CN118005806B

CN118005806B - Chimeric antigen receptor, microglia expressing chimeric antigen receptor differentiated from human pluripotent stem cells and their applications

Info

Publication number: CN118005806B
Application number: CN202410178272.4A
Authority: CN
Inventors: 金子兵; 许佳
Original assignee: BEIJING INSTITUTE OF OPHTHALMOLOGY; Beijing Tongren Hospital
Current assignee: BEIJING INSTITUTE OF OPHTHALMOLOGY; Beijing Tongren Hospital
Priority date: 2024-02-08
Filing date: 2024-02-08
Publication date: 2026-01-30
Anticipated expiration: 2044-02-08
Also published as: CN118005806A

Abstract

The invention discloses a chimeric antigen receptor, microglia expressing the chimeric antigen receptor differentiated by human pluripotent stem cells and application thereof. The chimeric antigen receptor CAR provided by the invention is a chimeric antigen receptor based on bisialoganglioside, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1. The invention also provides a preparation method of the microglial cell expressing the chimeric antigen receptor by differentiating the human pluripotent stem cell, which comprises the following steps of infecting the human pluripotent stem cell with the slow virus, carrying out positive clone screening, selecting positive clone cells for culture identification, differentiating the human pluripotent stem cell expressing the chimeric antigen receptor into microglial cells, and obtaining the microglial cell expressing the chimeric antigen receptor. The microglial cell expressing the chimeric antigen receptor, which is differentiated by the human pluripotent stem cell, has no immunogenicity when being used for human treatment, has no unique advantages such as immune rejection and the like.

Description

Chimeric antigen receptor, microglia expressing chimeric antigen receptor differentiated from human pluripotent stem cells and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to chimeric antigen receptor, microglial cells expressing the chimeric antigen receptor differentiated by human pluripotent stem cells and application thereof.

Background

Gliomas are solid tumors originating from brain glial cells, the most common primary intracranial tumors, with 5-year mortality being next to pancreatic and lung cancers in systemic tumors. The pathogenesis of gliomas is unknown, and the treatment means still depend on surgery, radiation therapy and chemotherapy. Microglia are immune cells that colonize the central nervous system, are glioma-associated macrophages, and play an important role in the development of the nervous system and in the progression of disease. If the functions of microglia cells can be enhanced through modification, the microglia cells can specifically kill tumor cells, improve the tumor microenvironment and reduce the side effects of cytokine storm, thus being beneficial to glioma patients.

The bissialoglioside GD2 is an antigen mainly expressed on gliomas (brain tumors, central neuroblastomas such as retinoblastomas, melanoma and the like), has low expression level and limitation in normal tissues, is an ideal tumor antigen for glioma immunotherapy, and is currently used for the immunotherapy of neuroblastomas by specific antibodies aiming at GD2, but because the antibody therapy mainly exists in peripheral blood, the antibody is difficult to accurately enter tumor tissues or tiny residual positions of tumors, the antibody is easy to degrade and cannot exist in the body for a long time, and the difficulty of the therapy is increased. Heretofore CN 106536563A

And CN 108948211a discloses that chimeric antigen receptors bound to GD2 all construct CAR T against GD2 on T cells by transgenic technology, although having a role in cancer treatment, the content of T cells in normal central nervous system and retina is small, and even T cells transferred from peripheral blood to central nervous system and retina in disease state are difficult to enter solid tumor, and difficult to exert therapeutic effect. Previously ZL 2023 1 1030988.1 discloses microglial cells expressing chimeric antigen receptors, but the microglial cells are cell lines, which are inherently immunogenic and thus difficult to use in humans to treat against serious immunological rejection problems, and examples are directed only to retinoblastomas and difficult to use in the treatment of central nervous system tumors.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide chimeric antigen receptors, microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells, and uses thereof.

The chimeric antigen receptor provided by the invention is a bisialoganglioside-based chimeric antigen receptor, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1.

The invention also provides a lentivirus comprising a chimeric antigen receptor.

The lentivirus containing the chimeric antigen receptor provided by the invention comprises the chimeric antigen receptor.

The invention also provides a preparation method of microglial cells expressing chimeric antigen receptors and differentiated by human pluripotent stem cells.

The preparation method of the microglial cell expressing the chimeric antigen receptor for the differentiation of the human pluripotent stem cell comprises the following steps of infecting the human pluripotent stem cell with the slow virus, carrying out positive clone screening, selecting positive clone cells for culture and identification, and differentiating the human pluripotent stem cell expressing the chimeric antigen receptor into the microglial cell to obtain the microglial cell expressing the chimeric antigen receptor.

Alternatively, the human pluripotent stem cells are human induced pluripotent stem cells.

Microglia expressing chimeric antigen receptor differentiated by the human pluripotent stem cells prepared by the method also belong to the protection scope of the invention.

The application of microglial cells expressing chimeric antigen receptor differentiated by human pluripotent stem cells in preparing medicaments for treating tumors expressing bisialoganglioside antigen also belongs to the protection scope of the invention.

Alternatively, the tumor is a human brain astrocytoma that expresses a bisialoganglioside antigen.

Further alternatively, the human brain astrocytoma is human brain astrocytoma U87MG cells.

Alternatively, the tumor is a retinoblastoma that expresses a bisialoganglioside antigen.

Further alternatively, the retinoblastoma is retinoblastoma Y79.

The chimeric antigen receptor provided by the invention is subjected to further optimization and transformation to specifically target tumor cells expressing GD2, and has strong capability of killing the tumor cells.

The microglial cell expressing chimeric antigen receptor, which is differentiated by the human pluripotent stem cell, can accurately identify and kill human brain astrocyte mother cell U87MG cells or retinoblastoma Y79, and has the unique advantages of no immunogenicity, no immune rejection problem and the like when being used for human treatment.

Drawings

For purposes of illustration and not limitation, the invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 is a schematic diagram of the construction of chimeric antigen receptors.

FIG. 2 is a diagram showing the construction of vector pCDH CAR-GD2 for expressing chimeric antigen receptor.

FIG. 3 is a diagram of the construction of vector pCDH CAR-CD19 expressing a chimeric antigen receptor.

FIG. 4 is an identification of human induced pluripotent stem cell differentiated microglial PCR products expressing CAR-GD2 and CAR-CD19 by electrophoresis.

FIG. 5 shows that human microglia inducing differentiation of pluripotent stem cells expressed microglial marker molecules IBA1 (purple) and CD68 (red) but did not express GD2 (green), scale 30 μm.

FIG. 6 is a graph of identification of human induced pluripotent stem cell differentiation microglia by flow cytometry to detect expression of CAR-GD2 and CAR-CD 19.

Figure 7 is an identification of flow cytometry to detect human induced pluripotent stem cell differentiated microglial cell-expressed CAR.

FIG. 8 shows that the flow cytometry detection shows that the retinoblastoma tumor cell Y79 expresses GD2 and does not express CD19, and that the human brain astroblastoma cell U87MG expresses GD2 and does not express CD19.

FIG. 9 is a live cell imaging of human CAR-GD2 and CAR-CD19 microglia cells killing retinoblastoma cells Y79 inducing differentiation of pluripotent stem cells.

Figure 10 is a graph showing the killing rate results of human induced pluripotent stem cell differentiated CAR-GD2 and CAR-CD19 microglial cells against human brain astrocytoma U87MG cells.

Detailed Description

The following description of the invention is intended only to illustrate various embodiments of the invention. Therefore, the specific modifications discussed should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it is to be understood that such equivalent embodiments are intended to be included in the invention. All references, including publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors, with no manufacturer noted.

A. General definition

As used herein, the term "comprising" is used in reference to a composition, method, and its corresponding components that are essential to the method or composition, but is still open to inclusion of unspecified elements, whether or not necessary.

The term "consisting of" means the compositions, methods, and corresponding components thereof as described herein, excluding any elements not recited in the description of the embodiments.

The term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

The term "cell" as used herein refers to a single cell, a cell line, or a culture derived from such cells.

Example 1 construction of chimeric antigen receptor

Chimeric Antigen Receptor (CAR) was constructed by total gene synthesis of signal peptide sequence, GD2 antigen binding domain, hinge region, transmembrane domain, costimulatory signaling domain, 2A sequence, enhanced Green Fluorescent Protein (EGFP), as shown in FIG. 1, signal peptide (SIGNAL PEPTIDE)

-Anti-GD2scFv-CD8 a transmembrane region (CD 8 a TM) -CD86-fcγr1-T2A-EGFP, the amino acid sequence being shown in SEQ ID No. 1:

MALPVTALLLPLALLLHAARPEVQLLQSGPELEKPGASVMISCKASGSSFTGY

NMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHL

KSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSEIVMTQ

SPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNR

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG

TCGVLLLSLVITLYCKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPER

SDEAQRVFKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQE

DRHLEEELKCQEQKEEQLQEGVHRKEPQGATEGRGSLLTCGDVEENPGPMVS

KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPV

PWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK

TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN

GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK。

Wherein:

the amino acid sequence of the signal peptide is shown in SEQ ID NO. 2:

MALPVTALLLPLALLLHAARP。

The amino acid sequence of a single-chain antibody (Anti-GD 2 scFv) against the tumor surface antigen GD2 is shown in SEQ ID NO. 3:

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSEIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK.

The linker sequence is a repeated sequence of a plurality of GGGGS (G4S), and 3 GGGGS are added between a heavy chain variable region and a light chain variable region to play a role in connection, so that the linker has flexibility and enables an antibody to easily contact an antigen when a CAR molecule is formed.

The function of the hinge region is to provide flexibility to overcome steric hindrance and to contribute to the length of the CAR so as to allow the antigen binding domain to contact the target epitope. The amino acid sequence of the hinge region is shown in SEQ ID NO. 4:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。

The transmembrane domain is a CD8 alpha transmembrane region (CD 8 alpha TM), and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO. 5:

IYIWAPLAGTCGVLLLSLVITLYC。

The co-stimulatory signaling domain is a combination of a CD86 signaling domain and an Fc gamma R1 signaling domain, namely a sequential combination of CD86-Fc gamma R1, and the amino acid sequence of the CD86-Fc gamma R1 is shown as SEQ ID NO. 6:

KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRV FKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQEDRH LEEELKCQEQKEEQLQEGVHRKEPQGAT.

the amino acid sequence of T2A is shown in SEQ ID NO. 7:

EGRGSLLTCGDVEENPGP。

the amino acid sequence of the enhanced green fluorescent protein is shown in SEQ ID NO. 8:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

in view of the fact that gliomas do not express the CD19 antigen, the present invention uses chimeric antigen receptors expressing CD19, including the scfv fragment of the anti-CD 19 antibody, the CD8 a transmembrane region and the microglia cell of the CD86 intracellular fragment, as control cells.

The control group cell CD19 chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain, a co-stimulatory signaling domain and a 2A sequence which are connected in series, wherein the amino acid sequence is shown as SEQ ID NO.9, and the specific steps are as follows:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGATEGRGSLLTCGDVEENPGP.

Example 2 chimeric antigen receptor vector construction

The invention is used for constructing a chimeric antigen receptor expression vector named pCDH-EF1 alpha-puro (purchased from Ningbo An Nuo Bode biomedical technology Co., ltd.) and uses a chimeric antigen sequence synthesized by a whole gene as a template, a primer is designed for amplification, and the sequence of an upstream primer (with EcoRI endonuclease sequence) is F:

GATTCGAATTCGCCGCCACCATGGCCCTCCCTGTCACCGCCCTGCT GC (SEQ ID NO. 10), and the downstream primer sequence (with XbaI endonuclease sequence) is R GAATTTCTAGATTACTTGTACAGCTCGTCCATGCCGAGAGT (SEQ ID NO. 11), and the amplified PCR product is electrophoresed and then cut into gel to recover the product. The PCR recovered product was digested with EcoRI and XbaI restriction enzymes, and the pCDH-EF 1. Alpha. -puro vector was digested with EcoRI and XbaI restriction enzymes, at 37℃for 2 hours. And (3) carrying out electrophoresis after enzyme cutting of the PCR product and the carrier, cutting glue, recovering the enzyme-cut product, and carrying out a connection reaction. The PCR cleavage product and the vector cleavage product were subjected to ligation reaction using T4 ligase at 16℃for 12 hours. The ligation product was transformed into Stbl3 (purchased from Tiangen Biochemical technologies (Beijing) Co., ltd.) by the following steps:

1. Melting the competent cells on ice, adding all the connection products into competent cells, mixing the walls of the flick tube uniformly, and standing on ice for 30 minutes.

2. Heat shock at 42 ℃ for 1min30s and rapidly incubating on ice for 2min.

3. 500 Μl of LB medium without antibiotics was added and shaken for 30 min at 37 ℃.

4. After centrifugation, 300. Mu.l of the supernatant was discarded, and the remaining suspension was spread evenly on LB-ampicillin positive agar plates and cultured overnight at 37 ℃. After 12h, observing the growth condition of the bacteria, selecting single large and full bacteria to shake for 12h in LB ampicillin positive culture medium, adding 20% glycerol into one part of the bacteria liquid for freezing and preserving, and delivering the other part of the bacteria liquid to company (Beijing qing biological technology Co., ltd.) for sequencing, and carrying out large-scale labeling on the identified correct bacteria after reviving and shaking to obtain pCDH CAR-GD2 and pCDH CAR-CD19. The vector maps are shown in fig. 2 and 3.

EXAMPLE 3 lentiviral packaging

This example uses the HEK-293T cell line to produce lentiviruses. pCDH CAR-GD2 and pCDH CAR-CD19 are lentiviral vectors, respectively, and can be packaged into lentiviruses after being transfected into 293T cells by adding lentiviral backbone plasmids, and the lentiviruses contain CAR. (reference Gene Ther.201110un; 18 (6): 531-8.).

(1) HEK293T cells in good growth (non-patent document describing HEK293T cells: gene Ther.201110un; 18 (6): 531-8) were digested with 0.25% pancreatin for 2 minutes, stopped by adding DMEM medium (available from Semer Feishan technology Co.) containing 10% FBS, and the bottom cells of the dishes were blown into single cells, counted 1X 10 ⁷, inoculated into 10cm dishes, and transfected for 12 hours to package viruses. In order to collect lentiviruses more, several dishes of cells can be cultured depending on the situation.

(2) Preparation of complexes of transfection reagents and plasmids

A. 35. Mu.g of the viral plasmid to be transfected (10. Mu. G PCDH CAR DNA vector plasmid, 10. Mu.g pMD2.G (available from Wohmmer brain science technologies Co., ltd.) and 15. Mu. g psPAX2 (available from Wohmmer brain science technologies Co.) were dissolved in Opti-MEM medium (available from Siemens Feisha technology Co.) in a total volume of 500. Mu.l, gently mixed, and allowed to stand for 5min to obtain a plasmid-containing mixture.

B. 100. Mu.l of the transfection reagent and 200. Mu.l of the enhancement reagent in lipofectamin2000 (available from the company Simer Feichi technology) were dissolved in Opti-MEM medium in a total volume of 500. Mu.l, gently mixed, and allowed to stand for 5min.

C. Adding the mixed solution containing the plasmid into the mixed solution containing the transfection reagent, and standing for 20min at room temperature after gently mixing the mixed solution with the plasmid so as to fully combine the DNA and the transfection reagent to form a stable transfection complex.

(3) The dishes were removed and the DNA transfection reagent mixture prepared above was added to HEK293T cells of step (1).

(4) After transfection, the medium was aspirated after 6h, washed once with PBS, 10ml of fresh DMEM medium containing 10% FBS was added and incubated in a 37℃5% CO2 incubator.

EXAMPLE 4 extraction and concentration of lentiviruses

Lentivirus extraction:

1) HEK293T cell supernatants 48h, 72h after transfection (0 h at transfection) were collected and aliquoted into 50ml centrifuge tubes. Centrifugation was performed at 3500rpm for 10min at room temperature to remove cells and large debris.

2) The supernatant was filtered through a 0.45 μm filter in an ultracentrifuge tube.

Ultracentrifugation concentrates virus:

3) A white viral pellet was observed on one side of the tube wall after centrifugation at 30,000rpm at 4℃for 2 h.

4) Discarding the supernatant, reversely buckling the centrifuge tube on sterilized absorbent paper, and removing the supernatant which is not discarded cleanly. Phosphate Buffer (DPBS) was added to each tube according to the amount of precipitation, 80. Mu.l to 120. Mu.l/tube, the tube mouth was sealed with a sealing film, and the precipitation was dissolved overnight at 4 ℃.

5) And packaging the virus according to the requirement, and storing in a refrigerator at-80 ℃.

Example 5 lentiviral titre assay

The copy number of the integrated viral genome in the cells was determined by quantitative PCR, and the amount of cells at the time of infection was determined based on the volume of virus added to infer the original viral titer. The viral genome was detected using WPRE sequences on the vector, which were absent in HEK293T cells.

1) The day before infection, 24 well plates were seeded with 1×10 ⁵ cells per well.

2) 10. Mu.l of virus, 1. Mu.l, and 0.1. Mu.l of virus were added to each cell culture well, followed by addition of Polybrene (Polybrene) (available from Merck). The virus may be diluted before addition.

3) After 24 hours of infection, the culture was continued with liquid change.

4) Cell supernatants were discarded 48h after infection. Collecting cells, and extracting 293T cell genome;

5) The obtained genome is used as a template, the reference gene and the viral sequence WPRE are quantified, and the ratio of the viral genome number to the cell genome number is calculated by a relative quantification method through a2 ^-ΔΔ Ct method.

As a result, lentiviruses used in the present invention had a titre of 1X 10 ⁸ TU/ml and the CAR-containing lentiviruses were designated as lenti CAR-GD2 and lenti CAR-CD19, respectively.

Example 6 preparation of CAR-microglia

Human induced pluripotent stem cells (reference PNAS Nexus.2022Aug

17;1 (4): Pgac162; stem Cell reports.2018Apr 10;10 (4): 1267-1281.) were added to the Stem Cell culture medium TeSR-E8 (from Stem Cell Corp.) and inoculated into 6-well plates with 5X 10 ⁵ cells per well, and after 24h of incubation, each was infected with the lenti-CAR GD2 and lenti-CAR CD19 (20. Mu.l/well), followed by addition of polybrene (10 ug/ml), and gently shaking the six-well plates to mix well. The virus supernatant was removed 6h after virus infection and the fresh stem cell medium TeSR-E8 (available from stem cells Co.). The slow virus vector has green fluorescent protein GFP and puromycin resistance, after cell culture for 48h, observing that cells expressing green fluorescence are in the infected slow virus cells, then adding puromycin into the infected slow virus cell holes and uninfected slow virus cell holes to screen, adding puromycin for the first day to see that some cells die, stopping screening when the uninfected slow virus cell holes die, continuing culture, when the infected slow virus cell holes can be cloned, selecting monoclonal cells to culture, when the monoclonal cells can be passaged, collecting cells to extract DNA, identifying PCR products, and sequencing. The PCR identification primer is F TTCTCAAGCCTCAGACAGTGGT (SEQ ID NO. 12);

R AGCGCATGCTCCAGACTGCCTT (SEQ ID NO. 13), and electrophoresis of the PCR products, as shown in FIG. 4, shows that the selected monoclonal stem cells contained specific CAR sequences.

The stem cells containing CAR-GD2 and CAR-CD19 were then differentiated into microglia (microglial differentiation method see patent document CN 114752565 A,CN114426951A and reference sci.china Life sci.2022.65 (6), 1057-1071.). The specific method is that when the cultured human induced pluripotent stem cells reach 80% density, the cells are digested with 0.5mM EDTA. To form Embryoid Bodies (EBs), hiPSCs were transferred to suspension plates containing embryoid body-simulating medium (containing 10. Mu.MY-27632). Embryoid body-simulating medium was carefully changed daily. On day 5, EBs were transferred in a mononuclear cell culture medium resuspended in 100mm cell culture dishes pre-coated with 0.1% gelatin to generate pre-medullary cells. The medium was changed weekly and hematopoietic progenitor cells appeared in the supernatant after 4 weeks of culture in monocyte medium. These hematopoietic progenitor cells were harvested by collecting the supernatant weekly for approximately two months. To differentiate and mature microglial cells, the collected hematopoietic progenitor cells are placed in a suspension culture plate containing microglial cell maturation medium, and after 1 week of culture microglial cell maturation can be tested. Microglia induced to differentiate by human pluripotent stem cells were immunofluorescent stained with specific markers IBA1 and CD68, and the results are shown in fig. 5, in which microglia expressed IBA1 (purple), CD68 (red) and GD2 antigen expressed by microglia were detected, and in which microglia induced to differentiate by human pluripotent stem cells did not express GD2 (green). The purity of microglial cells differentiated by stem cell identification by flow cytometry was high, with a proportion of more than 90%, as shown in figure 6, by staining with microglial cell specific markers CD11b and CD 45. The CAR molecules contained EGFP with green fluorescence, and further identified the EGFP-expressing situation by flow cytometry, as shown in fig. 7, thus obtaining human induced pluripotent stem cell differentiation-derived microglia expressing CAR-GD2 and control CAR-CD 19.

Example 7 in vitro killing experiments of human CAR-GD2 microglia inducing pluripotent stem cell differentiation

Retinoblastoma Y79 (from the company toyoku biology) was detected by labeling Y79 cells with specific flow antibodies of GD2 (from the company daceae biotechnology limited) and CD19 (from the company daceae biotechnology limited), followed by flow cytometry, as shown in fig. 8, Y79 expressed GD2 and not CD19. Flow cytometry detection method after collecting Y79 cells 200g centrifugation for 5 min, discarding supernatant, re-suspending with phosphate buffer DPBS (available from Simer F.), centrifuging for 5 min 200g, discarding supernatant, re-suspending cells with cell labelling buffer (available from Daidae Biotechnology Co.) to 1X 10 ⁷ cells/ml and counting 100ul of cells with 5ul GD2 flow antibody and 5ul CD19 flow antibody, incubating at room temperature for 20 min in the absence of light. Subsequently, 500ul of the cell marker buffer was added, and the mixture was centrifuged at 300g for 5 minutes, and the supernatant was discarded, and the cells were resuspended with 500ul of the cell marker buffer for flow cytometry detection. The CAR-GD2 microglia and the CAR-CD19 microglia are respectively co-cultured with the Y79 cells, and the killing property of the CAR-microglia on the tumor cells Y79 is observed through living cell imaging, as shown in fig. 9, the CAR-microglia has green fluorescence, Y79 staining CELLTRACE VIOLET expresses blue fluorescence, the upper part of the picture in fig. 9 shows the picture when the CAR-microglia and the Y79 are co-cultured, the uniform distribution of the cells can be seen, the lower part of the picture in fig. 9 is the picture when the CAR-microglia and the Y79 are co-cultured for 100 hours, the CAR-GD2 microglia obviously kills the Y79 cells, and almost no residual cells exist in the Y79.

Example 8 in vitro killing experiments of human CAR-GD2 microglia inducing pluripotent stem cell differentiation

Human brain astrocytoma U87MG cells have been reported to be malignant gliomas, expressing GD2 (Cancers (Basel).2020Oct 31;12 (11): 3211.). We examined the case of the purchased U87MG cells (purchased from mirror image of the company, the cell technologies of the sea) expressing GD2 and CD19, labeling the U87MG cells with specific flow antibodies to GD2 and CD19, and then examining the cells by flow cytometry, and as shown in fig. 8, the flow histogram revealed that human brain astrocytoma U87MG cells expressed GD2 in a ratio of 89% or more, and hardly expressed CD19. After co-culturing human embryonic stem cell differentiated CAR-GD2 microglia, CAR-CD19 microglia with U87MG cells (labeled blue fluorescence CELLTRACK VIOLET (available from sameidie science) at a ratio of 10:1, 5:1, respectively) for 24h, significant death of U87MG cells co-cultured with CAR-GD2 microglia was found by live cell imaging counting, with a significantly lower proportion of residual U87MG cells than the control group (fig. 10).

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of microglial cells expressing chimeric antigen receptors for human pluripotent stem cells is characterized by comprising the steps of infecting human pluripotent stem cells with lentiviruses comprising chimeric antigen receptors with amino acid sequences shown as SEQ ID NO. 1, carrying out positive clone screening, selecting positive clone cells for culture identification, differentiating the human pluripotent stem cells expressing chimeric antigen receptors into microglial cells to obtain microglial cells expressing chimeric antigen receptors, and inducing the human pluripotent stem cells into human pluripotent stem cells.

2. The human pluripotent stem cell-differentiated microglial cell expressing chimeric antigen receptor prepared by the method of claim 1.

3. The use of human pluripotent stem cell differentiated microglial cells expressing chimeric antigen receptor according to claim 2 for the preparation of a medicament for the treatment of a tumor expressing a bissialoganglioside antigen, said tumor being human brain astrocytoma expressing a bissialoganglioside antigen or retinoblastoma expressing a bissialoganglioside antigen.

4. The method according to claim 3, wherein the human brain astrocytoma is human brain astrocytoma U87MG cells.

5. The method according to claim 3, wherein said retinoblastoma is retinoblastoma Y79.