Engineered car-t cells secreting membrane protein degraders and uses thereof

Abstract

This invention discloses an enhanced CAR-T cell (CARTAC) capable of secreting a targeted membrane protein degrader and its applications. While retaining specific killing function, this cell can expand locally in tumors and continuously secrete the bispecific adaptor molecule scTAC. scTAC can recruit E3 ubiquitin ligases, mediate ubiquitination of tumor cell surface membrane proteins (such as PD-L1), and achieve targeted degradation via the lysosomal pathway, effectively reversing the immunosuppressive microenvironment, overcoming antigen heterogeneity, and eliminating antigen-negative tumor cells through the bystander effect. This invention also utilizes the efficient endocytosis properties of EGFR to design scTAC-dual, which can further enhance degradation efficiency. In various lung cancer mouse models, CARTAC-dual exhibited excellent tumor infiltration capacity, low exhaustion levels, and sustained antitumor activity, without systemic toxicity. This invention integrates the targeted killing and protein degradation delivery functions of CAR-T cells, not only enhancing the control effect on solid tumors but also possessing target scalability, making it suitable for the universal upgrading of various CAR-T products.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and tumor immunotherapy, specifically relating to a genetically engineered chimeric antigen receptor T cell (CAR-T) capable of secreting a bifunctional protein—a single-chain tandem aptamer (scTAC)—mediating the lysosomal degradation of key membrane proteins (such as PD-L1 and EGFR) in the tumor microenvironment. This invention further relates to methods for preparing this cell, pharmaceutical compositions, gene constructs, viral vectors, and their use in the preparation of drugs for treating antigenically heterogeneous solid tumors. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in hematologic malignancies, but its efficacy in solid tumors remains limited by multiple obstacles, including heterogeneity of tumor antigen expression, immunosuppressive microenvironment, insufficient T-cell infiltration, and functional exhaustion.

[0003] Current strategies primarily focus on enhancing the cytotoxic ability of CAR-T cells or co-expressing auxiliary molecules such as cytokines and checkpoint blocking antibodies. However, these methods are mostly "passive regulation," unable to continuously reshape the tumor microenvironment, and struggle to eliminate antigen-negative escape clones. Furthermore, traditional antibody therapies can only reversibly block target protein function, failing to completely eliminate its expression.

[0004] The development of targeted protein degradation (TPD) technology has provided a revolutionary alternative to the traditional "occupation-driven" inhibition model. In particular, extracellular TPD (eTPD) technologies, such as LYTAC, TransTAC, and AbTAC, use bispecific molecules to link target membrane proteins (POIs) to lysosomal sorting receptors (such as CI-M6PR) or transmembrane E3 ubiquitin ligases (such as RNF43), inducing their endocytosis-lysosomal degradation. This strategy not only completely eliminates key targets such as PD-L1 and EGFR, avoiding protein rebound, but also promotes antigen cross-presentation, activates adaptive immunity, and exerts an "in situ cancer vaccine" effect. Among these, AbTAC shows unique promise in targeting immune checkpoints and oncogenic receptors due to its genetically encoded, catalytically degradable, and potentially tissue-selective advantages.

[0005] However, existing eTPD molecules rely on systemic administration, which generally suffers from short serum half-life, limited tumor tissue penetration, high off-target toxicity risk, and difficulty in maintaining effective local concentrations, severely limiting their therapeutic window and clinical application potential. Currently, there is no technology capable of achieving targeted delivery, sustained expression, and in-situ release of eTPD effector molecules in the tumor microenvironment.

[0006] Given the specific homing, persistent intratumoral survival, and local expansion capabilities of CAR-T cells, engineering them into "living biofactories" that secrete eTPD molecules can achieve in situ, continuous, and targeted delivery of degradation factors. This strategy not only holds promise for efficiently clearing immune checkpoint molecules (such as PD-L1) and oncogenic receptors (such as EGFR), but also for covering antigenically heterogeneous tumor populations through the bystander effect, thereby overcoming a key bottleneck in the treatment of solid tumors using existing therapies. Summary of the Invention

[0007] This invention aims to address key issues faced by existing CAR-T cell therapy in solid tumor treatment, including: Antigen heterogeneity leading to immune escape; persistent expression of immune checkpoints (such as PD-L1) and oncogenic receptors (such as EGFR); restricted T cell infiltration and functional exhaustion; lack of long-acting, localized, and self-amplifying therapeutic mechanisms.

[0008] To this end, the present invention provides an engineered T cell platform that secretes membrane protein degrading agents—CARTAC (CAR-T cells secreting bifunctional single-chain targeting chimera), namely CAR-T cells that can secrete bifunctional scTAC molecules to achieve in situ biosynthesis and bystander-mediated degradation of membrane proteins, thereby establishing a self-reinforcing anti-tumor immune cycle.

[0009] To address the aforementioned technical problems, this invention provides an engineered enhanced CAR-T cell capable of secreting targeted membrane protein degrading agents. The core idea is to endow CAR-T cells with "dual functions": retaining their inherent targeted killing ability while adding the function of locally secreting protein degrading agents to modify the tumor microenvironment and expand the killing range. More importantly, this invention utilizes CAR-T cells as a live cell carrier to achieve targeted, in-situ, and continuous delivery of the degrading agent.

[0010] The specific technical solution described in this invention is as follows: This invention provides a single-stranded tandem aptamer (scTAC) comprising: (a) At least one binding domain that targets a tumor-associated membrane protein (such as an anti-PD-L1 or anti-EGFR scFv or nanobody). (b) An E3 ubiquitin ligase recruitment domain (such as anti-RNF43 or ZNFR3 scFV). (b) and the linker peptide sequence, which enables the two to form a functional fusion.

[0011] Furthermore, the amino acid sequence of the single-chain tandem aptamer scTAC is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5 or SEQ ID NO.6; the scTAC binds to the target membrane protein and recruits the E3 ligase, inducing its ubiquitination modification, and enters the lysosome for degradation in a clathrin-dependent manner dependent on EGFR, thereby achieving catalytic, non-cellular autonomous clearance of the target protein.

[0012] In a preferred embodiment of the present invention, the scTAC is a multispecific structure (scTAC-dual) that simultaneously targets PD-L1, RNF43, and EGFR, utilizing the inherently efficient endocytosis properties of EGFR to promote the co-internalization and accelerated degradation of PD-L1.

[0013] The present invention also provides a nucleic acid molecule encoding the single-stranded tandem aptamers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5 or SEQ ID NO.6.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0015] The present invention also provides a lentiviral or retroviral vector carrying the above-mentioned nucleic acid molecules.

[0016] The present invention also provides a CAR-T cell expressing the above-mentioned single-stranded tandem aptamer, which can achieve targeted homing and initial killing of tumors by expressing a CAR that specifically recognizes tumor antigens (such as HER2).

[0017] The CARTAC cells continuously secrete functional scTACs in the tumor microenvironment, mediating the "bystander effect" and degrading PD-L1 or EGFR on neighboring antigen-negative tumor cells, effectively clearing antigen escape variants.

[0018] The present invention also provides the use of the above-mentioned single-stranded tandem aptamers, nucleic acid molecules, lentiviruses or retrovirus vectors or CAR-T cells in the preparation of drugs for the prevention and / or treatment of solid tumors, wherein the solid tumors are non-small cell lung cancer and / or colorectal cancer.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. Originality: This invention is the first to combine extracellular targeted protein degradation (eTPD) with CAR-T cells to construct a "living biofactory" that can secrete degradation factors.

[0020] 2. Catalytic clearance: scTAC can act repeatedly to achieve efficient and sustained degradation of membrane proteins such as PD-L1 and EGFR, which is superior to traditional blocking antibodies.

[0021] 3. Overcoming antigenic heterogeneity: Eliminating antigen-negative tumor cells through the bystander effect, preventing immune escape.

[0022] 4. Synergistic dual mechanism: Simultaneously inhibits immune checkpoint (PD-L1) and oncogenic pathway (EGFR) to enhance anti-tumor efficacy.

[0023] 5. Self-amplification effect: Forms a positive feedback loop, enhancing T cell persistence and tumor control capabilities.

[0024] 6. Local delivery and systemic safety: scTACs are mainly secreted at the tumor site, avoiding systemic exposure and reducing the risk of toxicity.

[0025] 7. Modular design: The scTAC structure can be flexibly replaced with the target domain or E3 recruitment domain, making it suitable for a variety of targets and indications. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the CARTAC platform provided by the present invention, showing that CAR-T cells simultaneously express HER2-CAR and secrete scTAC. scTAC binds to PD-L1 and recruits E3 ligase, inducing its ubiquitination and lysosomal degradation. In the figure, a is a schematic diagram of CARTAC design, b is a Western blot detection of PD-L1 degradation levels of P-R_scTAC and P-Z_scTAC, and UT represents the untreated control group.

[0027] Figure 2 Figure 1 shows the results of the first-generation CAR-T cell-level protein degradation and cytotoxicity function test. Figure 2 shows the CAR expression detected by flow cytometry, Figure 3 shows the expression level of P-R_CARTAC and P-Z_CARTAC secreted proteins detected by Western blotting, Figure 4 shows the degradation level of PD-L1 by the first-generation CAR-T in the transwell assay, and Figure 5 shows the in vitro cell cytotoxicity test of the first-generation CAR-T.

[0028] Figure 3 The figures show the in vivo functional validation results of the first-generation CARTAC. Figure a is a schematic diagram of the animal experiment design process, figure b is the tumor growth curve of the animal experiment, figure c is the survival curve of mice in each group of the animal experiment, and figure d is the weight change of mice in each group of the animal experiment.

[0029] Figure 4For the structural design, optimization, and mechanism verification of scTAC-dual, Figure a shows the experimental design for the target protein module location test; Figure b shows the flow cytometry detection and statistical graph of scTAC-dual's degradation of EGFR protein; Figure c shows the flow cytometry detection and statistical graph of scTAC-dual's degradation of PD-L1 protein; Figure d shows the Western blotting detection of PRE-scTAC's degradation of PD-L1 and EGFR; Figure e shows the Western blotting analysis of the blocking effect of inhibitors MG132 and Bafilomycin A1 on protein degradation; and Figure f shows the Western blotting analysis of the blocking effect of inhibitors TAK243, Chlorpromazine (Chlor), Methyl-β-cyclodextrin (Cyclod), and Amiloride hydrochloride dihydrate (Amil) on protein degradation.

[0030] Figure 5 This study presents functional assays for the second-generation CARTAC-dual, including in vitro protein degradation, cytotoxicity, and prevention of T cell exhaustion. Figure a shows the flow cytometry analysis of CAR expression; figure b shows the Western blotting analysis of PR-E_scTAC-dual secreted protein expression levels; figure c shows the degradation levels of EGFR and PD-L1 by CARTAC-dual; figure d shows the in vitro cell-killing level of CAR-T cells as detected by Incucyte; and figure e shows the flow cytometry analysis of CAR-T cell exhaustion after long-term co-culture with oxygen.

[0031] Figure 6 To demonstrate the significant inhibition of tumor growth and prolongation of survival by the second-generation CARTAC-dual (PR-E_CARTAC) in various lung cancer models, Figure a shows the schematic design of the NCI-H1975 non-small cell lung cancer subcutaneous xenograft model; Figure b shows the tumor growth curve of the NCI-H1975 non-small cell lung cancer subcutaneous xenograft model; Figure c shows the tumor survival curve of the NCI-H1975 non-small cell lung cancer subcutaneous xenograft model; Figure d shows the schematic design of the PC-9 non-small cell lung cancer subcutaneous xenograft model; Figure e shows the tumor growth curve of the PC-9 non-small cell lung cancer subcutaneous xenograft model; Figure f shows the T cell infiltration detection in the PC-9 non-small cell lung cancer subcutaneous xenograft model; and Figure g shows the schematic design of the NCI-H1975 non-small cell lung cancer metastatic tumor model. Figure h shows IVIS imaging of the NCI-H1975 non-small cell lung cancer metastasis model; Figure i shows the tumor growth curve of the NCI-H1975 non-small cell lung cancer metastasis model; Figure j shows the mouse survival curve in the NCI-H1975 non-small cell lung cancer metastasis model.

[0032] Figure 7To eliminate tumor growth and alleviate CAR-T cell exhaustion in a heterogeneous tumor model using second-generation CARAC-dual cells, the following figures are presented: a) Schematic diagram of the experimental procedure for the heterogeneous subcutaneous tumor model; b) Tumor growth curve in the heterogeneous SW48 model; c) Representative tumor image collected on day 26; d) Schematic diagram of the heterogeneous tumor model used for tissue collection; e) Western blot analysis of representative scTAC secreted in vivo by CAR-T cells and PR-E_CARTAC cells; f) Quantitative analysis of mean fluorescence intensity (MFI) of EGFR (left) and PD-L1 (right) on the tumor cell surface; g) Quantitative analysis of IFN-γ concentration in tumor lysis buffer; h) Tumor infiltration CD3... + Representative flow cytometry scatter plots and quantitative analysis of T cells. Figure i shows the CD3 infiltrating tumor cells. + CAR + The left figure shows a representative flow cytometry scatter plot of T cells and a quantitative analysis of the data (right figure). The right figure shows a representative flow cytometry scatter plot of tumor-infiltrating CAR-T cell exhaustion markers (PD-1, LAG3, TIM3) and a quantitative analysis of the data (n=5). Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0034] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available products and can be purchased on the market. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Example 1: Construction and validation of secretory targeting chimera (scTAC) scTAC molecular design: Construct a single-stranded bispecific molecule containing two single-stranded variable fragments (scFv) linked by a flexible linker, one targeting a membrane protein (such as PD-L1), and the other binding a transmembrane E3 ubiquitin ligase (such as RNF43 or ZNRF3). Figure 1 a).

[0036] Molecular Construction and Expression: The gene sequences encoding the above-mentioned scTACs, PD-L1-RNF43 scTAC (abbreviated as P-R_scTAC, amino acid sequence as shown in SEQ ID NO.1) and PD-L1-ZNF43 scTAC (abbreviated as P-Z_scTAC, amino acid sequence as shown in SEQ ID NO.2), were fused with the CD8 secretion signal peptide and V5 tag sequence, respectively, and cloned into the mammalian expression vector pcDNA3.1 to construct two plasmids. The plasmids were transfected into 50 mL of Expi293F cells. Cells were cultured at 37℃ and 5% CO2 for 4-5 days before harvesting. After protein expression, the cell culture supernatant was centrifuged, filtered through a 0.45 µM filter, and purified using Ni-NTA affinity chromatography (Smart Lifesciences, catalog number: SA004025). The chromatography column was washed with PBS, and the protein was eluted with PBS containing 300 mM imidazole. The eluted proteins were dialyzed into PBS using a dialysis cassette with a 10 kDa molecular weight cutoff. Proteins were stored at 4°C or flash-frozen and then stored at -80°C. The purity and integrity of all proteins were verified by SDS-PAGE electrophoresis. Secreted scTAC proteins (including P-R_scTAC and P-Z_scTAC) were collected and analyzed by Western blotting. Figure 1 As shown in b. After sequencing verification confirms correctness, it is used for subsequent lentivirus packaging.

[0037] Degradation Function Validation: Purified scTAC proteins (including P-R_scTAC and P-Z_scTAC, with gradient concentrations of 0.04, 0.12, 0.36, 1.1, 3.3, and 10 nM, respectively) were co-incubated with PD-L1-expressing tumor cells (e.g., HCT15) for 24 hours. The mean fluorescence intensity (MFI) of PD-L1 on the cell surface was detected by flow cytometry. Total cellular protein was extracted, and the total PD-L1 protein level was detected by Western blotting using GAPDH as an internal control. The results showed that increasing scTAC protein concentration significantly reduced both cell surface and total PD-L1 protein levels, with a maximum degradation efficiency exceeding 75%. (Results are as follows...) Figure 1 As shown in b, both P-R_scTAC and P-Z_scTAC exhibited significant degradation activity, demonstrating the effective clearance of the target protein PD-L1 by this molecular architecture. Specifically, P-R_scTAC showed superior degradation activity against PD-L1 compared to P-Z_scTAC, indicating that scTAC molecules with the endocytic degradation module RNF43 as their core have better target protein clearance efficiency. The RNF43 module is the preferred structural molecule for this single-stranded tandem aptamer.

[0038] Example 2: Preparation and functional characterization of first-generation CARTAC cells Preparation of engineered T cells: Human primary T cells were obtained by co-transduction with two lentiviral vectors from peripheral blood mononuclear cells isolated from healthy donors, yielding first-generation CAR-TAC cells co-expressing CAR and scTAC. The first lentiviral vector encodes a second-generation CAR targeting HER2. The second lentiviral vector encodes P-R_scTAC (as shown in SEQ ID NO. 3) or P-Z_scTAC (as shown in SEQ ID NO. 4) from Example 1. T cells transduced only with the CAR vector were used as a control.

[0039] Basic characterization: Flow cytometry confirmed that engineered T cells (referred to as first-generation CARTAC cells) expressed CAR on their surface, and scTAC secretion vector transduction did not affect CAR expression levels or the proportion of T cell subsets. Western blotting confirmed the continuous detection of secreted scTAC protein in the CARTAC cell culture supernatant. Figure 2 (a-2b).

[0040] To verify whether the scTAC protein secreted by first-generation CARTAC cells can act on distant tumor cells via paracrine signaling, a Transwell co-culture system was used for verification. First-generation CARTAC cells were placed in the upper chamber, and HER2-positive tumor cells (HCT15 ATCC CCL-225) were placed in the lower chamber. After co-culturing at 37°C and 5% CO2 for 72 hours, HCT15-HER2 cells in the lower chamber were collected, and the mean fluorescence intensity of PD-L1 on the cell surface was detected by flow cytometry. The results are as follows: Figure 2 As shown in Figure c, the expression of PD-L1 on the surface of lower ventricular tumor cells was significantly reduced, demonstrating that the secreted scTAC has functional activity.

[0041] Enhanced in vitro killing effect: P-R_scTAC or P-Z_scTAC CAR-AC cells (effector cells E) were co-cultured with HER2-positive tumor cells (HCT15, HT29) at different effector-to-target ratios (E:T = 5:1, 2.5:1, 1:1, 1:2.5, 1:5). Compared with control CAR-T cells, P-R_scTAC CAR-AC cells exhibited stronger tumor cell killing activity at low effector-to-target ratios. Figure 2 d).

[0042] Example 3: In vivo antitumor effect of first-generation CARTAC cells A xenograft model was established in NSG mice by subcutaneous inoculation with HCT15 tumor cells. On day 0, NSG mice were subcutaneously injected with 1 × 10⁻⁶ HCT15 tumor cells. 6HCT15 cells were transplanted. Seven days after transplantation, mice with similar tumor sizes were randomly divided into four groups (n=5): (1) PBS blank control group, which received sterile PBS via tail vein injection; (2) CAR-T negative control group, which received 1×10 HCT15 cells via tail vein injection. 6 (3) P-RCARTAC group, 1×10 HER2-CAR-T cells were injected via tail vein. 6 (3) CARTAC cells co-expressing HER2-CAR and P-R_scTAC; (4) P-ZCARTAC group, with 1×10 cells injected via tail vein. 6 CARTAC cells co-expressing HER2-CAR and P-Z_scTAC were used. Tumor growth was measured every three days from the date of cell injection, and the survival rate and weight changes of mice in each group were recorded.

[0043] The results are as follows Figure 3 The results showed that CAR-T therapy inhibited tumor growth compared to the PBS group; while the P-RCARTAC and P-ZCARTAC treatment groups showed stronger tumor growth inhibition effects than the control CAR-T group, and the survival time of mice was prolonged. The body weight of mice in all groups remained stable during treatment, and no obvious toxicity was observed.

[0044] Example 4: Design and optimization of the dual-targeting single-stranded tandem aptamer scTAC-dual Molecular design: To simultaneously degrade PD-L1 and EGFR, a dual-targeting scTAC-dual was constructed, with two structures designed, such as... Figure 4 As shown in Figure a, ER-P_scTAC-dual has the amino acid sequence shown in SEQ ID NO. 5 and the nucleotide sequence shown in SEQ ID NO. 6. From the N-terminus to the C-terminus, it consists of: EGFR nanobody sequence - RNF43 scFv - PD-L1 nanobody sequence; PR-E_scTAC-dual has the amino acid sequence shown in SEQ ID NO. 7 and the nucleotide sequence shown in SEQ ID NO. 8, with the PD-L1 nanobody at the N-terminus and the EGFR nanobody at the C-terminus, linked by an RNF43 module. PR-E_scTAC-dual protein was collected and purified according to the method described in Example 1.

[0045] In vitro degradation efficiency: Non-small cell lung cancer cells NCI-H1975 (CRL-5908) were treated with purified ER-P_scTAC-dual and PR-E_scTAC-dual proteins (final concentrations of 1, 10, and 100 nM). Western blot and flow cytometry analyses showed that both molecules simultaneously and efficiently degraded PD-L1 (>90%) and EGFR (approximately 60%), exhibiting superior degradation efficiency compared to single-target scTAC molecules. Furthermore, the degradation capacity of PR-E_scTAC-dual was superior to that of ER-P_scTAC-dual. Figure 4 b、 Figure 4 c). To further evaluate the degradation efficiency and concentration dependence of PR-E_scTAC-dual protein on target proteins, different final concentrations (0.4, 1.2, 3.7, 11, 33, 100 nM) of PR-E_scTAC-dual molecules were co-incubated with NCI-H1975 non-small cell lung cancer cells for 24 hours. After incubation, total cellular protein was extracted, and the levels of PD-L1 and EGFR total protein were detected by Western blotting using GAPDH as an internal control. The results are as follows: Figure 4 As shown in Figure d, the PR-E_scTAC-dual molecule can effectively degrade and clear both PD-L1 and EGFR target proteins, with a particularly strong ability to degrade PD-L1. Degradation mechanism study: After pretreatment of NCI-H1975 cells with a pathway inhibitor, followed by treatment with PR-E_scTAC-dual protein at a final concentration of 100 nM, the results are as follows... Figure 4 As shown in e and 4f, the lysosomal inhibitor Bafilomycin A1 (0.2, 1 μM) completely blocked PR-E_scTAC-mediated degradation, while the proteasome inhibitor MG132 (1, 5 μM) had no significant effect. The ubiquitin activator inhibitor TAK123 (1, 2 μM) and the clathrin-mediated endocytosis inhibitor Chlorpromazine (10, 30 μM) significantly reversed the degradation effect, while the inhibitors methyl-β-cyclodextrin (10, 100 μM) and amiloride hydrochloride (10, 100 μM) had no inhibitory effect on the degradation effect. These results indicate that the degradation of the dual-target PR-E_scTAC-dual protein depends on ubiquitination and the clathrin-mediated endocytosis-lysosomal pathway.

[0046] Example 5: Preparation and efficacy of second-generation CARTAC cells (secreting scTAC-dual) Cell construction: The gene encoding PR-E_scTAC-dual was constructed into a lentiviral vector, which was then co-transduced with a HER2-CAR vector into human primary T cells to obtain second-generation CARTAC cells (PR-E_CARTAC). After culture, the expression levels of CAR and PR-E_scTAC-dual secreted protein were detected by flow cytometry and Western blotting. Figure 5 a, 5b).

[0047] In vitro functional validation: Transwell assays confirmed that second-generation CARTAC cells were placed in the upper chamber and NCI-H1975 cells in the lower chamber. After co-culturing at 37°C and 5% CO2 for 72 hours, cells from the lower chamber were collected, and the expression levels of PD-L1 and EGFR on the cell surface were detected by flow cytometry. The results showed that PRE-scTAC-dual secreted by PRE-E_CARTAC cells effectively degraded PD-L1 and EGFR on the surface of lower chamber tumor cells (NCI-H1975). In long-term co-culture experiments, PRE-E_CARTAC cells showed significantly better killing effects against various EGFR-mutant NSCLC cell lines than control CAR-T cells. Figure 5 c, 5d).

[0048] Delaying T cell exhaustion: One day before the start of repeated stimulation screening, 2 × 10 5 One NCI-H1975-Luc cell line was seeded into each well of a 6-well plate and cultured in RPMI 1640 medium. After 24 hours, the medium for the NCI-H1975-Luc cells was removed, and 1 × 10⁶ cells were added to each well. 5 CAR-T cells (control group) or PR-ECARTAC cells were co-cultured with NCI-H1975-Luc cells in 2 mL of X-VIVO 15 medium (containing 5% human AB serum and 50 U / ml IL-2). After 48 hours, an appropriate amount of NCI-H1975-Luc cells was added to each co-culture system to re-establish a 1:4 effector cell to target cell (E:T) ratio. This step was repeated every 3 days for a total of six target cell stimulations. At the end of the simulation experiment, CAR-T cells were collected for evaluation of cytotoxicity and immune checkpoint expression. In the in vitro continuous antigen exposure model, compared with control CAR-T cells, PR-E_CARTAC cells showed stronger proliferative capacity, higher cytotoxicity, and lower expression levels of exhaustion markers (PD-1, TIM3, LAG-3) after repeated stimulation. Figure 5 e).

[0049] Example 6: In vivo therapeutic effects of second-generation CARTAC cells and overcoming antigenic heterogeneity Anti-solid tumor efficacy: NSCLC xenograft models of NCI-H1975 or PC9TM (kindly provided by Professor Xiao Yibei of China Pharmaceutical University) were established in NSG mice. On day 0, NSG mice were subcutaneously injected with 1 × 10⁻⁶ mmol / L. 6 One HER2-positive NCI-H1975 cell or 4 × 10⁶ 6 HER2-positive PC9TM cells. Seven days after transplantation, mice with similar tumor sizes were randomly divided into three groups (n=6~8 mice / group). (1) PBS blank control group, sterile PBS was injected into the tail vein; (2) CAR-T negative control group, 1×10 6 One HER2-CAR-T cell or 3×10 6 HER2-positive PC9TM cells; (3) PR-ECARTAC group, 1×10 HER2-positive PC9TM cells were injected via tail vein. 6 Or 3×10 6 PR-ECARTAC cells were administered intravenously. Tumor growth was monitored every three days. Results were as follows: Figure 6 a-6f showed that its anti-tumor effect and survival prolongation effect were significantly better than those of control CAR-T cells. Tumor tissue analysis showed that the PR-ECARTAC treatment group had a greater number of tumor-infiltrating CAR-T cells and exhibited less exhaustion phenotype. Figure 6 a-6f).

[0050] Anti-metastatic effect in a lung metastasis model: 1×10⁻⁶ mice were injected via tail vein. 6 A metastatic tumor model was established using NCI-H1975-Luc cells. Seven days after inoculation, tumor colonization was confirmed by a small animal in vivo imaging system, and the animals were randomly divided into three groups (n=7 / group). The groups received PBS, control CAR-T cells, or PR-ECARTAC cells (cell count: 3×10⁶). 6 (Number of mice) were observed for mouse survival rate and tumor growth, and the results are as follows: Figure 6 As shown in g-6j, the PR-E_CARTAC treatment group effectively controlled the growth of multi-organ metastases and significantly prolonged the survival of mice.

[0051] Overcoming antigenic heterogeneity: HER2-positive (SW48-WT, 3×10) 6 (1 x 10⁻⁶) and HER2 knockout SW48 tumor cells (SW48-HER2-KO, 1 x 10⁻⁶) constructed and preserved in the laboratory. 6 (4 × 10⁻⁶ mice) were subcutaneously injected with a 3:1 mixture of 10⁻⁶ (units) of 4 × 10⁻⁶ (units) ... 6One sample of tumors mimicking antigenic heterogeneity was administered. Patients were treated with PBS, control CAR-T cells, or PR-ECARTAC cells, with an inoculation quantity of 1.5 × 10⁶ cells. 6 (n=7~8). Tumor growth was observed up to day 26 of inoculation treatment, and the results were as follows: Figure 7 As shown in b and 7c. After treatment, the tumor size in the control CAR-T group was inhibited, while PRE-E_CARTAC treatment achieved more durable and effective tumor control, and some mice experienced complete tumor regression.

[0052] Following the above method, a heterogeneous tumor model for tissue collection was constructed, with an inoculum size of 3 × 10⁻⁶ for treatment. 6 Mice were sacrificed and tumor tissue was collected on day 14 of treatment. Figure 7 d). Total tumor protein was provided for Western blot analysis. Results showed that scTAC protein bands were detected only in the PR-E_CARTAC group tumor tissue, confirming that CARTAC cells continuously secrete scTAC-dual protein in vivo. Figure 7 e). Tumor single-cell suspensions were prepared, and the mean fluorescence intensity of EGFR and PD-L1 on the surface of tumor cells was detected by flow cytometry. The results showed that EGFR expression on the surface of tumor cells in the PR-E_CARTAC group was significantly reduced ( Figure 7 f). Tumor tissue lysate was extracted, and IFN-γ concentration was detected by ELISA. The results showed that the IFN-γ level was significantly increased in the PR-E_CARTAC group ( Figure 7 g), indicating that the microenvironment has shifted to a pro-inflammatory state.

[0053] Further flow cytometry analysis showed that the PR-E_CARTAC group had CD3 infiltrating tumors. + T cells and CD3 + CAR + The number of T cells in the CAR-T group was significantly higher than that in the control CAR-T group, while the expression levels of exhaustion markers PD-1, LAG-3, and TIM-3 on the surface of infiltrating CAR-T cells were significantly lower than those in the control CAR-T group. The results are as follows: Figure 7 As shown in h-7j.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-chain tandem aptamer, characterized in that, Include: (a) At least one binding domain that targets a tumor-associated membrane protein; (b) An E3 ubiquitin ligase recruitment domain; (c) A linker peptide or adapter sequence for mediating the functional link between the binding domain and the E3 ubiquitin ligase recruitment domain.

2. The single-chain tandem aptamer as described in claim 1, characterized in that, The targeted tumor-associated membrane proteins are PD-L1 and / or EGFR.

3. The single-chain tandem aptamer as described in claim 1, characterized in that, The recruitment domain of the E3 ubiquitin ligase is RNF43 or ZNRF3.

4. The single-chain tandem aptamer as described in claim 1, characterized in that, The amino acid sequences of the single-chain tandem aptamers are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5 or SEQ ID NO.

7.

5. A nucleic acid molecule encoding the single-stranded tandem aptamer of claim 4, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.

8.

6. A lentiviral or retroviral vector, characterized in that, It carries the nucleic acid molecule as described in claim 5.

7. A CAR-T cell expressing a single-stranded tandem aptamer as described in any one of claims 1-4.

8. A method for constructing CAR-T cells according to claim 7, characterized in that, The specific steps are as follows: introduce the nucleic acid molecule described in claim 5 or the lentivirus or retrovirus vector described in claim 6 into T cells.

9. The use of a single-stranded tandem aptamer as described in claim 1, a nucleic acid molecule as described in claim 5, a lentiviral or retroviral vector as described in claim 6, or a CAR-T cell as described in claim 7 in the preparation of drugs for the prevention and / or treatment of solid tumors, characterized in that, The solid tumor is non-small cell lung cancer and / or colorectal cancer.

Images