Method for enhancing anti-tumor activity of CAR (Chimeric Antigen Receptor) cells and application thereof

Abstract

The invention relates to the field of gene engineering, in particular to a method for enhancing anti-tumor activity of CAR cells and application of the method. The CAR cells are equipped with INSR'metabolic armor ', so that the modified CAR cells can maintain stable expression of CAR molecules on the surface of a cell membrane and resist shedding of the CAR molecules induced by tumor-related fibroblasts (CAFs), the killing function of the CAR cells is remarkably enhanced in vitro, and an excellent anti-tumor effect is shown in vivo. The invention successfully solves the key bottleneck of functional failure of CAR cells in solid tumor TME, and provides a brand new strategy with great transformation potential for developing more effective solid tumor cell immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a method and its application for enhancing the antitumor activity of CAR cells by overexpressing insulin receptor (INSR). Background Technology

[0002] Chimeric antigen receptor (CAR) cell therapy has shown great potential in the treatment of solid tumors, particularly for malignant gastrointestinal tumors such as gastric cancer, colorectal cancer, and pancreatic cancer. Conventional CAR-T / NK cells (e.g., CAR-T / NK cells targeting mesothelin MSLN) can effectively recognize and kill tumor cells in vitro.

[0003] However, in the actual tumor microenvironment (TME), its efficacy is significantly reduced. Our previous research found that tumor-associated fibroblasts (CAFs) are a key immunosuppressive component in the TME. CAFs can induce a large outflow of cholesterol from CAR-T / NK cells by secreting a metabolite called 27-hydroxycholesterol (27HC). Since the stability of CAR molecules depends on cholesterol-rich lipid raft structures on the cell membrane, cholesterol depletion leads to the physical detachment of CAR molecules from the surface of T / NK cells, causing CAR-T / NK cells to lose their targeting ability and ultimately leading to treatment failure.

[0004] Therefore, existing technologies have a key drawback: the lack of effective strategies to protect CAR-T / NK cells from the metabolic attack of CAFs in the TME, thus failing to maintain their sustained killing function in vivo.

[0005] Chinese patent document CN117355602A discloses a low-immunogenic cell containing engineered HLA-E or HLA-G, and Chinese patent document CN114195901A discloses a bispecific recombinant protein and its uses. However, no method or application has been reported regarding enhancing the antitumor activity of CAR-T / NK cells by overexpressing the insulin receptor (INSR). Summary of the Invention

[0006] This invention aims to solve the technical problem of functional failure of existing CAR-T / NK cells in the solid tumor microenvironment due to the metabolic inhibition of CAFs, and provides a universal "metabolic armor" strategy. By overexpressing human insulin receptor (INSR) in CAR-T or CAR-NK cells, cholesterol depletion in the tumor microenvironment is antagonized, thereby maintaining CAR receptor stability and enhancing anti-tumor activity.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] The core of this invention is a genetically engineered immune effector cell (CAR-T or CAR-NK), comprising:

[0009] 1. First exogenous gene: A nucleic acid sequence encoding a chimeric antigen receptor (CAR). The CAR targets tumor-associated antigens (including but not limited to MSLN, Claudin18.2, HER2, CEA, etc.).

[0010] 2. Second exogenous gene: Nucleic acid sequence encoding human insulin receptor (INSR).

[0011] 3. Co-expression characteristics: The expression level of the INSR is significantly higher than that of unmodified immune cells of the same type, and it can activate the PI3K-AKT-SREBP2 signaling pathway in the tumor microenvironment (especially in a high concentration of 27HC environment) to promote endogenous cholesterol synthesis.

[0012] (1) For NK cells (such as NK92 and its derivatives): a transposon system (such as the PiggyBac system) is used. Transposon plasmids containing the human INSR gene are constructed and transfected into NK cells that have stably expressed CAR by electroporation. Stable lines (such as MSLN-CAR-NK92[PB-INSR]) are obtained by antibiotic selection.

[0013] (2) For primary T cells: the ProteanFect mRNA transfection system was used. In vitro transcribed INSR mRNA and CAR mRNA were co-transfected to prepare "metabolic armor" CAR-T cells.

[0014] Mechanism of action: This invention is based on the discovery of the “27HC-cholesterol-lipid raft” axis.

[0015] Inhibition mechanism: 27HC secreted by CAFs activates the LXR nuclear receptor, upregulating ABCA1 / ABCG1 and leading to cholesterol efflux. Cholesterol loss causes the disintegration of cell membrane lipid rafts, and CAR proteins anchored to the lipid rafts detach with microvesicles.

[0016] Protective mechanism: Overexpressed INSR acts as "metabolic armor," potently activating AKT and the downstream SREBP2 pathway by sensing environmental or autocrine / paracrine insulin signals, significantly upregulating cholesterol synthases (HMGCR, HMGCS1, etc.). This enhanced synthetic capacity (open source) counteracts 27HC-induced efflux (throttling), maintaining membrane cholesterol homeostasis and lipid raft integrity, thereby locking in the CAR receptor.

[0017] Based on the above technical solutions, the first aspect of the present invention provides the application of a gene encoding the human insulin receptor (INSR) in enhancing the anti-tumor activity of CAR cells.

[0018] Furthermore, the application of the gene encoding human INSR in the preparation of modified CAR cells for anti-tumor activity.

[0019] Furthermore, the CAR can be a conventional structure, such as containing a single-chain variable region (scFv) targeting a tumor-associated antigen (e.g., MSLN), a hinge region, a transmembrane region, one or more co-stimulatory domains (e.g., 4-1BB), and an activation domain (e.g., CD3ζ).

[0020] Furthermore, the target of the CAR can be any target suitable for the treatment of solid tumors, including but not limited to MSLN, Claudin18.2, EGFR, HER2, CEA, etc.

[0021] Furthermore, the CAR cells mentioned are CAR-T, CAR-NK, or CAR-M (macrophages), etc.

[0022] Furthermore, the aforementioned enhancement of CAR cell anti-tumor activity is achieved by introducing the gene sequence encoding human INSR into CAR cells through genetic engineering and ensuring its stable high-level expression.

[0023] Furthermore, the genetic engineering methods described herein employ PiggyBac transposon or mRNA transfection, and can also use lentiviral vectors, AAV vectors, or other non-viral vectors to achieve stable high expression of INSR.

[0024] Furthermore, the application of reagents that overexpress INSR in enhancing the antitumor activity of CAR cells.

[0025] In a second aspect, the present invention provides a genetically engineered CAR cell, wherein the CAR cell contains a gene sequence encoding a chimeric antigen receptor (CAR) and a gene sequence encoding a human insulin receptor (INSR).

[0026] Furthermore, the CAR-targeted tumor-associated antigens include, but are not limited to, MSLN, Claudin18.2, HER2, CEA, etc.

[0027] A third aspect of the present invention provides a method for constructing CAR cells as described above, wherein a gene sequence encoding human INSR is introduced into CAR cells through a gene delivery system and expressed at a stable high level.

[0028] Furthermore, the gene delivery system can be selected from transposon systems, mRNA transfection, lentiviral vectors, AAV vectors, or other non-viral vectors to achieve stable high expression of INSR.

[0029] Furthermore, the CAR cells mentioned are CAR-T, CAR-NK, or CAR-M (macrophages), etc.

[0030] Furthermore, the INSR expression cassette was stably integrated into the genome of CAR-NK cells using the PiggyBac transposon system; and CAR mRNA and INSR mRNA were transiently transfected together into primary T cells using mRNA transfection.

[0031] In one embodiment of the present invention, the construction method includes the following steps:

[0032] (1) Construction of the MSLN-CAR-NK92 cell line:

[0033] Plasmid Design: A second-generation CAR plasmid targeting human MSLN was constructed. This CAR structure comprises an anti-MSLN scFv fragment, a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ activation domain. A FLAG tag is attached to the N-terminus of the scFv fragment for detection. This CAR sequence was cloned into the pLVX-Puro lentiviral vector (WO2022 135578 A1).

[0034] Lentiviral packaging and transduction: HEK293T cells were co-transfected with the MSLN-CAR-pLVX-Puro vector and packaging plasmids psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). Viral supernatants were collected at 48 and 72 hours, filtered, and concentrated. NK-92 cells were then infected with the concentrated virus.

[0035] Screening and purification: After transduction, MSLN-CAR-NK92 cells stably expressing CAR were selected using puromycin (e.g., 1 µg / mL). Flow cytometry was used to detect the FLAG tag on the cell surface to confirm CAR expression efficiency.

[0036] (2) Construction of the “metabolic armor” cell line MSLN-CAR-NK92[PB-INSR]:

[0037] Plasmid preparation: Prepare the PiggyBac transposon expression vector (H_INSR) (Genomeditech, GM-106640) and the Super PiggyBac transposase expression vector (Genomeditech, GM-48338) encoding the human INSR gene.

[0038] Co-transfection: The two plasmids were co-transfected into the MSLN-CAR-NK92 cells obtained in step one at an optimized ratio (e.g., 3:1). Electroporation was preferred as the transfection method to improve the transfection efficiency of NK cells.

[0039] Screening and Validation: After transfection, cells were screened using G418 (500 µg / mL) to obtain cells that stably integrated the INSR gene into the genome. Finally, the level of INSR protein on the cell surface was detected by flow cytometry to verify successful overexpression of INSR.

[0040] (3) Construction of the original INSR+MSLN-CAR-T "metabolic armor":

[0041] A CAR encoding mesothelin (MSLN) targeting CAR (containing anti-MSLNscFv, CD8α hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ signaling domain) and codon-optimized mRNA for the human insulin receptor (INSR) were synthesized using an in vitro transcription (IVT) system. Prior to transfection, the synthesized mRNA was purified using a standard spin-column purification method, and its quality was assessed.

[0042] Using the Proteanfect Max transfection kit, synthesized CAR mRNA was transiently transfected into primary T cells, either alone or in combination with INSR mRNA. Twenty-four hours post-transfection, the expression of CAR and INSR proteins was detected and confirmed by flow cytometry using antibodies targeting the FLAG tag (for CAR detection) and human INSR.

[0043] In a fourth aspect, the present invention provides the use of the modified CAR cells as described above in the preparation of antitumor drugs.

[0044] Furthermore, the tumor in question is a solid tumor (TME).

[0045] Furthermore, the solid tumors mentioned include, but are not limited to, gastric cancer, pancreatic cancer, or colon cancer.

[0046] The advantages of this invention are:

[0047] Compared with existing technologies, this invention equips CAR cells with INSR "metabolic armor," enabling the modified CAR-T / NK cells to maintain stable expression of CAR molecules on the cell membrane surface, effectively resisting CAF-induced CAR molecule shedding, thereby significantly enhancing their killing function in vitro and exhibiting excellent anti-tumor effects in vivo, mainly reflected in:

[0048] 1. Resistance to CAFs-induced CAR molecule shedding: In a simulated microenvironment co-cultured with CAFs, the CAR-T / NK cells of this invention can maintain stable expression of CAR molecules on the cell membrane surface, while conventional CAR-T / NK cells will shed a large amount of CAR molecules. Figure 3 ).

[0049] 2. Significantly enhanced in vitro killing function: In a co-culture system containing CAFs, the CAR-T / NK cells of this invention exhibit a significantly enhanced tumor cell killing ability compared to conventional CAR-T / NK cells. Figure 4 ).

[0050] 3. Superior in vivo anti-tumor effect: In mouse models simulating peritoneal implantation of gastric cancer and PDX of colon cancer, mice treated with CAR-T / NK cells of this invention showed significantly suppressed tumor burden and significantly prolonged survival, with effects far superior to the conventional CAR-T / NK cell therapy group. Figure 5 ).

[0051] In summary, this invention successfully addresses the key bottleneck of CAR cell functional failure in solid tumor TME, providing a novel strategy with great translational potential for developing more effective cell immunotherapy for solid tumors. Attached Figure Description

[0052] Figure 1 : A schematic diagram of the core mechanism of this invention.

[0053] Figure 2 : Validation of INSR overexpression.

[0054] Figure 3 This invention resists CAR shedding mediated by 27HC from CAFs.

[0055] Figure 4 : The in vitro antitumor effect of this invention.

[0056] Figure 5 : The anti-tumor effect of this invention in vivo. Detailed Implementation

[0057] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0058] Example: Construction and validation of "metabolic armor" CAR-T / NK cells with high INSR expression

[0059] This embodiment aims to provide a novel CAR-T / NK cell to resist metabolic inhibition dominated by CAFs in the TME, and its technical solution includes:

[0060] 1. Experimental Materials

[0061] (1) Primary cells: human peripheral blood T cells

[0062] (2) Cell lines: human NK-92 cells (ATCC, Cat# CRL-2407) and HEK293T cells (ATCC, Cat# CRL-3216).

[0063] (3) Plasmids, vectors and mRNA:

[0064] MSLN-CAR lentiviral expression vector (pLVX-Puro) (Genomeditech, GM-2465);

[0065] MSLN-CAR plasmid (WO2022 135578 A1);

[0066] Lentiviral packaging plasmids: psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259).

[0067] Human INSR gene PiggyBac transposon expression vector (H_INSR) (Genomeditech, GM-106640);

[0068] Super PiggyBac transposase expression vector (Genomeditech, GM-48338);

[0069] MLSN-CAR mRNA (SEQ ID NO.1) and human INSR mRNA (ID: 3643 NM_000208.4).

[0070] (4) Main reagents: Puromycin, G418, lentivirus transfection reagent (such as Lipofectamine 3000), electroporation transfection system, Proteanfect MAX (Nanoportal Biotec, PT02) anti-FLAG tag antibody, anti-INSR antibody.

[0071] 2. Experimental Methods

[0072] (1) Construction of the MSLN-CAR-NK92 cell line:

[0073] Plasmid Design: A second-generation CAR plasmid targeting human MSLN was constructed. This CAR structure comprises an anti-MSLN scFv fragment, a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ activation domain. A FLAG tag is attached to the N-terminus of the scFv fragment for detection. This CAR sequence was cloned into the pLVX-Puro lentiviral vector (WO2022 135578 A1).

[0074] Lentiviral packaging and transduction: HEK293T cells were co-transfected with the MSLN-CAR-pLVX-Puro vector and packaging plasmids psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). Viral supernatants were collected at 48 and 72 hours, filtered, and concentrated. NK-92 cells were then infected with the concentrated virus.

[0075] Screening and purification: After transduction, MSLN-CAR-NK92 cells stably expressing CAR were selected using puromycin (e.g., 1 µg / mL). Flow cytometry was used to detect the FLAG tag on the cell surface to confirm CAR expression efficiency.

[0076] (2) Construction of the “metabolic armor” cell line MSLN-CAR-NK92[PB-INSR]:

[0077] Plasmid preparation: Prepare the PiggyBac transposon expression vector (H_INSR) (Genomeditech, GM-106640) and the Super PiggyBac transposase expression vector (Genomeditech, GM-48338) encoding the human INSR gene.

[0078] Co-transfection: The two plasmids were co-transfected into the MSLN-CAR-NK92 cells obtained in step one at an optimized ratio (e.g., 3:1). Electroporation was preferred as the transfection method to improve the transfection efficiency of NK cells.

[0079] Screening and Validation: After transfection, cells were screened using G418 (500 µg / mL) to obtain cells that stably integrated the INSR gene into the genome. Finally, the level of INSR protein on the cell surface was detected by flow cytometry to verify successful overexpression of INSR.

[0080] (3) Construction of the original INSR+MSLN-CAR-T "metabolic armor":

[0081] A CAR encoding mesothelin (MSLN) targeting CAR (containing anti-MSLNscFv, CD8α hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ signaling domain) and codon-optimized mRNA for the human insulin receptor (INSR) were synthesized using an in vitro transcription (IVT) system. Prior to transfection, the synthesized mRNA was purified using a standard spin-column purification method, and its quality was assessed.

[0082] Following the manufacturer's instructions, the synthesized CAR mRNA, alone or in combination with INSR mRNA, was transiently transfected into primary T cells using the Proteanfect Max transfection kit. Twenty-four hours post-transfection, the expression of CAR and INSR proteins was detected and confirmed by flow cytometry using antibodies targeting the FLAG tag (for CAR detection) and human INSR. Transfected cells typically exhibit transient protein expression characteristics, with expression levels peaking between 24 and 48 hours, suitable for downstream functional assays.

[0083] 3. Experimental Results

[0084] Figure 1 This is a schematic diagram of the core mechanism of the present invention. The diagram clearly compares the different fates of conventional CAR-T / NK cells (left) and the "metabolic armor" CAR-T / NK cells of the present invention (right) after being attacked by 27HC secreted by CAFs. The left side shows cholesterol depletion, CAR shedding, and tumor survival; the right side shows the entire process of INSR activating endogenous cholesterol synthesis, maintaining CAR stability, and ultimately clearing the tumor.

[0085] Using the methods described above, INSR overexpression target cells were successfully constructed. Figure 2 To validate INSR overexpression, cells were first washed with FACS buffer and then incubated with a fluorescein-conjugated antibody on ice for 30 minutes in the dark. Data were acquired using a BD FACS Calibur flow cytometer and analyzed using FlowJo software (v10). Results showed that, compared with parental MSLN-CAR-NK92 / T cells, the expression level of INSR on the surface of MSLN-CAR-NK92 [PB-INSR] cells modified with a transposon system and primary CAR-T cells directly transfected with mRNA was significantly increased by flow cytometry analysis using anti-INSR antibody.

[0086] This invention resists CAR shedding mediated by 27HC from CAFs. Figure 3Flow cytometry was used to detect CAR expression in T / NK cells. The top image shows conventional CAR-T / NK cells, where the expression of CAR is significantly reduced in the presence of CAF-derived 27HC. The bottom image shows the present invention, where CAR expression is no longer affected in the presence of 27HC. This invention resists CAF-mediated CAR shedding. In a simulated microenvironment co-cultured with CAFs, the CAR-NK cells of this invention maintain stable expression of CAR molecules on the cell membrane surface, while conventional CAR-NK cells experience significant CAR detachment.

[0087] This invention possesses significantly enhanced in vitro killing capabilities. Figure 4 This image shows the in vitro antitumor effects of the present invention. The top image shows the killing effect of conventional CAR and the present invention on the gastric cancer SNU16 cell line using the EuTDA method, with results indicating that the killing ability of the present invention is significantly increased. The bottom image shows the killing effect of the present invention on gastric / pancreatic / colon cancer PDO using the calcein release assay, with results showing that the experimental group treated with the present invention (far right) had significantly reduced calcein content (calcein and tumor viability are negatively correlated). In a co-culture system containing CAFs, the CAR-NK cells of the present invention have a much higher tumor cell killing ability than conventional CAR-NK cells.

[0088] This invention exhibits remarkable in vivo anti-tumor effects. Figure 5 This image illustrates the in vivo antitumor effects of the present invention. The top image is an in vivo bioluminescence imaging of cNKG mice with peritoneal tumors after NK cell immunotherapy. The results show that in tumor models with CAFs, the experimental group (far right) receiving CAR-NK cell therapy with the "metabolic armor" of the present invention showed significantly suppressed tumor fluorescence signals. The bottom image is a tumor growth curve of PDX colon cancer after T cell immunotherapy, showing that tumor growth was significantly inhibited after receiving CAR-T cell therapy with the "metabolic armor" of the present invention (bottom red curve). This in vivo experiment demonstrates the potent antitumor activity of the present invention. In a mouse model simulating peritoneal metastasis of gastric cancer, mice receiving CAR-NK cell therapy with the present invention showed significantly suppressed tumor burden and significantly prolonged survival, with effects far superior to the conventional CAR-NK cell therapy group.

[0089] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of the gene encoding human INSR in enhancing the anti-tumor activity of CAR cells.

2. The application according to claim 1, characterized in that, The application of the gene encoding human INSR in the preparation of modified CAR cells for anti-tumor treatment.

3. The application according to claim 1, characterized in that, The targets of the CAR include, but are not limited to, MSLN, Claudin18.2, EGFR, HER2, and CEA.

4. The application according to claim 1, characterized in that, The CAR cells mentioned are CAR-T, CAR-NK, or CAR-M cells.

5. The application according to claim 1, characterized in that, The aforementioned enhancement of CAR cell anti-tumor activity is achieved by introducing the gene sequence encoding human INSR into CAR cells through genetic engineering and ensuring its stable high-level expression.

6. The application according to claim 1, characterized in that, The tumor in question is a solid tumor.

7. A genetically engineered CAR cell, characterized in that, The CAR cells contain gene sequences encoding chimeric antigen receptor CAR and human INSR.

8. A method for constructing CAR cells as described in claim 7, characterized in that, It involves using a gene delivery system to introduce the gene sequence encoding human INSR into CAR cells and stabilize its high-level expression.

9. The construction method according to claim 8, characterized in that, The gene delivery system is selected from transposon systems, mRNA transfection, lentiviral vectors, AAV vectors, or other non-viral vectors.

10. The use of the modified CAR cell as described in claim 7 in the preparation of an antitumor drug.

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