

<170> SIPOSequenceListing 1.0

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<211> 63

<212> DNA

<213> Artificial Sequence

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 2

<211> 357

<212> DNA

<213> Artificial Sequence

<400> 2

ctgcatgtag gctgtgctgg aggattcgtc tacagtcaat gtgaccttgt ccttgaactc 60

ttgattgtag ttagtataaa tataagaagg ataaatattt ccgatccact caaggccttg 120

tccaggcctc tgcttcaccc agtttatcca gtagttggtg aaggtgtagc cagaagcctt 180

gcaggacagc ttcacagccc caggcctcac cagttcagcc ccctgctgca gcttgacctg 240

ctcagcagcc cgacatctga ggactctgcg gtctattact gtacaagatc cccttatggt 300

tacgacgagt atggtctgga ctactggggc caaggcacca cggtcaccgt ctcctca 357

<210> 3

<211> 336

<212> DNA

<213> Artificial Sequence

<400> 3

ggcgaaggag gatgtgctca actccatgta tacagtcatc cagacctggt ccttcaattc 60

agtccagttg taagtcatcc agtataccca attaatataa gtgatccatt gagtagtttg 120

agtgaagatt tgctccatcc agtataccca gtagttggag aaggagtagc cagaagtctc 180

agaggccaag aagacagtcc aagaccacat caatactggg gactcctcca actcgacctg 240

ctgagtagtc caacaagtga agacagtgcc gtatacgacg aatacggaag aagtccatac 300

ggctacgatg aatacttgga ctactggcat ccagta 336

<210> 4

<211> 372

<212> DNA

<213> Artificial Sequence

<400> 4

ctgcatagtc cacaattcca tcaattctcc ccactcagtg acccacttgt ttccagactg 60

ttgattgtag ttagtataaa tgctagagta attaatataa gtgatgtagt taaggccttg 120

acaagtccat tgctccatcc agtaacaagt gtagttggag aaggagtagc taggattctt 180

tccataagtg ttagtagtcc aaggccacat gactactggc cacttctcca actccaattc 240

ctgagtgttt ccacaagtga agattccgct gtatactact gtacaagaag tccatacgga 300

tacgacgaat acggcgtata cctttgggga caaggaacaa cagtaacagt tagtagtgaa 360

gcccttacat cc 372

<210> 5

<211> 342

<212> DNA

<213> Artificial Sequence

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gacatcgagc tcactcagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60

atgaactgca agtccagtca gagtctgtta aacagtagaa atcaaaagaa ctacttgacc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tatactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatgtttat 300

ccgctcacgt tcggtgctgg gaccaagctg gaaataaaac gg 342

<210> 6

<211> 360

<212> DNA

<213> Artificial Sequence

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gctagtacaa ggacacaaag tcaaaagagt cttacaggac aaccaggaga attcggcaca 60

atgaactgca agtcgtccca gagtctcctg actatgagga actccctcct ctacctgacc 120

tggtaccagc agaagccagg gcagccacca accgtgaggt tcaccggagc aagtaccagg 180

gagtctgggg tgccagacag gttcaccgga tccggatctg gaaccgactt ggtgtacacc 240

atctcctccg tccagaccaa gctcctcgca ttcgggtact gccagaacga ctacgtgtac 300

ccactcacct tcggggcagg gaccaagctc gagatcaaga ggaagtcccc aaccgtggtg 360

<210> 7

<211> 324

<212> DNA

<213> Artificial Sequence

<400> 7

tccctgactc tcactgactc taaggtgtcc ctcactgtga ctgcaggaga gaaggtcagt 60

aggaattgca agagtaccga cacttggtac aatgcatccg actacaagaa ctacctcact 120

tggtacgacc tggcatcctc cgtgccacct aaactgagtg gggtgtgggc aagtaccagg 180

gagtccgagg acctggacta ctgcacagga tctggcagtg gaacagactt cacactcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcaggatga ttatgttgat 300

ccgctcacgt tcggtgaaat aaaa 324

<210> 8

<211> 201

<212> DNA

<213> Artificial Sequence

<400> 8

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct t 201

<210> 9

<211> 123

<212> DNA

<213> Artificial Sequence

<400> 9

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 10

<211> 126

<212> DNA

<213> Artificial Sequence

<400> 10

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 11

<211> 555

<212> DNA

<213> Artificial Sequence

<400> 11

cccctctccc tccccccccc ccgttactgg ccgaagccgc ttggaataag gccggtgtgc 60

gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa 120

acctggccct gtcttcttga cgagcattcc gggtctttcc cctctcgcca aaggaatgca 180

aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 240

gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 300

ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 360

gagttggata gttgtggaaa gagtcaaatg gctcacctca agcgtattca acaaggggct 420

gaaggatgcc cagaaggtac cccattgtat gggatctctg gggcctcggt gcacatgctt 480

tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt 540

ttccttaaaa cacga 555

Claims

1. A CAR vector, characterized in that: the chimeric antigen receptor comprises a leader peptide SP, a B7-H3 single-chain antibody, a transmembrane region TM, a costimulatory domain CD28-4-1BB and a signal transduction domain CD3 zeta;

2. The CAR vector according to claim 1, characterized in that: the nucleotide sequence of the heavy chain variable region VH is shown in any one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequence of the light chain variable region VL is shown in any one of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

3. The CAR vector according to claim 2, characterized in that: the amino acid sequence of the flexible Linker is GGGGSGGGGSGGS.

4. A CAR vector according to any of claims 1 to 3, characterized in that: the vector is a lentiviral vector.

5. The CAR vector according to claim 4, characterized in that: the lentiviral vector is pCDH-CMV-MCS-EF 1-Puro.

6. A CAR vector according to claim 4, characterized in that: a method of making a CAR vector that targets B7-H3 expression proteins of solid tumor cells, comprising the steps of:

storing the gene encoding the chimeric antigen receptor on a PUC19 plasmid;

carrying out enzyme digestion on the PUC19 plasmid preserved with the chimeric antigen receptor, and separating to obtain a target fragment containing the chimeric antigen receptor;

connecting the target fragment containing the chimeric antigen receptor with a restriction enzyme vector, transforming, and extracting to obtain a recombinant plasmid, namely the CAR vector.

7. A CAR-T cell for targeting a B7-H3 expression protein of a solid tumor cell, characterized in that: comprising a T lymphocyte having bound thereto a CAR vector according to any one of claims 1 to 6.

8. A method of constructing a CAR-T cell expressing the protein B7-H3 of claim 7 that targets a solid tumor cell, comprising: the method comprises the following steps:

packaging the CAR vector into a lentivirus;

infecting T lymphocytes with the lentivirus suspension to obtain CAR-T cells.

9. The method of constructing a CAR-T cell of claim 8, wherein: the slow virus packaging method comprises the following steps: the CAR vector is packaged into lentivirus by taking a PSPAX2 plasmid, a pMD2G plasmid and the CAR vector as a three-plasmid packaging system and 293T cells as packaging cells.

10. Use of a CAR-T cell, characterized in that: the CAR-T cell is used for targeting B7-H3 expression protein of a solid tumor cell, wherein the solid tumor cell comprises any one of melanoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, osteosarcoma, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma and glioma.