Application of timosaponin AIII in preparation of curative effect enhancer for CAR-T cell therapy and product for overcoming drug resistance

Abstract

The invention discloses application of timosaponin AIII in preparation of a curative effect enhancer for CAR-T cell therapy and a product for overcoming drug resistance. The invention proves that TAIII is an allosteric inhibitor of an adenosine A2A receptor, and blocks a downstream signal channel through allosteric combination with A2AR, so that expression of a transcription factor FoxP3 is inhibited, Tregs in a CAR-T product is selectively cleared, immunosuppression of the Tregs on effector T cells is relieved, meanwhile, the proportion of central memory T cells is increased, and amplification, durability and cytokine secretion functions of the CAR-T cells are enhanced. The invention further provides a CAR-T cell composition containing the TAIII, and the TAIII and the CAR-T cells are combined or used as a pretreatment agent, so that the tumor removal capability can be remarkably enhanced, and recurrence after treatment can be effectively prevented. The invention provides a safe and effective new strategy for enhancing the anti-tumor activity of the CAR-T cells and overcoming the drug resistance of the CAR-T therapy.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to the application of Anemarrhena saponin AIII in the preparation of efficacy enhancers for CAR-T cell therapy and products for overcoming drug resistance. Background Technology

[0002] Chimeric antigen receptor (CAR)-T cell therapy has demonstrated significant efficacy in treating relapsed / refractory B-cell malignancies. However, this therapy still faces significant challenges, with approximately 60% of patients eventually experiencing relapse or developing resistance in clinical practice. Previous studies have shown that immunosuppressive molecules and cellular subsets are key factors limiting the efficacy of CAR-T therapy. The immunosuppressive tumor microenvironment (ITME), including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and hypoxia-driven molecules such as adenosine and IL-10, significantly weakens the activity and persistence of effector T cells, representing a major obstacle to the efficacy of CAR-T cell therapy.

[0003] Treg cells maintain self-tolerance and immune homeostasis by overexpressing the transcription factor FoxP3, which is crucial for Treg function. Tumor-infiltrating Treg cells (TITRs) have a unique immunosuppressive effect in the tumor microenvironment, weakening anti-tumor immune responses and thus promoting tumor immune escape. Two recent studies, through comprehensive single-cell analysis, have revealed the role of CAR-T cells in mediating CAR-T drug resistance. Using single-cell proteomics and transcriptomics, Good et al. demonstrated that higher levels of Helios... + FoxP3 + CAR-Treg cells are closely associated with poor clinical outcomes, providing insights into why in vivo CAR-T cell expansion is not always correlated with clinical response. Based on single-cell transcriptomics and TCR sequencing data, Haradhvala et al. analyzed samples from 32 patients who received CAR-T cell infusion therapy and found that high levels of CAR-Tregs led to clinical non-response, and that adding only 5% CAR-Tregs to CAR-T cell products was sufficient to reduce the expansion of conventional CAR-T (CAR-Tconv) and induce relapse in NSG mice implanted with JeKo-1 lymphoma. These findings reveal the immunosuppressive effect of CAR-Tregs on CAR-T therapy and suggest that effective Treg clearance strategies can optimize CAR-T efficacy.

[0004] Current strategies under development primarily involve combinations of immune checkpoint inhibitors (ICIs) and targeted therapies, such as CpG oligonucleotides combined with low-dose anti-OX40 / CTLA-4 therapy, or blocking chemokine pathways like anti-CCR4. Additionally, antibodies targeting Treg cell surface receptors, such as anti-APRIL and anti-CD122, have been explored. These neutralizing monoclonal antibodies (mAbs) are often used in combination with ICIs and have shown potential to restore immune tolerance in animal models. However, no clinically effective strategy has yet been developed to selectively clear CAR-Tregs to enhance CAR-T efficacy. A significant limitation is that existing anti-Treg antibodies lack selective targeting capabilities, potentially leading to unintended clearance of anti-tumor effector T cell populations or adverse reactions. Furthermore, due to limitations in monotherapy or inadequate drug delivery methods, some strategies have shown limited or negligible efficacy. Therefore, new methods to improve Treg targeting specificity and enhance anti-tumor efficacy are still under development.

[0005] Natural products, characterized by their structural diversity and abundance of bioactive compounds, are a vital source for drug discovery and lead compound development for treating major diseases. Compared to synthetic compounds, natural products typically exhibit better bioavailability, biodegradability, and safety profiles. Numerous successful cases demonstrate that lead drugs can be discovered from natural product extracts and further optimized into more effective medications. With increasing emphasis on functional assays and phenotypic screening, natural product-based drug discovery has regained renewed interest. The combination of CAR-T therapy with small molecule immunomodulators offers advantages such as high efficiency, flexibility, and personalization. Recent studies suggest that using small molecule immunomodulators to clear Tregs and enhance the cytotoxicity of CAR-T cells against tumor cells may be a promising strategy for overcoming treatment resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a novel application of Anemarrhena asphodeloides saponin AIII (TAIII) in enhancing the anti-tumor activity of CAR-T cells and preventing recurrence. TAIII, a natural steroidal saponin, has been proven to be a novel allosteric inhibitor of adenosine A2A receptor (A2AR). It competitively binds to the cholesterol-sensitive motif of A2AR, blocking the downstream cAMP-PKA-CREB signaling pathway, thereby inhibiting the expression of transcription factor FoxP3 and thus suppressing the differentiation and function of regulatory T cells (Tregs). Simultaneously, it selectively eliminates regulatory T cells (CAR-Tregs) in CAR-T products, relieving their immunosuppression of effector T cells. This effect is A2AR-dependent and antigen-specific, effectively relieving Treg-mediated immunosuppression, while simultaneously increasing the proportion of central memory T cells and the secretion of effector cytokines, optimizing CAR-T cell quality, and enhancing their proliferation and persistence. In preclinical models, TAIII, when used in combination with CAR-T cells or as a pretreatment agent, can significantly enhance tumor clearance, prolong survival, and prevent recurrence, providing a safe and effective new strategy for enhancing the anti-tumor activity of CAR-T cells and overcoming CAR-T therapy resistance.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides the application of Anemarrhena saponin AIII in the preparation of efficacy enhancers for CAR-T cell therapy and / or in the preparation of products that overcome drug resistance to CAR-T cell therapy.

[0008] Specifically, the CAS number for Anemarrhenasaponin AIII is 41059-79-4, and its chemical structural formula is shown in the attached figure. Figure 1 As shown in d.

[0009] Furthermore, the therapeutic enhancer has at least one of the following effects: A1) Inhibits A2AR activity and FoxP3 transcription, reduces the proportion of regulatory T cells, and relieves immunosuppression on effector T cells; A2) Increase the proportion of central memory T cells and the secretion of cytokines IFN-γ, IL-2, and TNF-α; A3) Enhances the cytotoxicity of CAR-T cells; A4) Enhance the efficacy of CAR-T cells in preventing tumor recurrence.

[0010] Furthermore, the CAR-T cells are treated with Anemarrhena saponin AIII before administration or are administered in combination with Anemarrhena saponin AIII during administration.

[0011] Furthermore, Anemarrhena saponin AIII competitively binds to the cholesterol-sensitive motif of A2AR, blocking the downstream cAMP-PKA-CREB signaling pathway, thereby inhibiting FoxP3 transcription and regulatory T cell differentiation, reducing the proportion of regulatory T cells, relieving their immunosuppression on effector T cells, and increasing the proportion of central memory T cells and cytokine secretion, thus enhancing the cytotoxicity of CAR-T cells and / or preventing tumor recurrence.

[0012] This invention also provides a method for preparing functionally enhanced CAR-T cells, which involves treating CAR-T cells with anemarrhena saponin AIII to obtain a therapeutically enhanced CAR-T cell population with a significantly reduced proportion of Tregs.

[0013] Furthermore, the concentration of Anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

[0014] The present invention also provides the application of Anemarrhena saponin AIII in combination with CAR-T cells and / or Anemarrhena saponin AIII in combination with PD-1 in the preparation of antitumor drugs, wherein the tumor is selected from hematologic malignancies or solid tumors, preferably, the solid tumors include lymphoma and / or colorectal cancer, and the hematologic malignancies include lymphocytic leukemia; preferably, the concentration of Anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

[0015] The present invention also provides a composition for CAR-T cell therapy, comprising anemarrhena saponin AIII and CAR-T cells, wherein the concentration of anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

[0016] This invention also provides the application of Anemarrhena saponin AIII in the preparation of selective A2AR allosteric inhibitors, wherein Anemarrhena saponin AIII affects A2AR function by competing for cholesterol and allosterically binding to key residues.

[0017] The present invention also provides a method for reducing the proportion of regulatory T cells in CAR-T cells in vitro for non-therapeutic purposes, wherein CAR-T cells are treated with anemarrhena saponin AIII, thereby significantly reducing the proportion of Tregs. Preferably, the concentration of anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

[0018] Beneficial effects: 1. Precise targeting and novel mechanism: This invention reveals for the first time that TAIII is a novel allosteric inhibitor of adenosine A2A receptor (A2AR). It competitively binds to the cholesterol-sensitive motif of A2AR, blocking downstream immunosuppressive signaling pathways, thereby precisely targeting and clearing the immunosuppressive components CAR-Tregs in CAR-T products.

[0019] 2. High selectivity and good safety: TAIII has A2AR-dependent and antigen-specific effects. It mainly eliminates inhibitory CAR-Tregs without impairing the function and activity of effector T cells, avoiding non-specific immune cell damage that may be caused by traditional antibody drugs, thus showing higher selectivity and potential safety.

[0020] 3. Significant efficacy and strong synergistic effect: In various preclinical models (such as colorectal cancer, lymphoma and leukemia models), TAIII can significantly enhance the tumor killing ability of CAR-T cells, promote the formation of central memory T cells, and prolong the survival time of CAR-T cells in vivo, thereby effectively inhibiting tumor growth and preventing recurrence after treatment.

[0021] 4. Dual Optimization for Enhanced Product Quality: TAIII can be used not only as a combination drug after CAR-T cell infusion, but also as a pretreatment during CAR-T cell preparation. This "dual" application strategy optimizes the quality of CAR-T products from the source, reducing their immunosuppressive properties and enhancing their "fighting power," representing an innovative production optimization process.

[0022] 5. Naturally derived with pharmaceutical potential: TAIII is a natural product with advantages such as a well-defined structure and good biocompatibility. Its entry into clinical trials as an anti-wrinkle agent signifies that its safety in humans has been preliminarily verified, laying a solid foundation for its rapid transformation into an adjuvant for tumor immunotherapy.

[0023] In summary, this invention provides a novel strategy for enhancing the efficacy of CAR-T therapy by selectively clearing CAR-Tregs. It has multiple advantages, including precise targeting, significant efficacy, high safety, and flexible application, providing a powerful tool for solving the clinical challenge of CAR-T therapy resistance. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is the experimental result of identifying Anemarrhena asphodeloides saponin AIII (TAIII) as a Treg function inhibitor through high-throughput functional screening in an embodiment of the present invention. Figure 1 a is a schematic diagram of the construction and screening process of the Treg functional reporter gene system; Figure 1 b is the preliminary screening results, showing TAIII as a candidate compound; Figure 1 c represents the detection result of IL-2 secretion level in human peripheral blood mononuclear cells (PBMCs) after TAIII treatment; Figure 1 d represents the chemical structural formula of TAIII; Figure 1 e represents the result of TAIII reversing FoxP3-mediated IL-2 transcriptional repression; Figure 1 f represents the detection results of TAIII inhibiting Treg differentiation and the expression of functional markers (FoxP3, IL2RA, CTLA4) mRNA in a dose-dependent manner; Figure 1 g and Figure 1 h represents the TAIII treatment for FoxP3 + The influence of the proportion of iTreg cells on flow cytometry results.

[0026] Figure 2 In this embodiment of the invention, TAIII eliminates FoxP3. + Experimental results showing that Treg cells enhance the in vitro antitumor efficacy of CAR-T cells. Among them, Figure 2 a is for TAIII treatment of FoxP3 in CAR-T products + Flow cytometry analysis results of the effect of Treg cell proportion; Figure 2 b and Figure 2 c represents the results of the detection of the effect of TAIII treatment on the ability of CAR-T cells to kill Raji or Nalm6 tumor cells and the secretion of related effector cytokines; Figure 2 d represents the results of monitoring the effect of TAIII on the killing dynamics of CAR-T cells using a real-time live-cell imaging system; Figure 2 e represents the results of detecting the effect of TAIII treatment on the secretion of effector cytokines after co-culturing CAR-T cells and Raji cells; Figure 2 f represents the results of detecting the effect of TAIII treatment on FoxP3 mRNA expression levels in CAR-T cells.

[0027] Figure 3 This presents the results of a mechanistic study of TAIII as a novel allosteric inhibitor of A2AR in this invention. Figure 3 a is a schematic diagram of the biotinylated TAIII probe (TAIII-P) used for target identification; Figure 3 b and Figure 3 c represents the detection results of the combination of TAIII and A2AR verified by the pull-down method; Figure 3 d is a pair Figure 3 Quantitative analysis of the results; Figure 3 e represents the detection results of the CREB reporter gene assay to assess the effect of TAIII on the A2AR signaling pathway; Figure 3 f and Figure 3g represents the detection results of the selective inhibitory effect of TAIII on A2AR evaluated by the LANCE cAMP experiment; Figure 3 h represents a computer-generated molecular docking model of TAIII and A2AR; Figure 3 i and Figure 3 j represents the detection results of the binding interaction between TAIII and key A2AR residues, verified by point mutation.

[0028] Figure 4 This represents the experimental results of TAIII activating T cell immune responses in an A2AR-dependent manner by impairing Treg function, as described in this embodiment of the invention. Figure 4 a represents the heatmap analysis results of the effect of TAIII treatment on the T cell transcriptome; Figure 4 b and Figure 4 c represents the validation results of the effect of TAIII treatment on the expression and secretion of T cell effector factor mRNA; Figure 4 d represents the detection results of the effect of TAIII treatment on the mRNA expression of key Treg biomarkers; Figure 4 e and Figure 4 f represents the detection results of the effect of TAIII treatment on Treg-inhibiting T cell function; Figure 4 g and Figure 4 h represents the detection results validating the specificity of TAIII action in A2AR-knockdown Treg; Figure 4 i represents the detection results of TAIII inhibiting CREB recruitment in the FoxP3 promoter region, as confirmed by CUT&RUN experiments.

[0029] Figure 5 This presents the experimental results of TAIII eliminating CAR-Tregs and enhancing CAR-T cell activity by blocking A2AR signaling in an embodiment of the present invention. Figure 5 a- Figure 5 e represents the detection results of the effect of TAIII on reversing the inhibition of CAR-T cell function by the A2AR agonist (NECA); Figure 5 f represents the validation results of A2AR knockdown CAR-T cell construction; Figure 5 g and Figure 5 h represents the test results verifying the TAIII-dependent effect in A2AR knockdown CAR-T cells.

[0030] Figure 6 This presents the experimental results of TAIII inhibiting colorectal cancer tumor growth by blocking A2AR-FoxP3+ Treg-mediated immunosuppression in an embodiment of the present invention. Figure 6 a- Figure 6 k represents the synergistic anti-tumor effect and immune microenvironment analysis results of TAIII combined with anti-PD-1 antibody in the MC38 syngeneic tumor model; Figure 6 l- Figure 6 n represents the analysis results of the CAR-T sensitization effect of TAIII combined with CAR-T in the MC38-mCD19-Luc colorectal cancer model.

[0031] Figure 7 This presents the experimental results of TAIII preventing relapse after CAR-T therapy in various preclinical models through targeting the A2AR-Treg signaling pathway, as described in this invention. Figure 7 a and Figure 7 b represents A2AR in patient-derived CAR-T products. + FOXP3 + Results of correlation analysis between Treg and clinical response; Figure 7 c- Figure 7 h represents the results of the efficacy analysis of TAIII combined with CAR-T in preventing relapse in the Raji-Luc lymphoma model; Figure 7 i- Figure 7 l represents the antitumor effect and T cell subset analysis results of TAIII combined with CAR-T in the Nalm6-Luc leukemia subcutaneous model; Figure 7 m and Figure 7 n represents the antitumor effect and in vivo expansion analysis results of TAIII pretreated CAR-T cells in the Nalm6-Luc leukemia model; Figure 7 o to Figure 7 r represents the validation results of the effect of TAIII treatment on enhancing the function of patient-derived CAR-T cells. Detailed Implementation

[0032] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0033] Cell lines and cell culture: HEK-293T and Jurkat T cell lines were purchased from the ATCC cell bank (Manassas, VA, USA). The Jurkat T cells used in this study were cultured in RPMI medium (Gibco, Thermo Fisher) at 37°C with 5% CO2 at a cell concentration of 2 × 10⁻⁶ cells / year. 5 Up to 5×10 5The culture medium was supplemented with 10% fetal bovine serum (FBS, VivaCell, CO4002) and 1% penicillin / streptomycin. Human T cells or CAR-T cells were cultured in X-VIVO medium (Lonza, 04-418Q) supplemented with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% non-essential amino acids (NEAA, Gibco, 11140050), 1% antibiotic-antifungal agent (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL), and magnetic beads CD3 / CD28 (Gibco, 1132D) to activate and expand human T cells. HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS (VivaCell, C04001) and 1% penicillin / streptomycin.

[0034] Biological reagents: The primary antibodies used for immunoblotting were as follows: β-actin (β-Actin, Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197T, 1:1000), phosphorylated CREB (Ser133; Cell Signaling Technology, 9198T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000). Secondary antibodies (rabbit anti-IgG antibody, M21002L) and (mouse anti-IgG HRP antibody, M21001L) were purchased from Abmart. Streptavidin magnetic beads were purchased from MedChemExpress (MCE, Shanghai). Cell viability assay kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay) was purchased from Vazyme (DD1101-02). Bright-Glo™ assay kit was purchased from Promega (E2620). Immobilon western HRP substrate was purchased from Millipore (WBKLS0500). LANCE TM The cAMP 384 detection kit was purchased from PerkinElmer (AD0262E). The one-step RT-qPCR SYBR Green kit was purchased from Yeasen (11143ES80).

[0035] The compound library included internally synthesized compounds as well as those commercially sourced from MCE and TargetMol (Shanghai). NECA was purchased from MCE (Shanghai). AZD4635, CPI-444, and Timosaponin AIII were purchased from TargetMol (Shanghai). Inhibitors were dissolved in DMSO to prepare 10 mM stock solutions, which were then serially diluted to a final DMSO concentration between 0.2% and 0.5% for cell experiments. All buffer solutions were prepared after pH adjustment using a Mettler Toledo (FE28-Standard) solution.

[0036] Example 1. Identification of Anemarrhena saponin AIII as a Tregs inhibitor using high-throughput screening. Given that the proportion and function of regulatory T cells (Tregs) are crucial to the efficacy of CAR-T therapy, and that the transcription factor FoxP3 is the most important functional marker of Tregs, exerting its effects by inhibiting the transcription of genes such as IL-2, a Treg functional evaluation system was established. The human IL-2 promoter region (-326 to +145, a total of 473 bp) containing the FoxP3a (i.e., FoxP3) binding site was cloned into the Lenti-PGL4 reporter vector. This system was packaged into a lentivirus, and the IL-2 promoter-firefly luciferase (Fluc) reporter gene carrying the puromycin resistance gene was transduced into Jurkat T cells. After puromycin selection, monoclonal cells were isolated, and their luciferase activity was detected under TCR stimulation. The best-performing clones were selected and then introduced into a lentiviral construct overexpressing the FoxP3a gene carrying the BSD resistance gene. After another round of TCR stimulation and screening, the best-performing monoclonal cell line was finally selected, allowing Treg function to be assessed using FLuc fluorescence intensity. Figure 1 a).

[0037] To identify potential Treg inhibitors, this invention performed high-throughput screening on a library of natural products and FDA-approved drugs (containing over 3000 compounds), normalizing the Fluc intensity relative to cell activity after 16 hours of treatment. Figure 1 a). In the initial screening, more than 50 compounds, including anemarrhena saponin AIII (TAIII, CAS No.: 41059-79-4), showed significant regulatory effects on the FOXP3–IL-2 axis. Figure 1 b). The top 10 candidate compounds were then subjected to secondary screening, including dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system and single-dose testing in human peripheral blood mononuclear cells (PBMCs). Figure 1 c), and its toxicity and efficacy were carefully considered. In this process, TAIII, a natural product derived from the traditional Chinese medicine Anemarrhena asphodeloides, became the lead candidate compound (structural formula shown). Figure 1 d), which exhibited potent dose-dependent efficacy and good safety. TAIII significantly reversed FoxP3-mediated IL-2 transcriptional repression in a dose-dependent manner ( Figure 1 e). TAIII is a steroidal saponin whose main reported properties include anticancer, anti-inflammatory, and antithrombotic effects. In inflammation models, TAIII can reduce the levels of cytokines such as IL-6, IL-1β, and TNF-α. However, its role in activating IL-2 or inhibiting human Tregs, as well as the molecular mechanisms underlying its influence on Treg ratios and function, remain largely unclear.

[0038] To verify the effect of TAIII on human Tregs, this invention isolated human CD4. + Naïve T cells were cultured under iTreg polarization conditions. The results showed that, compared with the DMSO control group, both 0.3 μM and 1 μM TAIII significantly impaired Treg differentiation and reduced the expression of key Treg functional markers (FoxP3, CD25 / IL2RA, CTLA4) in a dose-dependent manner. Figure 1 f), and ultimately reduced FoxP3 + The proportion of protein-positive cells was measured using FoxP3 during late differentiation (days 5-8). + Similar results were observed in the proportion of iTreg cells. Figure 1 These findings suggest that TAIII is a novel natural product capable of inhibiting the differentiation and development of Tregs and reducing their proportion.

[0039] 2. TAIII removes FoxP3 + Treg cells enhance the anti-tumor efficacy of CAR-T cells. To investigate whether TAIII can enhance the antitumor efficacy of CAR-T cells, this invention first prepared CD19 CAR-T cells by transducing T cells isolated from human peripheral blood mononuclear cells (PBMCs) using a lentiviral CAR construct. Baseline T cell phenotypes were then assessed during CAR-T cell preparation. These phenotypes included the proportions of CD8, CD4, naive T cells (Tnaive), central memory T cells (Tcm), effector T cells (Teff), terminally differentiated effector memory T cells (Temra), and regulatory T cells (Tregs), all analyzed by flow cytometry three days after CAR transduction. To assess phenotypic changes induced by in vitro TAIII exposure, CD19-CAR-T cells were treated with DMSO or TAIII for 72 hours, followed by flow cytometry analysis. The results showed that CD4... + T cells, CD8 + The ratio of T cells to effector T cells remained largely unchanged, while the TAIII-treated group showed an increase in terminally differentiated effector memory T cells and a corresponding decrease in naive T cells. Notably, TAIII treatment led to a significant reduction in the proportion of Tregs (approximately 40%). Figure 2 (a) This suggests that TAIII may enhance the efficacy of CAR-T cells by reducing the immunosuppressive Treg population.

[0040] Given these phenotypic changes, the effect of TAIII on CAR-T cell-mediated tumor cell killing was next evaluated in a co-culture cytotoxicity assay. CAR-T cells were co-cultured with Raji or Nalm6 cells at effector cell to target cell ratios (E:T) of 1:2 and 1:4, with treatment groups including those with and without TAIII. Compared with the control group using CAR-T cells alone, TAIII significantly enhanced CAR-T cell-mediated killing of Raji or Nalm6 cells in a dose-dependent manner. Figure 2 b, c). Specifically, TAIII enhanced the cytotoxicity of CAR-T cells, achieving a killing rate of approximately 80% against Nalm6 cells. Figure 2 c). Furthermore, in the TAIII-treated CAR-T group, the secretion of T cell effector cytokines (including IFN-γ, IL-2, and TNF-α) was significantly upregulated. Figure 2 b, c).

[0041] To further investigate the kinetic characteristics of the TAIII effect, in a co-culture cytotoxicity assay (E:T = 1:2 or 1:4), the CAR-T cell-mediated killing effect was monitored every 2 hours using Incucyte® cytolight technology via immunofluorescence for 36 hours. The results showed that TAIII significantly enhanced the activity of CD19 CAR-T cells against Raji cells in a time-dependent manner, with the effect observed as early as 4 hours. Figure 2 d).

[0042] Next, we investigated whether TAIII enhances the cytotoxicity of CAR-T cells by regulating Tregs. In experiments where CAR-T cells were co-cultured after activation with Raji cells (E:T=1:1) (with or without TAIII), we observed a significant upregulation of T cell effector cytokines (including IFN-γ and TNF-α) secretion in the TAIII-treated CAR-T group. Figure 2 e), and the expression of the key Treg-related transcription factor FoxP3 was significantly downregulated ( Figure 2 f), which is consistent with the reduction observed in CAR-T cells treated with TAIII alone. These results indicate that TAIII reduces FoxP3 + The proportion of CAR-Treg cells and targeting Tregs to enhance the cytotoxicity of CAR-T cells.

[0043] To specifically assess whether TAIII works by regulating Tregs, CD4 was sorted from human PBMCs. + CD25 + CD127 low Treg-depleted CAR-T cells (ΔTreg-CAR-T) were prepared by reducing their frequency to 1.28%. The resulting CAR-T cells were co-cultured with Nalm6-Luc cells for 24 hours, and target cell killing was assessed by measuring luciferase activity. At different E:T ratios, Treg depletion enhanced the short-term killing effect of CAR-T cells, confirming the negative impact of Tregs on CAR-T cell function. This highlights the crucial role of CAR-Tregs in regulating CAR-T-mediated cytotoxicity. Purified CD4+ was used... + or CD8 + ΔTreg-CAR-T cells were co-cultured with Raji-Luc cells for 24 hours. As mentioned earlier, CD8... + CAR-T cells are primarily responsible for short-term cytotoxicity. Notably, regardless of the subset, TAIII treatment did not alter the cytotoxic capacity of ΔTreg-CAR-T cells, indicating that the function-enhancing effect of TAIII requires the presence of Tregs.

[0044] Parallel experiments using parental CAR-T cells (containing 5.18% Tregs) further supported this conclusion. In parental CAR-T cells, TAIII significantly enhanced cytotoxicity, indicating that the functional enhancement effect of TAIII depends on the presence of Tregs. ELISA analysis showed that ΔTreg- or CD4- in TAIII-treated and DMSO-treated cells... + There were no significant differences in the secretion of IL-2, IFNγ, or TNFα among ΔTreg-CAR-T cells. Although TAIII secretion was present in CD8... + ΔTreg-CAR-T cells showed a moderate increase in cytokine secretion, but this was not sufficient to enhance cytotoxic activity.

[0045] The results demonstrate that TAIII primarily enhances CAR-T cell efficacy by alleviating Treg-mediated immunosuppression, rather than by directly acting on effector T cells. This targeting mechanism provides a therapeutic advantage by selectively counteracting immunosuppressive components within the CAR-T population while preserving the intrinsic function of T cells.

[0046] 3. TAIII is a novel selective A2AR allosteric inhibitor. To explore the intrinsic mechanism of TAIII-mediated Treg inhibition, direct targets of TAIII were identified using a reverse screening method. A biotinylated TAIII probe with a photoactivating group (Biotin-TAIII-P) was designed and synthesized using click chemistry. Figure 3 a) This probe incorporates a photosensitive "fishhook" structure for proximity labeling and combined with LC-MS analysis. Using this method, the adenosine A2A receptor (A2AR) was identified as a key immunosuppressive target for TAIII. To further validate this finding, the SwissTargetPrediction online tool was used for prediction, which showed that G protein-coupled receptors (GPCRs) are the most prominent class of potential targets for TAIII. Among them, A2AR (ADORA2A) and A1AR (ADORA1) ranked highly. This computational prediction also indicates that A2AR is a very promising candidate target. A2AR is a well-known immune checkpoint molecule expressed on various immune cells, especially T cells. Studies have shown that A2AR plays a crucial role in the tumor immunosuppressive microenvironment and promotes tumor immune escape, making it a potential target for immunotherapy. Although the immunosuppressive function of A2AR through Tregs has been confirmed, the molecular mechanisms regulating Treg activity remain largely unknown. Furthermore, whether blocking the A2AR signaling pathway can improve the preparation of CAR-T cells was previously unknown.

[0047] Subsequent experiments verified the binding of TAIII to A2AR. Due to the low A2AR expression level in 293T cells, an A2AR-overexpressing 293T cell line stably expressing A2AR-GFP (293T-A2AR-OE) was constructed. The effect of TAIII on A2AR protein stability at different temperatures was investigated using cell thermal transfer assay (CETSA). As expected, TAIII significantly enhanced the thermostability of A2AR. Furthermore, microthermophoresis further confirmed the binding of TAIII to A2AR. Subsequently, a biotin-streptavidin pull-down assay was used for target binding experiments, showing a dose-dependent interaction between TAIII and A2AR in both the overexpression system and primary human T cells. Figure 3 b). To further verify the target binding characteristics of A2AR, a Biotin-TAIII-P competition experiment was performed 4 hours after TAIII treatment. The results showed that 5 μM TAIII could label approximately 50% of A2AR ( Figure 3 (c, d), thus confirming that A2AR is the direct target of TAIII.

[0048] Adenosine receptors belong to the rhodopsin-like G protein-coupled receptor family and have four subtypes: A1R, A2AR, A2BR, and A3R, each coupled to different intracellular small G proteins. A1R and A3R are coupled to Gi proteins, while A2AR and A2BR are coupled to Gs proteins. Ligand binding to A2AR triggers its coupling with Gs proteins, which in turn activates adenylate cyclase to produce cAMP and initiates the classical cAMP-PKA-CREB signaling pathway, which mediates various immunosuppressive responses. To evaluate the selectivity of TAIII for each adenosine receptor subtype, expression vectors for A1R, A2AR, A2BR, and A3R were constructed and overexpressed in 293T cells. Intracellular cAMP levels after activation of A2AR with 0.1 μM of the adenosine receptor agonist 5-(N-ethylcarboxamido)adenosine were measured using the LANCE cAMP assay. The results showed that TAIII exhibited high selectivity for A2AR, with minimal or no effect on other subtypes. The activity of TAIII was then compared with that of the phase II A2AR antagonist AZD4635 using the CREB reporter gene assay and the LANCE cAMP assay. In different cell models, TAIII showed potency comparable to AZD4635, and cell viability was maintained at all tested concentrations, suggesting that TAIII may not be toxic to T cells. Figure 3e, f). To rule out off-target effects of TAIII on the A2AR-cAMP / PKA / CREB pathway, we evaluated the sensitivity of TAIII in A2AR-overexpressing cells and parental cells. TAIII showed higher sensitivity in A2AR-overexpressing cells, and its potency was comparable to other A2AR antagonists (e, f). Figure 3 g). In summary, these findings suggest that TAIII is a selective A2AR inhibitor.

[0049] To investigate the binding mode of TAIII and A2AR, computer-aided structural analysis was used to study the crystal structure of the A2AR complex (PDB ID: 4EIY). Figure 3 h). Studies have shown that the cholesterol-sensitive motif (CCM) in the transmembrane region of A2AR is highly conserved. The cholesterol content in the cell membrane can regulate the ligand-binding pocket of A2AR, thereby affecting its function. Cholesterol depletion or CCM mutations disrupt the A2AR structure, impairing ortho-ligand binding and downstream signal transduction. As a steroidal saponin, TAIII has a core structure similar to cholesterol. Previous studies have shown that replacing cholesterol with TAIII in the lipid bilayer can enhance drug delivery efficiency, highlighting the structural similarity between the two. Given that membrane cholesterol is crucial for A2AR activation, it is speculated that TAIII may affect A2AR function by competing for cholesterol binding. Structural modeling analysis shows that the polyhydroxy head of TAIII extends into the polar pocket formed by Q38, W32, Y43, and K122, achieving stable binding; its hydrophobic portion aligns with the non-polar region of the cell membrane along the transmembrane helix, consistent with the phospholipid bilayer model structure. Figure 3 h). To verify this hypothesis, point mutations were introduced into three key CCM residues (reported to bind cholesterol), and the contribution of these residues to the binding of A2AR to TAIII was investigated using CREB-luciferase reporter gene assays. The W129A mutation retained some activity, which is an important consideration when interpreting the sensitivity of TAIII to these assays. Compared to wild-type A2AR, all mutations reduced CREB activity, and the mutants did not respond even with increased TAIII doses. Figure 3 (i, j), indicating that TAIII affects A2AR function by competing for cholesterol and allosterically binding to these key residues.

[0050] 4. TAIII activates T cell responses in an A2AR-dependent manner by impairing Treg function. Adenosine suppresses immune cells in the tumor microenvironment by inhibiting T cell activation and cytotoxicity. Furthermore, extracellular adenosine, as an important mediator of Treg function, contributes to the immunomodulatory activity of Tregs. To evaluate the efficacy of TAIII in enhancing T cell immune responses via A2AR signaling, PBMCs were isolated from healthy donors and co-cultured with NECA (a potent adenosine agonist) or TAIII under pan-T cell culture conditions. T cell transcriptomic analysis showed that, in the presence of NECA, TAIII treatment significantly upregulated immune-activating genes (including TNF, GZMB, IFN-γ, and IL-2) while downregulating immunosuppressive genes (such as FoxP3, IKZF2 (Helios), IKZF4 (Eos), and CCR8). Figure 4 a). Furthermore, Reactome pathway enrichment analysis showed that it enriched immunosuppression-related genes and pathways in Tregs. To verify these findings, ELISA and qRT-PCR experiments were performed, demonstrating that TAIII effectively counteracted NECA-mediated inhibition of IL-2 and IFN-γ expression, thereby enhancing T cell activation. In addition, TAIII reduced FoxP3 transcription in T cells in a dose-dependent manner, with efficacy exceeding that of AZD4635 ( Figure 4 (b and c). During the assessment of differentiation, FoxP3... + Similar results were observed when the proportion of iTreg cells was adjusted. NECA (5 μM) upregulated the expression of Treg differentiation and functional markers (FoxP3, CTLA4, CD25, and TGF-β), effects of which were significantly inhibited by TAIII treatment. Figure 4 d). Furthermore, TAIII significantly inhibited the NECA-mediated immunosuppressive effect of Tregs on effector T cells ( Figure 4 e, f).

[0051] Delivering shRNA targeting A2AR to primary human iTregs via lentivirus significantly reduced FoxP3 protein levels and increased IL-2 transcription, while the non-targeted control showed no such effect. Figure 4 g). To rule out off-target effects, we treated A2AR-knockdown Tregs with TAIII and confirmed by qPCR and LANCE cAMP assays that TAIII-mediated FoxP3 inhibition and cAMP production were significantly reduced by A2AR knockdown, indicating that TAIII regulates FoxP3 by specifically targeting A2AR. + Tregs ( Figure 4 h).

[0052] To investigate the mechanism of TAIII-mediated FoxP3 transcriptional repression, cut & run analysis showed that in human iTregs, the occupancy of CREB in the FoxP3-CNS-2 promoter region was significantly reduced. Figure 4 i), this confirms that TAIII inhibits FoxP3 by blocking the A2AR signal. + Tregs enhance T cell activity.

[0053] 5. TAIII enhances the in vitro antitumor activity of CAR-T cells by blocking A2AR. To investigate whether TAIII improves CAR-T cell function through the A2AR signaling pathway, a co-culture cytotoxicity experiment was conducted. Figure 5 In the E:T ratio, the ratio is 1:2 or 1:4. NECA increases FoxP3. + The proportion of CAR-Tregs weakened the cytotoxicity of CAR-T cells, while TAIII treatment restored the killing function of NECA-treated CAR-T cells against Nalm-6 or Raji cells by upregulating effector cytokines (including IL-2, IFN-γ, and TNF-α). Figure 5 (ac). Given that the proportion of CAR-Tregs is crucial in determining patient response and outcome to CAR-T therapy, and considering the ability of TAIII to impair Treg function and differentiation, this study investigated whether TAIII reduces FoxP3 through the A2AR signaling pathway. + CAR-Tregs. In a co-culture system of CAR-T cells and Raji cells, regardless of whether TAIII was added to the culture medium, NECA significantly upregulated FoxP3 protein levels and FoxP3. + The frequency of Tregs occurrence, and TAIII treatment can completely reverse these effects of NECA without affecting total CD4. + T cells and cytotoxic CD8 + The proportion of T cells ( Figure 5 d). This selective regulatory effect further demonstrates the target specificity of TAIII. These findings suggest that TAIII enhances the cytotoxicity of CAR-T cells by blocking A2AR signaling.

[0054] Considering that central memory T cells (Tcm) not only possess anti-tumor activity but also prevent relapse, the effects of TAIII on the Tcm cell population (CD62L) were further investigated through co-culture and FACS analysis (E:T=1:1, 1:2, or 1:4). + CD45RO + The results showed that NECA stimulation significantly reduced the proportion of Tcm cells, while TAIII treatment completely restored Tcm levels under all conditions. Figure 5 e).

[0055] To validate these findings, single-cell RNA sequencing (scRNA-seq) analysis was performed on cultured CAR-T cells alone or in co-culture systems (with or without TAIII treatment) using 10× Genomics technology. By clustering and quantifying the ratio of B cells (Raji) to T cells (CAR-T), it was observed that TAIII significantly enhanced the killing effect on Raji cells (a subset of B cells) (E:T=1:1), consistent with previous results. After re-clustering the pooled T cell single-cell data, CD4+ was isolated. + T cells were further identified, and a FOXP3 was further identified. high CD4 + CTLA4 + Tregs subpopulation. Under co-culture conditions, TAIII treatment reduced CD4+. + The proportion of this Treg subset in T cells decreased by more than 40%. Similarly, in the CAR-T group alone, after TAIII treatment, FOXP3... high CD4 + CTLA4 + Treg cell clusters and A2AR + CD4 + Tregs were significantly reduced (>40%), highlighting the sustained inhibitory effect of TAIII on the Treg population. These findings support the regulatory role of TAIII on the Treg population in the context of NECA stimulation. Notably, scRNA-seq analysis revealed cytotoxic CD8... + Significant changes in T cells. In CAR-T / Raji co-culture, CD8... + CAR-T cells co-cultured with tumor cells exhibited enhanced cytotoxicity, which was further amplified in the TAIII-treated group. This effect was particularly pronounced in the key cytotoxic subset (CD8). + GZMB + TeX and CD8 + GZMB + Differences in cytotoxicity were observed among Tregs. CellChat analysis revealed the strongest interaction between these subsets and Tregs, indicating that this enhanced cytotoxicity is a secondary effect of Treg reduction. Furthermore, TAIII treatment significantly upregulated the expression of IFN-γ and TNF-α, further confirming that TAIII enhances CAR-T cell cytotoxicity by regulating Tregs, thereby indirectly affecting CD8+. + T cells.

[0056] Furthermore, bulk RNA-seq results showed that TAIII treatment reversed the effects of NECA by enriching T cell proliferation and activation, cytotoxic cytokines, and Treg immunosuppression-related genes and pathways. Reactome pathway enrichment analysis also confirmed this, consistent with previous observations in T cells. Importantly, in TAIII-treated CAR-T cells, downstream A2AR effector CREB1 and Treg functional markers (such as FoxP3, CTLA4, and CD25) were significantly downregulated, while cytotoxic cytokines such as TNF were significantly increased, indicating that cells developed resistance to adenosine-mediated transcriptional changes, consistent with previous T cell data. In addition, fatty acid metabolism pathways, particularly cholesterol metabolism pathways, were significantly enriched after TAIII treatment, consistent with the role of TAIII in regulating lipid metabolism. Given that membrane cholesterol homeostasis regulates A2AR activity and is crucial for Treg stability and function, and that TAIII has the ability to compete with cholesterol, these results suggest that TAIII regulates A2AR activity by modulating cholesterol metabolism and allosterically binding to A2AR. In addition, TAIII treatment enhances T cell immune responses by upregulating TNF expression, while downregulating the transcription of FoxP3, CTLA4, and CD25 in CAR-T cells, thereby significantly counteracting the effects of NECA.

[0057] To confirm that the effect of TAIII is A2AR-dependent and to rule out potential off-target effects, an A2AR knockdown (KD) experiment was performed in CAR-T cells. In short, A2AR knockdown was achieved using shRNA (GFP) targeting A2AR. + CD19-CAR-T cells were transduced and sorted by FACS. Knockdown efficiency was confirmed at the protein and mRNA levels by Western blotting and qRT-PCR, respectively. Subsequently, A2AR-KD CAR-T cells were co-cultured with Nalm6-Luc cells, and the results showed that compared with the SCR control (GFP... - Compared to TAIII, A2AR knockdown enhanced the cytotoxicity of CAR-T cells, while TAIII did not further enhance this cytotoxic effect, confirming that the effect of TAIII depends on the A2AR signaling pathway. Figure 5 Furthermore, A2AR-KD CAR-T cells exhibited higher IFN-γ and IL-2 secretion, but were unresponsive to both NECA and TAIII treatments, further supporting the A2AR-dependent effect of TAIII. Figure 5 These findings are consistent with experimental data from A2AR-KD Tregs (in which TAIII has no effect on Treg activity), providing strong evidence that A2AR is a key mediator of TAIII regulation of Treg and CAR-T cell function.

[0058] In summary, TAIII plays an important role in enhancing CAR-T cell function by regulating A2AR signaling and may help prevent relapse after CAR-T therapy.

[0059] 6. TAIII combined with PD-1 reverses A2AR-FoxP3 + Treg-mediated immunosuppression to inhibit solid tumor growth A recent study showed that PD-1 blockade therapy expands Tregs in tumors, thereby limiting the efficacy of immunotherapy. To investigate whether TAIII could enhance the antitumor activity of PD-1 therapy, we treated mice (C57BL / 6J) bearing MC38 tumors with isotype control, anti-PD-1 monotherapy (5 mg / kg, twice weekly), TAIII monotherapy (10 mg / kg, intraperitoneal injection), or TAIII combined with PD-1 therapy for 24 days. Figure 6 a). Compared with monotherapy, TAIII significantly enhanced the efficacy of anti-PD-1 therapy, and weight monitoring showed no significant side effects (a). Figure 6 Furthermore, TAIII significantly increased the production of anti-tumor cytokines IFN-γ and IL-2 in tumor tissues and serum (bf). Figure 6 g). Similarly, Western blot analysis of resected tumors showed a significant reduction in A2AR downstream signaling (pCREB). Figure 6 (h), demonstrating that the observed therapeutic effect was mediated by blocking the A2AR pathway.

[0060] Flow cytometry analysis of tumor-infiltrating lymphocytes showed that the proportion of central memory T cells (Tcm) was significantly increased in the combination therapy group compared with the anti-PD-1 monotherapy group. Figure 6 i), indicating that TAIII+ anti-PD-1 therapy enhances T cell responses by promoting Tcm differentiation. Consistent with this, immunohistochemical staining showed FoxP3 in tumor-infiltrating lymphocytes. + Tregs decreased significantly, while CD8 + Increased T cells ( Figure 6 (jk), further confirming the enhanced anti-tumor efficacy of combination therapy. In summary, these results highlight the role of TAIII in enhancing the efficacy of PD-1 immunotherapy by reducing intratumoral Tregs through combination therapy.

[0061] Furthermore, to verify the broader applicability of TAIII in enhancing the efficacy of CAR-T cells against colorectal cancer, we evaluated its effects in the MC38-mCD19-Luc subcutaneous xenograft model. 1×10⁻⁶ tv / L C57BL / 6J immunocompetent mice were subcutaneously injected with TAIII. 6MC38-mCD19-Luc cells were administered intraperitoneally on day 6 with cyclophosphamide (CTX) 4.5 mg + fludarabine phosphate (FLU) 0.75 mg / 200 μL / mouse. On day 7, the mice were treated with the optimal dose of CAR-T cells, TAIII (10 mg / kg, once daily), or a combination thereof. Figure 6 (l). Continuing until day 29, CAR-T monotherapy induced partial tumor regression, while TAIII combination therapy achieved the most significant tumor suppression effect in this study, without significant toxicity (proven by weight stability). Figure 6 The above results indicate that in colorectal cancer models, TAIII combined with anti-PD-1 therapy not only effectively inhibits tumor growth, reduces immunosuppressive Treg cells, and enhances anti-tumor immunity, but also exhibits the most significant tumor regression effect when combined with CAR-T therapy. This confirms that TAIII has a broad synergistic effect and application potential in improving the efficacy of immunotherapy for colorectal cancer.

[0062] 7. TAIII prevents relapse after CAR-T therapy in various preclinical models by targeting the A2AR-Treg signaling pathway. Given FoxP3 + To assess the importance of CAR-T retinoid levels in predicting prognosis after CAR-T infusion, we analyzed clinical samples from a database of patients receiving CAR-T therapy. CD19 CAR-T products were collected before infusion from B-lymphoblastic leukemia patients who achieved long-term remission (R, over 8 years, n = 25) or had no objective response to treatment (NR, n = 6), and single-cell RNA sequencing analysis was performed (GSE262072). Analysis showed that A2AR levels were high in the total CAR-T cell population. + FOXP3 + The proportion of cells was significantly correlated with the percentage of cytotoxic cells, cytokine production, and clinical response, consistent with the findings of Good and Haradhvala et al. Figure 7 ab). Specifically, the A2AR signaling pathway and Tregs-related pathways (such as NF-κB1, TGF-β1) and CD8 + The T-cell-related gene CRTAM is closely associated with tumor response after CAR-T infusion. Figure 7 (b) This makes A2AR-FoxP3 a potential biomarker for predicting treatment response and suggests that TAIII blockade of A2AR may enhance the efficacy of CAR-T cell therapy in patients.

[0063] The results of this invention further demonstrate that TAIII effectively reduces the proportion and function of Tregs by blocking A2AR signaling, thereby potentially enhancing the efficacy of CAR-T cells in preventing tumor recurrence. To verify this hypothesis, the effect of TAIII on tumor recurrence after CAR-T cell therapy was studied in vivo. Raji-Luc cells were intravenously injected into NSG immunodeficient mice, followed by CAR-T cell therapy two days later. From day 3 (after Raji cell engraftment) to day 58, treatment was initiated with or without TAIII (once daily). Figure 7 c). Both the CD19-CAR-T cell-only group and the combination therapy group effectively cleared Raji cells within one week. However, the CAR-T cell-only group experienced relapse two weeks after remission, while no relapse was observed in the combination therapy group. Figure 7 d,e). Compared with the vector control group and the CAR-T group alone, TAIII combined with CAR-T significantly prolonged the survival of mice ( Figure 7 f). Furthermore, under all conditions, the combination therapy significantly upregulated CAR-T and Tcm cell populations while downregulating FoxP3 in bone marrow and blood. + Tregs ( Figure 7 These findings suggest that TAIII combination therapy is an effective and cost-effective treatment strategy for preventing relapse after lymphoma CAR-T cell therapy.

[0064] To validate this finding and demonstrate the broader applicability of TAIII in enhancing CAR-T cell efficacy, we further evaluated its effects in a Nalm6-Luc subcutaneous xenograft model. TAIII was administered subcutaneously to NSG immunodeficient mice. 6 Nalm6-Luc cells were administered, followed by treatment with either the optimal dose of CAR-T cells or UNT control (untransduced control cells) (infused on days 3 and 8, respectively). TAIII (10 mg / kg, once daily) was administered in combination starting on day 3 post-inoculation and continued until day 38. Figure 7 i). CAR-T monotherapy induced partial tumor regression, while TAIII combination therapy achieved the most significant tumor suppression effect in this study without significant toxicity (proven by weight stabilization). In contrast, TAIII did not enhance the anti-tumor activity of UNT cells (i). Figure 7 (j, k) indicates that its effect is antigen-specific rather than non-specific. Notably, in the TAIII combination therapy group, mice carrying Nalm6 tumors showed elevated CD4 levels in peripheral blood, bone marrow, and spleen samples on day 14 after CAR-T implantation. + CAR +The proportion of Treg cells was significantly reduced, but no significant change was observed in the CD8 / CD4 ratio. Specifically, compared with the CAR-T-only group, the combination therapy group (TAIII+CAR-T) had a significantly lower total CAR concentration in peripheral blood on day 7 after CAR-T engraftment. + T cells and CAR + CD62L + The proportion of T (Tcm) cells increased significantly. Figure 7 These findings suggest that TAIII selectively targets Tregs in vivo and modulates their population to enhance CAR-T cell proliferation and persistence. This may explain the improved antitumor efficacy observed in the combination therapy group. These results are consistent with our observations in the Raji-Luc model, reinforcing the role of TAIII as an effective adjuvant for CAR-T therapy and its potential to improve prognosis in solid tumors. In conclusion, these findings support the universality of the immunomodulatory effects of TAIII.

[0065] To assess whether TAIII pretreatment enhances the in vivo antitumor activity of CAR-T cells, we treated mice carrying Raji-Luc cells with CD19-CAR-T cells (with or without TAIII pretreatment (1 μM, 36 h)) on day 6 post-tumor implantation. The percentage of Raji-Luc cells in the TAIII pretreatment group (which showed reduced FoxP3 levels both in vitro and in vivo) was significantly lower than that in the CAR-T-only group and the vector control group. Compared to the CAR-T-only group, the pretreatment group exhibited greater potency and earlier tumor clearance.

[0066] To further confirm the enhancing effect of TAIII pretreatment on CAR-T cell-mediated cytotoxicity and its broader applicability, we used a Nalm6-Luc subcutaneous tumor model. NSG mice were subcutaneously injected with 1×10⁻⁶ TAIII. 6 One Nalm6-Luc lymphocytic leukemia cell was administered, and six days later, 5 × 10⁸ cells were infused. 6 CAR-T cells or CAR-T cells pretreated with TAIII (1 μM, 72 h). As expected, TAIII pretreatment significantly enhanced the in vivo antitumor activity of CAR-T cells. Compared with the CAR-T group alone, the TAIII pretreatment group showed a rapid reduction in tumor burden and earlier tumor clearance, highlighting the enhancing effect of TAIII pretreatment on CAR-T cell-mediated cytotoxicity. Figure 7 Subsequently, we used flow cytometry to track T cell frequency and phenotype in peripheral blood (days 4, 7, and 14 post-CAR-T infusion), bone marrow, and spleen (day 14 post-CAR-T infusion). Results showed that, compared to the CAR-T-only group, the TAIII pretreatment group exhibited significant expansion of CAR-T cells in both peripheral blood and bone marrow, as well as increased CAR cell count.+ FoxP3 + A significant reduction in Tregs ( Figure 7 From day 4 to day 7, we observed CD3+ levels in the peripheral blood of the TAIII pretreatment group. + T, CD4 + T, CD8 + The total number of T cells, Tcm cells, and terminally differentiated effector memory T cells (Temra) increased. Furthermore, the CD4 / CD8 ratio remained unchanged in the combination therapy group compared to the CAR-T therapy-only group. Notably, the sustained changes in the T cell population suggest that TAIII regulation of Tregs provides long-term benefits, enhancing the persistence and function of CAR-T cells well after the initial treatment phase. These results are consistent with our findings in the Raji-Luc model and further underscore the universality of the TAIII effect.

[0067] In addition, we collected CD19 CAR-T products from 8 patients with uncontrolled disease progression following CAR-T infusion (Table 1) and evaluated them with TAIII in a co-culture model.

[0068] Table 1. Clinical characteristics of patients with malignant tumors of the lymphatic system The patient's CAR-T cells were co-cultured with Raji-Luc cells at an E:T ratio of 1:1 to 1:4, with or without TAIII treatment (1 μM, 24h). Figure 7 o). Compared with the CAR-T group alone, TAIII significantly enhanced CAR-T cell-mediated killing of Raji-Luc cells, with a killing efficiency several times higher in some patients (o). Figure 7 To determine whether TAIII enhances CAR-T cell cytotoxicity by targeting Tregs, we co-cultured Raji cell-activated CAR-T cells with TAIII (E:T = 1:2). TAIII treatment significantly increased the secretion of T cell-derived cytokines (IL-2, IFN-γ). Figure 7 q), and significantly reduced FoxP3 expression levels ( Figure 7 r), indicating that FoxP3 +CAR-Tregs were reduced. To investigate whether TAIII improves patient-derived CAR-T function through the A2AR signaling pathway, we conducted a co-culture experiment (E:T=1:2) and found that NECA treatment significantly impaired the cytotoxicity of CAR-T cells. However, TAIII restored the cytotoxic function of NECA-treated CAR-T cells, enabling them to effectively kill Raji cells. These findings suggest that, in preclinical models, TAIII pretreatment and its combination with CAR-T cells enhance antitumor activity, accelerate tumor clearance, and reduce recurrence compared to CAR-T monotherapy.

[0069] To examine whether the immunomodulatory effects of TAIII depend on immune cells (such as T cells), Raji-Luc cells were intravenously injected into NSG-immunodeficient mice, followed by treatment with or without TAIII. TAIII had minimal effect on Raji cell growth in T-cell-deficient mice, with no significant survival benefit. Furthermore, no significant weight loss was observed, confirming the safety of TAIII in immunodeficient mice.

[0070] To further investigate the role of TAIII in modulating Tregs during in vivo CAR-T cell therapy, we conducted additional experiments using a Nalm6-Luc xenograft model and ΔTreg-CAR-T cells (Tregs reduced to 1.28%). Nalm6-Luc cells were intravenously injected into NSG immunodeficient mice, followed by CAR-T cell infusion on day 2. TAIII treatment (once daily) was initiated simultaneously and continued until day 18. Our study design ensured the inclusion of appropriate controls, such as UNT cell controls. Consistent with our in vitro findings, depletion of Tregs eliminated the therapeutic benefit of TAIII. While CAR-T monotherapy induced partial tumor regression, the addition of TAIII to ΔTreg-CAR-T cells did not provide further tumor suppression or significantly improve survival. These results suggest that TAIII enhances antitumor efficacy by specifically targeting Tregs within the CAR-T cell population. By selectively modulating Treg activity without impairing other immune components, TAIII improves CAR-T function while maintaining systemic immune homeostasis, thereby improving treatment outcomes.

[0071] In summary, these findings reveal that TAIII enhances CAR-T cell efficacy and reduces relapse when used as a pretreatment or in combination therapy by targeting Tregs, providing a promising strategy for optimizing CAR-T treatment outcomes and production.

[0072] The experimental method used in this embodiment is as follows: Synthesis of TAIII probe: HATU (38 mg, 0.1 mmol) and DIEA (26 mg, 0.2 mmol) were added to a 2 mL DMF mixture containing anemarrhenasaponin AIII (74 mg, 0.1 mmol) and 3-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)propionic acid (17 mg, 0.1 mmol). The mixture was stirred at room temperature in the dark for 2 hours. Subsequently, the reaction was quenched by adding water (10 mL), and the mixture was extracted with dichloromethane (CH2Cl2). The combined organic phases were dried over anhydrous Na2SO4. After solvent evaporation, the residue was purified by rapid column chromatography to obtain TAIII probe (7 mg, 8% yield) as a white solid.

[0073] Click Chemistry and Proximity-Labeled Proteomics: 293T cells were cultured in 10 cm culture dishes to 80-90% confluence. Cells were washed with PBS and lysed with 1 mL NP-40 lysis buffer. Centrifugation (12,000 rpm; 10 min; 4°C) was performed to remove insoluble components from the cell lysates. DMSO or TAIII-P was added to the cell lysates, and the mixture was incubated at 4°C for 2 h. All samples were then irradiated under 365 nm UV light for 30 min using a UV crosslinker. Subsequently, biotin-azide (10 μM), TCEP (1 mM), TBTA (100 μM), and CuSO4 (1 mM) were added sequentially to the lysis buffer, and the mixture was incubated for another 1 h at room temperature in the dark. Afterward, the adjacent labeled proteins were precipitated with acetone, air-dried, and then resuspended in 1.5% SDS solution. The sample was incubated with avidin-agarose beads at room temperature for 2 hours, then centrifuged, the supernatant was discarded, and the beads were washed three times with PBS. Finally, proteomics analysis based on LC-MS / MS or Western blotting analysis was performed. The generated raw mass spectrometry data have been stored in the PRIDE public database, accession number PXD060766.

[0074] Cell heat transfer assay: 293T cells overexpressing A2AR were collected and centrifuged to remove the precipitate after three freeze-thaw cycles (12,000 rpm; 10 min; 4 °C). TAIII or DMSO was added to the cell lysate in a dose-dependent manner and incubated for 1 hour. Subsequently, the sample was aliquoted into 100 μL aliquots into 0.2 mL PCR tubes and briefly heated in a PCR instrument at the specified temperature for 3 min, followed by cooling to room temperature for 3 min. The samples were then centrifuged (12,000 rpm; 10 min; 4 °C) to separate soluble proteins from cell debris and aggregates. The supernatant was transferred to new tubes and analyzed by Western blotting.

[0075] LANCE cAMP assay: Using LANCE TM The Camp 384 kit (PerkinElmer, AD0262) was used to detect cAMP levels, following the manufacturer's instructions. A simplified procedure is as follows: Cells were collected and resuspended in stimulation buffer (PBS containing 0.1% BSA and 0.5 mmol / L IBMX) to a cell concentration of 6 × 10⁻⁶ cells / day. 5 Cells / mL. Then, 5 μL of cell suspension was transferred to each well of a 384-well plate. Cells were then stimulated for 30 min with TAIII dilution in the presence of 0.1 μM NECA. The assay mixture was then added, and the plate was incubated at room temperature in the dark for 60 min. Finally, fluorescence intensities at 340 / 665 nm and 340 / 615 nm were read; the ratio of fluorescence intensity at 665 nm to 615 nm was inversely proportional to cAMP concentration.

[0076] Activation and expansion of pan-T cells: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human peripheral blood by density gradient centrifugation. To induce PBMCs into pan-T cells, PBMCs were centrifuged at 1×10⁻⁶ cells / mL. 6 Dynabeads Human T-Activator CD3 / CD28 (Gibco, 11131D) were seeded at a concentration of 1 / mL in suitable tissue culture plates or dishes, with a bead-to-cell ratio of 1:1. 100 IU / mL of IL-2 was also added to the culture medium. Dynabeads were removed on day 5, and pan-T cells were collected 48 hours after bead removal for further analysis.

[0077] To assess cytokine secretion and mRNA expression, PBMCs or T cells were charged at 1×10⁻⁶ mcg / mL. 5 Cells were cultured at a concentration of [cell / mL] in 96-well or 6-well plates. Cells were activated with Dynabeads at a 1:1 ratio and treated with a specified concentration of TAIII or AZD4635 in the presence of 5 μM NECA. After 16 hours of treatment, PBMCs were collected for RT-qPCR analysis (Yeason); after 48 hours of culture, the secretion levels of IFN-γ and IL-2 were analyzed using an ELISA kit (MultiSciences).

[0078] Treg cell induction: Human PBMCs were obtained from blood samples of healthy donors (n=3) after obtaining approval from the Ethics Committee of Shanghai Blood Center. Monocytes were first isolated from fresh umbilical cord blood (n=3) using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation, followed by the isolation of naïve CD4+ T Cells using the Human Naïve CD4+ T Cell Isolation Kit II (Miltenyi Biotec, 130-094-131). + T cells were then activated using Dynabeads HumanT-Activator CD3 / CD28 (Gibco, 11131D) and cultured under the previously described Treg induction conditions: supplemented with IL-2 (100 IU / ml) and TGF-β1 (5 ng / ml), along with the designated drug treatment. After 5 days, TGF-β1 was removed from the culture medium, and differentiation efficiency was assessed by flow cytometry (FACS) prior to subsequent experiments. The PBMCs and naïve T cells used in this experiment were derived from different healthy donors (ethics approval number: YXLL-2023-17), ensuring that the data reflected inter-individual variability and were representative of the population. This approach enhanced the robustness and generalizability of the research results.

[0079] Treg inhibition assay: CD4+ was isolated from healthy adult PBMCs using a BD FACSAria III flow cytometer. + CD25 high CD127 low CD45RA - Effector T cells (Teff). Teff cells were incubated with 2.5 μM CFSE (Invitrogen, C34570) at 37°C for 20 min, followed by the addition of five times the volume of the original chromosomes in complete culture medium, and incubation for another 5 min. The supernatant was then removed by centrifugation, and the cells were resuspended in preheated culture medium. These stained cells were stimulated with CD3 / CD28 Dynabeads (bead-to-cell ratio 1:4) and then co-cultured with a specified proportion of Treg cells in 200 μL of medium in 96-well round-bottom plates (20,000 Teff cells per well). After 96 hours of co-culture, the proliferation peak was analyzed by flow cytometry (FACS).

[0080] Flow cytometry (FACS): For staining cell surface proteins, cells are collected and incubated with fluorescently labeled antibodies at room temperature in the dark for 30 minutes. The following fluorescently labeled antibodies are used for flow cytometry analysis: For human T cell staining, appropriate flow cytometry gating strategies are applied to distinguish different cell populations, including CD4+. + T(CD4-FITC, BD222346; CD4-Alexa Fluor 700, BD561030), CD8 + T (CD8-PC5.5, BD560662), CD3 + T cells (CD3-PE, BD561803), CAR⁺ T cells (CAR-Alexa Fluor 647, BIOSWAN, 200102), regulatory T cells (Treg), naive T cells (Tnaive), central memory T cells (Tcm), effector T cells (Teff), and terminally differentiated effector memory T cells (Temra). CAR-T cells are gating based on the expression of CD19-CAR (CD19-BV421, BD562440) which binds to CD3 or CD4. Tregs are defined as CD4+. + FoxP3 + Cells. For intranuclear FoxP3 staining, after surface staining, cells were fixed and perforated using Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions, followed by anti-FoxP3-PE-Cy7 (eBioscience, 25-4776-41) or FoxP3-PE (BDPharmingen). TM Staining was performed on cells (560082). Human T cell subsets were classified according to surface markers as follows: CD45RO⁻CD62L⁺ (naive T), CD45RO⁺CD62L⁺ (central memory T), CD45RO⁺CD62L⁻ (effective T), and CD45RO⁻CD62L⁻ (terminally differentiated effector memory T). For Treg clearance assays, native Tregs (nTregs) in PBMCs were identified as CD45RO⁻. + CD25 + CD127 lowCells. For mouse T cell subsets, CD44 was used instead of CD45RO. CD45RO-PE (BD 555493), CD62L-BV421 (BD 563862), and CD44-BV421 (BD 563970) were used. Cell proliferation was tracked using CFSE (Invitrogen, C34554), and cell viability was assessed using Fixable Viability Stain 660 (BD 564405) or Fixable Viability Stain 520 (BD 564407). For mouse T cell staining, anti-mouse CD45-FITC (BD 553080), CD3-APC-Cy7 (BD 557596), CD4-APC (BD 553051), CD8-PC5.5 (BD 551162), FoxP3-PE (BD 560408), CD44-BV421 (BD 563970), and CD62L-Alexa Fluor700 (BD 560517) were used. Data were acquired using Beckman CytoFLEX and analyzed using FlowJo V10.8.1 software.

[0081] Micro-thermophoretic kinetic analysis: Micro-thermophoretic kinetic analysis was performed according to the previously described method. The brief procedure is as follows: A2AR-overexpressing 293T cells were lysed using RIPA lysis buffer supplemented with PMSF, and centrifuged (12,000 rpm, 10 min, 4 °C) to remove the pellet. Then, 10 μL of cell lysis buffer was incubated with 10 μL of serially diluted TAIII or AZD4635 at room temperature for 30 min. Subsequently, the mixture was added to a capillary tube provided by Nano-Temper, and thermophoretic kinetics were scanned using a Monolith instrument (Nano-Temper).

[0082] Human samples used for scRNA-seq and bulk RNA-seq: Human CAR-T products were obtained with the approval of the Ethics Review Committee of Tongji Hospital (Clinical Trial No.: NCT02537977, Sponsor: Shanghai Tongji Hospital, Tongji University School of Medicine). Clinical characteristics of patients with B-cell lymphoblastic malignancies are shown in Table 1. Single-cell RNA sequencing and bulk RNA sequencing were performed by Novogene according to the manufacturer's instructions. Raw data generated in this study have been stored in the GEO database, accession numbers GSE289388 and GSE302847.

[0083] CUT&RUN Analysis: The CUT&RUN analysis was performed and analyzed by Novogene Corporation, following the previously described methodology.

[0084] Molecular modeling: The crystal structure of A2AR was obtained from the protein database (PDB ID: 4EIY). Water molecules and the original ligands were manually removed using PyMol software. Molecular docking was performed using Autodock (version 4.2.6) to predict the binding conformation of anemarrhenasaponin AIII. First, the initial file was prepared using the prepare_ligand4.py and prepare_receptor4.py scripts in AutoDockTools 1.5.6, including adding charges and hydrogen atoms. Next, a 60×60×60Å grid box with a spacing of 0.375Å was set to surround the entire binding site. The optimal binding conformation was searched using a Lamarckian genetic algorithm, with the following docking settings: 100 docking experiments were performed, the population size was 300 individuals, the crossover rate was 0.8, and the local search rate was set to 0.06. All other parameters during the docking process were set to their default values.

[0085] CAR-T Cell Preparation: CD19 CAR-T cells are generated from human T cells. Brief Procedure: Human pan-T cells (CD3+ T cells) were isolated from PBMCs (n = 3) using a human pan-T cell isolation kit (Miltenyi Biotec, 130-096-535) and co-incubated with CD3 / CD28 cross-linked antibody magnetic beads. On day 3, the CAR lentiviral construct was introduced into the T cells at an MOI of 5-10, followed by in vitro expansion for 7 days in medium containing 100 U / ml IL-2. During expansion, the medium was changed every other day to maintain a cell density of 1×10⁻⁶ cells / day. 6 / mL. CAR-T cells and Raji cells (E:T ratio of 2:1 or 4:1) were co-cultured for 16 hours under specified conditions, followed by flow cytometry analysis. The amino acid sequence of CD19 CAR is: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* CAR-T cell function assay: To assess the cytotoxic function of CAR-T cells, 2×10⁻⁶ cells were used. 4 Raji cells and CD19-CAR-T cells were co-cultured in 96-well round-bottom plates in 1640 medium containing 5% FBS at a specified ratio, and treated with 1 μM TAIII in the presence or absence of 5 μM NECA. After 16 hours of co-culture, cells were collected for RT-qPCR and flow cytometry analysis. The frequency of CD19-positive cells was considered as the residual Raji cell frequency, and the CAR-T cell killing percentage was calculated as (initial frequency - residual frequency) ÷ initial frequency. After 16 hours of co-culture, the culture supernatant was collected, and the secretion levels of IL-2, IFN-γ, and TNF-α were measured by ELISA (Lianke Biotechnology). After 48 hours of co-culture, CD62L was measured. + CD45RO + Frequency of Tcm cell subsets.

[0086] Real-time Imaging Analysis of CAR-T Cell Killing Process: The killing process of CD19-CAR-T cells was monitored using the Incucyte® S3 Live Cell Analysis System (Sartorius). In this assay, Raji cells were stained with Incucyte® cytolight rapid red dye (Sartorius, #4706) before being seeded into 96-well plates (10,000 cells per well). Different numbers of CAR-T cells were then added to achieve the specified E:T ratio, and the recommended concentration of Incucyte® Annexin V green dye (Sartorius, #4642) was added to the culture medium to label apoptotic cells. During the 36-hour co-culture, fluorescence images of each well were collected every 2 hours at 10x zoom; co-localization of red and green fluorescent regions indicated apoptotic Raji cells. Data were analyzed using the Incucyte® Spheroid analysis software module.

[0087] A2AR shRNA knockdown assay: A2AR-shRNA pLKO.1-GFP-puro lentiviral particles targeting A2AR were purchased from Tsingke Biotechnology Co., Ltd. Treg cells were co-incubated with the viral particles in 5-8 μg / ml polybrene (Hanbio) for 24 hours, followed by fresh culture. 72 hours after viral infection, cells were selected using 0.3 μg / ml puromycin for 48 hours. After selection, cells were cultured for another 3-5 days and collected. GFP-positive and GFP-negative cell populations were separated by sorting, and protein and mRNA levels were measured by Western blotting (immunoblotting) and quantitative PCR (qPCR), respectively.

[0088] The shRNA sequence used is as follows: A2AR-sh1:GCCTCTTCATTGCCTGCTTCG A2AR-sh2:GCCAAGTGGCCTGTCTCTTTG A2AR-sh3:GCTAAGGGCATCATTGCCATC Construction of the Treg functional screening system: To construct the Treg functional screening system, the human IL-2 promoter region (-326 to +145, a total of 473 bp) containing the FoxP3 binding site was cloned into the Lenti-PGL4 delivery vector. This system was packaged into lentivirus using Hanbio, and then, following the lentivirus transduction protocol, the IL-2 promoter-Fluc reporter gene was introduced into Jurkat T cells in the presence of 5-8 μg / ml polygel (Hanbio). After incubation for 24 hours, the medium was replaced with fresh medium. 72 hours after viral infection, selection was performed for 48 hours using 0.3 μg / ml puromycin. After puromycin selection, single-clonal cells were isolated. The best-performing single-clonal cell lines were selected by detecting the luciferase activity of each single-clonal cell after TCR stimulation. Subsequently, a FoxP3a overexpression lentiviral construct containing a BSD resistance tag was introduced into the selected clones. After another round of TCR stimulation and selection, single-clonal cell lines were selected based on their final performance. Treg function was assessed by measuring the fluorescence intensity of firefly luciferase (FLuc).

[0089] Animal models: (1) Homologous transplantation model: Six-week-old male C57BL / 6J mice (Jicui Yaokang, Nanjing, China) were housed in IVC cages in an SPF-grade facility and used in accordance with the animal care regulations of the Hefei Institutes of Physical Science, Chinese Academy of Sciences (Hefei, China). After one week of acclimatization, 1×10⁻⁶ mice harvested during the exponential growth period were transplanted. 6 One MC38 cell (suspended in PBS) was injected into the subcutaneous space of the right inguinal region of mice. When the tumor volume reached approximately 50 mm... 3 Oral administration began daily. TAIII was administered daily via intraperitoneal injection, dissolved in 5% DMSO / 0.5% methylcellulose / 0.4% Tween 80 solution, at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg, respectively; anti-PD-1 antibody (5 mg / kg, dissolved in physiological saline, twice weekly) was administered; its carrier served as a control. Reference compound AZD4635 was dissolved in 5% DMSO corn oil and administered orally twice daily at a dose of 50 mg / kg. Body weight and tumor volume were measured every two days. Tumor volume (mm) 3 The formula for calculating width is: [(width) 2 [×length) / 2], where width (W) is defined as the smaller of the two measurements, and length (L) is defined as the larger one. Harvested tumor-draining lymph nodes or tumor tissue were further analyzed by immunohistochemistry (IHC), stained with Foxp3 and CD8 antibodies according to the manufacturer's instructions. The simplified procedure is as follows: After fixing the tumor-draining lymph nodes or tumor tissue in 4% neutral buffered formalin and embedding it in paraffin, it was cut into 4 μm thick sections and then stained with FoxP3 or CD8 antibodies.

[0090] (2) Non-invasive in vivo bioluminescence model: 5.5-week-old NSG mice were intravenously injected with 3×10 6 10 Raji-Luc or Nalm6-Luc cells (Shanghai Southern Model Biotechnology Center, product number NM-B07-1). Two or six days later, administer 6 × 10⁶ cells intravenously. 6 CAR-T cells prepared on days 6-10. In a non-invasive in vivo bioluminescent model of lymphoma (n=5 or 6), the efficacy of CAR-T cells alone or in combination with TAIII (5 mg / kg, intraperitoneal injection, HKI) against Raji-Luc was evaluated. + or Nalm6-Luc + Effects on cell growth. In a preconditioning model, mice injected with Raji-Luc cells 6 days prior were infused with either TAIII-pretreated (1 μM, 36 h) or untreated CD19-CAR T cells. Luciferase-positive lymphoma burden was measured every 3 days or weekly using Bright-Glo assay and Luminoskan, and bioluminescent images of representative mice with matched initial lymphoma burdens were captured. Survival curves were compared using the log-rank Mantel–Cox test. The percentages of Luc(CD19) and CAR-T cells, as well as other T cell subsets, in blood, bone marrow, or spleen samples from transplanted mice (n=5 or 6) were determined by flow cytometry using specified antibodies. Body weight of transplanted mice was measured every two days.

[0091] In the Nalm6-Luc subcutaneous tumor model, NSG mice were subcutaneously injected with 1×10 6 One Nalm6-Luc lymphoma cell. Three days later, on days 3 and 8, 3 × 10⁻⁶ cells were administered intravenously. 6 10 CD19 CAR-T cells or UNT (untransduced) cells were administered to NSG mice, with either added or unadded (w / o) 10 mg / kg TAIII (once daily) as the treatment group (n = 6). Tumor size was measured after treatment. Subsequent assays, including tumor imaging, tumor growth inhibition rate calculation, tumor tissue staining, and ELISA, were performed according to similar procedures used in syngeneic transplantation models. For the Nalm6-Luc subcutaneous tumor model using pretreated cells, 1 × 10⁻⁶ cells were subcutaneously injected into NSG mice (n = 3). 6 One Nalm6-Luc lymphocytic leukemia cell. Six days later, 5 × 10⁶ cells were infused. 6CAR-T cells or CAR-T cells pretreated with TAIII (1 μM, 72 h). Correlation analysis was performed on luciferase-positive leukemia burden measured by Bright-Glo assay and Luminoskan. Total fluorescence intensity was quantified and plotted. T cell frequency and phenotype in peripheral blood (days 4, 7, and 14 post-CAR-T infusion), bone marrow, and spleen (day 14 post-CAR-T infusion) were assessed by flow cytometry.

[0092] ELISA Detection of Tumor Samples: ELISA detection of tumor samples was performed as described above. The simplified procedure includes two main steps: ① Tumor Tissue Preparation: Tumor tissue was immediately removed after euthanasia and placed on ice to minimize protein degradation. The tissue was minced and homogenized in optimized protein extraction tissue lysis buffer. After homogenization, the sample was centrifuged at 4°C to collect the supernatant containing soluble proteins. ② ELISA Procedure: Using a commercially available ELISA kit (Linke Biotechnology), the supernatant was analyzed according to the manufacturer's instructions to quantify IL-2, IFN-γ, and other cytokines of interest in the tumor tissue homogenate and serum samples.

[0093] Statistical analysis: Each experiment was independently repeated three times, with each experiment containing three technical replicates. The results shown in the figure represent representative data from the three independent experiments. Data are expressed as mean ± standard deviation (SD) or mean ± standard error (SEM) (as shown in the figure caption). Student's t-test, Mann-Whitney U test, or Kruskal-Wallis test were performed using GraphPad Prism (version 9.0) or IBM SPSS Statistics (version 24.0) software to determine statistical significance. # or * P<0.05, ## or ** P<0.01, ### or *** P<0.001, #### or **** P<0.0001).

[0094] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Application of Anemarrhena saponin AIII in the preparation of efficacy enhancers for CAR-T cell therapy and / or in the preparation of products to overcome drug resistance in CAR-T cell therapy.

2. The application according to claim 1, characterized in that, The efficacy enhancer and / or product for overcoming CAR-T cell therapy resistance has at least one of the following effects: A1) Inhibits A2AR activity and FoxP3 transcription, reduces the proportion of regulatory T cells, and relieves immunosuppression on effector T cells; A2) Increase the proportion of central memory T cells and the secretion of cytokines IFN-γ, IL-2, and TNF-α; A3) Enhances the cytotoxicity of CAR-T cells; A4) Enhance the efficacy of CAR-T cells in preventing tumor recurrence.

3. The application according to claim 1, characterized in that, The CAR-T cells were treated with Anemarrhena saponin AIII before administration or administered in combination with Anemarrhena saponin AIII during administration.

4. The application according to claim 1, characterized in that, Anemarrhena saponin AIII competitively binds to the cholesterol-sensitive motif of A2AR, blocking the downstream cAMP-PKA-CREB signaling pathway, thereby inhibiting FoxP3 transcription and regulatory T cell differentiation, reducing the proportion of regulatory T cells, relieving their immunosuppression on effector T cells, and increasing the proportion of central memory T cells and cytokine secretion, thus enhancing the cytotoxicity of CAR-T cells and / or preventing tumor recurrence.

5. A method for preparing functionally enhanced CAR-T cells, characterized in that, CAR-T cells were treated with Anemarrhena saponin AIII to obtain a therapeutically enhanced CAR-T cell population with a significantly reduced proportion of Tregs.

6. The method according to claim 5, characterized in that, The concentration of Anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

7. The application of Anemarrhena saponin AIII in combination with CAR-T cells and / or Anemarrhena saponin AIII in combination with PD-1 in the preparation of antitumor drugs, characterized in that, The tumor is selected from hematologic malignancies or solid tumors. Preferably, the solid tumors include lymphomas and / or colorectal cancer, and the hematologic malignancies include lymphocytic leukemia.

8. A composition for CAR-T cell therapy, characterized in that, This includes anemarrhena saponin AIII and CAR-T cells, wherein the concentration of anemarrhena saponin AIII is not less than 0.3 μM or not more than 1 μM.

9. The application of Anemarrhena saponin AIII in the preparation of selective A2AR allosteric inhibitors, characterized in that, Anemarrhena saponin AIII affects A2AR function by competing for cholesterol and allosterically binding to key residues.

10. A method for reducing the proportion of regulatory T cells in CAR-T cells in vitro for non-therapeutic purposes, characterized in that, CAR-T cells were treated with Anemarrhena saponin AIII, which significantly reduced the proportion of Tregs. Preferably, the concentration of Anemarrhena saponin AIII was not less than 0.3 μM or not more than 1 μM.

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