Anti-CD97 chimeric antigen receptor, gene, expression vector, T cell and application

Abstract

The present application provides an anti-CD97 chimeric antigen receptor, gene, expression vector, T cell and application, which relate to the field of tumor treatment technology. The anti-CD97 chimeric antigen receptor selects CD97 as the specific antigen of brain glioma, and selects Th9 cells that can efficiently kill and eliminate solid tumors and effectively inhibit tumor metastasis and recurrence as cells for constructing CAR-T. The obtained CD97-CAR-T cells can exist for a long time. CD97-CAR-T cells can target GSCs to achieve precise treatment, achieve a therapeutic effect on GBM, reduce the drug resistance of CAR-T treatment, and improve the therapeutic effect of CAR-T on solid tumors.

Description

Technical Field

[0001] The present application relates to the technical field of tumor treatment, and in particular to an anti-CD97 chimeric antigen receptor, gene, expression vector, T cell and application. Background Art

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) has been shown to be effective in improving hematologic malignancies, but its efficacy against solid tumors is currently suboptimal. One major reason for this is the weak specificity of tumor-specific antigen recognition, while another is the loss of antigens and poor persistence of infused CAR-T cells. The main causes of this poor persistence are twofold: programmed cell death and exhaustion. Therefore, identifying specific targets in solid tumors and improving the therapeutic efficacy of CAR-T cells are crucial for their treatment.

[0003] Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, and the median overall survival of GBM patients is less than 16 months. A large number of molecular-level research results have shown that GBM stem cells (GSCs) are the key cells that cause anticancer drug resistance, radiotherapy resistance, invasion and spread. In the existing technology, conventional targets for GSCs are CD133, CD44 and CD15, etc. However, these targets have shortcomings such as low specificity and unstable expression, and cannot be used as good targets for GSCs. Therefore, the selection of suitable GSC-enriched and stable markers is conducive to the construction of CAR-T cells targeting GSCs to function. Summary of the Invention

[0004] The purpose of this application is to provide an anti-CD97 chimeric antigen receptor, which selects CD97 as the specific target of GSC, aiming to solve the problem that the existing GSC target is not specific enough and the prepared CAR-T cells have poor therapeutic effects.

[0005] To achieve the above objectives, the first aspect of the present application provides an anti-CD97 chimeric antigen receptor, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The second aspect of the present application further provides an anti-CD97 chimeric antigen receptor expression gene, which encodes the above-mentioned anti-CD97 chimeric antigen receptor.

[0007] Preferably, the nucleotide sequence of the anti-CD97 chimeric antigen receptor expression gene is shown in SEQ ID NO.7.

[0008] The third aspect of the present application further provides an expression vector, wherein the expression vector is inserted with the above-mentioned anti-CD97 chimeric antigen receptor expression gene, and the expression vector can, after transfecting a host cell, enable the host cell to express the above-mentioned anti-CD97 chimeric antigen receptor.

[0009] Preferably, the expression vector is a lentiviral expression vector.

[0010] The fourth aspect of the present application further provides a T cell expressing an anti-CD97 chimeric antigen receptor, wherein the T cell is capable of expressing the anti-CD97 chimeric antigen receptor as described above.

[0011] Preferably, the T cells are Th9 cells.

[0012] A fifth aspect of the present application further provides a method for preparing T cells expressing an anti-CD97 chimeric antigen receptor, comprising:

[0013] Constructing a lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene;

[0014] performing lentiviral packaging on the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene;

[0015] Polarization to obtain Th9 cells;

[0016] The Th9 cells are transfected with the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene.

[0017] The sixth aspect of the present application further provides a pharmaceutical composition, the active ingredients of which include the above-mentioned T cells expressing anti-CD97 chimeric antigen receptor.

[0018] In a seventh aspect, the present application also provides the use of the above-mentioned anti-CD97 chimeric antigen receptor, the above-mentioned anti-CD97 chimeric antigen receptor expression gene, the above-mentioned expression vector, and the above-mentioned T cells expressing anti-CD97 chimeric antigen receptor in the preparation of drugs for treating brain gliomas.

[0019] Compared with the prior art, the advantages of this application include:

[0020] The anti-CD97 chimeric antigen receptor provided in this application selects CD97 as the specific antigen for brain glioma, and selects Th9 cells that can efficiently kill and eliminate solid tumors and effectively inhibit tumor metastasis and recurrence as cells for constructing CAR-T. The obtained CD97-CAR-T cells can target GSCs to achieve precise treatment and realize the therapeutic effect on GBM. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0022] Figure 1 Schematic diagram of the structure of the anti-CD97 chimeric antigen receptor of the present application;

[0023] Figure 2 Figure 2 is the flow cytometry results of CD97 and other GSC markers;

[0024] Figures 3A to 3F This is the result of screening for CD97 downstream targets in GSCs;

[0025] Figures 4A to 4D This is the result diagram showing that CD97 regulates downstream gene expression through mTORC2 signaling;

[0026] Figure 5 CD97 high Expression of GSC and CD97 - / low Graph showing the effects of expressing GSC;

[0027] Figure 6 This is a diagram showing the killing effect of CD97-CAR Th9 at the cellular level;

[0028] Figure 7 This is a diagram showing the killing effect of CD97-CAR Th9 at the animal level;

[0029] Figure 8 This is a graph showing the CD97-CAR T cell expansion results of Comparative Example 1. DETAILED DESCRIPTION

[0030] As used herein:

[0031] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0032] The conjunction "consisting of" excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed, excluding materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0033] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0034] In these examples, parts and percentages are by mass unless otherwise indicated.

[0035] "Parts by mass" refers to the basic unit of measurement used to express the mass ratio of multiple components. One part can represent any unit of mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, this means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, we could say that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number representing a multiplication factor). It's important to note that, unlike parts by mass, the sum of the mass of all components is not limited to 100 parts.

[0036] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0037] The first aspect of the present application provides an anti-CD97 chimeric antigen receptor, such as Figure 1 As shown, it includes: CD8SP, scfv CD97, human CD8 hinge, human CD8TM, human 4-1BB and human CD3ζ connected in sequence. The amino acid sequence of the anti-CD97 chimeric antigen receptor is shown in SEQ ID NO.1.

[0038] CD8 SP is a signal peptide that is located outside the cell after expression. Its function is to guide different proteins to different parts of the cell. The amino acid sequence of CD8 SP is: MALPVTALLLPLALLLHAARP (SEQ ID NO. 2).

[0039] scFv CD97 is an anti-CD97 single-chain antibody that is located extracellularly after expression. The anti-CD97 chimeric antigen receptor can target CD97 through scFv CD97, thereby targeting tumor cells with high CD97 expression. The amino acid sequence of scFv CD97 is: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGINVVWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQYNSSPLTFGAGTKLELKSSGGGGSGGGGGGSSRSSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNIHWVKQTPGQGLEWIGAISPGNGDTSYNRKFKGKATLTADISSSTAYLQLSSLTSEDSAVYFCARFYGGSYWYFDVWGAGTTVTVSS (SEQ ID NO. 3).

[0040] The human CD8 hinge is the hinge region of CD8, located between the scFv CD97 and the cell membrane after expression. This region provides the CAR molecule with greater flexibility and facilitates binding to the target antigen. The human CD8TM is the transmembrane region of CD8, located at the cell membrane after expression. The amino acid sequence of the human CD8 hinge plus the human CD8TM is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 4).

[0041] Among them, human 4-1BB is located in the cell cytoplasm after expression, and the amino acid sequence of human 4-1BB is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO.5).

[0042] Among them, human CD3ζ is located in the cell cytoplasm after expression, and the amino acid sequence of human CD3ζ is: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO.6).

[0043] The second aspect of the present application further provides an anti-CD97 chimeric antigen receptor expression gene, which encodes the above-mentioned anti-CD97 chimeric antigen receptor.

[0044] Preferably, after codon optimization, the nucleotide sequence of the anti-CD97 chimeric antigen receptor expression gene is shown as SEQ ID NO.7.

[0045] Among them, the nucleotide sequence of CD8 SP is: ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO. 8).

[0046] Among them, the nucleotide sequence of scfv CD97 is: GATATCGTGATGACCCAGAGCCAGAAGTTTATGTCCACATCCGTGGGCGATAGGGTGTCCGTGACATGCAAGGCCTCCCAGAACGTGGGCATCAACGTGGTGTGGTACCAGCAGAAGCCCGGCCAGAGCCCTAAGGCCCTGATCTACTCCGCCAGCTACAGATTCAGCGGCGTGCCTGACAGATTCACTGGCTCCGGCTCCGGCACCGATTTTACCCTGACCATCAGCAATGTGCAGTCCGAGGATCTGGCCGAGTTCTTTTGCCAGCAGTACAATAGCAGCCCCCTGACCTTCGGCGCCGGCACAAAGCTGGAGCTGAAGAGCAGCGGCGGCGGCGGAAGCGGAGGAGGAGGAGGCGGAAGCTCCAGGAGCTCCCAGGTGCAGCTGCAGCAGCCCGGCGCTGAGCTGGTGAAGCCTGGCGCTTCCGTGAAGATGAGCTGTAAGGCCAGCGGCTACACATTCACAAGCTACAATATCCACTGGGTGAAGCAGACCCCTGGCCAGGGCCTGGAGTGGATCGGCGCTATCTCCCCCGGCAATGGCGACACCTCCTACAATAGAAAGTTTAAGGGCAAGGCCACACTGACCGCCGACATCAGCTCCTCCACAGCCTACCTGCAGCTGTCCAGCCTGACCAGCGAGGATTCCGCCGTGTACTTCTGTGCCAGATTCTACGGCGGCTCCTACTGGTACTTCGACGTGTGGGGCGCCGGCACCACCGTGACAGTGTCCAGC(SEQ ID NO.9).

[0047] Among them, the nucleotide sequence of human CD8 hinge+human CD8TM is: ACCACGACGCCAGCGCCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO.10).

[0048] The nucleotide sequence of human 4-1BB is: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO. 11).

[0049] Among them, the nucleotide sequence of human CD3ζ is: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCT CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ IDNO.12).

[0050] The third aspect of the present application further provides an expression vector, wherein the expression vector is inserted with the above-mentioned anti-CD97 chimeric antigen receptor expression gene, and the expression vector can, after transfecting a host cell, enable the host cell to express the above-mentioned anti-CD97 chimeric antigen receptor.

[0051] Preferably, the expression vector is a lentiviral expression vector.

[0052] Lentiviral vectors are derived from the human immunodeficiency virus-1 (HIV-1). Lentiviral vectors contain the genetic information required for packaging, transfection, and stable integration, and are the primary component of the lentiviral vector system. Lentiviral expression vectors carrying exogenous genes, with the assistance of lentiviral packaging plasmids and cell lines, are packaged into infectious viral particles. These particles then infect cells or living tissues, achieving expression of the exogenous gene in these cells or living tissues.

[0053] The fourth aspect of the present application further provides a T cell expressing an anti-CD97 chimeric antigen receptor, wherein the T cell is capable of expressing the anti-CD97 chimeric antigen receptor as described above.

[0054] Preferably, the T cells are Th9 cells.

[0055] Th9 cells are a type of CD4+ T cell subset with significant anti-solid tumor effects. CD4+ T cells can be divided into Th cell subsets such as Th1, Th2, Th3, Treg, Tr1, Tfh, Th17, Th9, and Th22 according to their functions. Th9 cells can not only efficiently kill and eliminate solid tumors, but also effectively inhibit tumor metastasis and recurrence. Research has found that they are not easily exhausted and have sufficient cytolytic properties against tumor cells.

[0056] A fifth aspect of the present application further provides a method for preparing T cells expressing an anti-CD97 chimeric antigen receptor, comprising:

[0057] Constructing a lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene;

[0058] performing lentiviral packaging on the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene;

[0059] Polarization to obtain Th9 cells;

[0060] The Th9 cells are transfected with the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene.

[0061] The sixth aspect of the present application further provides a pharmaceutical composition, the active ingredients of which include the above-mentioned T cells expressing anti-CD97 chimeric antigen receptor.

[0062] In a seventh aspect, the present application also provides the use of the above-mentioned anti-CD97 chimeric antigen receptor, the above-mentioned anti-CD97 chimeric antigen receptor expression gene, the above-mentioned expression vector, and the above-mentioned T cells expressing anti-CD97 chimeric antigen receptor in the preparation of drugs for treating brain gliomas.

[0063] The anti-CD97 chimeric antigen receptor provided in this application selects CD97 as the specific antigen for brain glioma, and selects Th9 cells that can efficiently kill and eliminate solid tumors and effectively inhibit tumor metastasis and recurrence as cells for constructing CAR-T. The obtained CD97-CAR-T cells can target GSCs to achieve precise treatment and realize the therapeutic effect on GBM.

[0064] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.

[0065] Example 1 Identification and Validation of GSC Surface Markers

[0066] 1. Identification of GSC surface markers

[0067] GSC surface markers were detected by flow cytometry, and the expression of CD97 was compared with traditional GSC markers such as CD133, CD44 and CD15. The flow cytometry contour results of each marker are shown in Figure 2. Figure 2 As shown, GSC1, GSC2, and GSC3 are GSC cells from different patients, U87 is a control cell, and NT is a blank control unstained cell.

[0068] according to Figure 2 The results showed that the CD133+ ratio of some GSCs ranged from 18% to 51.9%, which is similar to the previous report (10% to 50%). In contrast, GSC1 showed only 3.5% CD133 expression ( Figure 2 ), GSC2 does not contain any cells with detectable CD133 expression. Therefore, CD133 is not suitable as a GSC surface marker, as its expression is unstable. CD44, another reported GSC marker, is highly expressed not only in both GSCs but also in a common glioma cell line (U87). Therefore, CD44 is also unsuitable as a GSC surface marker, as its expression is not specific. CD15 has also been reported as a GSC enrichment marker, but it was not expressed in the GSCs we tested.

[0069] In contrast, in GBM cell lines, the proportion of CD97-positive cells was generally significantly higher in GSCs, whereas it was not expressed in U87 cells ( Figure 2 ), these results suggest that CD97 may be a better surface marker for GSCs.

[0070] 2. Screening of CD97 downstream targets in GSCs

[0071] To understand the regulatory role of CD97, we used a comprehensive approach to comprehensively determine the biological function of CD97 expression. After CD97 knockout, we performed transcriptome analysis of the three GSCs using RNA sequencing (RNA-seq) and found that a total of 131 genes were significantly downregulated by CD97 knockdown in all GSCs ( Figure 3A ).

[0072] Each gene identified as downregulated after CD97 knockout was carefully examined for its significance in biological processes. Therefore, the Gene Ontology (GO) biological processes of these commonly downregulated genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The results showed that positive regulation of cell-cell adhesion, nucleobase-containing compound metabolism, and hormone biosynthesis were the only three signaling pathways significantly enriched in CD97 knockout GSCs ( Figure 3B Among them, the gene ratio of cell-cell adhesion was the highest, including poly (rC) binding protein 1 (PCBP1), basic protein (BSG), Rho-GTPase activating protein 1 (ARHGAP1), basic leucine zipper and W2 domain-containing protein 1 (BZW1), basic leucine zipper and W2 domain-containing protein 2 (BZW2) and Drebrin (DBN1) ( Figure 3A and Figure 3B ).

[0073] Downregulation of mRNA expression of these cell-cell adhesion-related genes was confirmed in CD97 knockdown GSCs ( Figure 3C To investigate the possible relationship between CD97 and these related genes, we analyzed clinical prognosis using a publicly available GBM dataset and found that patients with high levels of CD97 and ARHGAP1 or BZW1 or BZW2 had a worse prognosis than those with low expression of both (TCGA database) ( Figure 3D ).

[0074] In addition, to evaluate the regulators of cell-cell adhesion-related genes, we used gene set enrichment analysis (GSEA) to analyze 50 signature signaling pathways with transcriptome results. Among the important signature signaling pathways enriched in the shCtrl group, we observed that the mTOR signaling pathway (including PI3K / AKT / mTOR, also known as mTORC1 and mTORC2) was enriched in the shCtrl group ( Figure 3E and Figure 3F Thus, among the candidate genes, we found that CD97 could regulate the expression of ARHGAP1, BZW1, and BZW2 through mTOR signaling.

[0075] 3. CD97 regulates downstream gene expression through mTORC2 signaling

[0076] To examine the function of CD97 downstream signaling pathways, silencing CD97 using shRNA significantly attenuated the phosphorylation of S6K (S371 and T389) and AKT (T308 and S473) in GSCs ( Figure 4A ). At the same time, the expression levels of ARHGAP1, BZW1, and BZW2 were significantly reduced in CD97 knockout GSCs ( Figure 4A ), these results prompted us to investigate the potential involvement of mTOR downstream of CD97.

[0077] Next, we performed small molecule inhibitor treatment in GSCs to explore the relevance of target genes to mTOR signaling in GSCs. Torin1 (an inhibitor of mTORC1 and mTORC2) and Akt inhibitor IV not only induced decreased phosphorylation of S6K (S371 and T389) and Akt (T308 and S473), but also induced decreased expression of the three target molecules. Subsequently, to understand the mechanism by which CD97-triggered signaling acts on its target genes, we blocked each signaling pathway with two different mTOR-specific inhibitors (rapamycin, an mTORC1 inhibitor; JR-AB2-011, an mTORC2 inhibitor) and analyzed the functional status of each pathway in GSCs.

[0078] The results showed that rapamycin treatment reduced the phosphorylation of S6K (S371 and T389) but did not change the expression levels of the three target proteins ( Figure 4B On the other hand, JR-AB2-011 treatment affected the phosphorylation of AKT (S473) and downregulated the expression of three target proteins ( Figure 4B ).

[0079] These results indicate that CD97 expression in GSCs leads to the induction of genes related to ARHGAP1, BZW1, and BZW2 by activating the mTORC2 / AKT (S473) pathway. In addition, based on cell proliferation assays and LDA, all inhibitors that selectively inhibit mTOR / AKT signaling therapeutically blocked cell growth and stemness properties in GSCs, further demonstrating that CD97 can activate both mTORC1 and mTORC2 signaling, but only mTORC2 signaling can regulate the expression of the three downstream target genes ( Figure 4C and Figure 4D ).

[0080] 4. CD97 high Role of expressing cells in regulating GSC proliferation, self-renewal, and tumorigenicity

[0081] To verify that CD97 identifies GSCs and plays a functional role in GBM initiation, we examined the characteristics of CD97-enriched GSCs. Based on the CD97 expression profile, GSC4s could be divided into two subpopulations; the top 38.5% were defined as CD97 by flow cytometry. high , the bottom 18.8% is defined as CD97 - / low ( Figure 5 (A)).

[0082] With CD97 - / low Compared with the isolated CD97 high The GSC population highly expressed the related genes CD133, Nestin, and CD44 at the mRNA and protein levels. Figure 5 (B) and (C)). As described above, we demonstrated that CD97 high GSCs regulate AKT phosphorylation (S473) associated with mTORC2 signaling and induce increased expression of ARHGAP1, BZW1, and BZW2 ( Figure 5 (D)).

[0083] To determine the in vivo tumorigenic potential of CD97-enriched GSCs, we established a high and CD97 - / low Importantly, the onset of tumors was significantly accelerated, as shown by MRI imaging 70 days after implantation, which showed that the tumors from CD97 high The tumor size of cells was significantly larger than that of cells derived from CD97 - / low Tumor size of cells ( Figure 5 (E)). Therefore, the Kaplan-Meier plot shows that compared with the injection of CD97 - / low Compared with the cell group (101.5 days), the CD97 high The overall survival of tumor-bearing animals in the cell group (86.5 days) was significantly shortened ( Figure 5 (F)).

[0084] These results indicate that CD97 high Tumors are propagated by ARHGAP1, BZW1, and BZW2. Collectively, these data strongly suggest that CD97 high The cells exhibit characteristics of GSCs in vitro and are highly tumorigenic in vivo.

[0085] Example 2 Preparation of CD97-CAR Th9 cells

[0086] 1. Construction of CD97-CAR lentiviral plasmid

[0087] Obtain a linearized vector using restriction enzyme digestion. Prepare the target gene fragment CD97-CAR by PCR amplification. When designing the amplification primers used, a homologous recombination sequence must be added to their 5' end. Use this primer to amplify the target gene fragment, and the sequences at the 5' and 3' ends of the amplified product are completely consistent with the sequences at both ends of the linearized cloning vector. Prepare a reaction system with the linearized vector and the target gene amplification product, and perform a recombination reaction to achieve in vitro circularization of the linearized vector and the target gene fragment. The recombinant product is directly transformed, and single clones on the plate are picked for PCR identification. The positive clones are sequenced and the results analyzed. The correct clone bacterial solution is expanded and extracted to obtain a high-purity plasmid for downstream viral packaging.

[0088] 2. Preparation of CD97-CAR Lentivirus

[0089] ① 24 h before transfection, trypsinize 293T cells in the logarithmic growth phase and adjust the cell density to about 5x10 cells in DMEM medium containing 10% serum. 6 Re-inoculate 10 cm cell culture dish with 15 ml of culture medium and culture in a 37°C, 5% CO2 incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection.

[0090] ②Change to serum-free culture medium 2 h before transfection.

[0091] ③ Add the prepared plasmids (20 μg of GV vector plasmid, 15 μg of pHelper1.0 vector plasmid, and 10 μg of pHelper 2.0 vector plasmid) to a sterile centrifuge tube, mix well with the transfection reagent, adjust the total volume to 1 ml, and incubate at room temperature for 15 minutes.

[0092] ④ Slowly add the mixed solution dropwise to the culture medium of 293T cells, mix well, and culture in a cell culture incubator at 37°C and 5% CO2.

[0093] ⑤ After culturing for 6 hours, discard the culture medium containing the transfection mixture, add 10 ml of PBS solution to wash once, gently shake the culture dish to wash away the residual transfection mixture, and then discard it.

[0094] ⑥ Slowly add 20 ml of cell culture medium containing 10% serum and continue culturing in a 37°C incubator with 5% CO2 for 48 hours.

[0095] ⑦ According to the cell status, collect the 293T cell supernatant 48 hours after transfection (transfection can be counted from 0 hours), centrifuge at 4°C and 4000g for 10 minutes to remove cell debris.

[0096] ⑧ Filter the supernatant with a 0.45 μm filter into an ultracentrifuge tube, set the centrifugation parameters to 25,000 rpm, the centrifugation time to 2 h, and the centrifugation temperature to 4°C.

[0097] ⑨ After centrifugation, discard the supernatant and try to remove the liquid remaining on the tube wall. Add virus preservation solution (PBS or cell culture medium can be used as an alternative), gently pipette and resuspend repeatedly. After sufficient dissolution, divide the solution into portions as required and transfect or store at -80℃.

[0098] 3. Polarized Th9 cells

[0099] ① First, PBMC cells were isolated from human peripheral blood and separated by a kit for separating CD4+ T cells (EasySep TM CD4+ T cells were purified using Human CD4+ T Cell Isolation Kit.

[0100] ②CD4+T cells were activated by CD3 (1μg / ml) and CD28 (2μg / ml), and IL-4 (10ng / ml), TGF-β (1ng / ml), and IFN-Y (10μg / ml) were added to culture for 5 days to polarize them into Th9 cells.

[0101] 4. Transfection preparation of CD97-CAR lentivirus

[0102] The CD97-CAR lentivirus was produced using 293T, and then CD97-CAR was transfected into 280,000 Th9 cells in a 24-well plate using lentiviral transfection. On the fourth day after lentiviral transfection, GFP expression was observed using an inverted fluorescence microscope and amplified.

[0103] Example 3: Killing effect of CD97-CAR Th9 at the cellular level

[0104] First, the expression of IL-9 was detected by RT-PCR. It can be seen that the expression of IL-9 in polarized Th9 cells can reach 10,000 times, which proves that we have successfully polarized Th9 cells ( Figure 6 (A)).

[0105] In order to detect whether CD97-CAR Th9 cells have the effect of specifically killing CD97-expressing tumor cells at the cellular level, U87 cells that do not express CD97, GSCs that express CD97, and CD97-CAR Th9 cells were co-cultured at an E:T ratio of 0.5:1 and then the killing effect was detected. It was found that CD97-CAR Th9 cells had the effect of specifically killing CD97-expressing tumor cells ( Figure 6 (B)).

[0106] Example 4 Therapeutic Effect of CD97-CAR Th9 at the Animal Level

[0107] To test the therapeutic effect of CD97-CAR Th9 in vivo, we constructed an orthotopic xenograft model and injected 100,000 GSC2-Lucifease overexpressing cells into the mouse brain. We also detected the luminescence of the tumor to reach 1×10 5 1×10 7 CD97-CAR Th9 cells were injected into the tail vein for the second time on day 17 (1×10 7 CD97-CAR Th9 cells were used to monitor the luminescence changes of mouse tumors during this period. We found that CD97-CAR Th9 treatment had a significant inhibitory effect on tumor growth ( Figure 7 (A)).

[0108] In addition, by monitoring the survival cycle of mice, it was found that the median survival of mice treated with CD97-CAR Th9 was 35 days, compared with the median survival of the control group of 22 days. It can be clearly seen that CD97-CAR Th9 treatment can prolong the survival cycle of mice ( Figure 7 (B)).

[0109] Comparative Example 1

[0110] Different from Example 2, the CD97-CAR lentivirus of Comparative Example 1 used ordinary T cells instead of Th9 cells. The results showed that although the transfection efficiency of CD97-CAR in PBMC cells was good ( Figure 8 (A) However, PBMC cells transfected with CD97-CAR lentivirus (CD3+ T cells account for 45-70% in PBMCs) cannot grow normally, while cells without any treatment and cells transfected with CAR lentivirus without CD97 molecules will not affect the normal growth of PBMC cells ( Figure 8 (B)), indicating that the CD97-CAR lentivirus of the present application is only applicable to Th9 cells, but not to ordinary T cells.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

[0112] Furthermore, those skilled in the art will appreciate that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and to form distinct embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is intended solely to enhance understanding of the overall background of this application and should not be construed as an admission or any implication that such information constitutes prior art known to those skilled in the art.

Claims

1. An anti-CD97 chimeric antigen receptor, characterized in that The amino acid sequence of the anti-CD97 chimeric antigen receptor is shown in SEQ ID NO.

1.

2. An anti-CD97 chimeric antigen receptor expression gene, characterized in that: The gene encodes the anti-CD97 chimeric antigen receptor according to claim 1.

3. The anti-CD97 chimeric antigen receptor expression gene according to claim 2, characterized in that: The nucleotide sequence of the anti-CD97 chimeric antigen receptor expression gene is shown in SEQ ID NO.

7.

4. An expression vector, characterized in that The expression vector is inserted with the anti-CD97 chimeric antigen receptor expression gene of claim 2, and after transfecting a host cell, the expression vector can cause the host cell to express the anti-CD97 chimeric antigen receptor of claim 1.

5. The expression vector according to claim 4, characterized in that The expression vector is a lentiviral expression vector.

6. A T cell expressing an anti-CD97 chimeric antigen receptor, characterized in that: The T cells are capable of expressing the anti-CD97 chimeric antigen receptor according to claim 1.

7. The T cell expressing anti-CD97 chimeric antigen receptor according to claim 6, characterized in that The T cells are Th9 cells.

8. A method for preparing T cells expressing the anti-CD97 chimeric antigen receptor according to claim 1, characterized in that: include: Constructing a lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene; performing lentiviral packaging on the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene; Polarization to obtain Th9 cells; The Th9 cells are transfected with the lentiviral plasmid expressing the anti-CD97 chimeric antigen receptor gene.

9. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition includes the T cells expressing the anti-CD97 chimeric antigen receptor according to claim 6 or 7.

10. Use of the anti-CD97 chimeric antigen receptor according to claim 1, the anti-CD97 chimeric antigen receptor expression gene according to claim 2, the expression vector according to claim 4, and the anti-CD97 chimeric antigen receptor-expressing T cell according to claim 6 in the preparation of a medicament for treating brain glioma.

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