A STAT3-mutated cell product and its uses

Abstract

This invention discloses a STAT3-mutated cell product and its uses, belonging to the interdisciplinary field of genetic engineering and tumor immunotherapy. Through saturation mutation screening, this invention is the first to discover that STAT3 gain-of-function mutations promote CAR-T anti-tumor responses. Furthermore, through cell and animal experiments, it is demonstrated that activating STAT3 can alleviate CAR-T cell immune exhaustion and significantly enhance the in vivo and in vitro killing ability of CAR-T cells against tumor cells, thereby improving the efficacy of anti-tumor therapy. This invention provides a new therapeutic target and strategy for the field of tumor immunotherapy, possessing significant scientific and clinical application value.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of genetic engineering and tumor immunotherapy, specifically involving the application of gain-of-function mutants of signal transducer and activator of transcription 3 (STAT3) in engineered T cells, and their use in the preparation of anti-tumor immunotherapy cell products. Background Technology

[0002] As the fourth generation of cancer treatment following surgery, radiotherapy, chemotherapy, and targeted therapy, tumor immunotherapy's core mechanism involves activating or enhancing the body's immune system to specifically recognize and eliminate tumor cells. Current technologies mainly encompass immune checkpoint inhibitors, tumor vaccines, adoptive cell therapy (such as CAR-T therapy), cytokine therapy, and co-stimulatory receptor agonists. Although these therapies offer advantages over traditional methods, such as strong targeting and low systemic toxicity, T-cell exhaustion severely restricts their clinical efficacy. T-cell exhaustion manifests as reduced effector function, upregulation of inhibitory receptors (PD-1, LAG-3, TIM-3), metabolic homeostasis dysregulation, and abnormal epigenetic remodeling. Particularly in chimeric antigen receptor T-cell (CAR-T) therapy, continuous antigen stimulation leads to T-cell metabolic dysfunction, altered epigenetic modifications, and the accumulation of terminal differentiation phenotypes, significantly weakening anti-tumor activity. Existing solutions such as PD-1 blockade (e.g., invention patents with publication numbers CN117756915A and CN113069529A) can only partially reverse the depletion state and carry the risk of immune-related adverse reactions. Summary of the Invention

[0003] This invention, through adenine base editing system (ABE)-mediated saturation mutation screening, has for the first time discovered that the histidine-to-arginine mutation at position 410 of the STAT3 gene (H410R) significantly enhances CAR-T cell effector function. This mutant, by reconstructing the JAK-STAT signaling network, upregulates the secretion of effector factors such as IFN-γ and TNF-α (in vitro experimental data) and reduces PD-1. + LAG-3 + Proportion of double-positive exhausted subsets. Animal models showed that mutant CAR-T significantly increased tumor volume inhibition rate in tumor-bearing mice. This invention demonstrates that STAT3 is a potential target for tumor immunotherapy.

[0004] This invention aims to provide a novel tumor immunotherapy target based on gain-of-function mutations in the STAT3 gene, and also discloses engineered T cells containing this mutated gene and their application in adoptive cell therapy.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] The first aspect of the present invention is to obtain a set of saturated mutation sites in the entire coding region of STAT3.

[0007] Furthermore, the series of STAT3 saturation mutation sites fully covers the entire 2310 bases of the human STAT3 gene's CDS region. Furthermore, the 2310 bases of the entire STAT3 gene CDS region are altered at the DNA or RNA level using gene editing tools. Furthermore, the gene editing tools include, but are not limited to, an adenine base editor, a cytosine base editor, and a saturation mutation library. Furthermore, the saturation mutation library targets the entire 2310 bases of the STAT3 gene's CDS region.

[0008] In this invention, the information of the gene STAT3 is as follows: the genomic coordinates of STAT3 (Gene ID: 6774) are NC_000017.11 (GRCh38.p13): g.40468453_40497750, and its encoded protein UniProtKB is numbered P40763. Relevant sequence information can be obtained from the NCBI Gene database.

[0009] A second aspect of the present invention establishes a T-cell screening system.

[0010] Furthermore, the T cells are a collection of T cells obtained by editing the STAT3 gene through library infection. Furthermore, the obtained T cells include primary T cells, activated T cells, and CAR-T cells. Library infection refers to packaging the library into a lentivirus and adjusting the MOI to 0.3 to infect T cells. Furthermore, the MOI of 0.3 is determined by flow cytometry, where the proportion of infected target cells (expressing mCherry) is 30% of the total cells. The screening system involves flow cytometry sorting of the edited T cells. Furthermore, the flow cytometry sorting strategy includes STAT3 transcriptional activity, effector cytokine secretion, and immune exhaustion levels. Furthermore, STAT3 transcriptional activity is detected by the Ser phosphorylation level at position 705 of STAT3; effector cytokine secretion is determined by the positivity of IFNγ and TNFα; and immune exhaustion levels are determined by PD-1 and LAG-3 levels.

[0011] A third aspect of the present invention provides a gene-edited T cell.

[0012] Furthermore, the gene-edited T cells are obtained through a second screening process, where the STAT3 gene's 410th histidine residue is mutated to arginine via ABE. The STAT3 gene function is activated in these T cells. Further, the gene-edited T cells are obtained by gene editing the STAT3 gene in target T cells to obtain STAT3-activated T cells. Further, the target T cells are chimeric antigen receptor T cells (CAR-T cells). Further, the CAR-T cells are CD19 CAR-T cells and GPC3 CAR-T cells.

[0013] In this invention, the chimeric antigen receptor T cell (CAR-T) refers to a genetically engineered T lymphocyte that expresses a synthetic receptor protein that specifically recognizes tumor-associated surface antigens. This receptor comprises:

[0014] The CAR-T cell pathway consists of an antigen-binding domain (scFv, such as a CD19 or GPC3-specific sequence), a transmembrane domain (derived from CD28 or CD8α), a co-stimulatory domain (derived from 4-1BB or CD28), and a T cell activation domain (CD3ζ chain). CARs bind to the target antigen structurally specifically via scFv in an MHC-independent manner, activating downstream signaling pathways in the TCR pathway and initiating tumor killing. Because CAR-T cells are independent of antigen presentation, they bypass the main mechanisms of tumor immune escape.

[0015] In a specific embodiment of the present invention, CD19 CAR-T refers to chimeric antigen receptor T cells that target human CD19 antigen, and GPC3 CAR-T refers to chimeric antigen receptor T cells that target human GPC3 antigen.

[0016] In this invention, the gene-edited T cells exhibit significantly enhanced in vitro and in vivo tumor cell killing ability. In T cells, the STAT3 H410R mutation increases the expression of effector molecules such as IFNγ and TNFα, as well as CD107a degranulation molecules, alleviating CAR-T cell depletion in the tumor microenvironment, thereby enhancing the tumor cell killing ability of the gene-edited T cells.

[0017] In the implementation plan, the gene-edited T cells are isolated from peripheral blood PBMCs donated by multiple human volunteers. Alternatively, T cells can be induced to differentiate from hematopoietic stem cells in vitro, or reprogrammed and re-differentiated using iPSC technology. T cells can be derived from: peripheral blood mononuclear cells, lymph node tissue, thymus tissue, bone marrow, tumors, tissues from infected sites, spleen tissue, umbilical cord blood, ascites, and pleural effusion. Isolation methods include density gradient centrifugation using FICOLL and magnetic negative selection to obtain T cells with a purity of over 99%.

[0018] Further, the preparation method includes: gene editing of the STAT3 H410R site of target T cells to obtain the gene-edited T cells described in the first aspect of the present invention. Further, the reagent for gene editing the STAT3 H410R site includes: a relevant RNA component for gene editing of the STAT3 gene. Further, the relevant RNA component includes ABE mRNA and phosphate-thioester-modified sgRNA. Further, the sequence of the sgRNA is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0019] In this invention, any reagent capable of gene editing or site-directed mutation of the STAT3 gene or activation of STAT3 activity is within the scope of protection of this invention, including but not limited to: CRISPR gene editing reagents, various versions of adenine base editors and cytosine base editing reagents, various versions of lead editing reagents, small molecule compounds, peptides, peptide mimics, matrix analogs, aptamers, antibodies, etc.

[0020] Based on the STAT3 H410R base editing sequence information provided by this invention, the required RNA or protein for editing can be synthesized through various methods, including but not limited to chemical synthesis and in vitro transcription, and delivered into T cells via electroporation or liposome technology. In some embodiments, gene editing systems (including but not limited to ABE8e, BE4max, etc.) and lead editing systems (including but not limited to PE7, etc.) can be used to edit the STAT3 gene in T cells. The sequences used include those provided by this invention, or sequences that, after sequence similarity comparison, have more than 70% similarity to the sequences provided by this invention are included in this invention.

[0021] Furthermore, other site substitution techniques are also applicable to this invention. In some embodiments, homologous recombination can be used to specifically target the STAT3 gene in T cells, replacing the wild-type sequence with the STAT3 mutant sequence to express the STAT3 gain-of-function mutant. Alternatively, lentiviruses or retroviruses can be used to directly infect T cells to express the STAT3 gain-of-function mutant. Or, the STAT3 mutant can be inserted into the T cell genome using the Piggybac transposon system or the Sleepy Beauty transposon system to achieve stable expression of the STAT3 mutant.

[0022] The fourth aspect of the present invention provides any of the following products:

[0023] (1) A gene-edited T cell population product, wherein the gene-edited T cell population comprises the gene-edited T cells described in the third aspect of the present invention;

[0024] (2) An activated gene-editing T cell, wherein the activated gene-editing T cell is obtained by activating the gene-editing T cell described in the third aspect of the present invention;

[0025] (3) An activated gene-editing T cell population, said activated gene-editing T cell population comprising said activated gene-editing T cells;

[0026] (4) A pharmaceutical composition comprising the gene-edited T cells, the gene-edited T cell population, the activated gene-edited T cells, and / or the activated gene-edited engineered T cell population as described in the third aspect of the present invention.

[0027] The present invention also provides an in vitro method for enhancing the anti-tumor effect of T cells, the method comprising: gene editing the STAT3 gene gain-of-function site of T cells.

[0028] In some embodiments, the pharmaceutical compositions of the present invention may also comprise any pharmaceutically acceptable carrier and / or excipient, and any formulation. Furthermore, they may include any form of administration, method of administration, and frequency of administration of any drug and cell product. This includes, but is not limited to, oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical, and intranasal administration. In specific embodiments, the dosage may be adjusted to be beneficial to the patient in light of the clinical situation.

[0029] Furthermore, the present invention also provides a method for treating tumors in a subject in need, the method comprising: administering to a subject in need a therapeutically effective amount of the gene-edited T cells described in the third aspect of the present invention, the gene-edited T cell population described in the third aspect of the present invention, activated gene-edited T cells, activated gene-edited T cell population, pharmaceutical composition and / or pharmaceutical preparation.

[0030] Furthermore, the tumor includes any tumor currently known in the art, as well as new types of tumors that may be discovered in the future, including but not limited to: solid tumors and non-solid tumors. Solid tumors include, but are not limited to: liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, melanoma, neuroblastoma, breast cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, kidney cancer, osteosarcoma, Hodgkin's lymphoma, chondrosarcoma, etc. Non-solid tumors include, but are not limited to: non-Hodgkin's lymphoma, multiple myeloma, leukemia, etc.

[0031] The fifth aspect of the present invention provides for application in any of the following aspects:

[0032] (1) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of reagents for promoting the efficacy of T cell anti-tumor immunotherapy;

[0033] (2) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of reagents for promoting the efficacy of CAR-T cell anti-tumor immunotherapy;

[0034] (3) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of T cell immunotherapy drugs;

[0035] (4) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of reagents to reduce T cell exhaustion;

[0036] (5) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of reagents to enhance T cell activity;

[0037] (6) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate STAT3 protein activity in the preparation of gene-edited T cells for the treatment of tumors;

[0038] (7) Application of gene editing of STAT3, expression of STAT3 mutants or substances that activate the activity of STAT3 protein in the preparation of drugs for the treatment of tumors.

[0039] Specifically, the use of the aforementioned gene-edited T cells, the aforementioned gene-edited T cell population, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumors.

[0040] Furthermore, the tumor may be a solid tumor or a non-solid tumor;

[0041] The solid tumor is selected from at least one of the following: liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, melanoma, neuroblastoma, breast cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, kidney cancer, osteosarcoma, Hodgkin's lymphoma, and chondrosarcoma.

[0042] The non-solid tumor is selected from at least one of non-Hodgkin's lymphoma, multiple myeloma, and leukemia.

[0043] Preferably, B-cell precursor acute lymphoblastic leukemia and liver cancer are used as examples in the embodiments of the invention, but are not limited thereto.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] This invention, through saturation mutation screening, first discovered that STAT3 gain-of-function mutations promote CAR-T anti-tumor responses. Furthermore, through cell and animal experiments, it was demonstrated that activating STAT3 can alleviate CAR-T cell immune exhaustion and significantly improve the in vivo and in vitro killing ability of CAR-T cells against tumor cells, thereby improving the efficacy of anti-tumor therapy. This invention provides new therapeutic targets and strategies for the field of tumor immunotherapy, and has significant scientific and clinical application value. Attached Figure Description

[0046] Figure 1 To enhance the specific killing effect of CD19 CAR-T cells against B-cell lymphoma by STAT3 H410R mutation; A: Schematic diagram of gene-edited CAR-T cell preparation and in vitro functional killing experiment; B: Structural information map of CD19 CAR and GPC3 CAR retroviral vectors; C: Sanger sequencing analysis of gene editing efficiency of STAT3 H410R site in sgNT and sgH410R transfected CD19 CAR-T cells; D: Flow cytometry quantitative analysis of the number of residual NALM6 tumor cells after 24 hours of co-culture (effect-to-target ratio = 1:10 and 1:20); E: Transcriptome sequencing heatmap showing the clustering characteristics of cytokine expression profiles in STAT3 wild-type / H410R mutant CD19 CAR-T cells; ** P < 0.01, *** P < 0.001; scatter plots represent parallel sample data.

[0047] Figure 2 To enhance the anti-hepatocellular carcinoma activity of GPC3 CAR-T cells by STAT3 H410R mutation; A: Schematic diagram of continuous tumor attack experiment design; B: Flow cytometry detection of GPC3 CAR-T cell transduction efficiency and statistical analysis; C: Sanger sequencing verification of STAT3 gene mutation efficiency in the sgH410R edited group; D: Western blot detection of the regulatory effect of H410R mutation on STAT3 phosphorylation level (pSTAT3); E: Detection of short-term (24 h) and long-term (96 h) cytotoxicity of mutant / wild-type GPC3 CAR-T cells against hepatocellular carcinoma cells under different effector-target ratios; * P < 0.05, ** P < 0.01, *** P < 0.001; Scatter dots represent parallel sample data.

[0048] Figure 3To investigate the potential mechanism by which the STAT3 H410R mutation enhances the killing ability of CAR-T cells; A: CD107a was quantitatively analyzed by flow cytometry to determine the degranulation level of CAR-T cells after co-culture; B: TNF-α and IFN-γ secretion in the co-culture supernatant was determined by ELISA; C: The expression dynamics of exhaustion markers of CAR-T cells in short-term (24 h) and long-term (92 h) co-culture were detected by time-series flow cytometry; * P < 0.05, ** P < 0.01, *** P < 0.001.

[0049] Figure 4 To enhance the in vivo antitumor efficacy of GPC3 CAR-T cells by STAT3 H410R mutation; A: Schematic diagram of CAR-T treatment experiment in Huh7 hepatocellular carcinoma-bearing mice; B: Tumor volume growth curve showing that the mutant CAR-T treatment group had a significant tumor-suppressing effect compared with the wild-type group; Statistical data were obtained by log-rank Mantel Cox test, and the results are expressed as mean ± standard deviation. The number of experimental animals in each group was n=4. * P < 0.05, ** P < 0.01. Detailed Implementation

[0050] The following embodiments are intended to exemplify the technical solutions and application scenarios of the present invention, and their descriptions should not be construed as limiting the scope of protection of the present invention. Those skilled in the art should understand that, without departing from the design principles and core technology of the present invention, necessary adjustments, equivalent substitutions, or adaptive improvements can be made to the implementation schemes. The scope of the present invention is determined by the claims and their equivalent technical solutions. All biological reagents involved in this invention are conventional commercially available products. When experimental operations are not explicitly specified, one of the following two methods is used by default: (1) standard experimental methods known in the art; (2) the recommended operating procedures in the instruction manuals of commercial reagents. All statistical analyses involved were completed using GraphPad Prism 10.0 software, and the significance criteria were P<0.05, P<0.01, and P<0.001.

[0051] Example 1: A STAT3-mutated cell product and its uses

[0052] Experimental materials:

[0053] (1) Laboratory animals

[0054] NSG mice were obtained from Nanjing Jicui Pharmaceutical Biotechnology Co., Ltd. All mice were maintained under specific pathogen-free conditions. All mouse experiments were conducted under the supervision of the Animal Ethics Committee (IACUC) of Zhejiang University Medical Center. All studies were conducted on animals aged 6 to 8 weeks.

[0055] (2) Cell line

[0056] The 293T, Nalm6, and Huh7 cell lines were obtained from the laboratory of Zhao Bin at the Institute of Life Sciences, Zhejiang University. The 293T and Huh7 cell lines were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. The Nalm6 cell line was cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. STR testing was performed on all cell lines, confirming they were free of mycoplasma.

[0057] Reagents and consumables:

[0058] The main reagents and consumables used in this embodiment are shown in Table 1 below.

[0059] Table 1: Main Reagents and Consumables

[0060]

[0061] 2. Experimental Methods

[0062] (1) Isolation of primary T cells

[0063] Collect a 4 mL sample of human venous blood in a vacuum collection tube containing sodium heparin, then gently invert the tube several times to mix thoroughly. Add 4 mL of Ficoll histopaque density gradient separation medium to a 15 mL centrifuge tube. Carefully spread the blood onto the surface of the histopaque medium using a 1 mL automated pipette. The layering process should be very slow to maintain the blood and histopaque medium as two distinct liquids. Immediately (without any delay), place the tube in a horizontal rotor and centrifuge at 100 × g for 30 minutes at 4°C. A fixed-angle rotor can also be used, but more care is required when separating the cells at the interface. Aspirate the white erythrocyte sedimentation rate (ESR) layer (approximately 1 mL) (peripheral blood mononuclear cells, PBMCs) that forms at the interface between the histopaque medium and the upper liquid. The cells in the interface layer must be aspirated immediately. If the test tube is left to stand for more than 10 minutes, the peripheral blood mononuclear cells at the interface layer will be disturbed and begin to settle. Wash the cells twice with 10 mL of sterile phosphate-buffered saline (PBS) (centrifuging at 100 × g for 10 minutes each time). The cell yield obtained from 4 mL of blood is approximately 10 8 Up to 10 9 Between [number] cells. T cells were isolated from PBMCs using a human T cell sorting kit (#17951). The specific steps were as follows: The isolated PBMCs were resuspended in PBS solution containing 2% FBS and diluted to 5 × 10⁻⁶. 7Cells / mL, final volume 0.5 mL. Transfer the sample to a flow cytometry tube and add 25 μL of antibody mixture, mix well and incubate at room temperature for 5 minutes. Vortex the magnetic beads for 30 seconds, add 20 μL of magnetic beads to the sample tube, add separation buffer, gently pipette 2-3 times to mix, and bring the volume to 2.5 mL. Place the flow cytometry tube in the magnetic pole and incubate at room temperature for 3 minutes. Pick up and invert the magnetic pole to transfer the liquid in the flow cytometry tube to a new sample tube. The isolated cells are ready for subsequent experiments.

[0064] (2) Preparation of CAR-T cells and gene-edited CAR-T cells

[0065] CD19 CAR (amino acid sequence as shown in SEQ ID NO.4), GPC3 CAR plasmid (amino acid sequence as shown in SEQ ID NO.5), and packaging plasmid were transfected into 293T cells, and the medium was changed after 24 hours. The viral supernatant produced 24 hours after medium change was collected, filtered, and added to T cells that had been activated for 48 hours. Suspension infection was performed, and fresh medium was added after 48 hours. The infection positivity rate was detected by flow cytometry. Following the experimental protocol provided by the electroporation kit (V4SP-3096), editor mRNA and sg NT or STAT3 H410R sgRNA (using the sequence shown in SEQ ID NO.1 in Table 2, i.e., STAT3H410R sgRNA#1) were electroporated into CAR-T cells, and editing efficiency was detected after 48 hours.

[0066] Table 2

[0067]

[0068] (3) Screening for STAT3 saturation mutations

[0069] Design of a STAT3 saturated mutant library. Lentiviral packaging and concentration: 293T cells were co-transfected using a second-generation lentiviral packaging system (psPAX2 packaging plasmid / pMD2.G envelope plasmid = 3:1) via the calcium phosphate method. Viral supernatants were collected at 24 and 48 hours post-transfection, filtered through a 0.45 μm PVDF filter, and then concentrated using a Beckman ultracentrifuge (24000×g, 4℃, 2 hours). The viral pellet was resuspended in RPMI 1640 medium containing 10% FBS, aliquoted, and stored at -80℃. Viral titer quantification: The concentrated virus solution was premixed with polybrene (final concentration 8 μg / mL) and incubated at room temperature for 5 minutes. Human primary T cells activated for 48 hours (CD3 / CD28 antibody stimulation) were infected with seven concentration gradients (n=3) of virus, ranging from 0-32 μL. After 1.5 hours of suspension infection at 37℃, the medium was replaced with complete medium.

[0070] Forty-eight hours post-infection, the percentage of mCherry-positive cells was detected by flow cytometry, and the functional titer was calculated using the formula: Titer (TU / mL) = Percentage of positive cells × Total number of cells / Virus volume (mL) × Dilution factor. MOI calibration: The dosage was adjusted according to the formula (Virus volume = MOI × Cell number / Titer = Titer MOI × Cell number) to ensure MOI = 0.30 ± 0.02. Use 1 × 10⁻⁶ cells. 8 GPC3CAR-T cells (CAR positivity ≥85%) were added to pre-titrated viral solution (MOI=0.3) and incubated at 37°C for 2 hours. Infection efficiency verification: Flow cytometry showed the proportion of mCherry-positive cells. Library coverage assurance: The initial cell number was calculated as N = (library capacity × 300) / infection efficiency to ensure monoclonal coverage >99%. Multidimensional phenotypic screening and mechanism analysis: Cells were co-incubated with Huh7 hepatocellular carcinoma cells at an effector-to-target ratio of 1:2 for 48 hours. Flow cytometry sorted out PD-1 and LAG-3 double-negative populations, TNF-α and IFN-γ double-positive populations, and a pSTAT3 (Ser705) high-expression population. Genomic DNA was extracted from the sorted populations, and sgRNA regions were amplified by PCR and deep sequenced. Bioinformatics analysis: The MAGeCK algorithm was used to identify significantly enriched sgRNAs (FDR < 0.05, |log2(fold change)| > 1), and key functional mutation sites were located.

[0071] (3) Flow cytometry detection of the absolute number of NALM6 tumor cells

[0072] Collect co-cultured cells, add 1 mL of 2% FBS / PBS solution, centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL of FVS780 dye at a ratio of 1:750 and incubate on ice for 15 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 50 μL of human Fc Block solution diluted 1:400 and incubate on ice for 20 minutes. Mix CD19 antibody and G4S antibody at a ratio of 1:750, add to sample tubes, and incubate on ice for 30 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add counting beads and perform flow cytometry analysis. Flow cytometry analysis was performed using a BD LSR Fortessa instrument (BD). Data analysis was performed using FlowJo software. All experiments were performed in at least two biological replicates.

[0073] (4) Flow cytometry detection of CD107a levels

[0074] Add 1 μL of CD107a-BV605 antibody to a co-cultured plate. After 1 hour of incubation, add 1 μM monensin to block endocytosis following CD107a molecule expression on the cell surface. After 4 hours, harvest the co-cultured cells, add 1 mL of 2% FBS / PBS solution, centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL of LVS780 dye at a 1:750 ratio and incubate on ice for 15 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 50 μL of human Fc blocking agent solution diluted 1:400 and incubate on ice for 20 minutes. Dilute G4S antibody at a 1:750 ratio, add to sample tubes, and incubate on ice for 30 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Resuspend the cells and analyze CD107a levels using a BD LSR Fortessa instrument (BD). Data were analyzed using FlowJo software. All experiments were performed in at least two biological replicates.

[0075] (5) Flow cytometry detection of PD-1 and LAG-3 levels

[0076] Collect co-cultured cells, add 1 mL of 2% FBS / PBS solution, centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL of FVS780 dye at a ratio of 1:750 and incubate on ice for 15 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. Add 50 μL of human Fc blocking agent solution diluted 1:400 and incubate on ice for 20 minutes. Mix PD-1 antibody, LAG-3 antibody, and G4S antibody at a ratio of 1:750, add to sample tubes, and incubate on ice for 30 minutes. Centrifuge at 300×g, 4°C for 5 minutes, and discard the supernatant. After resuspending, analyze PD-1 and LAG-3 levels using a BD LSR Fortessa (BD) system. Data were analyzed using FlowJo software. All experiments were performed in at least two biological replicates.

[0077] (6) ELISA detection of IFNγ and TNFα levels

[0078] Sample addition: Add 100 μL / well of diluted cytokine standard to the standard wells, 100 μL / well of sample to the sample wells, and 100 μL / well of Dilution Buffer R (1×) to the blank control wells. Add detection antibody: Add 50 μL / well of biotin-labeled antibody working solution. Mix well, cover with sealing film, and incubate at room temperature for 2 hours. Wash plate: Remove liquid from wells, add 300 μL / well of 1× washing buffer working solution; let stand for 1 minute, then discard the liquid. Repeat 3 times, drying on filter paper each time. Add enzyme: Add 100 μL / well of streptavidin-HRP working solution. Cover with sealing film and incubate at room temperature for 20 minutes. Wash plate: Repeat the above steps. Develop color: Add 100 μL / well of TMB and incubate at room temperature in the dark for 20 minutes. The reaction can be terminated by observing the color intensity in the wells. The wells with the highest calibration curve value should be dark blue, and the blank wells should show no obvious blue color. A good result is usually achieved after 20 minutes of color development. To terminate the reaction: Quickly add 100 μL / well of stop solution. To read the plate: 10 minutes after terminating the reaction, perform a dual-wavelength reading on a microplate reader. Set the detection wavelength to 450 nm and the reference wavelength to 610 nm–630 nm. The corrected OD value is the measured value at the detection wavelength minus the measured value at the reference wavelength.

[0079] (7) Tumor model

[0080] Cells resuspended in PBS were mixed with matrix gel at a 1:1 ratio and injected subcutaneously into the ventral side of NSG mice. Each mouse received 100 μL of Huh7 cells (3 × 10⁶ cells). 6 Each mouse was assessed for tumor size and weight every 3 days after it developed tumor cells. Tumor size was calculated as (length × width × height) / 2. Tumors reaching 2000 mm were counted. 3 The mice were considered the endpoint of the experiment and were euthanized.

[0081] 3. Experimental Results

[0082] (1) STAT3 gain-of-function mutations enhance the specific killing effect of CD19 CAR-T cells on B-cell lymphoma.

[0083] Based on the previously established STAT3 saturation mutation screening platform, this study identified that the H410R mutation significantly enhances STAT3 transcriptional activity. To verify the effect of this mutation on CD19 CAR-T cell function, we first constructed a gene-edited CAR-T cell preparation and in vitro killing evaluation system (…). Figure 1 A). CD19 CAR and GPC3 CAR sequences were successfully transduced into T cells using a retroviral vector (A). Figure 1 (B in the text), and achieves approximately 90% STAT3 H410R site editing efficiency through precise electroporation technology ( Figure 1(C in the middle).

[0084] In vitro co-culture experiments showed that, compared with wild-type, the survival rate of NALM6 cells in the H410R mutant group was significantly reduced (p<0.01). Figure 1 The D in the sequence suggests that this mutation can effectively enhance the killing efficacy of CD19 CAR-T cells against B-cell lymphoma. Further transcriptome sequencing revealed that the H410R mutation significantly upregulated the expression levels of multiple effector cytokines (such as IFN-γ and TNF-α) (fold change >2). Figure 1 The presence of E in the sample confirmed its functional enhancement effect at the molecular mechanism level.

[0085] (2) STAT3 H410R mutation enhances the anti-hepatocellular carcinoma activity of GPC3 CAR-T cells.

[0086] To explore the potential application of this mutation in the treatment of solid tumors, we established a dynamic co-culture model of GPC3 CAR-T cells and liver cancer cells. Figure 2 (A) A T-cell population with a CAR positivity rate >80% was obtained by optimizing transduction conditions. Figure 2 (B in the middle), and maintains approximately 90% H410R site editing efficiency ( Figure 2 C in the text). Western blot analysis showed that the H410R mutation significantly increased phosphorylation at the STAT3 Ser705 site (C). Figure 2 The presence of D in the figure indicates that it can effectively activate the STAT3 signaling pathway. In vitro killing experiments using a gradient effect-to-target design and quantitative analysis based on a luciferase reporter system showed that the mutant group significantly improved the killing efficiency against Huh7 hepatocellular carcinoma cells (D). Figure 2 The study of E in the study confirmed the synergistic effect of this mutation on the treatment of solid tumors.

[0087] (3) Molecular mechanism by which STAT3 H410R mutation enhances the killing ability of CAR-T cells

[0088] Mechanistic studies showed that flow cytometry analysis of CD107a expression levels revealed significantly increased degranulation activity in the H410R mutant group. Figure 3 (A in the text); ELISA detection confirmed a significant increase in the secretion of effector factors IFN-γ and TNF-α ( ). Figure 3 (B in the text). Dynamic monitoring revealed that the expression levels of exhaustion markers (PD-1, LAG-3) in the mutant group decreased by more than 30% during 96 hours of continuous killing. Figure 3 (C in the text). The above results indicate that the H410R mutation enhances anti-tumor function through a triple mechanism: promoting the release of cytolytic granules; enhancing the secretion of effector factors; and delaying the process of T cell exhaustion.

[0089] (4) STAT3 H410R mutation enhances the in vivo antitumor efficacy of GPC3 CAR-T cells.

[0090] In the Huh7 subcutaneous hepatocellular carcinoma model (n=4 / group), after a single infusion of H410R mutant GPC3 CAR-T cells ( Figure 4 In the A group, the tumor volume in the treatment group was significantly reduced compared to the wild-type group. Figure 4 (B in the text). These results not only validate the therapeutic advantages of STAT3 gain-of-function mutations, but more importantly, reveal the transformative potential of the STAT3 signaling axis as an optimized target for CAR-T cell therapy.

Claims

1. A gene-edited T cell, characterized in that, The gene-edited T cells were obtained by editing the endogenous STAT3 gene in T cells using a gene-editing system, changing the 410th amino acid from histidine to arginine; The gene editing system described herein contains sgRNA that targets the 410th amino acid codon of the STAT3 gene; The T cells are CAR-T cells; The antigen targeted by the CAR-T cells is selected from CD19 or GPC3; The sequence of the sgRNA is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The gene-edited T cell according to claim 1, characterized in that, The gene editing system is an adenine base editor.

3. Use of the gene-edited T cells according to claim 1 or 2 in the preparation of a medicament for treating tumors; wherein the tumor is liver cancer, non-Hodgkin's lymphoma, or leukemia.

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