Immune cell product for improving cell therapy effect

Abstract

The invention discloses an immune cell product for improving the effect of a cell therapy, which is used for remarkably improving the treatment effect of cell therapies such as CAR-T (Chimeric Antigen Receptor-T) on solid tumors by blocking the interaction between tumor extracellular vesicles (EVs) and immune cells. The molecular weight of the PEG is 5K-100K Da, and the PEG is modified to the surface of an immune cell through chemical coupling or physical adsorption, so that the adhesion rate of EVs can be reduced by more than 50%, and the tumor killing activity can be enhanced by more than 2 times. The product improves the tumor inhibition rate to 89% in a human head and neck squamous cell carcinoma animal model, and has significant clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an immune cell product that enhances the efficacy of cell therapy. Background Technology

[0002] Immunotherapy is a biomedical technology that treats cancer, autoimmune diseases, or infectious diseases by modifying, expanding, or activating the patient's / donor's immune cells to specifically recognize and eliminate disease targets. Its core lies in reconstructing the "recognition-attack-memory" functional chain of the immune system, including chimeric antigen receptor (CAR)-T cell immunotherapy, CAR-natural killer cell therapy, CAR-macrophage therapy, and TCR-T cell therapy. Currently, research primarily focuses on CAR-T cell therapy, an immunotherapy method that uses genetic engineering to modify the patient's own T cells, enabling them to specifically recognize and kill cancer cells. The basic procedure involves isolating T cells from the patient, using gene editing technology to express chimeric antigen receptors, expanding them in vitro, and then reinfusing them into the patient to achieve anti-tumor effects.

[0003] As of April 2025, 12 CAR-T products have been approved globally (such as Kymriah, Yescarta, and Carvykti), covering B-cell lymphoma, multiple myeloma, and other diseases, and have achieved good progress. However, this therapy still faces several challenges, especially in solid tumors where its efficacy remains unsatisfactory. Specific treatment resistance mechanisms include: lack of specific targets, the complexity and heterogeneity of solid tumors themselves, the complex tumor microenvironment, and the complex immunosuppressive microenvironment of solid tumors. Tumor cell extracellular vesicles are nanoscale vesicles with a double-membrane structure secreted by tumor cells, carrying a large amount of biological information from the parent cell, such as proteins, nucleic acids, and lipids.

[0004] In the treatment of solid tumors, CAR-T cells often become ineffective due to EV-mediated immunosuppression in the tumor microenvironment. Current technologies such as antibody blocking and gene editing present challenges including off-target risks, toxicity, and difficulties in clinical translation. (1) Antibody blocking targeting EV surface proteins: Representative technology: pretreatment of extracellular vesicles with monoclonal antibodies such as anti-PD-L1 / CD63 / TSG101. Its drawbacks include: due to the complex in vivo environment and safety considerations, it is not possible to apply antibodies in large quantities in vivo, and the application of antibodies may induce Fc receptor-mediated phagocytosis, and it cannot block extracellular vesicles derived from all types of tumor cells.

[0005] (2) Gene editing to silence EV secretion mechanism: Representative technology: Knocking out the Rab27a / nSMase2 gene to inhibit EV biogenesis. Limitations: It is difficult to achieve specific gene knockout in solid tumor tissues; off-target risks exist with gene interference technologies such as CRISPR. (3) Small molecule inhibitors interfere with EV release, such as GW4869 (an nSMase inhibitor) for cell preparation. Defects include: significant cytotoxicity, short half-life, and due to lack of targeting, may inhibit the function of CAR-T cells, exacerbating the depletion of CAR-T cells and endogenous T cells.

[0006] 4) Gene Editing for Novel CAR-T Cells: Developing novel CAR-T cells through gene editing is a common method for CAR-T cell modification. However, this method has drawbacks such as off-target effects and genomic instability. Gene editing technologies, such as CRISPR / Cas9, carry off-target risks, potentially leading to the insertion, deletion, or mutation of non-target genes (e.g., inactivation of tumor suppressor genes or activation of proto-oncogenes), which may induce secondary tumors. Furthermore, overactivation may trigger toxic reactions: enhanced CAR-T cells (e.g., those carrying IL-15 secretory elements) may exacerbate cytokine release syndrome (CRS) or neurotoxicity. Additionally, the use of non-humanized editing tools (such as bacterial-derived Cas9) can induce host antibody responses.

[0007] Therefore, in order to solve the above-mentioned technical problems, it is necessary to provide an immune cell product that can improve the efficacy of cell therapy. Summary of the Invention

[0008] The purpose of this invention is to provide an immune cell product that improves the efficacy of cell therapy. This invention is the first to propose modifying the surface of immune cells with PEG to form a physical barrier that blocks EV adhesion, while enhancing the killing activity of immune cells, thus providing a new solution for the treatment of solid tumors.

[0009] To achieve the aforementioned objective, the present invention adopts the following technical solution: In a first aspect of the invention, an immune cell product for improving the efficacy of cell therapy is provided, the product comprising polyethylene glycol-modified immune cells, wherein:

[0010] The immune cells are CAR-T cells, CAR-Macrophage, CAR-NK cells, or TILs cells; The polyethylene glycol is modified onto the surface of the immune cells through chemical coupling or physical adsorption.

[0011] Further, the molecular weight of the polyethylene glycol is 5K-100K Da. Preferably, the molecular weight of the polyethylene glycol is 5K-10K, and most preferably, the molecular weight of the polyethylene glycol is 5K.

[0012] Furthermore, the chemical coupling can be performed in one of the following ways: NHS ester-mediated lysine-targeted modification; Maleimide-mediated cysteine ​​targeting modification.

[0013] Furthermore, the modification method specifically includes: PEG-NHS ester was dissolved in PBS to obtain a PEG solution; The PEG solution is mixed with the immune cells.

[0014] In one specific embodiment, the PEG solution is mixed with CAR-T cells; wherein the number of CAR-T cells is (1-9) × 10⁻⁶. 6 The working concentration of the PEG solution is 80-120 nM.

[0015] Prior to mixing, the CAR-T cells were activated using CD3 / CD28 magnetic beads. The volume ratio of CD3 / CD28 magnetic beads to cells was 1-3:1.

[0016] Furthermore, the immune cells are one type of CAR-T cells, CAR-NK cells, or TILs cells that target MSLN, HER2, or EGFR.

[0017] Furthermore, compared to the unmodified immune cells, the polyethylene glycol-modified immune cells exhibit reduced adhesion to tumor cell extracellular vesicles and increased tumor cell killing activity by at least 1.5 times.

[0018] Furthermore, the PEG-modified immune cells exhibit more than a twofold increase in their inhibitory efficiency against solid tumors in vivo.

[0019] Furthermore, the product also includes a pharmaceutically acceptable carrier or cryoprotectant.

[0020] In a second aspect of the invention, the use of the aforementioned immune cell product for enhancing the efficacy of cell therapy in the preparation of a medicament for treating solid tumors is provided.

[0021] Furthermore, the solid tumor includes at least one of pancreatic cancer and glioblastoma.

[0022] In a third aspect of the invention, there is provided a combination of an immune cell product and an immune checkpoint inhibitor in the preparation of a medicament for treating solid tumors, (a) the immune cell product being the polyethylene glycol-modified immune cells described above; and (b) the immune checkpoint inhibitor being a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody. In a fourth aspect of the invention, a pharmaceutical composition for treating solid tumors is provided, comprising: (i) the polyethylene glycol-modified immune cells; and (ii) an immune checkpoint inhibitor, which is a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention provides an immune cell product to enhance the efficacy of cell therapy. The polyethylene glycol-modified immune cells form a steric hindrance layer, dynamically covering the cell surface. This charge shielding effect simultaneously addresses three core issues: blocking efficiency, safety, and universality (covering multiple EV subsets such as CD9 / CD63 / CD81), effectively improving the effectiveness of CAR-T cells. PEG modification is applied to the immune cell surface via chemical coupling or physical adsorption, reducing EV adhesion and enhancing cytotoxic activity. In vitro experiments: 10K PEG modification increased the cytotoxicity of CAR-T cells against CAL27 cells overexpressing mesothelin (MSLN) (CAL27-MSLN cells) from 35% to 85%. Animal models: Tumor growth inhibition rate increased from 42% to 89%. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 PEG-modified CAR-T cells block the adhesion of tumor extracellular vesicles to them. a, PEG modification a) Schematic diagram of co-culturing MSLN-CAR-T cells with tumor cells; b) Laser confocal microscopy experiments showed that PEG-modified CAR-T cells significantly inhibited the adhesion of small extracellular vesicles (sEVs) derived from tumor cells (CAL27-MSLN cells) to CAR-T cells; c) Flow cytometry experiments showed that PEG modification effectively inhibited the adhesion of tumor cell-derived exosomes to CAR-T cells.

[0026] Figure 2PEG modification enhances the ability of MSLN-CAR-T cells to resist tumor cell extracellular vesicles. a) After PEG modification, MSLN-CAR-T cells were co-cultured with tumor cell extracellular vesicles for 48 hours, and the expression of the proliferation marker Ki-67 was detected in the lost cells. b) After PEG modification, MSLN-CAR-T cells were co-cultured with tumor cell extracellular vesicles for 48 hours, and the expression of the cytotoxic marker GzmB was detected in the lost cells. c) PEG modification effectively inhibited the transfer of antigens to CAR-T cells by extracellular vesicles derived from tumor cells (CAL27-MSLN cells). d) PEG modification effectively inhibited the self-cannibalization of CAR-T cells mediated by tumor cell-derived extracellular vesicles.

[0027] Figure 3 PEG-modified MSLN-CAR-T cells inhibit their ability to cytosolic tumor cell antigens. a, Left panel: Flow cytometry showing the transfer of DiO fluorescent dye from DiO-labeled CAL27-MSLN cells to co-cultured (4 hours) MSLN-CAR-T cells pretreated with 10K PEG or untreated. Right panel: Percentage of DiO-positive MSLN-CAR-T cells under specified treatment. b, Left panel: Flow cytometry showing the MSLN level on the cell surface of MSLN-CAR-T cells after co-culturing with CAL27-MSLN cells for 4 hours. Right panel: Percentage of MSLN-positive CAR-T cells under specified treatment. c, Percentage of DiO-positive untransduced (UTD) T cells under specified treatment. d, Percentage of MSLN-positive untransduced (UTD) T cells under specified treatment, analyzed by flow cytometry. e, Left panel: Flow cytometry view of apoptotic MSLN-CAR-T cells after the co-culture experiment described in panel a. Right panel: Percentage of apoptotic MSLN-CAR-T cells under specified treatments.

[0028] Figure 4PEGylation of MSLN-CAR-T cells enhances their in vivo therapeutic effect. a) Schematic diagram illustrating animal experiments: After constructing a CAL27 animal model overexpressing MSLN, PBS, untransfected T cells (UTD T cells), MSLN-CAR-T cells, and PEG-modified MSLN-CAR-T cells were injected when the tumor size reached approximately 150 mm³. b) Tumor growth curves showing tumor growth under different treatments. c) Tumor weight harvested from mice receiving the specified treatments. d) Survival curves of mice receiving the specified treatments. e) Representative immunofluorescence images of PEG-treated and untreated MSLN-CAR-T cells in tumor tissue. f) Quantitative analysis of the number of MSLN-CAR-T cells in tumors of each group of mice based on immunofluorescence. g) Quantitative analysis of the number of MSLN-CAR-T cells in tumors of each group of mice based on flow cytometry. h and i, the percentages of Ki-67+ (h) and granzyme B (GzmB)+ (i) CAR-T cells quantified by flow cytometry.

[0029] Figure 5 PEG-treated CAR-T cells combined with anti-PD-1 antibody (anti-PD-1 Ab) enhance anti-tumor efficacy. a, Flow cytometry shows the binding of PD-1 antibody on the surface of PEG-treated or untreated MSLN-CAR-T cells. The right side shows the quantitative analysis results of flow cytometry. b, Schematic diagram showing NSG mice carrying CAL27-MSLN tumors treated with MSLN-CAR-T cells or PEG-treated MSLN-CAR-T cells, with or without anti-PD-1 Ab treatment, using six mice in each group. c, Growth curves of CAL27-MSLN tumors in mice with specified treatments. d, Weight of CAL27-MSLN tumors with specified treatments. e, Survival curves of mice carrying CAL27-MSLN tumors and with specified treatments. f, Representative immunofluorescence images of MSLN-CAR-T cells in tumor tissues of different treatment groups. g and h, quantitative analysis of the number of CAR-T cells in tumors: number of cells per square centimeter (g) and per gram (h) of tumor tissue. i and j, Ki-67 quantification by flow cytometry. + (i) and granzyme B (GzmB) + (j) Percentage of CAR-T cells.

[0030] Figure 6 This is a map of the CAR vector. Detailed Implementation

[0031] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0032] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0033] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0034] The following will provide a detailed description of an immune cell product for improving the efficacy of cell therapy, in conjunction with embodiments and experimental data.

[0035] Example 1: Preparation of 5K PEG-modified CAR-T cells

[0036] 1. CAR-T cell preparation: T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donors and activated with CD3 / CD28 magnetic beads; subsequently, lentiviral transduction was performed to create a CAR construct targeting MSLNs (scFv: CD8α hinge region + 4-1BB co-stimulatory domain; the CAR vector map is shown below). Figure 6 The sequence shown is referenced in Addgene: CAT#:217990, and the nucleotide sequence is shown in SEQ ID NO.2. The specific preparation method is as follows: (1) Day 0: T cell isolation and activation

[0037] PBMC isolation: PBMCs are isolated from peripheral blood using density gradient centrifugation methods such as Ficoll-Paque™ PLUS.

[0038] T-cell enrichment: Purified T-cells were further enriched from PBMCs using a T-cell negative selection magnetic bead kit, removing unwanted cells such as B cells, monocytes, and NK cells. The number of viable cells was counted using a cell counting chamber (trypan blue exclusion method).

[0039] T cell activation: Resuspend the enriched T cells in preheated complete T cell culture medium and adjust the cell density to 0.5-1×10⁶ cells / day. 6Add anti-CD3 / CD28 magnetic beads at a ratio of 1:1 to 3:1 (optimization required) and mix gently. Add IL-2 (final concentration 300-600 IU / mL). Seed cells in cell culture plates / flasks and incubate at 37°C in a 5% CO2 incubator.

[0040] (2) Days 1-2: Lentiviral transduction preparation

[0041] Virus plate coating (if using Retrolectin): Dilute Retrolectin to a working concentration (e.g., 10-20 µg / mL) with PBS. Add to a non-tissue culture plate (e.g., 500 µL / well in a 24-well plate), and coat overnight at 4°C or incubate at room temperature for 2 hours. After coating, discard the Retrolectin solution and block with PBS containing 2% BSA for 30 minutes. After blocking, wash once with PBS.

[0042] (3) Days 2-3: Lentiviral transduction

[0043] Cell preparation: Examine the activated T cells. At this point, the cells should begin to aggregate into clusters and increase in size.

[0044] Transduction procedure: Remove cells from the incubator, gently resuspend cells, and count them. Centrifuge to collect cells, discard the old culture medium, and resuspend cells in fresh, preheated complete culture medium (containing IL-2) to a final volume of 0.5–1 × 10⁻⁶. 6 High density of cells / mL.

[0045] For retrolectin-coated plates: Add the prepared viral suspension (MOI = 3-10, requires pre-experiment optimization) to the coated wells. Centrifuge at 500-1000 xg for 30 minutes ("centrifugation-based plating" can improve transduction efficiency). After centrifugation, carefully aspirate a portion of the viral supernatant, leaving approximately 0.5 mL, and then add the cell suspension to the wells.

[0046] For the Polybrene method: Mix cells, virus solution, and Polybrene (final concentration 5-8 µg / mL) directly and add to the wells. Gently mix and incubate at 37°C in a 5% CO2 incubator.

[0047] Culture: 16-24 hours after transduction, carefully observe the cell status and replenish with fresh, preheated complete culture medium (containing IL-2) or perform a half-volume medium change to remove residual viral fragments and reduce polybrene toxicity.

[0048] (4) Days 4-7: CAR-T cell expansion: Change half or all of the medium every 2-3 days according to the cell density, and always maintain the cell density at 0.5-2×10⁻⁶. 6 Maintain a concentration of cells / mL and supplement with fresh IL-2. Observe cell morphology, aggregation, and culture medium color daily.

[0049] Magnetic bead removal (optional): Around days 5-7 of culture, when cells are proliferating in large numbers, CD3 / CD28 magnetic beads can be removed by magnetic racking to avoid T cell exhaustion caused by continuous stimulation.

[0050] (5) Days 7-14: Harvest and quality inspection to obtain activated CAR-T cells.

[0051] Harvesting cells: When the cell number reaches the expected level (usually after 7-14 days of culture), collect the cell suspension and centrifuge.

[0052] Cell counting and cryopreservation: Resuspend cells in serum-free cryopreservation solution, count them, and then cryopreserve them in liquid nitrogen or use them directly in subsequent experiments.

[0053] (6) Quality inspection: Transduction efficiency: A small number of cells were taken and the CAR positivity rate was detected by flow cytometry (e.g., using anti-idiotype antibodies or antibodies targeting scFv).

[0054] Cell viability: Assay using trypan blue staining, viability should be >90%.

[0055] Sterility testing: Ensure that cell cultures are free from microbial contamination such as bacteria and fungi.

[0056] 2. PEG modification: 5 kDa PEG-NHS ester (purity >99%) was dissolved in PBS (pH 8.5) and mixed with the obtained activated CAR-T cells at a ratio of 10 mg / mL. The mixture was incubated at 4°C for 2 hours. After terminating the reaction, the cells were washed three times, and the modification efficiency (coverage >80%) was verified by flow cytometry.

[0057] Example 2: Preparation of CAR-T cells modified with 10K PEG

[0058] 1. CAR-T cell preparation is the same as in Example 1.

[0059] 2. PEG modification: 10 kDa PEG-NHS ester (purity >99%) was dissolved in PBS (pH 8.5) and mixed with activated CAR-T cells at a ratio of 10 mg / mL. The mixture was incubated at 4°C for 2 hours. After terminating the reaction, the cells were washed three times, and the modification efficiency (coverage >80%) was verified by flow cytometry.

[0060] Example 3: Preparation of 100K PEG-modified CAR-T cells

[0061] 1. CAR-T cell preparation is the same as in Example 1.

[0062] 2. PEG modification: 100 kDa PEG-NHS ester (purity >99%) was dissolved in PBS (pH 8.5) and mixed with activated CAR-T cells at a ratio of 10 mg / mL. The mixture was incubated at 4°C for 2 hours. After terminating the reaction, the cells were washed three times, and the modification efficiency (coverage >80%) was verified by flow cytometry.

[0063] Experimental Example 1: Effect of PEG molecular weight on CAR-T cell function

[0064] 1. Experimental Objective

[0065] To explore the effects of different molecular weight PEGs (1K, 5K, 10K, 100K Da) on the adhesion blocking effect and antitumor activity of CAR-T cells EVs, and to screen the optimal molecular weight range.

[0066] 2. Experimental Materials

[0067] PEG material: 5K, 10K, 100K Da PEG-NHS ester (Sigma-Aldrich), purity >99%.

[0068] Cells and reagents: Preparation of MSLN-CAR-T cells; CAR carrier design and viral packaging (1) Targeted structure design Modular element: Targeting domain: The nucleotide sequence of scFv (single-chain antibody) against antigens such as MSLN is shown in SEQ ID NO.1: Signaling domain: Mouse CD8α hinge region + transmembrane region (TM) Co-stimulatory domain: 4-1BB or CD28 reporter gene: Whether to insert EGFP / T2A self-cleaving peptide as needed (for transduction efficiency tracking) The CAR vector's spectrum (e.g.) Figure 6 (as shown in the image) and the entire sequence reference Addgene: CAT#:217990, the nucleotide sequence is shown in SEQ ID NO.2.

[0069] (2) Virus production and purification Retrovirus (mouse system): Packaging cells: Phoenix Eco cells (ATCC CRL-3214); Virus concentration: 70,000×g ultracentrifugation, titer ≥1×10⁻⁶. 8 IU / mL Lentiviral (human system): using the third-generation VSV-G envelope system, PEG-it TM Concentrated titer > 5 × 10 7 TU / mL (3) CAL27-derived EVs (NTA verified particle size 100-150 nm); Flow cytometry antibodies: CD63-FITC (EVs label), CD3-APC (CAR-T label).

[0070] Table 1

[0071]

[0072] 3. Experimental Procedure

[0073] PEG modification: CAR-T cells were incubated in 100 nM PEG solution at 37°C for 20 minutes and washed 3 times.

[0074] EV adhesion detection: PKH26-labeled CAL27-MSLN EVs (50 μg / mL) were co-incubated with modified CAR-T for 4 hours, and the adhesion rate was detected by flow cytometry (FITC-EVs vs. PE-CAR-T).

[0075] Tumor killing experiment: Modified CAR-T cells and CAL27-MSLN cells were co-cultured at a 1:1 ratio, and the killing efficiency was detected by LDH release assay.

[0076] 4. Results

[0077] Table 2

[0078] The PEG group significantly reduced EV adhesion (p<0.001) and improved killing efficiency (p<0.01). Due to excessive steric hindrance, the adhesion rate of the 100K PEG group rebounded and the killing efficiency decreased (comparable to the effect of the antibody blocking regimen).

[0079] PEG materials with molecular weights of 5K, 10K, and 100K can all effectively reduce the adhesion of exosomes derived from oral squamous cell carcinoma to T cells. Interestingly, PEG materials enhance the killing effect of HER2-CAR-T cells on tumor cells, suggesting that the promoting effect of PEG materials on CAR-T cells may depend on blocking the inhibitory effect of extracellular vesicles. Since 100K PEG materials weaken the killing ability of CAR-T cells on tumor cells in in vitro experiments, and 10K PEG materials have a more significant promoting effect on the killing ability of CAR-T cells, the effect of 10K PEG materials on the adhesion of extracellular vesicles to CAR-T cells was investigated in Example 1 of this application. It was found that CAR-T cells modified with 10K PEG materials showed a more reduced adhesion ability to tumor extracellular vesicles. Figure 1 b,c).

[0080] Experimental Example 2: The blocking effect of PEG-modified CAR-T cells on the adhesion of tumor extracellular vesicles (EVs)

[0081] 1. Experimental Objective

[0082] To verify the inhibitory effect of PEG modification on the adhesion of EVs on the surface of CAR-T cells.

[0083] 2. Experimental Materials

[0084] Cell source: CAR-T cells: Human MSLN-CAR-T cells (see references: Wenqun Zhong et al, Nature Cancer, 2025; Wenqun Zhong et al, Cancer Research, 2023, the scFv sequence used is shown in SEQ ID NO.2); Tumor cells: CAL27-MSLN (human head and neck squamous cell carcinoma cell line, EVs carrying PD-L1 / TGF-β).

[0085] Reagents: PEG-NHS ester (10K Da, purity >99%, Sigma-Aldrich); EVs fluorescently labeled with dye PKH26 (Sigma-Aldrich).

[0086] 3. Experimental Procedure

[0087] CAR-T cell preparation: Isolate healthy donor PBMCs, activate them with CD3 / CD28 magnetic beads, transduce the MSLN-CAR construct using lentivirus, and amplify to 1×10⁻⁶. 6 / mL.

[0088] 4. PEG modification: The 5K Da PEG-NHS ester prepared in Example 1 and the 10K Da PEG-NHS ester prepared in Example 2 were dissolved in PBS (pH 8.5), mixed with CAR-T cells at 100 nM, incubated at 4°C for 2 hours, and washed 3 times.

[0089] 5. EV separation: EVs were enriched in CAL27-MSLN cell culture supernatant by ultracentrifugation (100,000×g, 90 min), and the particle size was verified by NTA (100-150 nm).

[0090] 6. EV adhesion experiment: PKH26-labeled CAL27-MSLN EVs (50 μg / mL) were co-incubated with PEG-modified / unmodified CAR-T cells for 4 hours, and the adhesion rate was detected by flow cytometry (FITC-EVs vs. PE-CAR-T).

[0091] 4. Experimental Results

[0092] Figure 1 a: PEG modification of the CAR-T cell surface forms a physical barrier, blocking EV adhesion.

[0093] Figure 1 c: The adhesion rate of EVs in the PEG-modified group decreased from 38.8% to 19.0% (inhibition rate 51.03%, p=0.0015).

[0094] Experimental Example 3: PEG Modification Inhibits CAR-T Cell Cytokinesis of Tumor Cells

[0095] 1. Experimental Objective

[0096] To verify the inhibitory effect of PEG modification on CAR-T cells cytotoxic tumor cells.

[0097] 2. Experimental Materials

[0098] Cell source: CAR-T cells: Human MSLN-CAR-T cells are described above; Tumor cells: CAL27-MSLN (human head and neck squamous cell carcinoma cell line, EVs carrying PD-L1 / TGF-β).

[0099] Reagents: PEG-NHS ester (10K Da, purity >99%, Sigma-Aldrich); Cell membrane fluorescent labeling dye DiO (Beyotime).

[0100] 3. Experimental Procedure

[0101] CAR-T cell preparation: Isolate healthy donor PBMCs, activate them with CD3 / CD28 magnetic beads, transduce the MSLN-CAR construct using lentivirus, and amplify to 1×10⁻⁶. 6 / mL.

[0102] 4. PEG modification: The 5K Da PEG-NHS ester prepared in Example 1 and the 10K Da PEG-NHS ester prepared in Example 2 were dissolved in PBS (pH 8.5), mixed with CAR-T cells at 100 nM, incubated at 4°C for 2 hours, and washed 3 times.

[0103] 5. DiO markers in tumor cells: CAL27-MSLN cells were incubated in 5 μM DiO solution at 37°C for 20 minutes and washed three times.

[0104] 6. Co-culture experiment: DiO-labeled CAL27-MSLN cells were co-incubated with PEG-modified / unmodified CAR-T cells at a ratio of 2:1 for 4 hours, and cell phagocytosis efficiency was detected by flow cytometry.

[0105] 4. Experimental Results

[0106] Figure 3 ab: PEG-modified CAR-T cells significantly enhance their cytotoxic effect on tumor cells.

[0107] Figure 3 cd:PEG modification of untransfected T cells (UTD T cells) has no significant effect on the cytokinesis of tumor cells.

[0108] Figure 3 e: PEG-modified CAR-T cells significantly inhibited CAR-T cell self-killing caused by cytokinesis.

[0109] Experiment Example 4: PEG Modification Enhances the Antitumor Activity of CAR-T Cells

[0110] 1. Experimental Objective

[0111] To verify the effect of PEG modification with 10K on enhancing the proliferation, killing and anti-exhaustion capabilities of CAR-T cells.

[0112] 2. Experimental Materials

[0113] Reagents: Tumor EVs: CAL27-MSLN-derived EVs (50 μg / mL); Flow cytometry antibodies: Ki-67-APC, GzmB-FITC, Annexin V-PE.

[0114] 3. Experimental Procedure

[0115] EV co-culture: Modified / unmodified CAR-T cells were co-cultured with CAL27-MSLN EVs at a 1:1 ratio for 48 hours.

[0116] Functional testing: Proliferation capacity: flow cytometry detection of Ki-67 + Cell ratio; Cytotoxicity: Flow cytometry detection of GzmB expression levels; Apoptosis rate: Annexin V was used to detect the apoptosis rate of CAR-T cells.

[0117] 4. Experimental Results

[0118] Figure 2 ab: PEG-modified Ki-67 + (42.8% vs. 35.93% of the unmodified group) and GzmB + (36.17% vs. 20.47%) A significant improvement; Figure 2 The antigen transfer rate mediated by cd:EVs decreased from 11.57% to 5.66%, and the self-cannibalization apoptosis rate decreased from 35.03% to 23.33%.

[0119] Experimental Example 5: In vivo antitumor effect of PEG-modified CAR-T cells

[0120] 1. Experimental Objective

[0121] To verify that PEG modification helps improve the therapeutic effect of CAR-T cells on solid tumors.

[0122] 2. Experimental Materials

[0123] Animal models: NOD / SCID mice were subcutaneously inoculated with CAL27 cells overexpressing MSLN (5 × 10⁻⁶ cells). 6 cells / each).

[0124] 3. Grouping and drug administration (animal experimental design, such as...) Figure 3 (as shown in a) Control group: PBS; UTD group: T cells that have not been transduced with CAR (5×10) 6 cells / each); Unmodified CAR-T group: MSLN-CAR-T cells (5×10⁻⁶) 6 cells / each); PEG-modified group: 10K PEG-modified CAR-T cells (5×10⁻⁶) 6 cells / each).

[0125] 4. Experimental Procedure

[0126] Treatment plan: When the tumor volume reaches ~150 mm³, administer via tail vein injection, and monitor tumor volume and survival every 4 days.

[0127] Testing indicators: Tumor growth curve: Tumor volume is measured using vernier calipers; Survival period: Recorded until the maximum tumor diameter >1.5cm or death; Tumor tissue examination: Detecting the infiltration and activation status of CAR-T cells in tumor tissue.

[0128] 5. Experimental Results

[0129] Figure 4 The tumor volume in the PEG-modified group was reduced by 42.73% compared to the unmodified group (p=0.0374), and the survival time was significantly prolonged. The level of CAR-T cell infiltration within the tumor increased by more than 100%, and the activation level increased by more than 100%. In vivo experiments demonstrated that PEG-modified CAR-T cells have superior tumor-suppressive effects.

[0130] Experimental Example 6: In vivo antitumor effect of PEG-modified CAR-T cells combined with PD-1 antibody

[0131] 1. Experimental Objective

[0132] To verify the effect of PEG-modified CAR-T cells combined with PD-1 antibody on the treatment of solid tumors.

[0133] 2. Experimental Materials

[0134] Animal models: NOD / SCID mice were subcutaneously inoculated with CAL27 cells overexpressing MSLN (5 × 10⁻⁶ cells). 6 cells / each).

[0135] 3. Grouping and drug administration (animal experimental design, such as...) Figure 3 (as shown in a) Control group: PBS; UTD group: T cells that have not been transduced with CAR (5×10) 6 cells / each); PD-1 antibody group: PD-1 antibody (200 μg / animal) Unmodified CAR-T group: MSLN-CAR-T cells (5×10⁻⁶) 6 cells / each); PEG-modified group: 10K PEG-modified CAR-T cells (5×10⁻⁶) 6 cells / each); Unmodified CAR-T+PD-1 antibody group: MSLN-CAR-T cells (5×10 6 (cells / animal) + PD-1 antibody (200 μg / animal) PEG-modified CAR-T+PD-1 antibody group: 10K PEG-modified CAR-T cells (5×10 6 (cells / animal) + PD-1 antibody (200 μg / animal).

[0136] 4. Experimental Procedure

[0137] Treatment plan: When the tumor volume reaches ~150 mm³, administer via tail vein injection, and monitor tumor volume and survival every 4 days.

[0138] Testing indicators: Tumor growth curve: Tumor volume is measured using vernier calipers; Survival period: Recorded until the maximum tumor diameter >1.5cm or death; Tumor tissue examination: Detecting the infiltration and activation status of CAR-T cells in tumor tissue.

[0139] 5. Experimental Results

[0140] Figure 5 The tumor volume in the PEG-modified CAR-T + PD-1 antibody group was reduced by 73.76% compared with the unmodified CAR-T group (p<0.0001), the survival time was significantly prolonged, the level of CAR-T cell infiltration in the tumor increased by more than 10 times, and the activation level was significantly increased by 0.5 times. In vivo experiments demonstrated that CAR-T cells pretreated with PEG and then combined with PD-1 antibody have a better tumor-suppressive effect.

[0141] Experiment 7: Optimization of Co-incubation Conditions for PEG and CAR-T Cells

[0142] 1. Experimental Objective

[0143] Optimize the co-incubation parameters (temperature, time, buffer) of 10K PEG with CAR-T cells to improve modification efficiency and maintain cell viability.

[0144] Table 4

[0145]

[0146] Experimental steps

[0147] PEG modification: CAR-T cells were co-incubated with PEG-NHS ester (100 nM) according to grouping conditions (temperature / time / buffer). Modification efficiency assay: Flow cytometry was used to detect surface PEG coverage (FITC-PEG vs. PE-CD3). Cell viability verification: CCK-8 assay was used to detect proliferation capacity (72 hours of culture); LDH release assay was used to detect cytotoxicity (co-cultured with tumor target cells).

[0148] Table 5

[0149] Condition C (25°C, 2 hours) achieves the optimal balance between coverage (62%), activity (88%), and lethality (87%). In summary, one or more technical solutions of this application possess at least the following advantages: 1. Technological breakthrough in PEG-modified immune cells

[0150] Table 6

[0151] 2. Synergistic effect of combination therapy (PEG-treated immune cells + immune checkpoint inhibitors)

[0152] Table 7

[0153] This application significantly improves the therapeutic efficacy of CAR-T and other cell therapies for solid tumors by blocking the interaction between tumor extracellular vesicles (EVs) and immune cells. In a human head and neck squamous cell carcinoma animal model, this product achieved a tumor inhibition rate of up to 89%, demonstrating significant potential for clinical translation.

[0154] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. An immune cell product for improving the effect of cell therapy, characterized by, The product comprises polyethylene glycol modified immune cells, wherein: The immune cells are CAR-T cells, CAR-NK cells or TILs cells. The polyethylene glycol is modified to the surface of the immune cells by chemical coupling or physical adsorption.

2. The immune cell product for improving the effect of cell therapy according to claim 1, characterized in that, The molecular weight of the polyethylene glycol is 5K-100K Da.

3. The immune cell product for improving the effect of cell therapy according to claim 1, characterized in that, The chemical coupling is one of the following ways: NHS ester mediated lysine targeted modification; Maleimide reaction mediated cysteine targeted modification.

4. The immune cell product for improving the effect of cell therapy according to claim 1, characterized in that, The method for modifying the chemical coupling comprises: Dissolving PEG-NHS ester in PBS to obtain a PEG solution; Mixing the PEG solution with the immune cells.

5. The immune cell product for improving the effect of cell therapy according to claim 1, characterized in that, The immune cells are one of the following: CAR-T cells, CAR-NK cells or TILs cells targeting MSLN, HER2 or EGFR.

6. The immune cell product for improving the effect of cell therapy according to claim 1, wherein The product further comprises a pharmaceutically acceptable carrier or a cryoprotective agent.

7. Use of an immune cell product for improving the effect of cell therapy according to any one of claims 1-6 in the preparation of a medicament for treating solid tumors.

8. Use according to claim 7, characterized in that, The solid tumors comprise at least one of the following: pancreatic cancer, glioblastoma.

9. Use of an immune cell product in combination with an immune checkpoint inhibitor in the manufacture of a medicament for the treatment of a solid tumor, characterized in that, (a) The immune cell product is the polyethylene glycol modified immune cells according to any one of claims 1-6; (b) The immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody or a CTLA-4 antibody.

10. A pharmaceutical composition for treating solid tumors, characterized by, Comprise: (i) The polyethylene glycol modified immune cells according to any one of claims 1-6; (ii) An immune checkpoint inhibitor which is a PD-1 antibody, a PD-L1 antibody or a CTLA-4 antibody.

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