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

Abstract

This invention discloses a chimeric antigen receptor (CAR), microglia expressing the CAR differentiated from human pluripotent stem cells, and their applications. The chimeric antigen receptor CAR provided by this invention is a bisialic acid ganglioside-based chimeric antigen receptor, and its amino acid sequence is shown in SEQ ID NO.1. This invention also provides a method for preparing microglia expressing the CAR differentiated from human pluripotent stem cells, comprising the following steps: infecting human pluripotent stem cells with the lentivirus; screening for positive clones, selecting positive clones for culture and identification; differentiating the human pluripotent stem cells expressing the CAR into microglia, thereby obtaining the microglia expressing the CAR. The microglia expressing the CAR differentiated from human pluripotent stem cells provided by this invention have unique advantages such as non-immunogenicity and no immune rejection issues in human treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to chimeric antigen receptors, microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells, and their applications. Background Technology

[0002] Gliomas are solid tumors originating from glial cells in the brain. They are the most common primary intracranial tumors, with a 5-year mortality rate second only to pancreatic and lung cancer among all cancers. The pathogenesis of gliomas is still unclear, and treatment currently relies on surgery, radiotherapy, and chemotherapy. Microglia are immune cells that colonize the central nervous system and are glioma-associated macrophages, playing a crucial role in nervous system development and disease progression. If microglia can be modified to enhance their function, enabling them to specifically kill tumor cells, improving the tumor microenvironment, and reducing the side effects of cytokine storms, it would benefit glioma patients.

[0003] Disialotetrahexosylganglioside GD2 is an antigen primarily expressed in gliomas (brain tumors, retinoblastomas, and other central neuroblastomas, melanomas, etc.). Its expression in normal tissues is low and limited, making it an ideal tumor antigen for glioma immunotherapy. Currently, specific antibodies against GD2 are available for neuroblastoma immunotherapy. However, because these antibodies are mainly found in peripheral blood, they are difficult to precisely penetrate tumor tissue or minimal residual tumor sites. Furthermore, antibodies are easily degraded and cannot persist in the body long-term, increasing the difficulty of treatment. (Previously, CN 106536563A)

[0004] CN 108948211A discloses that chimeric antigen receptors binding to GD2 are constructed on T cells using transgenic technology to create CAR T cells targeting GD2. While this has some effect in cancer treatment, the number of T cells in the normal central nervous system and retina is low. Even when T cells migrate from peripheral blood to the central nervous system and retina in a disease state, it is difficult for them to enter solid tumors and exert a therapeutic effect. Previously, ZL 2023 1 1030988.1 disclosed microglia expressing chimeric antigen receptors. However, these microglia are cell lines, and because cell lines themselves are immunogenic, their use in humans would cause severe immune rejection, making them unsuitable for human treatment. Furthermore, the examples only target retinoblastoma and are difficult to use for the treatment of central nervous system tumors. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide chimeric antigen receptors, microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells, and their applications.

[0006] The chimeric antigen receptor provided by this invention is a chimeric antigen receptor based on disialic acid ganglioside, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1.

[0007] The present invention also provides a lentivirus containing a chimeric antigen receptor.

[0008] The lentivirus containing a chimeric antigen receptor provided by the present invention includes the chimeric antigen receptor.

[0009] This invention also provides a method for preparing microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells.

[0010] The present invention provides a method for preparing microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells, comprising the following steps: infecting human pluripotent stem cells with the lentivirus; screening for positive clones, selecting positive clone cells for culture and identification; differentiating human pluripotent stem cells expressing chimeric antigen receptors into microglia, thereby obtaining the microglia expressing chimeric antigen receptors.

[0011] Optionally, the human pluripotent stem cells are human induced pluripotent stem cells.

[0012] Microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells prepared by the method are also within the scope of protection of this invention.

[0013] The application of microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells in the preparation of drugs for treating tumors expressing disialic acid ganglioside antigens also falls within the scope of protection of this invention.

[0014] Optionally, the tumor is a human brain astrocytoma expressing disialotetrahexosylganglioside antigen.

[0015] Further, optionally, the human brain astrocytoblastoma is a human brain astrocytoblast U87MG cell.

[0016] Optionally, the tumor is a retinoblastoma expressing disialic acid ganglioside antigen.

[0017] Further optionally, the retinoblastoma is retinoblastoma Y79.

[0018] The chimeric antigen receptor provided by this invention, after further optimization and modification, specifically targets tumor cells expressing GD2, exhibiting a strong ability to kill tumor cells.

[0019] The microglia expressing chimeric antigen receptors differentiated from human pluripotent stem cells provided by this invention can accurately recognize and kill human brain astrocytes U87MG cells or retinoblastoma Y79 cells, and have unique advantages such as no immunogenicity and no immune rejection problems when used for human treatment. Attached Figure Description

[0020] For illustrative and not limiting purposes, the invention will now be described with reference to preferred embodiments thereof, particularly the accompanying drawings, in which:

[0021] Figure 1 This is a diagram illustrating the construction of a chimeric antigen receptor.

[0022] Figure 2 This diagram shows the construction of the vector pCDH CAR-GD2, which expresses the chimeric antigen receptor.

[0023] Figure 3 This diagram shows the construction of the vector pCDH CAR-CD19, which expresses the chimeric antigen receptor.

[0024] Figure 4 Electrophoretic identification results of PCR products from human induced pluripotent stem cells differentiated into microglia expressing CAR-GD2 and CAR-CD19.

[0025] Figure 5 Microglia differentiated from human induced pluripotent stem cells expressed the microglia markers IBA1 (purple) and CD68 (red), but not GD2 (green); scale bar 30 μm.

[0026] Figure 6 Image for identifying microglia differentiated from human induced pluripotent stem cells expressing CAR-GD2 and CAR-CD19 by flow cytometry.

[0027] Figure 7 Image for identifying CAR expression in microglia differentiated from human induced pluripotent stem cells by flow cytometry.

[0028] Figure 8 To detect the expression of GD2 but not CD19 in Y79 retinoblastoma tumor cells by flow cytometry; and to detect the expression of GD2 but not CD19 in U87MG human astrocytoma cells.

[0029] Figure 9 Live-cell imaging of CAR-GD2 and CAR-CD19 microglia, induced by human pluripotent stem cell differentiation, killing Y79 retinoblastoma cells.

[0030] Figure 10Results of the killing rate of human brain astrocytoblastoma U87MG cells by CAR-GD2 and CAR-CD19 microglia differentiated from human induced pluripotent stem cells. Detailed Implementation

[0031] 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 should be understood that such equivalent embodiments will be included in the invention. All references cited in this invention, including publications, patents, and patent applications, are incorporated herein by reference in their entirety. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques or conditions described in the literature in the art, or according to product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased from legitimate channels.

[0032] A. General Definition

[0033] As used herein, the term "comprising" is used when referring to a composition, a method, and its respective components that are essential to said method or composition, but is open to including unspecified elements, whether or not they are necessary.

[0034] The term "composed of" refers to compositions, methods and their respective components as described herein, excluding any elements not described in the embodiments described herein.

[0035] The terms “about” or “approximately” mean within 20% of a given value or range, preferably within 10%, and more preferably within 5%.

[0036] As used in this article, the term "cell" refers to a single cell, a cell line, or a culture derived from such cells.

[0037] Example 1: Construction of chimeric antigen receptor

[0038] A chimeric antigen receptor (CAR) was constructed by synthesizing a whole-genome signal peptide sequence, a GD2 antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signal transduction domain, a 2A sequence, and enhanced green fluorescent protein (EGFP). Figure 1 As shown, this is the signal peptide.

[0039] -Anti-GD2scFv-CD8α transmembrane region (CD8αTM)-CD86-FcγR1-T2A-EGFP, amino acid sequence as shown in SEQ ID NO.1:

[0040] MALPVTALLLPLALLLHAARPEVQLLQSGPELEKPGASVMISCKASGSSFTGY

[0041] NMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHL

[0042] KSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSEIVMTQ

[0043] SPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNR

[0044] FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

[0045] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG

[0046] TCGVLLLSLVITLYCKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPER

[0047] SDEAQRVFKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQE

[0048] DRHLEEELKCQEQKEEQLQEGVHRKEPQGATEGRGSLLTCGDVEENPGPMVS

[0049] KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPV

[0050] PWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK

[0051] TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN

[0052] GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0053] in:

[0054] The amino acid sequence of the signal peptide is shown in SEQ ID NO.2:

[0055] MALPVTALLLPLALLLHAARP.

[0056] The amino acid sequence of the single-chain antibody against tumor surface antigen GD2 (Anti-GD2scFv) is shown in SEQ ID NO.3:

[0057] EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK.

[0058] The linker sequence consists of multiple repeating sequences of GGGGS (G4S). Three GGGGS sequences are added between the variable regions of the heavy chain and the variable region of the light chain to act as a linker, providing flexibility and making it easier for the antibody to contact the antigen when forming the CAR molecule.

[0059] The hinge region functions to provide flexibility to overcome spatial obstacles and contributes to the length of the CAR, allowing the antigen-binding domain to access the target epitope. The amino acid sequence of the hinge region is shown in SEQ ID NO.4:

[0060] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0061] The transmembrane domain is the CD8α transmembrane region (CD8αTM), and its amino acid sequence is shown in SEQ ID NO.5:

[0062] IYIWAPLAGTCGVLLLSLVITLYC.

[0063] The co-stimulatory signal transduction domain is a combination of the CD86 signal transduction domain and the FcγR1 signal transduction domain, i.e., a sequential combination of CD86-FcγR1. The amino acid sequence of CD86-FcγR1 is shown in SEQ ID NO.6.

[0064] KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRV FKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQEDRH LEEELKCQEQKEEQLQEGVHRKEPQGAT.

[0065] The amino acid sequence of T2A is shown in SEQ ID NO.7:

[0066] EGRGSLLTCGDVEENPGP.

[0067] The amino acid sequence of the enhanced green fluorescent protein is shown in SEQ ID NO.8:

[0068] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0069] Since gliomas do not express CD19 antigen, this invention uses microglia expressing a chimeric antigen receptor for CD19, including the anti-CD19 antibody scfv segment, the CD8α transmembrane region, and the CD86 intracellular segment, as control cells.

[0070] The CD19 chimeric antigen receptor in the control group cells consists of a signal peptide, an antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction domain, and a tandem 2A sequence. The amino acid sequence is shown in SEQ ID NO.9, as detailed below:

[0071] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKWKKKKRPRNSYKCGTNTMERE ESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGATEGRGSLLLTCGDVEENPGP.

[0072] Example 2: Construction of chimeric antigen receptor vector

[0073] In this invention, the vector selected for constructing the chimeric antigen receptor expression was named pCDH-EF1α-puro (purchased from Ningbo Annoroad Biotechnology Co., Ltd.). The fully synthesized chimeric antigen sequence was used as a template, and primers were designed for amplification. The upstream primer (containing the EcoRI restriction enzyme sequence) has the sequence F:

[0074] GATTC GAATTC GCCGCCACCATGGCCCTCCCTGTCACCGCCCTGCT GC (SEQ ID NO.10); Downstream primer sequence (with XbaI restriction enzyme sequence) is R: GAATT TCTAGATTACTTGTACAGCTCGTCCATGCCGAGAGT (SEQ ID NO.11), after amplification, the PCR product was electrophoresed, and the product was recovered by gel excision. The recovered PCR product was digested with EcoRI and XbaI restriction endonucleases, and the pCDH-EF1α-puro vector was also digested with EcoRI and XbaI restriction endonucleases. The digestion conditions were 37℃ for 2 hours. After digestion, the PCR product and vector were electrophoresed, and the digested products were recovered by gel excision and ligation reaction was performed. The PCR digested product and vector digested product were ligated using T4 ligase at 16℃ for 12 hours. The ligation product was transformed using Stbl3 competent strain (purchased from Tiangen Biotech (Beijing) Co., Ltd.). The transformation steps were as follows:

[0075] 1. Melt the competent cells on ice, add all the ligation product to the competent cells, gently tap the tube wall to mix, and place on ice for 30 minutes.

[0076] 2. Heat shock at 42℃ for 1 min 30 s, then quickly place on ice and incubate for 2 min.

[0077] 3. Add 500 μl of antibiotic-free LB medium and shake on a shaker at 37°C for 30 minutes.

[0078] 4. After centrifugation, discard 300 μl of supernatant. Resuspend the remaining culture and evenly spread it onto LB ampicillin-positive agar plates. Incubate overnight at 37°C. After 12 hours, observe bacterial growth. Select individual, large, and plump bacteria and transfer them to LB ampicillin-positive medium. Shake for 12 hours. Add 20% glycerol to one portion of the culture and freeze. Send the other portion to our company (Beijing Qingke Biotechnology Co., Ltd.) for sequencing. Resuscitate the correctly identified bacteria, gently shake, and then perform large-scale sequencing, labeling them as pCDH CAR-GD2 and pCDH CAR-CD19. Vector chromatograms are shown below. Figure 2 and Figure 3 As shown.

[0079] Example 3: Lentiviral Packaging

[0080] In this embodiment, the HEK-293T cell line was used to produce lentivirus. pCDH CAR-GD2 and pCDH CAR-CD19 are lentiviral vectors, which are then transfected into 293T cells with lentiviral backbone plasmids and packaged into lentiviruses containing CAR. (Reference: Gene Ther. 2011 Jun; 18(6):531-8.)

[0081] (1) HEK293T cells in good growth condition (the non-patent literature describing HEK293T cells is: GeneTher. 2011 Jun; 18(6):531-8.) were digested with 0.25% trypsin for 2 minutes, and DMEM medium containing 10% FBS (purchased from Thermo Fisher Scientific) was added to stop the digestion. The cells at the bottom of the culture dish were pipetted into single cells and counted at 1×10⁻⁶. 7 Inoculate into 10cm dishes and transfect and package the virus after 12 hours. To collect more lentivirus, multiple dishes of cells can be cultured, depending on the actual situation.

[0082] (2) Preparation of the complex of transfection reagent and plasmid

[0083] a. Dissolve 35 μg of the viral plasmid to be transfected (10 μg pCDH CAR DNA vector plasmid, 10 μg pMD2.G (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) and 15 μg psPAX2 (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.)) in Opti-MEM medium (purchased from Thermo Fisher Scientific Co., Ltd.), with a total volume of 500 μl. Mix gently and let stand for 5 min to obtain a mixture containing plasmid.

[0084] b. Dissolve 100 μl of the transfection reagent and 200 μl of the enhancement reagent from the liposome transfection reagent Lipofectamin 2000 (purchased from Thermo Fisher Scientific) in Opti-MEM medium, with a total volume of 500 μl. Mix gently and let stand for 5 min.

[0085] c. Add the plasmid-containing mixture to the transfection reagent mixture while gently mixing, and then let it stand at room temperature for 20 minutes to allow the DNA and transfection reagent to fully combine and form a stable transfection complex.

[0086] (3) Take out the culture dish and add the prepared DNA transfection reagent mixture to the HEK293T cells in step (1).

[0087] (4) Change the medium after transfection: After 6 hours, remove the medium, wash once with PBS, add 10 ml of fresh DMEM medium containing 10% FBS, and incubate in a 37℃ 5% CO2 incubator.

[0088] Example 4: Extraction and Concentration of Lentiviral Viruses

[0089] Lentiviral extraction:

[0090] 1) Collect HEK293T cell supernatant at 48h and 72h post-transfection (0h at transfection) and aliquot them into 50ml centrifuge tubes. Centrifuge at 3500rpm at room temperature for 10min to remove cells and large debris.

[0091] 2) Filter the supernatant through a 0.45 μm filter membrane into an ultracentrifuge tube.

[0092] Ultra-high speed centrifugation to concentrate viruses:

[0093] 3) Centrifuge at 30,000 rpm and 4°C for 2 hours. White virus precipitate can be observed on one side of the tube wall.

[0094] 4) Discard the supernatant. Invert the centrifuge tube onto sterile absorbent paper to remove any remaining supernatant. Add 80-120 μl of phosphate-buffered saline (DPBS) to each tube, depending on the amount of precipitate. Seal the tube with sealing film and incubate at 4°C overnight to dissolve the precipitate.

[0095] 5) Disassemble the virus as required and store it in a refrigerator at -80℃.

[0096] Example 5: Lentiviral titer determination

[0097] The copy number of integrated viral genome in cells was determined using quantitative PCR. The initial viral titer was inferred based on the volume of virus added and the number of cells at infection. The viral genome was detected using the WPRE sequence on the vector; the WPRE sequence was not present in HEK293T cells.

[0098] 1) One day before infection, administer 1×10 mg per well. 5 1 cell per 24-well plate.

[0099] 2) Add 10 μl, 1 μl, and 0.1 μl of virus to one cell culture well each, followed by the addition of polybrene (purchased from Merck). The virus can be diluted before addition.

[0100] 3) Change the culture medium and continue culturing 24 hours after infection.

[0101] 4) Discard the cell supernatant 48 hours after infection. Collect the cells and extract the genome from 293T cells;

[0102] 5) Using the obtained genome as a template, quantify the internal reference gene and viral sequence WPRE, and use a relative quantification method with 2 -ΔΔ The Ct method is used to calculate the ratio of viral genome number to cellular genome number.

[0103] Results: The lentivirus titer used in this invention was 1×10⁻⁶. 8 The lentiviruses containing CAR were named lenti CAR-GD2 and lenti CAR-CD19, respectively, at TU / ml.

[0104] Example 6: Preparation of CAR-microglia

[0105] Human induced pluripotent stem cells (Reference: PNAS Nexus. 2022 Aug)

[0106] 17;1(4):pgac162;Stem Cell Reports.2018Apr 10;10(4):1267-1281.) Add stem cell culture medium TeSR-E8 (purchased from a stem cell company) and seed in 6-well plates, 5×10⁶ cells per well. 5 Cells were cultured for 24 hours and then infected with lenti-CAR GD2 and lenti-CAR CD19 (20 μl / well), respectively. Polybrene (10 μg / ml) was added immediately, and the six-well plate was gently shaken to mix thoroughly. Six hours after viral infection, the viral supernatant was removed, and fresh stem cell culture medium (TeSR-E8, purchased from a stem cell company) was used. The lentiviral vector contained GFP and puromycin resistance. After 48 hours of cell culture, cells expressing green fluorescence were observed in the lentivir-infected cells. Puromycin was then added to both lentivir-infected and uninfected cell wells for selection. Some cell death was observed on the first day after adding puromycin. Selection was stopped when all cells in the uninfected cell wells had died, and culture continued. When clones could be selected from the lentivir-infected cell wells, single clones were selected and cultured. When the single clones were ready for passage, cells were collected, DNA was extracted, and PCR products were identified and sequenced. The PCR primer was F: TTCTCAAGCCTCAGACAGTGGT (SEQ ID NO. 12).

[0107] R: AGCGCATGCTCCAGACTGCCTT (SEQ ID NO.13), the PCR product was electrophoresed, and the electrophoresis diagram is shown below. Figure 4 As shown, the selected monoclonal stem cells contain a specific CAR sequence.

[0108] Subsequently, stem cells containing CAR-GD2 and CAR-CD19 were differentiated into microglia (for microglia differentiation methods, see patent documents CN 114752565 A, CN114426951A and reference Sci. China Life Sci. 2022. 65(6), 1057-1071.). Specifically, when the cultured human induced pluripotent stem cells reached 80% confluence, the cells were digested with 0.5 mM EDTA. To form embryoid bodies (EBs), hiPSCs were transferred to suspension culture plates containing embryoid body medium (containing 10 μMY-27632). The embryoid body medium was carefully changed daily. On day 5, the EBs were resuspended in mononuclear cell medium and transferred to 100 mm cell culture dishes pre-coated with 0.1% gelatin to generate premyeloid cells. The medium was changed weekly, and hematopoietic progenitor cells appeared in the supernatant after 4 weeks of mononuclear cell culture. These hematopoietic progenitor cells were harvested by collecting the supernatant weekly, a process that lasted approximately two months. To induce microglia differentiation and maturation, collected hematopoietic progenitor cells were placed in suspension culture plates containing microglia maturation medium. After one week of culture, the microglia matured and were ready for experiments. Microglia differentiated from human induced pluripotent stem cells were immunofluorescently stained with the specific markers IBA1 and CD68. The results are as follows: Figure 5 As shown, microglia express IBA1 (purple) and CD68 (red). Simultaneously, the expression of GD2 antigen in microglia was detected, revealing that microglia differentiated from human induced pluripotent stem cells do not express GD2 (green). Staining with microglia-specific markers CD11b and CD45, flow cytometry analysis confirmed high purity of the stem cell-differentiated microglia, exceeding 90%. Figure 6 As shown. CAR molecules contain EGFP and exhibit green fluorescence. Further flow cytometry was used to identify EGFP expression, as shown... Figure 7 As shown, microglia expressing CAR-GD2 and control group CAR-CD19 were obtained in this way to differentiate into human induced pluripotent stem cells.

[0109] Example 7: In vitro killing experiment of CAR-GD2 microglia differentiated from human induced pluripotent stem cells.

[0110] Retinoblastoma Y79 cells (purchased from Fenghui Biotechnology Co., Ltd.) were labeled with specific flow cytometry antibodies against GD2 (purchased from Dakwei Biotechnology Co., Ltd.) and CD19 (purchased from Dakwei Biotechnology Co., Ltd.), and then detected by flow cytometry. Figure 8As shown, Y79 cells express GD2 but not CD19. Flow cytometry detection method: Y79 cells were collected, centrifuged at 200g for 5 minutes, the supernatant was discarded, and the cells were resuspended in phosphate-buffered saline (DPBS) (Thermo Fisher Scientific). After centrifugation at 200g for 5 minutes, the supernatant was discarded, and the cells were resuspended again in cell labeling buffer (Dake Biotechnology Co., Ltd.) and counted to a cell count of 1×10⁶ cells / year. 7 Cells / ml were incubated with 100 μL of GD2 and CD19 flow cytometry antibodies at room temperature for 20 minutes in the dark. Then, 500 μL of cell labeling buffer was added, and the cells were centrifuged at 300 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 500 μL of cell labeling buffer for flow cytometry analysis. CAR-GD2 microglia and CAR-CD19 microglia were co-cultured with Y79 cells, and the killing effect of CAR-microglia on Y79 tumor cells was observed using live-cell imaging. Figure 9 As shown, CAR-microglia exhibit green fluorescence, while Y79-stained CellTrace violets express blue fluorescence. Figure 9 The upper images show CAR-microglia co-cultured with Y79 cells, demonstrating uniform cell distribution. Figure 9 The images below show CAR-GD2 microglia co-cultured with Y79 cells for 100 hours. It can be seen that CAR-GD2 microglia significantly kill Y79 cells, with almost no Y79 cells remaining.

[0111] Example 8: In vitro killing experiment of CAR-GD2 microglia differentiated from human induced pluripotent stem cells.

[0112] Previous literature (Cancers (Basel). 2020 Oct 31; 12(11):3211.) reports that U87MG cells in human astrocytoblastoma are malignant gliomas expressing GD2. We tested the expression of GD2 and CD19 in purchased U87MG cells (purchased from Mirror Qidian (Shanghai) Cell Technology Co., Ltd.). U87MG cells were labeled with GD2 and CD19-specific flow cytometry antibodies, and then detected by flow cytometry. Figure 8As shown in the flow cytometry histogram, human astrocytoma U87MG cells express GD2 at a rate of over 89%, with almost no CD19 expression. After co-culturing U87MG cells (labeled with blue fluorescent Celltrack violet, purchased from Thermo Fisher Scientific) with CAR-GD2 microglia and CAR-CD19 microglia differentiated from human embryonic stem cells at ratios of 10:1 and 5:1, respectively, for 24 hours, live-cell imaging and counting revealed that U87MG cells co-cultured with CAR-GD2 microglia showed significant cell death, with a significantly lower proportion of remaining U87MG cells compared to the control group. Figure 10 ).

[0113] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for the preparation of human pluripotent stem cell differentiated microglia expressing chimeric antigen receptors, characterized in that: The method comprises the following steps: infecting human pluripotent stem cells with a lentivirus comprising a chimeric antigen receptor comprising an amino acid sequence as shown in SEQ ID NO. 1; performing positive clone screening, picking positive clone cells for culture and identification; differentiating the human pluripotent stem cells expressing the chimeric antigen receptor into microglial cells, i.e. obtaining the microglial cells expressing the chimeric antigen receptor; and the human pluripotent stem cells are human induced pluripotent stem cells.

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

3. Use of the human pluripotent stem cell differentiated microglial cells expressing the chimeric antigen receptor of claim 2 in the preparation of a drug for treating a tumor expressing a disialo ganglioside antigen; the tumor is human brain astrocytoma expressing a disialo ganglioside antigen or retinoblastoma expressing a disialo ganglioside antigen.

4. Use according to claim 3, characterized in that: The human brain astrocytoma is human brain astrocytoma U87MG cells.

5. Use according to claim 3, characterized in that: The retinoblastoma is retinoblastoma Y79.

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