CN112877346B

CN112877346B - Encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid and immune cell with gene and application thereof

Info

Publication number: CN112877346B
Application number: CN202010808765.3A
Authority: CN
Inventors: 刘明录; 许淼; 冯建海; 金海锋; 张传鹏; 王亮
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shanghai Xingrui Yida Biotechnology Co.,Ltd.
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2023-01-03
Anticipated expiration: 2040-08-12
Also published as: CN112877346A

Abstract

The invention discloses an encoding gene of an anti-B7-H3 chimeric antigen receptor, a preparation method, a plasmid with the gene, an immune cell and application thereof, wherein the encoding gene of the anti-B7-H3 chimeric antigen receptor is characterized by at least comprising an artificial nucleotide sequence for expressing IL15 and CCL21. The encoding gene of the anti-B7-H3 chimeric antigen receptor provided by the invention enhances the killing effect of CIK cells on solid tumor cells.

Description

Encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid and immune cell with gene and application thereof

Technical Field

The invention relates to the technical field of genes, in particular to a coding gene of a B7-H3-resistant chimeric antigen receptor, a preparation method, a plasmid with the gene, an immune cell and application thereof.

Background

In the treatment of malignant tumors, conventional therapies include surgical treatment, radiotherapy, chemotherapy, traditional Chinese medicine treatment and immunotherapy. Immunotherapy approaches anti-tumor therapy is currently in clinical trials around the world, such as leukemia or lymphoma. Two are already FDA approved for marketing. At present, CAR-T cells aiming at CD19 and CD22 have made great progress in treating tumors in the blood system and have obvious curative effect, but CAR-T cell treatment aiming at solid tumors is slow in progress and poor in effect, and the lack of safe and effective specific tumor antigen targets is one of important hindering factors.

Therefore, the development of the anti-B7-H3 chimeric antigen receptor, the preparation method and the application thereof not only have urgent research values, but also have good economic benefits and industrial application potentials, which is the basis and the motivation for the completion of the invention.

Disclosure of Invention

The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.

Specifically, the technical problems to be solved by the present invention are: provides a coding gene of an anti-B7-H3 chimeric antigen receptor, a preparation method, a plasmid with the gene, an immune cell and application thereof, so as to improve the treatment effect of the immune cell on solid tumors.

In order to solve the technical problems, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides an anti-B7-H3 chimeric antigen receptor-encoding gene comprising at least an artificial nucleotide sequence that expresses IL15 and CCL21.

In the present invention, as a preferred technical scheme, the anti-B7-H3 chimeric antigen receptor encoding gene comprises an antigen binding region, a transmembrane structural region, a costimulatory signaling region and a T cell signaling region functional domain.

In the present invention, the preferred embodiment of the anti-B7-H3 chimeric antigen receptor comprises a sequence of linked

(1) Leader nucleic acid artificial sequence (SEQ ID NO. 2)

(2) Anti-B7-H3 antigen single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO. 3)

(3) CD8Hinge region nucleic acid artificial sequence (SEQ ID NO. 4)

(4) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO. 5)

(5) CD226 intracellular domain nucleic acid artificial sequence (SEQ ID NO. 6)

(6) CD3 zeta intracellular region nucleic acid artificial sequence (SEQ ID NO. 7)

(7) T2A-IL15-CCL21 nucleic acid artificial sequence (SEQ ID NO. 8).

In the invention, as a preferred technical scheme, the nucleotide sequence table of the B7-H3-resistant chimeric antigen receptor encoding gene is shown in SEQ ID NO. 1.

In a second aspect, the present invention provides a method for preparing a gene encoding an anti-B7-H3 chimeric antigen receptor, comprising the steps of:

(1) Synthesizing the whole expression frame by committing the biological engineering (Shanghai) company of engineering biology, inserting the expression frame into pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro to obtain pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 according to the sequence of the nucleic acid artificial sequence of the Leader of the fusion gene fragment, the nucleic acid artificial sequence of Anti-B7-H3, the nucleic acid artificial sequence of CD8, the nucleic acid artificial sequence of a linker, the nucleic acid artificial sequence of CD8TM, the nucleic acid artificial sequence of CD226, the nucleic acid artificial sequence of CD3 zeta and the nucleic acid artificial sequence of T2A-IL15-CCL 21;

(2) Carrying out double enzyme digestion on pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21, cutting off an agar part of a DNA fragment containing CAR (B7-H3) -IL15-CCL21 by using agar electrophoresis, treating by using a sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, carrying out air separation, eluting the DF column, and collecting a centrifugal substance to obtain a purified CAR (B7-H3) -IL15-CCL21 DNA fragment, namely the anti-B7-H3 chimeric antigen receptor coding gene.

More specifically, the steps are as follows:

synthesizing the whole expression frame by committee biotechnology (Shanghai) Limited according to the sequence of fusion gene fragment Leader-scFv (Anti-B7-H3) -CD8-CD226-CD3 zeta-T2A-IL 15-CCL21, and inserting the expression frame into a T vector pUC57 to obtain pUC57-scFv (B7-H3) -T2A-scFv (CTLA 4);

pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 was subjected to Fast Digest AsiSI and Fast Digest NotI double digestion at 37 ℃ for 20min, and 100. Mu.l of the digestion system: 10 × buffer:10 mul; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; supplementing the volume with deionized water;

cutting off the agar part containing the DNA fragment of CAR (B7-H3) -IL15-CCL21 by using agar electrophoresis, and placing the cut agar part in a centrifuge tube;

dissolving DNA from the agar by adopting a DNA extraction kit, firstly adding 500 mu l DF buffer into the centrifuge tube, acting for 10 minutes at 55 ℃, shaking once every 2-3 minutes until the agar is completely dissolved;

then sucking the agar solution into DF Column, sleeving a Collection Tube, centrifuging at 8000rpm for 1min, and pouring off the filtrate;

then adding 500 mul Wash Buffer into the DF Column, centrifuging at 8000rpm for 1 minute, and pouring off the filtrate; DF Column was centrifuged at 12000rpm for 2 minutes to ensure ethanol was removed;

and finally transferring the DF Column to another clean micro-centrifuge tube, adding 30 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging the micro-centrifuge tube for 2 minutes at 14000rpm, and obtaining the purified CAR (B7-H3) -IL15-CCL21 DNA fragment as the liquid in the micro-centrifuge tube.

In a third aspect, the present invention provides a plasmid having an anti-B7-H3 chimeric antigen receptor, comprising the steps of: respectively inserting pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI site into the sequence of fusion gene fragment Leader-scFv (B7-H3) -CD8-CD226-4-1BB-CD3 zeta-T2A-Leader-scFv (CTLA 4) -CD8-CD3 zeta, transforming the insertion site into E.coli (DH 5 alpha), extracting plasmids by using a plasmid extraction kit of Qiagen company after PCR identification of bacteria liquid, and naming the recombinant plasmid with correct sequencing result as pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 after the plasmids are correctly sequenced.

In more detail, the steps are as follows:

the pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI site is inserted into the fusion gene fragment Leader-scFv (Anti-B7-H3) -CD8-CD226-CD3 zeta-T2A-IL 15-CCL21 in the order, and is named as pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21,

at the same time, the pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 and pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vectors were subjected to a double enzyme digestion with Fast Digest asiSI (purchased from ThermoFisher Co.) and Fast Digest NotI (purchased from ThermoFisher Co.) at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mul; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the DNA fragment of CAR (B7-H3) -IL15-CCL21 and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment were excised by agarose electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean microcentrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified CAR (B7-H3) -IL15-CCL21 DNA fragment and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment in the microcentrifuge tube.

The two DNA fragments were ligated overnight at 16 ℃ to form pLent-scFv (B7-H3) -T2A-scFv (CTLA 4). The connecting system is as follows: 10 × buffer:1 mul; t4 ligase: 1 mul; purified CAR (B7-H3) -IL15-CCL21 DNA:4 mu l of the solution; linearized pLent-EF1 α -FH-CMV-GFP-P2A-Puro DNA:4 μ l.

Coli (DH 5 α) was transformed with the above-described pLent-EF1 α -CAR (B7-H3) -IL15-CCL21.

The method comprises the following specific steps: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB. A single colony is picked for overnight culture the next day, and a plasmid extraction and purification kit (purchased from Qiagen company) is adopted to extract a pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 plasmid, and the specific steps are as follows: (1) 1.5ml of the bacterial solution was centrifuged at room temperature 10000 Xg for 1min. (2) The supernatant was removed, 250. Mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mul of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain clear lysate. Preferably, the incubation is carried out at room temperature for 2min. (4) Add 350. Mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) Discarding the filtrate, adding 500. Mu.l Buffer HBC, centrifuging at 10000 Xg for 1min, cleaning the absorption column, and removing residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. Mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1min. (8) The filtrate was discarded, and 750. Mu.l of Wash Buffer was added to Wash the column. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. Mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of known concentration, and the comparison results showed that the plasmid concentration of pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 was 328 ng/. Mu.l.

The pLent-EF 1. Alpha. -CAR (B7-H3) -IL15-CCL21 plasmid described above was sequenced by committee of Biotechnology engineering (Shanghai) Co., ltd. Sequencing is carried out correctly for later use.

In a fourth aspect, the present invention provides an immune cell having a gene encoding an anti-B7-H3 chimeric antigen receptor, said immune cell being selected from autologous CIK cells.

In the present invention, as a preferred embodiment, the immune cells are obtained by the following preparation method:

taking autologous peripheral blood of a patient, and separating peripheral blood mononuclear cells; after 24 hours of induction culture, adding recombinant interleukin 2, 50ng/ml OKT-3 and 5 percent of autologous plasma of the patient for induction and further culture for 24 hours; and (4) continuing culturing, wherein the positive rate of CD3+ is more than 80%, and the double positive rate of CD3+ CD56+ is more than 20%, so that the CIK with successful induction is obtained.

In a fifth aspect, the invention provides the use of an anti-B7-H3 chimeric antigen receptor for the treatment of solid tumors.

In the present invention, as a preferred embodiment, the pharmaceutical form to be used includes a kit.

After the technical scheme is adopted, the invention has the beneficial effects that:

B7-H3 (CD 276) belongs to a B7 super family member, is a type I transmembrane protein highly expressed in various malignant tumors, such as liver cancer, pancreatic cancer and prostate cancer with the expression rate of more than 93 percent, breast cancer and osteosarcoma with the expression rate of more than 90 percent, colon cancer and ovarian cancer with the expression rate of more than 80 percent, endometrial cancer and cervical cancer with the expression rate of more than 70 percent, non-small cell lung cancer and bladder cancer with the expression rate of more than 50 percent, and the like. B7-H3 (CD 276) is not only highly overexpressed in a variety of human solid tumors and positively correlated with cancer severity and poor prognosis, while B7-H3 belongs to an immune checkpoint molecule of the B7-CD28 pathway, which not only regulates innate and adaptive immunity, but also promotes cancer cell invasiveness through various non-immune functions, compared to other immune checkpoints. Therefore, B7-H3 can be used as a new target for future solid tumor immunotherapy.

Infiltration, accumulation and survival of chimeric antigen receptor T (CAR-T) cells in solid tumors is critical for tumor clearance. There is therefore a need for more efficient chimeric antigen receptor T (CAR-T) cells for the treatment of malignancies, in particular solid tumors. Research shows that chemotactic factors CCL21 and IL-15 are secreted by the lymphatic tissue, wherein CCL21 can recruit peripheral T lymphocytes, dendritic cells, B cells and the like to enter the lymphatic tissue, and IL-15 plays an important role in the proliferation and the stability of the T cells.

Based on the above thought, the invention designs a chimeric antigen receptor which can fuse and express IL-15 and CCL21 anti-B7-H3 (CD 276) for treating solid tumors.

The encoding gene of the anti-B7-H3 chimeric antigen receptor provided by the invention enhances the killing effect of T cells on solid tumors.

Drawings

FIG. 1 is a schematic diagram of the lentiviral expression vector pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21.

FIG. 2 is a schematic diagram of the CAR module of CAR (B7-H3).

FIG. 3 is a graph showing the results of flow cytometry for detecting the positive expression rate of CD3+ and CD56+ in T cells.

FIG. 4 is a graph showing the results of flow assay of chimeric antigen receptor expression.

FIG. 5 is a graph of anti-B7-H3 chimeric antigen receptor T cell cytokine release assay.

FIG. 6 is a schematic diagram of the study of the killing activity of T cells targeting the B7-H3 chimeric antigen receptor.

Detailed Description

The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.

Example 1

An anti-B7-H3 chimeric antigen receptor encoding gene, wherein at least an artificial nucleotide sequence for expressing IL15 and CCL21 is contained. Also included are antigen binding regions, transmembrane domains, costimulatory signaling regions, and functional domains of the T cell signaling region.

In this embodiment, the anti-B7-H3 chimeric antigen receptor comprises a sequence of linked

(1) Leader nucleic acid artificial sequence (SEQ ID NO. 2)

(3) CD8Hinge region nucleic acid artificial sequence (SEQ ID NO. 4)

(4) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO. 5)

(5) CD226 intracellular region nucleic acid artificial sequence (SEQ ID NO. 6)

(7) T2A-IL15-CCL21 nucleic acid artificial sequence (SEQ ID NO. 8).

That is, the nucleotide sequence table of the gene encoding the anti-B7-H3 chimeric antigen receptor of this example is shown in SEQ ID NO. 1.

Example 2

Preparation example of anti-B7-H3 chimeric antigen receptor-encoding gene.

The method for preparing the anti-B7-H3 chimeric antigen receptor encoding gene of the embodiment comprises the following steps:

In this embodiment, the detailed steps are as follows:

pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 was subjected to Fast Digest AsiSI and Fast Digest NotI double digestion at 37 ℃ for 20min, and 100. Mu.l of the digestion system: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; supplementing the volume with deionized water;

and finally transferring the DF Column to another clean microcentrifuge tube, adding 30 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging the microcentrifuge tube for 2 minutes at 14000rpm, and obtaining the purified CAR (B7-H3) -IL15-CCL21 DNA fragment as the liquid in the microcentrifuge tube, wherein the DNA fragment is shown in figure 2.

Example 3

Examples of plasmids having a gene encoding a chimeric antigen receptor against B7-H3.

The plasmid with the anti-B7-H3 chimeric antigen receptor coding gene is prepared by adopting a method comprising the following steps: respectively inserting pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI site into the sequence of fusion gene fragment Leader-scFv (B7-H3) -CD8-CD226-4-1BB-CD3 zeta-T2A-Leader-scFv (CTLA 4) -CD8-CD3 zeta, transforming the insertion site into E.coli (DH 5 alpha), extracting plasmids by using a plasmid extraction kit of Qiagen company after identifying the plasmids by bacterial liquid PCR, and naming the recombinant plasmid with correct sequencing result as pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 as shown in FIG. 1 after the plasmids are correctly sequenced.

In this embodiment, the more detailed steps are:

inserting pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vector (Invitrogen) NotI-AsiSI site into the fusion gene fragment Leader-scFv (Anti-B7-H3) -CD8-CD226-CD3 zeta-T2A-IL 15-CCL21 in the sequence, and naming pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21,

at the same time, pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 and pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vectors were subjected to a double enzyme digestion of Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher) at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the DNA fragment of CAR (B7-H3) -IL15-CCL21 and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment were excised by agarose electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean microcentrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, and centrifuging for 2 minutes at 14000rpm, wherein the liquid in the microcentrifuge tube is the purified CAR (B7-H3) -IL15-CCL21 DNA fragment and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment.

The specific steps of the transformation are as follows: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB. A single colony is picked for overnight culture the next day, and a plasmid extraction and purification kit (purchased from Qiagen company) is adopted to extract a pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 plasmid, and the specific steps are as follows: (1) 1.5ml of the bacterial solution was centrifuged at room temperature 10000 Xg for 1min. (2) The supernatant was removed, 250. Mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mul of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain clear lysate. Preferably, the incubation is carried out at room temperature for 2min. (4) Add 350. Mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) The filtrate was discarded, 500. Mu.l Buffer HBC was added, 10000 Xg was centrifuged for 1min, and the column was washed to remove residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. Mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1min. (8) The filtrate was discarded, and 750. Mu.l of Wash Buffer was added to Wash the column. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. Mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of known concentration, and the comparison results showed that the plasmid concentration of pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 was 328 ng/. Mu.l.

Example 4

Examples of immune cells having a gene encoding a chimeric antigen receptor against B7-H3.

An immune cell having a gene encoding an anti-B7-H3 chimeric antigen receptor, said immune cell being selected from autologous CIK cells.

Generally, immune cells are prepared by the following method: the pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 plasmid was transfected into the cell line 293T, and CIK cells were then infected with the recombinant lentivirus described above.

In more detail, the present embodiment adopts the following detailed steps:

(1) Preparation of plasmids

at the same time, pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 and pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro vectors were subjected to a double enzyme digestion of Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher) at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the DNA fragment of CAR (B7-H3) -IL15-CCL21 and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment were excised by agarose electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean microcentrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified CAR (B7-H3) -IL15-CCL21 DNA fragment and the linearized pLent-EF1 alpha-FH-CMV-GFP-P2A-Puro DNA fragment in the microcentrifuge tube.

The two DNA fragments were ligated overnight at 16 ℃ to form pLent-scFv (B7-H3) -T2A-scFv (CTLA 4). The connecting system is as follows: 10 × buffer:1 mul; t4 ligase: 1 mul; purified CAR (B7-H3) -IL15-CCL21 DNA:4 mul; linearized pLent-EF1 α -FH-CMV-GFP-P2A-Puro DNA:4 μ l.

The specific steps of the transformation are as follows: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added for slow shaking for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin. A single colony is picked for overnight culture the next day, and a plasmid extraction and purification kit (purchased from Qiagen company) is adopted to extract a pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 plasmid, and the specific steps are as follows: (1) 1.5ml of the bacterial solution was centrifuged at room temperature 10000 Xg for 1min. (2) The supernatant was removed, 250. Mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mul of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain clear lysate. Preferably, the incubation is carried out at room temperature for 2min. (4) Add 350. Mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) The filtrate was discarded, 500. Mu.l Buffer HBC was added, 10000 Xg was centrifuged for 1min, and the column was washed to remove residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. Mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1min. (8) The filtrate was discarded, and 750. Mu.l of Wash Buffer was added to Wash the column. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. Mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of known concentration, and the comparison results showed that the plasmid concentration of pLent-EF1 alpha-CAR (B7-H3) -IL15-CCL21 was 328 ng/. Mu.l.

The same procedure was used to prepare the B7-H3-CAR (Leader-scFv (B7-H3) -CD8-CD226-CD3 ζ) plasmid.

(2) Lentiviral packaging, viral titer detection

The lentivirus packaging cell line 293T was inoculated into a DMEM +10% FBS-containing 6-well plate, cultured at 37 ℃ under 5% CO2 conditions, and transfected after the anchorage rate was 70% to 80%. The Leader-scFv (B7-H3) -CD8-CD226-CD3 zeta-IL 15-CCL21 plasmid (IL 15-CCL 21-B7-H3-CAR) and Leader-scFv (B7-H3) -CD8-CD226-CD3 zeta (B7-H3-CAR) plasmids were co-transfected with lentivirus packaging plasmids into 293T cells, respectively, by changing the 6-well plates to 2ml of fresh medium before transfection. After 24h after transfection, cells are obviously enlarged and spherical, cell nucleuses are enlarged and become round, and the wall-attaching capability is reduced and the cells are easy to fall off. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72h, the supernatant was collected, filtered to remove bacteria, and stored in a low temperature freezer at-80 ℃ for further use. According to Lenti-X ^TM Go Stix ^TM The kit (product of Beijing Huaxia ocean technology Co., ltd.) determines the virus titer, and the result shows that the titer of the recombinant lentivirus Leader-scFv (B7-H3) -CD8-CD226-CD3 zeta-IL 15-CCL21 is 2.66 multiplied by 10 ⁶ pfu/mL, titer of 2.78X 10 for recombinant lentivirus Leader-scFv (B7-H3) -CD8-CD226-CD3 ζ ⁶ pfu/ml。

(4) Lentiviral-infected CIK (cytokine induced killer) cells and amplification culture of infected CIK cells

Peripheral blood mononuclear cells were isolated from 75ml of patient autologous peripheral blood using TBD sample density separation medium (purchased from tianjin tertiary ocean organisms). After induction culture with a medium (purchased from CORNING, 88-551-CM) containing 1000IU/ml of recombinant interferon alpha 2a (purchased from sheng yang sansheng) for 24 hours, 1000IU/ml of recombinant interleukin 2 (purchased from sheng yang sansheng) was added, 50ng/ml of OKT-3 and 5% of autologous plasma of the patients were added for further culture for 24 hours. Every two days, the solution was added at double rate, and cultured to day 14, and the positive expression rate of CD3+ and CD56+ in T cells was examined by flow cytometry (CD 3-FITC, CD16/CD56-PE antibody purchased from BECKMAN, A07735). The positive rate of CD3+ is more than 80%, the double positive rate of CD3+ CD56+ is more than 20%, the CIK is regarded as successful induction, and the CIK is reserved for virus infection.

As shown in FIG. 3, the CD3+ positive rate is 97.6%, and the CD3+ CD56+ double positive rate is 27.1%, i.e. the CIK induction is successful, and can be used for subsequent experiments.

CIK cells were infected with each of the above lentiviruses at MOI = 5. After the infected cells were cultured in a CO2 incubator at 37 ℃ and 5% for 8 hours, the cells were collected, the virus solution and polybrene were added again, the cells were centrifuged at 1000g and 32 ℃ for 90 minutes again, and then the cells were cultured in a CO2 incubator at 37 ℃ and 5% again, and multiple infection was repeated in this manner, thereby increasing the infection efficiency of T cells. 2ml of culture supernatant was aspirated off, 2ml of fresh CORNING Medium was added, and the expansion culture was continued for 17 days until the cells were expanded to a sufficient amount. As shown in fig. 4, the expression of the chimeric antigen receptor was detected by cell flow, and the GFP and CAR were co-expressed, so that the positive cells for detecting GFP were positive cells expressing the chimeric antigen receptor. The positive rate of the virus-infected T cells which express IL-15 and CCL21 anti-B7-H3 chimeric antigen receptor (IL-15-CCL 21-B7-H3-CAR) in a fusion way is 27%, and the positive rate of the virus-infected T cells which express B7-H3 chimeric antigen receptor (B7-H3-CAR) is 29% by taking uninfected T lymphocytes as a negative control.

Example 5

The application of anti-B7-H3 chimeric antigen receptor in killing and inhibiting solid tumor is provided.

Research on in-vitro killing activity of anti-B7-H3 chimeric antigen receptor modified immune cells

IL-15-CCL21-B7-H3-CAR-T cells (1X 10) ⁵ ) And B7-H3-CAR-T cells (1X 10) ⁵ ) Respectively with pre-plated renal cancer cells 786-0 or lung cancer cellsCell H23 (1X 10) ⁵ ) The cells were co-cultured in a plate without exogenous cytokines, and after 24 hours, cell supernatants were collected and assayed for cytokine (interferon γ (IFN- γ) and interleukin 2 (IL-2) content by ELISA. The above experiments were repeated 3 times. The results are shown in FIG. 5, in which both IL-15-CCL21-B7-H3-CAR-T cells and B7-H3-CAR-T cells were activated, and IL-15-CCL21-B7-H3-CAR-T cells released more cytokines (IFN-. Gamma., IL-2) and had stronger killing activity than B7-H3-CAR-T cells.

Example 6

Therapeutic effect of CAR-CIK cells on solid tumors

1. Renal cancer cell 786-0 or lung cancer cell H23 tumor cell 1 × 10 ⁵ Concentrations per well were plated in RTCA well plates and incubation was monitored overnight.

2. IL-15-CCL21-B7-H3-CAR-T cells and normal anti-B7-H3-CAR-T cells were added to pre-monitored overnight RTCA well plates, respectively, at different ratios (E: T is 1.

3. E: t is 1: the best results were obtained at 5, as shown in FIG. 6: the proliferation of target cells without adding CAR-T cells was not affected, the curve rose, whereas tumor cells with both IL-15-CCL21-B7-H3-CAR-T cells and B7-H3-CAR-T cells were killed, the curve dropped, and IL-15-CCL21-B7-H3-CAR-T cells had a stronger ability to kill tumor cells than normal anti-B7-H3-CAR-T cells.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

Sequence listing

<110> Shandong Xingyi Biotechnology Ltd

<120> encoding gene of anti-B7-H3 chimeric antigen receptor, preparation method, plasmid with the gene, immune cell and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1644

<212> DNA

<213> Homo sapiens

<400> 1

atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60

ccccaggtcc agctggtaca gtctggagct gaagtcaaga aacctgggag ctcagtgaag 120

gtatcctgca aggcttctgg atacacattc actaactatg ttatgcactg ggtgagacag 180

gcacctggtc aaggactcga gtggatggga tatattaatc cttacaatga tgatgtcaag 240

tacaatgaga agttcaaagg ccgagtcaca attactgcag acgagtccac tagcacagcc 300

tacatggaac tcagcagctt gcgatcggag gacactgcag tctattactg tgcaaggtgg 360

gggtattatg gtagcccact ttattacttc gattactggg gccaaggtac actggtcaca 420

gtctcctcag gaggtggggg cagtggtggc gggggatctg gaggtggagg ttccgaaatt 480

gtgttgacgc agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540

tgcagggcca gttcgagact tatctacatg cactggtacc agcagaaacc tggccaggct 600

cccaggctcc tcatctatgc tacatccaac ttggcctctg gcatcccagc caggttcagt 660

ggcagtgggt ctgggacaga cttcactctc accatcagca gactggagcc tgaagatttt 720

gcagtgtatt actgtcagca gtggaatagc aatcctccga cgttcggcca agggaccaag 780

gtggaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca acagaaggag aaggagagag 1020

agaagagatc tatttacaga gtcctgggat acacagaagg cacccaataa ctatagaagt 1080

cccatctcta cctctcaacc taccaatcaa tccatggatg atacaagaga ggatatttat 1140

gtcaactatc caaccttctc tcgcagacca aagactagag ttaagagagg ccggaagaag 1200

ctgctgtaca tcttcaagca gcccttcatg cggcccgtgc agaccaccca ggaagaggac 1260

ggctgcagct gtcggttccc cgaggaagaa gaaggcggct gcgaactgag agtgaagttc 1320

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1380

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1440

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1500

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1560

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1620

cacatgcagg ccctgccccc tcgc 1644

<210> 2

<211> 63

<212> DNA

<213> Homo sapiens

<400> 2

atggcactgc cagtgaccgc cctgctgctg cctctggccc tgctgctgca cgcagccaga 60

ccc 63

<210> 3

<211> 729

<212> DNA

<213> Homo sapiens

<400> 3

caggtccagc tggtacagtc tggagctgaa gtcaagaaac ctgggagctc agtgaaggta 60

tcctgcaagg cttctggata cacattcact aactatgtta tgcactgggt gagacaggca 120

cctggtcaag gactcgagtg gatgggatat attaatcctt acaatgatga tgtcaagtac 180

aatgagaagt tcaaaggccg agtcacaatt actgcagacg agtccactag cacagcctac 240

atggaactca gcagcttgcg atcggaggac actgcagtct attactgtgc aaggtggggg 300

tattatggta gcccacttta ttacttcgat tactggggcc aaggtacact ggtcacagtc 360

tcctcaggag gtgggggcag tggtggcggg ggatctggag gtggaggttc cgaaattgtg 420

ttgacgcagt ctccagccac cctgtctttg tctccagggg aaagagccac cctctcctgc 480

agggccagtt cgagacttat ctacatgcac tggtaccagc agaaacctgg ccaggctccc 540

aggctcctca tctatgctac atccaacttg gcctctggca tcccagccag gttcagtggc 600

agtgggtctg ggacagactt cactctcacc atcagcagac tggagcctga agattttgca 660

gtgtattact gtcagcagtg gaatagcaat cctccgacgt tcggccaagg gaccaaggtg 720

gaaatcaaa 729

<210> 4

<211> 135

<212> DNA

<213> Homo sapiens

<400> 4

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 5

<211> 72

<212> DNA

<213> Homo sapiens

<400> 5

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 6

<211> 309

<212> DNA

<213> Homo sapiens

<400> 6

aacagaagga gaaggagaga gagaagagat ctatttacag agtcctggga tacacagaag 60

gcacccaata actatagaag tcccatctct acctctcaac ctaccaatca atccatggat 120

gatacaagag aggatattta tgtcaactat ccaaccttct ctcgcagacc aaagactaga 180

gttaagagag gccggaagaa gctgctgtac atcttcaagc agcccttcat gcggcccgtg 240

cagaccaccc aggaagagga cggctgcagc tgtcggttcc ccgaggaaga agaaggcggc 300

tgcgaactg 309

<210> 7

<211> 336

<212> DNA

<213> Homo sapiens

<400> 7

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 8

<211> 3441

<212> DNA

<213> Homo sapiens

<400> 8

gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg accaactccg 60

ggtggcaggc gcccggggga atcccagctg actcgctcac tgccttcgaa gtccggcgcc 120

ccccgggagg gaactgggtg gccgcaccct cccggctgcg gtggctgtcg ccccccaccc 180

tgcagccagg actcgatgga ggtacagagc tcggcttctt tgccttggga ggggagtggt 240

ggtggttgaa agggcgatgg aattttcccc gaaagcctac gcccagggcc cctcccagct 300

ccagcgttac cctccggtct atcctactgg ccgagctgcc ccgccttctc atggggaaaa 360

cttagccgca acttcaattt ttggtttttc ctttaatgac acttctgagg ctctcctagc 420

catcctcccg cttccggagg agcgcagatc gcaggtccct ttgcccctgg cgtgcgactc 480

cctactgcgc tgcgctctta cggcgttcca ggctgctggc tagcgcaagg cgggccgggc 540

accccgcgct ccgctgggag ggtgagggac gcgcgtctgg cggccccagc caagctgcgg 600

gtttctgaga agacgctgtc ccgcagccct gagggctgag ttctgcaccc agtcaagctc 660

aggaaggcca agaaaagaat ccattccaat atatggccat gtggctcttt ggagcaatgt 720

tccatcatgt tccatgctgc tgacgtcaca tggagcacag aaatcaatgt tagcagatag 780

ccagcccata caagatcgta ttgtattgta ggaggcattg tggatggatg gctgctggaa 840

accccttgcc atagccagct cttcttcaat acttaaggat ttaccgtggc tttgagtaat 900

gagaatttcg aaaccacatt tgagaagtat ttccatccag tgctacttgt gtttacttct 960

aaacagtcat tttctaactg aagctggcat tcatgtcttc attttgggct gtttcagtgc 1020

agggcttcct aaaacagaag ccaactgggt gaatgtaata agtgatttga aaaaaattga 1080

agatcttatt caatctatgc atattgatgc tactttatat acggaaagtg atgttcaccc 1140

cagttgcaaa gtaacagcaa tgaagtgctt tctcttggag ttacaagtta tttcacttga 1200

gtccggagat gcaagtattc atgatacagt agaaaatctg atcatcctag caaacaacag 1260

tttgtcttct aatgggaatg taacagaatc tggatgcaaa gaatgtgagg aactggagga 1320

aaaaaatatt aaagaatttt tgcagagttt tgtacatatt gtccaaatgt tcatcaacac 1380

ttcttgattg caattgattc tttttaaagt gtttctgtta ttaacaaaca tcactctgct 1440

gcttagacat aacaaaacac tcggcatttc aaatgtgctg tcaaaacaag tttttctgtc 1500

aagaagatga tcagaccttg gatcagatga actcttagaa atgaaggcag aaaaatgtca 1560

ttgagtaata tagtgactat gaacttctct cagacttact ttactcattt ttttaattta 1620

ttattgaaat tgtacatatt tgtggaataa tgtaaaatgt tgaataaaaa tatgtacaag 1680

tgttgttttt taagttgcac tgatatttta cctcttattg caaaatagca tttgtttaag 1740

ggtgatagtc aaattatgta ttggtggggc tgggtaccaa tgctgcaggt caacagctat 1800

gctggtaggc tcctgccagt gtggaaccac tgactactgg ctctcattga cttccttact 1860

aagcatagca aacagaggaa gaatttgtta tcagtaagaa aaagaagaac tatatgtgaa 1920

tcctcttctt tatactgtaa tttagttatt gatgtataaa gcaactgtta tgaaataaag 1980

aaattgcaat aactggcata taatgtccat cagtaaatct tggtggtggt ggcaataata 2040

aacttctact gataggtaga atggtgtgca agcttgtcca atcacggatt gcaggccaca 2100

tgcggcccag gacaactttg aatgtggccc aacacaaatt cataaacttt catacatctc 2160

gtttttagct catcagctat cattagcggt agtgtattta aagtgtggcc caagacaatt 2220

cttcttattc caatgtggcc cagggaaatc aaaagattgg atgcccctgg tatagaaaac 2280

taatagtgac agtgttcata tttcatgctt tcccaaatac aggtatttta ttttcacatt 2340

ctttttgcca tgtttatata ataataaaga aaaaccctgt tgatttgttg gagccattgt 2400

tatctgacag aaaataattg tttatatttt ttgcactaca ctgtctaaaa ttagcaagct 2460

ctcttctaat ggaactgtaa gaaagatgaa atatttttgt tttattataa atttatttca 2520

cctgaaggcc gagggagcct gctgacatgt ggcgatgtgg aggaaaaccc aggaccaaca 2580

gacccccaac ttgcagctgc ccacctcacc ctcagctctg gcctcttact caccctctac 2640

cacagacatg gctcagtcac tggctctgag cctccttatc ctggttctgg cctttggcat 2700

ccccaggacc caaggcagtg atggaggggc tcaggactgt tgcctcaagt acagccaaag 2760

gaagattccc gccaaggttg tccgcagcta ccggaagcag gaaccaagct taggctgctc 2820

catcccagct atcctgttct tgccccgcaa gcgctctcag gcagagctat gtgcagaccc 2880

aaaggagctc tgggtgcagc agctgatgca gcatctggac aagacaccat ccccacagaa 2940

accagcccag ggctgcagga aggacagggg ggcctccaag actggcaaga aaggaaaggg 3000

ctccaaaggc tgcaagagga ctgagcggtc acagacccct aaagggccat agcccagtga 3060

gcagcctgga gccctggaga ccccaccagc ctcaccagcg cttgaagcct gaacccaaga 3120

tgcaagaagg aggctatgct caggggccct ggagcagcca ccccatgctg gccttgccac 3180

actctttctc ctgctttaac caccccatct gcattcccag ctctaccctg catggctgag 3240

ctgcccacag caggccaggt ccagagagac cgaggaggga gagtctccca gggagcatga 3300

gaggaggcag caggactgtc cccttgaagg agaatcatca ggaccctgga cctgatacgg 3360

ctccccagta caccccacct cttccttgta aatatgattt atacctaact gaataaaaag 3420

ctgttctgtc ttcccaccca a 3441

Claims

1. An anti-B7-H3 chimeric antigen receptor-encoding gene characterized in that,

comprising sequential connections

(1) Leader nucleic acid artificial sequence shown in SEQ ID NO.2

(2) Anti-B7-H3 antigen antibody single chain Fv antibody (scFv) nucleic acid artificial sequence shown in SEQ ID NO.3

(3) CD8Hinge region nucleic acid artificial sequence shown in SEQ ID NO.4

(4) CD8 transmembrane region nucleic acid artificial sequence shown in SEQ ID NO.5

(5) CD226 intracellular domain nucleic acid artificial sequence shown in SEQ ID NO.6

(6) CD3 zeta intracellular region nucleic acid artificial sequence shown in SEQ ID NO.7

(7) T2A-IL15-CCL21 nucleic acid artificial sequence shown in SEQ ID NO. 8.

2. An immune cell characterized by: has the anti-B7-H3 chimeric antigen receptor-encoding gene of claim 1, and the immune cells are selected from autologous CIK cells.

3. The immune cell of claim 2, wherein: the autologous CIK cells are obtained by the following method:

taking autologous peripheral blood of a patient, and separating peripheral blood mononuclear cells; after 24 hours of induction culture, adding recombinant interleukin 2, 50ng/mL OKT-3 and 5 percent of autologous plasma of the patient for induction and further culture for 24 hours; and (4) continuing culturing, wherein the positive rate of CD3+ is more than 80%, and the double positive rate of CD3+ CD56+ is more than 20%, so that the CIK cell successfully induced is obtained.

4. Use of the anti-B7-H3 chimeric antigen receptor-encoding gene of claim 1 in the preparation of a medicament for the therapeutic killing and inhibition of solid tumor cells.