Oncolytic virus-t cell chimera for cancer therapy and use thereof

Abstract

An oncolytic virus-T cell chimera (ONCOTECH) for cancer therapy and a use thereof. Oncolytic adenoviruses (OAs) are engineered to bear the gene editing capability of a Cas9 genome editor, and the OAs are anchored onto the surface of a T cell by means of a T-cell specific antigen presented by a layer of biological or microcapsule membrane, so as to form an ONCOTECH. During intravenous injection of the ONCOTECH, the T cell facilitates the delivery of the OAs into a homologous antigen-expressing tumor, and then, the OAs competitively released from the T cell lyse tumor cells and disrupt a programmed death-ligand 1 (PDL1) gene by means of Cas9-mediated gene editing. This therapeutic modality is conducive to reversing the drug resistance of tumors to oncolysis and T cells, improving antigen presentation and activating endogenous tumor specific T cell responses, and inducing a long-lasting immune memory effect, thereby significantly enhancing the therapeutic effect in a mouse tumor model that simulates a series of clinical indicators.

Description

Oncolytic virus-T cell chimera for tumor treatment and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Application No. 202410085145.X, filed on January 20, 2024. Said application No. 202410085145.X is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the field of biomedicine, and more specifically, to an oncolytic virus-T cell chimera for tumor treatment and applications thereof. Background Art

[0004] Oncolytic virus therapy is an emerging cancer immunotherapy modality. Oncolytic viruses (OVs) selectively replicate in tumor cells, inducing a tumor-specific inflammatory response and killing tumor cells without infecting cells in normal tissues. For example, Talimogene laherparepvec (T-VEC, Imlygic), the first OV-based drug approved by the US Food and Drug Administration, is developed from a genetically modified herpes simplex virus type 1 (HSV-1) and has shown effectiveness in treating advanced melanoma. Following intratumoral injection, T-VEC lyses tumor cells, subsequently releasing potent danger signals (DAMPs and PAMPs), tumor-derived antigens, and expressing the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to promote antitumor immune responses. Although intratumoral injection ensures direct viral delivery to local tumor lesions and avoids potential systemic neutralization, systemic delivery of oncolytic viruses is more practical for clinical indications and can easily target multiple tumor sites, especially for widely metastatic tumors. In contrast to intratumoral administration, many previous studies have failed to demonstrate significant antitumor activity of oncolytic viruses via intravascular administration, which may be due to a combination of neutralizing antibodies, antiviral cytokine responses, complement-mediated viral inactivation, and clearance by liver Kupffer cells or erythrocytes. In addition, many recent preclinical and clinical studies have revealed that oncolytic virus therapy upregulates the expression of immune checkpoints (such as PDL1) in a variety of cancer cells and immune cells, because viral infection induces a proinflammatory response, leading to enhanced expression of cytokines and chemokines (including type I and type II TNFs), which upregulate the expression of PDL1 and related proteins. Therefore, OVs encoding specific biomacromolecules for PDL1 or combined therapy with PDL1 antibodies and OVs have been widely studied to alleviate the immunosuppressive tumor microenvironment (TME), which may also form tumor resistance to oncolytic virus therapy. Given the limited systemic binding of OVs and immune checkpoint inhibitors (ICIs) to tumors, the combination of oncolytic virus therapy and checkpoint blockade immunotherapy is often limited, so new strategies are needed to deliver OVs and ICIs to tumors in a targeted manner to address these current problems.

[0005] Current strategies to address these problems rely primarily on living carrier cells capable of carrying OVs for systemic delivery. T cells or mesenchymal stem cells have been explored as carrier cells to deliver OVs systemically (meaning via intravenous injection) by exploiting their tumor tropism. Alternatively, biomaterials or nanocarriers, such as liposomes or cell membranes, which can encapsulate and release OVs, have been used as delivery vehicles to enhance their systemic delivery capabilities. Although these approaches are feasible for protecting OVs from neutralization in the circulatory system, they also have disadvantages. Specifically, cell-based strategies rely heavily on the endocytosis of OVs by host carrier cells, which is generally limited to specific types of viruses. For OVs internalized by host cells, it is still unclear how these carrier cells excrete these viruses contained in the host carrier cells after reaching the tumor. OV-infected host cells have been shown to have lower proliferation capacity and gradually reduced survival ability, which may impair their killing function to target tumors. Compared with cell-based carriers, although biomaterial-based carriers, either organic or inorganic, can be customized by chemical or physical means to load OVs and release OVs in response to specific stimuli in the TME, most of them are hampered by clinical applicability, rapid clearance (by the endothelial reticular system), and nonspecific uptake by healthy tissues. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present application provides a tumor-targeted OV delivery system, which can deliver a CRISPR-Cas9 genome editor by integrating genetically engineered OVs with T cells. This Cas9 editor is controlled by sgRNA targeting the PDL1 gene to knock out PDL1 in tumor cells. This strategy utilizes the tumor targeting of T cells and uses biofilms to protect OVs from systemic neutralization. Specifically, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides an oncolytic virus-T cell chimera, which is obtained by fixing oncolytic virus OVs on the outer surface of carrier T cells by engineering biomembranes or vesicles carrying T cell-specific antigens.

[0008] In one embodiment, the T cells include one or more of TCR-T cells, CAR-T cells, and tumor-infiltrating lymphocytes (TILs) derived from human PDAC pancreatic cancer tumors.

[0009] In one embodiment, the tumor-targeted oncolytic virus delivery system is obtained by attaching the oncolytic virus to the T cell through the biophysical interaction between the T cell receptor TCR and pMHC-I, or the chimeric antigen receptor CAR and the tumor cell surface antigen.

[0010] In one embodiment, the oncolytic virus comprises one or more of an oncolytic adenovirus and an oncolytic herpes virus HSV-1.

[0011] In one embodiment, the specific preparation method of the oncolytic virus-T cell chimera comprises the following steps:

[0012] In step 1, eOA was encapsulated with the B16OVA cell membrane expressing H-2Kb-SIINFEKL by liposome extrusion technology to form M@eOA;

[0013] Step 2: M@eOA binds to TCR and MHC-I-OVA 257-264 The biophysical interaction between them is anchored in OT-1CD8+T;

[0014] Or the specific preparation method of the oncolytic virus-T cell chimera comprises the following steps:

[0015] In step 1, eOA was encapsulated with the tumor cell membrane overexpressing specific tumor antigens (EphA2, PSCA, GD2) through liposome extrusion technology to form M@eOA;

[0016] Step 2: M@eOA is anchored to CAR-T cells through biophysical interactions between CAR and specific tumor antigens (EphA2, PSCA, GD2);

[0017] Or the specific preparation method of the oncolytic virus-T cell chimera comprises the following steps:

[0018] Step 1: After eOA infects 293T cells overexpressing PSCA, the cells are treated with relaxin B, and the eOA-containing vesicles in the supernatant are collected. The eOA-containing nanovesicles (MV@eOA) are obtained by liposome extrusion technology.

[0019] Step 2: MV@eOA is anchored to CAR-T cells through the biophysical interaction between CAR and tumor antigen PSCA;

[0020] Or the specific preparation method of the oncolytic virus-T cell chimera comprises the following steps:

[0021] In step 1, eOA is encapsulated with patient-derived autologous tumor cell membranes through liposome extrusion technology to form M@eOA;

[0022] In the second step, M@eOA is anchored on TIL cells through the biophysical interaction between TCR and pMHC-I of autologous tumor.

[0023] In one embodiment, the oncolytic adenovirus includes an engineered oncolytic adenovirus, wherein the engineered oncolytic adenovirus is obtained by cloning Cas9, sgRNA targeting a target gene, and EGFP into an oncolytic adenovirus.

[0024] In a preferred embodiment, the clone is obtained by cloning Cas9, sgRNA targeting the target gene, and EGFP into the shuttle plasmid of OA, and then transforming the shuttle plasmid into an oncolytic adenovirus.

[0025] In a more preferred embodiment, the shuttle plasmid comprises pDC315-hTERT-E1A.

[0026] In a second aspect, the present invention provides a pharmaceutical composition for treating cancer, comprising the oncolytic virus-T cell chimera according to any one of claims 1 to 8.

[0027] In one embodiment, the pharmaceutical composition further comprises an anti-CTLA4 antibody.

[0028] The third aspect of the present invention provides the use of the above-mentioned oncolytic virus-T cell chimera in the preparation of a drug for cancer treatment.

[0029] In one embodiment, the tumor comprises a primary or disseminated tumor that expresses the antigen.

[0030] In a preferred embodiment, the tumor comprises melanoma, lung cancer, pancreatic cancer or glioblastoma.

[0031] A fourth aspect of the present invention provides the use of the above-mentioned oncolytic virus-T cell chimera in combination with an anti-CTLA4 antibody in the preparation of a cancer treatment drug.

[0032] Compared with the prior art, the present invention has the following beneficial technical effects and significant improvements:

[0033] OVs are first protected by an engineered biomembrane that presents T cell-specific antigens, and the camouflaged membrane-covered OVs are further physically attached to the surface of the carrier T cells through antigen-receptor interactions to recognize T cell receptors (TCRs) or chimeric antigen receptors (CARs), rather than being internalized by T cells. This anchoring strategy does not affect the effector function of T cells, and once the carrier T cells reach the surface of tumor cells and recognize their cognate tumor-specific antigens through competitive antigen-receptor interactions, the OVs are easily released. OAs are then internalized by cancer cells and promote specific viral infection. After infection, oncolytic virus constructs encoding Cas9 editors can destroy the PDL1 gene of tumor cells and infiltrating immune cells, thereby downregulating their PDL1 expression levels, which greatly helps to alleviate the tumor immunosuppressive microenvironment and facilitate the killing effect of T cell therapy and oncolytic therapy on tumors. Therefore, oncolytic virus-T cell chimeras (ONCOTECH) represent an innovative therapeutic platform that is not only suitable for systemic targeted delivery of OVs, but also helps to reshape the TME. It could potentially be expanded into a universal platform for integrating different types of T cells (including TCR-T and CAR-T cells) with OVs (including oncolytic viruses such as oncolytic adenovirus OA and oncolytic herpes virus HSV-1) to combine viral therapy and cell therapy for a wide range of cancer treatment applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0035] Figure 1 shows tumor PDL1 analysis and construction of engineered oncolytic adenovirus (eOA). a, Overall survival of patients with melanoma, pancreatic cancer, or glioma with high or low levels of PDL1 was analyzed by gene expression profile interaction analysis (GEPIA). Source data were isolated from TCGA, and n is the number of patients included in the survival analysis. Survival analysis was performed using the log-rank (Mantel-cox) test. b, Representative images of pancreatic cancer patient tumor immunohistochemistry (brown) PDL1 used to establish patient-derived xenografts (PDX) in NSG mice. PDX mouse models were treated with saline and injected intratumorally (it) with OA (1×10 10 VP), intravenous (iv) injection of CAR-T cells (1×10 7 cells), OA (it) and CAR-T cells (iv) were co-treated. Scale bar: 100 μm. N = 5 biologically independent mice, 3 IHC sections were randomly analyzed for each tumor. 0 (-, negative), 1 (+, weak), 2 (++, moderate), 3 (+++, strong). c, Flow cytometry gating strategy (top) and EpCAM+ Representative flow cytometry images of PDL1 expression in tumor cells after the indicated treatments (bottom). d, PDL1 expression in B16OVA tumor-bearing mice after the indicated treatments. e, Quantitative analysis of B16OVA and SW1990 cells at increasing MOIs after infection of GFP-positive cells with eOA for 48 hours. Untransduced cells (MOI 0) served as controls. f, Western blot analysis of Cas9 protein levels and indel mutations in the PDL1 gene locus (bottom) in B16OVA cells 48 hours after eOA infection (top). g, Western blot analysis of Cas9 protein levels and indel mutations in the human PDL1 gene locus (bottom) in SW1990 cells 48 hours after infection with eOA at increasing MOIs. h, Schematic diagram of the eOA-PLK1 design and indel mutations in the PLK1 gene locus after infection of B16F10 cells with eOA for 48 hours at increasing MOIs, as determined by T7E1. i. Quantification of Ad5 gene copy number in different cell lines 48 hours after eOA infection by qRT-PCR. j. Cell viability of B16OVA, L929, SW1990, and HUVEC cells 72 hours after the indicated treatments. k. Western blot analysis of PDL1 levels in B16OVA cells 72 hours after the indicated treatments. l. Cell viability of B16OVA cells 72 hours after the indicated treatments. m. Schematic diagram of C57BL / 6 mice bearing B16OVA tumors treated with eOA (top). T7E1 (bottom left) and western blot (bottom right) assays were used to analyze PDL1 gene / protein levels after the indicated treatments. n. Tumor volume (left) and survival curves after the corresponding treatments (N = 5 biologically independent mice). Red arrows (fh) indicate cleaved DNA fragments. Data are presented as mean ± sd (be,i,l) and sem (n). N = 3 (e,i), N = 5 (b,c,d,j), and N = 6 (l) biologically independent samples. (c, i, l) One-way ANOVA with Tukey post hoc analysis, (n) two-way ANOVA with Bonferroni post hoc test, and survival curves (a, n) were performed using the log-rank (Mantel-Cox) test. P values ​​are shown in the figures.

[0036] Figure 2 shows the scheme of engineering oncolytic adenovirus (eOA) genome editing of PDL1 to effectively improve viral therapy and / or adoptive T cell therapy.

[0037] Figure 3 shows the generation and characterization of ONCOTECH. a, Schematic diagram of the generation of cell membrane-encapsulated eOA (M@eOA, left) and the release mechanism of TM@eOA (right), M@eOA is competitively released from TM@eOA after interacting with tumor cells expressing pMHC-I. b, TEM images of eOA and M@eOA subjected to immunogold analysis using anti-Ad5 antibodies. Scale bar 100 nm. c, Dot blot method (left), quantitative analysis of the loading capacity of TM@eOA using anti-Ad5 antibodies (right). M@eOA contains 100 μg of B16OVA cell membrane (M) and 1×10 10 VP eOA. d, SEM images of T cells and TM@eOA. Scale bar 1 μm. e, Fluorescence microscopy of TM@eOA and T cells. T cells were stained with AF647 anti-CD8 antibody, eOA was labeled with TRITC, and M was labeled with DiO. Scale bar 2 μm. Pearson correlation coefficient between M / eOA and CD8 / eOA (right, N = 6 biologically independent samples). f, Representative fluorescence images of TM@eOA (T cells-CFSE, eOA-Cy5) recognized by B16OVA-mCherry cells. Scale bar 5 μm. g, Flow cytometric analysis of vector T cells (1×10 6(c) Recognition of SIINFEKL-tetramer-APC with different weights of M@eOA. h, Representative flow cytometric plots and MFI analysis of M@eOA release from TM@eOA after 30-minute incubation with B16OVA cells, TRITC-stained eOA, n = 5 biologically independent samples. i, CLSM images of B16OVA cells after 6-hour incubation with TM@eOA (left). Scale bar, 10 μm. Pearson correlation coefficient between M and eOA (right). (n = 6 biologically independent samples). j, Dot blot assay (top), quantitative analysis of TM@eOA stored at different temperatures and + / - serum for 24 hours (bottom). k, l, Representative cytometric histograms of in vitro expansion of TM@eOA cells (k) and CD69-expressing T cells (l) after stimulation with anti-CD3 / CD28 microbeads. m, Flow cytometric analysis of IFNγ and GZMB release from T cells or TM@eOA stimulated with ConA for 48 hours. n, Quantification of adenoviral gene copy number by qRT-PCR after incubation of B16OVA cells with OA, M@eOA, or TM@eOA (200 VP / cell). o, Western blot analysis of Cas9 in B16OVA cells (top) and T7E1 gene disruption assay for PDL1 (bottom); red arrows indicate cleaved DNA fragments. p, Quantitative tumor killing analysis of B16OVA cells after indicated treatments (N = 5 biologically independent samples). N = 3 biologically independent samples (c, fh, jn). Data are expressed as mean ± SD. (f, m) Unpaired two-tailed t-test was used, (j, n, p) one-way ANOVA with Tukey's post hoc test was used, and (k) two-way ANOVA with Bonferroni's post hoc test was used. P values ​​are indicated in the figures. NS indicates no statistically significant difference (p > 0.05).

[0038] Figure 4 shows the characteristics of TM@eOA. a. Dot blot and quantitative analysis of anti-Ad5 antibody against eOA (1×10 10 VP) Encapsulation effect of cancer cell membrane (CM). CM@eOA was denatured with RIPA buffer, N = 3 biological independent samples. b, Cell counting analysis of surface-bound hybrid membranes (M) obtained by mixing CM with different weight percentages of DMG-PEG. N = 3 biological independent samples. c, Size and potential analysis of different membrane@eOA formulations. M represents CM mixed lipid mixture, N = 3 biological independent samples. d, TEM images of eOA and M@eOA. Scale bar 200 nm. e, Dot blot analysis of hybrid membrane (M) encapsulating eOA, M@eOA was denatured with RIPA buffer, N = 3 biological independent samples. f, Different weights of M@eOA in T cells (1×10 7Flow cytometry images of TM@eOA (hM@eOA cells), N = 3 biologically independent samples. g, Representative flow cytometry images and MFI analysis of M@eOA release from TM@eOA after 30 minutes of incubation with H-2Kb-SIINFEKL tetramer, TRITC-stained eOA, N = 5 biologically independent samples. h, Representative CLSM images of TM@eOA used for stability analysis of 48 hM@eOA viral vectors labeled with DiI, and T cells stained with Hoechst 33342. Scale bar, 10 μm. i, Representative CLSM images of TM@eOA cross-sections (left) and 3D models reconstructed from Z-stacks (right). Scale bar, 5 μm. j, Quantitative analysis of Ad5 antibody fluorescence intensity on the surface of vector T cells, N = 5 biologically independent samples. k, Analysis of T7E1 gene mutation frequency in vector T cells after 3 days of incubation at 37°C. l, m, Quantification of T cell viability (l) and relative eOA intensity (m) in TM@eOA cells by 7-AAD and dot blot analysis after incubation at 37°C with or without agitation (100 rpm) for 24 hours. N = 3 biologically independent samples. n, Flow cytometry analysis of TM@eOA cell apoptosis using Annexin V-FITC and PI. T cells without M@eOA served as a control. N = 3 biologically independent samples. o, Representative cytometry histograms of T cell proliferation stimulated by anti-CD3 / anti-CD28 microbeads and IL-2. p, Percentage of cell migration of T cells and TM@eOA stimulated by MCP-1 in Transwell culture dishes. N = 3 biologically independent samples. q, ELISA analysis of IL-2 release by T cells or TM@eOA after 48 hours of ConA stimulation. N = 3 biologically independent samples. r, Representative images of GFP expression in B16OVA cells 24 hours after treatment with M@eOA or TM@eOA, respectively. The percentage of GFP-positive cells was analyzed by cytometry. N = 3 biologically independent samples. s, Representative images of B16OVA cells 48 hours after treatment. Scale bar, 50 μm. Data are expressed as mean ± SD. (m, n, q, r) were analyzed using an unpaired two-tailed t-test, (b, g, j) were analyzed using a one-way ANOVA with a Tukey post hoc test, and (l, p) were analyzed using a two-way ANOVA with a Bonferroni post hoc test. P values ​​are indicated in the figures. None. No statistically significant differences were observed (P > 0.05).

[0039] Figure 5 shows the ONCOTECH-mediated oncolytic virus therapy and immunogenic cell death (ICD) cascade. a, Schematic diagram of the ONCOTECH anti-tumor mechanism. b, Immunoblotting analysis of PDL1, CRT and HSP70 protein levels after treatment (left). Quantitative analysis of ATP and HMGB1 in the supernatant 48 hours after treatment (center right). c, Representative cytometry images and quantification of SIINFEKL H-2Kb expression in BMDCs. d, Representative cytometry histogram of T cell proliferation rate on day 5. e, Cytometry quantitative analysis of OT-1CD8 + f, CFSE expression in OT-1CD8 T cells after 72 hours of co-culture with pretreated BMDCs. + Representative cytometry histograms of T cell expansion (left). OT-1CD8 + Representative cytometry images and quantitative analysis of T cell cytokine release (right). g, Schematic diagram of co-culture of multicellular spheroids with BMDM, BMDC, T cells and B16OVA-mCherry on a 3D spheroid plate. Microenvironment and killing function were evaluated by fluorescence imaging and cytometry. h, Quantitative analysis of the ratio of M1 / M2 (CD86 / CD206) BMDM cells (left), the percentage of H-2Kb SIINFEKL positive cells in BMDC (middle), and CD4 + Tregs (Foxp3 + ) percentage (right). i. Quantitative analysis of endogenous CD8 + T cells (TCR-Vα2 - , left) and exogenous CD8+ T cells (TCR-Vα2 + , right) Percentage of IFNγ positivity. j, Representative spheroid imaging after 10 days of treatment with mCherry. The volume of multicellular spheroids was analyzed every two days. Scale bar 500 μm. Data in (bi) are expressed as mean ± SD, and (j) are expressed as mean ± sem. N = 3 biologically independent samples (cf, hj). (be, h, i) One-way ANOVA with Tukey post hoc test, (f, j) Two-way ANOVA with Bonferroni post hoc test, P values ​​are shown in the figures. NS indicates no statistically significant difference (P>0.05);

[0040] Figure 6 shows the genome editing and tumor cell killing effects mediated by TM@eOA. a. In vitro co-culture time schedule for Figures 5 and 8. b. Representative protein levels (cleaved caspase-3, caspase-1, cleaved GSDMD) in B16OVA cells after the indicated treatments, with GAPDH as a control. c. Gene mutations at the PDL1 gene locus after indicated treatments. Red arrows indicate cleaved DNA fragments. d. OT-1TCR-Vα2 + CD8 + Representative cytometry and quantitative analysis of IFNγ, TNFα, and GZMB in T cells. (e) After 72 hours of pretreatment of B16OVA cells with TM@eOA, T+M@eOA, or M@eOA in plate 1, supernatants were collected from plate 1 and analyzed for viral potency by dot blot analysis (center, N = 3 biologically independent samples). The same volume of supernatant was used to treat B16OVA cells in plate 2. 72 hours later, cytotoxicity was measured using CCK-8 assay (right, N = 5 biologically independent samples). Data are presented as mean ± SD. (e) One-way analysis of variance with Tukey's post hoc test. P values ​​are indicated in the figure.

[0041] Figure 7: ONCOTECH reshapes the immunosuppressive TME. a, Schematic diagram of the experimental design. b, Representative fluorescence images and quantitative analysis of B16OVA tumor-bearing mice 24 hours after intravenous injection. M@eOA was labeled with Cy7, and vector T cells were labeled with DiR. c, Representative fluorescence images of B16OVA tumor-bearing mice after tail vein injection of TM@eOA (T cells were labeled with DiD, and eOA was labeled with Cy7). d, Representative B16OVA tumor sections after 72 hours of treatment. Tumors were harvested and stained for adenovirus type 5 (Ad5, red). Scale bar, 500 μm. t-SNE (t-distributed random neighbor embedding) of immune subsets and tumor cell clusters (left). Representative t-SNE plots (center) and normalized PDL1 expression plots (right) between the TM@eOA and saline groups. f, Volcano plot of all differentially expressed DEGs. Differential gene expression was compared with Sleuth using the Wald test with FDR-adjusted Q values. h, Immunoblot analysis of PDL1, CRT and HSP70 protein expression, quantification of ATP and HMGB1 levels after treatment. 48 hours after im treatment, H-2Kb-SIINFEKL positive CD11c cells (i), CD3 + CD8 + TCR-Va2 - SIINFEKL tetramer content of cells (j), M1 / M2 ratio analysis (k), in CD3 + TCR-Va2 -The ratio of endogenous CD4 and CD8 T cells in SW (1), the ratio of tumor Treg (m). n, PCA principal component analysis diagram summarized by el diagram. o, p, OT-1 (TCR-Va2 per gram of B16OVA tumor + CD8 + )T cell number (o), and the OT-1CD8 + T cell proliferation (p). q, exogenous OT-1CD8 + T cells and endogenous CD8 + Quantitative analysis of IFNγ and GZMB expression in T cells, and GSEA enrichment analysis of DEGs between the saline and TM@eOA groups (p values ​​were analyzed using the Jami-Hochberg correction). Tumor samples were collected on day 20 (en, qr), N = 5, and biologically independent samples (b, eh-q). Data are expressed as mean ± standard deviation. One-way ANOVA with an unpaired two-tailed t-test was used in (g, q) and (q), and a Tukey post hoc test was used in (e, hp). P values ​​are shown in the graphs.

[0042] Figure 8 shows that M@eOA treatment reshapes the immunosuppressive tumor microenvironment in vivo. a. TCR Vα2 in blood 6 hours after intravenous injection + Cytometry and quantitative analysis of eOA-Cy7 in T cells. b. Cy7-eOA and vector DiD-OT-1CD8 24 hours after administration + Quantitative analysis of T cell distribution in different organs. c, CLSM images of tumor sections 24 hours after intravenous injection of T+M@eOA or TM@eOA. eOA was labeled with Cy7, and vector T cells were labeled with DiD. Scale bar, 200 μm. d, Representative images of B16OVA tumor sections 72 hours after TM@eOA-GFP treatment. The left panel shows GFP expression in eOA-GFP-infected cells. The blue signal is DAPI. Scale bar, 300 μm. Quantitative analysis of GFP expression after eOA-GFP infection (right), N = 5 biologically independent samples. e, Genetic mutations in the PDL1 locus in tumors after the indicated treatments (top) and in different organs after TM@eOA treatment (bottom). f, Quantitative analysis of PDL1 expression in different cell types. N = 5 biologically independent mice. g, Gating strategy for PDL1 expression in different subpopulations. The gating strategy represents six experimental replicates. Data are expressed as mean ± SD. (a) Paired two-tailed t-test, (f) one-way ANOVA with Tukey's post hoc test, P values ​​are shown in the figures. NS indicates no statistically significant difference (P>0.05).

[0043] Figure 9 shows the immune composition of the immune microenvironment after TM@eOA treatment. a. Immunoblotting analysis of cleaved caspase-3, caspase-1, and cleaved GSDMD protein levels. GAPDH was used as a control. b. Representative cell images and quantitative analysis of CD11c+ dendritic cells in lymph nodes (CD80+CD86+). N = 5 biologically independent mice. cg, SIINFEKL-H-2Kb+ gates CD11c+ cells in lymph nodes (c), SIINFEKL-tetramer-APC gates CD3+ cells in tumors. + CD8 + TCR-Vα2 - Gating of cells (d), TAMs (CD45 + CD11b + f / 4 / 80 + M1 (CD86 + CD206 - ) / M2(CD86 - CD206 + ) ratio (e), CD4 + and CD8 + T cells (CD3 + TCR-Vα2 - Cell gating strategy, f) and Tregs (Foxp3 + CD3 + CD4 + Cell gating strategy, representative cytometry images of g). h. Endogenous TCR-Vα2 - CD8 + Representative flow cytometry images of T cells and quantitative analysis of cytotoxic effector (IFNγ, GZMB) levels. N = 5 biologically independent samples. i. Intratumoral OT-1TCR-Vα2 + CD8 + Representative flow cytometry plots of T cell effector cytotoxicity (IFNγ, GZMB) levels. j, Heat map analysis of differentially expressed genes (DEGs) in B16OVA tumors after the indicated treatments (T cells, M@eOA, TM@eOA) with saline as the control. Red represents upregulation, and blue represents downregulation. Significant differentially expressed genes were identified with an absolute log2 (fold change) > 1 and a corrected P < 0.05. k, Venn diagram of the heat map of DEGs in T cell-treated mice, M@eOA, and TM@eOA (left). Quantitative analysis of the number of DEGs in tumors after different treatments (right). Data represent mean ± SD. P values ​​were determined by one-way ANOVA with Tukey post hoc analysis (b) and two-way ANOVA with Bonferroni post hoc analysis (h). P values ​​are indicated in the figures.

[0044] Figure 10 shows the immune composition of the immune microenvironment after TM@eOA treatment. a. Immunoblotting analysis of cleaved caspase-3, caspase-1, and cleaved GSDMD protein levels. GAPDH was used as a control. b. Lymph node CD11c + Dendritic cell CD80 + CD86 + Representative cell images and quantitative analysis. N = 5 biologically independent mice. cg, SIINFEKL-H-2Kb + CD11c in lymph nodes + Cell gating (c), MHC-ISIINFEKL-tetramer-APC against CD3 in tumor + CD8 + TCR-Vα2 - Cell gating strategy (d), TAMs (CD45 + CD11b + f / 4 / 80 + M1 (CD86 + CD206 - ) / M2(CD86 - CD206 + ) ratio (e), CD4 + and CD8 + T cells (CD3 + TCR-Vα2 - Cell gating, f) and Tregs (Foxp3 + CD3 + CD4 + Cell gating, representative cytometry images of g). h, endogenous TCR-Vα2 - CD8 + Representative flow cytometry images of T cells and quantitative analysis of cytotoxic effector (IFNγ, GZMB) levels. N = 5 biologically independent samples. i. Intratumoral OT-1TCR-Vα2 + CD8 +Representative flow cytometry plots of T cell effector cytotoxicity (IFNγ, GZMB) levels. j, Heat map analysis of differentially expressed genes (DEGs) in B16OVA tumors after the indicated treatments (T cells, M@eOA, TM@eOA) with saline as the control. Red represents upregulation, and blue represents downregulation. Significant differentially expressed genes were identified with an absolute log2 (fold change) > 1 and a corrected P < 0.05. k, Venn diagram of the heat map of DEGs in T cell-treated mice, M@eOA, and TM@eOA (left). Quantitative analysis of the number of DEGs in tumors after different treatments (right). Data represent mean ± SD. P values ​​were determined by one-way ANOVA with Tukey post hoc analysis (b) and two-way ANOVA with Bonferroni post hoc analysis (h). P values ​​are indicated in the figures.

[0045] Figure 11 shows the anti-tumor effect of ONCOTECH in primary and disseminated mouse models. a, using OT-1CD8 + Schematic diagram of the experimental design and timeline of the subcutaneous T cell therapy for the B16OVA tumor model. Representative bioluminescence images of subcutaneous B16OVA tumors after treatment (N = 5 biologically independent mice). c, B16OVA tumor growth curve (left) and tumor-bearing mouse survival curve (right). d, Quantitative analysis of CD45 expression in the responder and non-responder groups after TM@eOA treatment. + Percentages of different cell populations in PBMCs. N = 10 or 20 biologically independent mice. B16OVA tumor growth curves after M@eOA treatment (N = 12 biologically independent mice). Mice with a complete antitumor response (CR, 9 mice) were challenged with B16OVA cells on day 60. CD8 was depleted with anti-CD8, anti-CD4, anti-NK1.1, or isotype antibodies on day 60 after B16OVA treatment. + T, CD4 +T, or NK cells in a subcutaneous B16OVA tumor model resistant to scB16OVA treatment. g. Timeline, indicating genetic mutations at the PDLI site in B16OVA tumor tissue after treatment. i, B16OVA tumor growth curve (left) and mouse survival curve (right) after treatment. N = 5 or 10 biologically independent mice. j. Experimental design and timeline for treatment of metastatic B16OVA tumor model. k, Western blot analysis of genetic mutations at the PDLI site and PDL1 in lung tissue of metastatic B16OVA tumors after treatment. I, In vivo bioluminescence images of mice bearing lung metastatic B16OVA tumors. m, Survival curves of mice after treatment in the metastatic B16OVA model. n, Experimental design and timeline for treatment of metastatic 4T1OVA tumors. o, Genetic mutations at the PDLI site in 4T1-OVA tumor tissue after treatment. p, Representative lung images of the 4T1-OVA spontaneous metastasis model. Scale bar, 5 mm. q, Representative H&E-stained images of lung metastases. Scale bar, 2 mm. r, Primary tumor volume and experimental endpoint on day 0 (left), total number of lung surface metastases (right), N = 10 biologically independent mice. OT-1CD8 + T cells were used in (a, g, j, n). All experiments were repeated twice independently with similar results. N = 5 biologically independent mice (c, d, f, m). Red arrows in the T7E1 assay indicate cleaved DNA fragments. Data are presented as mean ± standard deviation (SD). (d) and mean ± SEM (c, fi, r). Unpaired two-tailed t-test was used in (r), and two-way ANOVA with Bonferroni post hoc test was used in (c, d, fi, r). Survival curves (c, i, m) were analyzed using the log-rank (Mantel-Cox) test. P values ​​are indicated on the graphs.

[0046] Figure 12 shows the long-term immune effect of B16OVA tumor-bearing mice. a, TM@eOA on NK cells or CD4 + Timeline of the antitumor effect of T cell depletion. b, c, Tumor volume (b) and survival curve (c). N = 5 biologically independent mice. d, Splenic effector memory T cells (CD3 + CD8 + CD44 + CD62L - , TEM) and central memory T cells (CD3 + CD8 + CD44 + CD62L + , Representative cell images and quantitative analysis of Tcm). N = 5 biologically independent mice. e, Gating strategy for lymphocyte populations after antibody treatment. C57BL / 6 mouse NK cells (f), CD4+ T cells (g) and CD8 + Representative flow cytometry plots (top) and quantitative analysis (bottom) of T cell (h) depletion. Graphs represent three experimental replicates. N = 3 biologically independent mice. Data represent mean ± SD (dh) and SEM (b). P values ​​were determined by two-way ANOVA with Bonferroni post hoc analysis (b, d, fh). Survival curves (c) were analyzed using the log-rank (Mantel-Cox) test, and P values ​​are indicated on the graphs.

[0047] Figure 13 shows the biosafety analysis and other in vivo anti-tumor experiments. a. Comparison of the anti-tumor effects of TM@eOA (Figure 11c) and T(iv)+eOA(iv) (Figure 1n) treatment in B16OVA tumor-bearing mice. B16OVA tumors were subcutaneously inoculated on day -7, and OT-1CD8 + T (intravenous injection) treatment. b. Body temperature of mice after administration. c. Serum inflammatory cytokine levels 7 days after treatment. d. Biosafety analysis of lactate dehydrogenase (LDH), alkaline phosphatase (AKP / ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in mice 7 days after administration. e. Schematic diagram of eOA expressing Cas9 and anti-PDL1scFv expressing OA-aPDL1, as well as the timeline of the anti-tumor effect of different OA in the subcutaneous B16OVA tumor model. f. Tumor volume (left) and survival curve (right) of B16OVA tumor-bearing mice after indicative treatment. g. Vector OT-1CD8 in lung metastases + Representative cytometry histograms of CFSE expansion of T cells. h, Expression vector OT-1CD8 in lung metastases. + Representative cytometry images and quantitative analysis of T cell cytokine production (IFNγ and GZMB). i, Photographs of lung tissue and corresponding H&E-stained sections from B16OVA tumor-bearing mice 20 days after treatment with the indicated treatments. Scale bar, 3 mm. N = 5 biologically independent mice. Data are presented as mean ± SD (b, d, h) and mean ± SEM (a, f). Survival curves were analyzed using one-way ANOVA with a Tukey's post hoc test (c, d, g, h) and two-way ANOVA with a Bonferroni post hoc test (a, b, f). P values ​​are indicated in the figures.

[0048] Figure 14 shows the analysis of human PBMC, CAR-T cells and humanized mice based on flow cytometry. a, Cell counting gating strategy for EphA2-CAR-T, GD2-CAR-T and PSCA-CAR-T. b, HLA-A2 +PBMC cell counting gating strategy. c. Schematic diagram of humanized mouse construction. d. hCD45 + Lymphocytes and hCD3 + Representative images of T cells reconstructing the human immune system 21 days after transplantation. e, Quantification of hCD45 percentage by cell counting after mouse hPBMC engraftment. N = 6 biologically independent mice, hCD45 percentage 7-21 days after transplantation. + The increase in PBMC cells indicated that the human immune system was successfully reconstituted in NSG mice.

[0049] Figure 15 shows the construction and characterization of CAR-TM@eOA in vitro and in vivo. a. Dot blot and quantitative analysis were performed on hybrid membrane (LN229CM: lipid, weight ratio (wt)) for eOA (1×10 10 b, The anti-Ad5 antibody loading capacity of M@eOA anti-GD2-CAR-T was evaluated by Dot blot and quantitative analysis. M@eOA contained 100 μg of A549 cell membrane (M) and 1×10 10 c, Cytometry analysis of the effects of CAR-TM@eOA and M@eOA with different weights on CAR-T cells (1×10 7 d, Schematic diagram of orthotopic LN229 glioblastoma treatment with anti-GD2-CAR-TM@eOA in humanized NSG mice, using LN229 cell membranes overexpressing GD2 in the M@eOA generation (M). e, f, Indel mutations of the PDL1 locus in LN229 tumor tissues after treatment (e) and immunoblot analysis of PDL1 (f). Red arrows indicate cleaved DNA fragments. g, CD4 + Quantitative analysis of Tregs in T cells. h, ELISA was used to detect the expression of cytokines (TNFα, IL12p70, IL-2) in tumor supernatant. i, CD8 + Representative cell images and quantitative analysis of GZMB and IFNγ expression in CAR-T cells. j, k, In vivo bioluminescence images (j) and survival curves (k) of LN229 orthotopic glioblastoma humanized mice after different treatments. 1, Schematic diagram of anti-EphA2-CAR-TM@eOA treatment of humanized NSG mouse metastatic tumor A549. + PBMC (1×10 7 Humanized NSG mice were injected intravenously with A549-Luc cells (1×10 5). M@eOA of A549 cell membrane overexpressing EphA2. m, n, Immunoblotting analysis of PDL1 locus gene mutation (m) and PDL1 (m) in lung metastatic tissue after treatment. Red arrows indicate cleaved DNA fragments. o, ELISA detection of cytokine (TNFα, IL12p70, IL-2) expression in tumor supernatant. p, CD8 + GZMB in CAR-T cells + Quantitative analysis of cells. q, r, In vivo bioluminescence images (q) and survival curves (r) of metastatic A549 tumor-bearing humanized mice after treatment with the indicated treatments. N = 3 biologically independent samples (ac), N = 5 biologically independent mice (g, k, or). Data are expressed as mean ± SD. (g, i, p) One-way ANOVA with Tukey's post hoc test was used for analysis, and survival curves (k, r) were analyzed using the log-rank (Mantel-Cox) test. P values ​​are indicated in the figures.

[0050] Figure 16 shows that engineered TIL-M@eOA gene edits PDL1 and kills tumor cells. a, Schematic diagram of the generation of patient tumor-derived TIL-M@eOA. b, Anti-adenovirus 5 antibody dot blot assay to detect the loading capacity of TIL-M@eOA. For TIL-M@eOA tumor cell membranes containing 100 μg of PDAC, there are 1×10 10 VP eOA and 1×10 6 CD8 + TILs. c, Fluorescence microscopy of TIL-M@eOA and T cells. T cells were stained with CFSE, eOA was labeled with TRITC, and the membrane was labeled with DiO. Scale bar, 5 μm. d, Schematic diagram of co-culture of TIL-M@eOA and PDAC cells. The TILs used to construct TIL-M@eOA and PDAC tumor cells were derived from resected tumor tissue from patients. e, T7E1 analysis of PDL1 gene mutations in PDAC cells. After 48 hours of co-culture, cells were harvested and magnetic cell sorting was used to isolate PDAC cells. Red arrows indicate cleaved DNA fragments. f, Representative histograms and quantitative analysis of PDAC cells after indicated treatments (N = 5 biologically independent samples). Data are presented as mean ± SD. One-way ANOVA with Tukey's post hoc test was performed in (f), and P values ​​are shown in the figure.

[0051] Figure 17 shows engineered microvesicles packaged with eOA assembled by CAR-T cells to enhance the anti-tumor function of human pancreatic cancer (PDAC) organoids. a, Schematic diagram of the strategy for loading eOA onto CAR-T cells using microvesicles packaged with eOA. b, Immunofluorescence microscopy images of PSCA-CAR-T-based CAR-TT-MV@eOA, stained with AF647-conjugated anti-CD3 antibody, MVs labeled with DiO, eOAs labeled with TRITC, and cell nuclei labeled with DAPI. In vitro T cell expansion of CAR-T cells in CAR-T-MV@eOA and CAR-TM@eOA was quantified by CFSE cleavage. CAR-T cells were stimulated with anti-CD3 / CD28 antibodies combined with IL-2. d, Quantitative analysis of activated T cells (GZMB) and CAR-TT-MV@eOA. Analysis of eOA replication capacity in SW1990 cells. f, Analysis of SW1990 cells after 48 hours of incubation with MV@eOA or CAR-T-MV@eOA. Red arrows indicate cleaved DNA fragments. g, Schematic diagram of multicolor 3D live CLSM imaging of PDAC patient-derived organoids (PDOs) using CAR-T-MV@eOA, combined with cytometry and ELISA analysis. h, 3D CLSM imaging of PDAC PDOs using CAR-T-MV@eOA. Dead organoid cells labeled with YOYO-3. jk Quantification of PDO cell death, cytometry analysis of the percentage of Ad5-positive PDO cells (j), and the MFI of PDL1 expression in PDO cells (k). i, CAR-T cell counts after co-culture with PDO cells. m, Quantitative analysis of cytokines (IFNγ, GZMB, released by vector = cells. N = 3 biologically independent samples and N = 6 biologically independent samples. Data are expressed as mean ± standard deviation. Unpaired two-tailed t-test was used in (d, e), one-way ANOVA with Tukey post hoc test was used in (c, i-l), and two-way ANOVA with Bonferroni post hoc test was used in (m). P values ​​are indicated on the graphs. NS indicates no statistically significant difference (P>0.05).

[0052] Figure 18 shows tumor technology treatment for humanized patient-derived xenograft (PDX) models. a, Schematic diagram of the experimental design of CAR-T-MV@eOA treatment of humanized PDX models based on PSCA-CAR-T. b, Representative bioluminescence images and quantitative analysis of different organs carrying PDX tumors after eOA virus labeling with CAR-T-MV or CAR-T-MV@CA7-NHS, and CAR-T cells labeled with DiD. c, Representative immunofluorescence images of (b) PDX tumor tissues and CAR-T cells of eOA virus. Scale bar, 200 μm. d, Representative GFP fluorescence imaging of eOA-GFP virus after administration of MV@eOA-GFP or CAR-T-MV@eOA-GFP. Scale bar, 1 mm. e, T7E1 detection of the frequency of gene mutations at the PDLI site in PDX tumor tissue after treatment (top). The red arrow indicates the cut DNA fragment. Tumor cells (CD45 - EpCAM + ) MFI value of PDL1 expression in vivo. f, Immunoblotting analysis of PDL1, CRT and HSP70 protein expression, and quantification of HMCB1 and ATP. g, Quantitative analysis of Foxp3 in CD4 + h, Number of CAR-T cells proliferating and infiltrating in PDX tumors after CAR-T or CAR-T-MV@eOA treatment. i, CAR-T cells activated in tumors after intravenous administration (hCD8 + CFSE-GZMB + ) and endogenous hCD8 + CFSE + GZMB + Quantitative analysis of T cells. j, PDX tumor growth curves (left) and survival curves (right) of humanized mice bearing PDX tumors after the indicated treatments. k, Schematic diagram of the experimental design for CAR-T-MV@eOA treatment with humanized PDX models. l, Gene mutation frequency of the PDL1 locus after T7E1 detection of PDX tumor tissues, tumor growth curves (left) and survival curves (right) of PDX tumor-bearing mice (b, em). Data in g are presented as mean ± SD, and data in (j, m) are presented as mean ± SD. Statistical significance was assessed using Tukey's one-way ANOVA (ei), two-way ANOVA with Bonferroni post hoc test, or log-rank (Mantel-Cox) test for survival curves (k), and P values ​​are indicated on the figures.

[0053] Figure 19 shows the construction and characterization of anti-PSCA-CAR-T-MV@eOA in vitro and in vivo. a, TEM image of MV@eOA. b, Dot blot quantification of MV / eOA (wt:VP). c, Quantitative analysis of CAR-T-MV@eOA by dot blot and anti-ad5 antibody. MV@eOA contained 200 μg of 293T-PSCA-MV and eOA containing 1 × 1010 VP. d, Quantitative analysis of anti-PSCA-scFv taken up by MV@eOA on the surface of CAR-T cells using recombinant PSCA-FITC protein. e, Dot blot analysis (top) and quantitative analysis (bottom) of CAR-TM@eOA cultured at different temperatures for 24 h with or without serum. f, RT-qPCR quantification of PSCA mRNA levels in PDAC tumor samples and corresponding peritumoral normal tissues (n = 5 biologically independent PDX tumors derived from the same human tumor sample). g, Western blot analysis of cleaved caspase 3, caspase 1, and cleaved GSDMD in PDX tumor tissue after indicated treatment. h, Representative percentage of Tregs (Foxp3+) among CD4+ T cells within the human PDX TME. i, j, Representative cytometry images of intratumorally activated CAR-T cells (hCD8+CFSE+GZMB+, i) and endogenous hCD8+CFSE-GZMB+ T cells (j) after drug administration. k, Cytokine profile (TNFα, IFNγ) in tumor supernatants assessed by ELISA. 1, Body temperature changes in humanized mice after indicated treatment. N = 3 biologically independent samples (a, e), N = 5 biologically independent mice (f, k, l). Data are expressed as mean ± SD. (e, f) One-way ANOVA with Tukey post hoc analysis, (k, l) Two-way ANOVA with Bonferroni post hoc test. P values ​​are indicated in the figures. There was no statistically significant difference in NS (P>0.05).

[0054] FIG20 is a diagram showing the structure of the shuttle plasmid pDC315-hTERT-E1A.

[0055] Figure 21 shows the location of the Bbs1 restriction site in the plasmid. DETAILED DESCRIPTION

[0056] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0057] Example 1

[0058] 1. PDL1 genome editing using oncolytic adenovirus (OA) to deliver CRISPR / Cas9

[0059] Bioinformatics analysis of patients with melanoma, pancreatic cancer, and glioblastoma showed that high PDL1 levels were generally associated with shorter survival (Figure 1a). As shown in Figures 1b and 1c, PDL1 expression increased in PDAC-derived xenograft (PDX) mouse models after CAR-T cell therapy or oncolytic virus therapy. The combination of oncolytic adenovirus and CAR-T cells resulted in the highest level of PDL1 expression. Similarly, in C57BL / 6 mice bearing KPC tumors, upregulated PDL1 expression was most significant when oncolytic virus therapy was combined with the adoption of CAR-T, TCR-T, or tumor-infiltrating lymphocyte (TIL) cells (Figure 1d), which is consistent with previous reports.

[0060] Since PDL1 overexpression is detrimental to both CAR-T and oncolytic virus therapy, we cloned Cas9 and EGFP into the OA shuttle plasmid pDC315-hTERT-E1A (purchased from Microbix Biosystems, the sequence is shown in SEQ ID NO. 1) and inserted hTERT-E1A into the multiple cloning site to obtain the plasmid pDC315-hTERT-E1a-CMV-Cas9-EGFP-pA-U6-sgRNA (the sequence is shown in SEQ ID NO. NO.2, the plasmid structure is shown in Figure 20), and then the plasmid pDC315-hTERT-E1a-CMV-Cas9-EGFP-pA-U6-sgRNA is digested with Bbsl enzyme, and then sgRNA primers targeting mouse PDL1 or human PDL1 or mouse PLK1 (can also be replaced with other target genes) (as shown in Table 1) are enzyme-ligated, and any pair of sgRNA primers after annealing is inserted into the Bbsl restriction site by Ligase enzyme (Figure 21) to obtain pDC315-hTERT-E1a-CMV-Cas9-EGFP-pA-U6-sgRNA, which is co-transfected with Admax (pBHGlox (delta) E1, 3Cre) adenovirus packaging plasmid at a mass ratio of 1:2 into 293A cells to generate engineered OA virus (eOA) carrying a gene targeting mouse PDL1 or human PDL1 or mouse PLK1.

[0061] Table 1 Examples of sgRNA primers for mouse and human

[0062] This approach allows for infection and knockdown of PDL1 or PLK1 in tumor cells while retaining oncolytic activity. We screened the multiplicity of infection (MOI) of eOA and found that when eOA-mediated infection reached approximately 80% GFP positivity, the equivalent MOI was 1.79 for B16OVA (ovalbumin-expressing mouse melanoma) and 1.22 for SW1990 (human pancreatic adenocarcinoma, PDAC) (Figure 1e). As a DNA vector, eOA was shown to efficiently deliver its encoded Cas9 and sgRNA into B16OVA and SW1990 cells, resulting in strong gene mutation efficiencies (indel, insertion, and deletion mutations) in PDL1 or PLK1, significantly reduced PDL1 levels (Figures 1f, g), and disruption of the PLK1 genomic locus (Figure 1h).

[0063] RT-PCR analysis of Ad5 gene copy number, which is directly related to adenoviral gene replication and tumor-selective oncolysis, showed that eOA-infected tumor cells (B16OVA and SW1990) had more viral copies than normal cells (Figure 1i). In addition, we observed that eOA-mediated infection only selectively induced oncolysis of tumor cells, without lysing normal cells (Figure 1j). In general, eOA has a strong ability to target the genome editing of target genes and retains selectivity and oncolytic activity for tumor cells. At the same time, we also noticed that B16OVA cells incubated with T cells and OA alone, or B16OVA cells incubated with T cells and OA together, upregulated the expression of PDL1 (Figure 1k). In contrast, when B16OVA cells were incubated with eOA or T+eOA (a mixture of T cells and eOA), the expression level of PDL1 decreased, and the combination of T cells and eOA appeared to be most effective in killing tumor cells (Figure 1l). In the presence of eOA (1×10 10 Virus particles, VP) and / or OT-1CD8+ T cells (TCR-T, 1×10 7 After iv injection of eOA (T cells), we found that both eOA and T+eOA treatment effectively destroyed the PDL1 genomic locus and led to a significant downregulation of PDL1 protein levels (Figure 1m). Compared with the groups using only OA or T cells, eOA or combined treatment led to more effective inhibition of B16OVA tumor growth and significantly prolonged the survival of tumor-bearing mice (Figure 1n). These results indicate that eOA-mediated genome editing targeting PDL1 can greatly promote anti-tumor responses in T cell therapy and oncolytic virus therapy.

[0064] 2. Anchoring eOAs on the T cell surface through antigen-receptor interaction.

[0065] To obtain oncolytic virus-T cell chimeras (ONCOTECH), we took advantage of the tumor-targeting properties of T cells, namely that T cell receptors (TCR) or chimeric antigen receptors (CAR) can selectively recognize tumor-specific antigens or peptide-major histocompatibility complex class I (pMHC-I). Therefore, we loaded eOAs onto the T cell surface in two steps. First, eOAs were lipofectamine-expressing H-2Kb-SIINFEKL (MHC-I-OVA) using a liposome extruder (LiposoFast LF-50, AVESTIN). 257-264 ) of the B16OVA cell membrane (B16 cells that overexpress OVA will present the H-2Kb-SIINFEKL fragment to the cell membrane surface through the cell's own MHC-I presentation system) to form M@eOA. Secondly, M@eOA is encapsulated through TCR and MHC-I-OVA 257-264 Biophysical interactions between OT-1 and CD8 + On T cells. When the T cell receptor contacts the tumor-specific antigen of the tumor cell, these anchored OVs can be competitively released from the T cell surface (TM@eOA, Figure 3a, left). In order to quantitatively analyze the eOAs encapsulated by the B16OVA cell membrane, we first used the Dotblot method to examine the encapsulation efficiency of M@eOA (Figure 4a). Viruses (1×10 10 The encapsulation efficiency of VP) increased with the mass of cell membrane (CM), and the encapsulation efficiency (~78%) reached saturation at 400μg of membrane. To improve the encapsulation efficiency, we further mixed lipids and tumor cell membranes to generate mixed membranes (M). We found that the presence of DMG-PEG inhibited the endocytosis of M@eOA by T cells (Figure 4b), while diluting the tumor cell membrane CM with the lipid mixture (DOPC:Chol:DMG-PEG=5:4:1) could improve the encapsulation capacity of eOA. The size of M@eOA is approximately 190.2nm and the surface potential is -12.4mV (Figure 4c, d). Immunogold staining results show that M@eOA can protect eOA from neutralization by Ad5 antibodies (Figure 3b). At the same time, the mixed membrane does not affect the loading of eOAs onto T cells (Figure 3c and Figure 4e). Scanning electron microscopy (SEM) images showed that M@eOA nanoparticles were successfully localized to the T cell surface (Figure 3d), and confocal laser scanning microscopy (CLSM) images showed that both eOA (labeled by Cy7) and mixed membranes (labeled by CM-DiI) colocalized with the T cell surface marker CD8 (Figure 3e), which was consistent with the flow cytometry results (Figure 4f).

[0066] Real-time CLSM images showed that the TM@eOA chimera (200 μg M@eOA at 1×107 T cells) can approach B16OVA tumor cells (Figure 3), which means that the presence of M@eOA does not affect the recognition of MHC-I-OVA by OT-1 CD8+ T cells. 257-264 ability (Figure 3g). We also evaluated the release of M@eOA from carrier T cells by labeling eOA with tetramethylrhodamine (TRITC). After incubation with B16OVA cells or OVA tetramers for 30 minutes, we found that the mean fluorescence intensity (MFI) of eOA-TRITC was significantly reduced (Figure 3h and Figure 4g), indicating that the release of M@eOA from TM@eOA was triggered by competitive interaction between tumor antigens and T cell receptors anchored by M@eOA or membrane fusion between tumor cells and M@eOA (Figure 3a, right). Confocal imaging images of TM@eOA-treated B16OVA cells further confirmed the release of eOA, which was then internalized by B16OVA cells (Figure 3i). In addition, we incubated TM@eOA for 24 to 48 hours under different conditions and found that neither serum conditions, body temperature nor fluid shear stress affected the amount of eOA loaded in T cells or the survival rate of T cells (Figure 3j and Figure 4h-n).

[0067] We further investigated whether the attachment of oncolytic viruses affects the effector function of T cells. Under stimulation with anti-CD3 / CD28 magnetic beads, T cells equipped with oncolytic viruses showed similar characteristics to native T cells (unanchored oncolytic viruses) in terms of proliferation rate and cell migration (Figure 3k and Figure 4o-q). The mean fluorescence intensity (MFI) of CD69 of native T cells, a marker of early T cell activation, was also comparable to that of the TM@eOA group after stimulation with anti-CD3 / CD28 magnetic beads (Figure 3l). In addition, the secretion of interferon gamma (IFNγ), granzyme B (GZMB), and interleukin 2 (IL-2) in the TM@eOA group (Figure 3m and Figure 4r) was higher than that in the native T cell group, which may be due to the TCR-MHC-I recognition effect stimulating the activation of downstream T cell signaling pathways. The above results confirm that attaching oncolytic viruses to T cells through the biophysical interaction between TCR and pMHC-I can improve T cell function. After infection, the copy number of the viral gene (Pol) in the TM@eOA group was comparable to that in the OA or M@eOA group, and the Cas9 expression level and indel frequency in the TM@eOA group were similar to those in the M@eOA group (Figure 3n, o). Surprisingly, TM@eOA was more cytotoxic to B16OVA cells than M@eOA, indicating their combined killing effect on tumor cells (Figure 3p and Figure 4s). These results indicate that attaching M@eOA to the T cell surface does not affect the basic characteristics of oncolytic viruses, and the combination of the two greatly promotes overall anti-tumor immunity.

[0068] 3. TM@eOA is a method to anchor eOAs on the surface of T cells to enhance anti-tumor responses.

[0069] We investigated the ability of TM@eOA to induce ICD in B16OVA tumor cells by measuring the release of damage-associated molecular patterns (DAMPs), including calmodulin (CRT), heat shock protein 70 (HSP70), adenosine triphosphate (ATP), and high-mobility group box 1 (HMGB1), following co-culture with tumor cells (Figures 5a and 6a). We found that TM@eOA treatment significantly increased the levels of CRT, HSP70, ATP, and HMGB1, indicating that this treatment induced a robust ICD effect (Figure 5b). As typical hallmarks of ICD, cleaved caspase-3, caspase-1, and cleaved gasdermin D (GSDMD) were all upregulated after TM@eOA treatment (Figure 6b). We also investigated the effects of TM@eOA on B16OVA cells using the T7 nuclease I (T7E1) assay and immunoblotting to determine whether the PDL1 genomic locus was disrupted. Our results showed that TM@eOA effectively disrupted the genomic locus of PDL1 and downregulated the expression of PDL1 in B16OVA cells (Figures 5b and 6c).

[0070] We then examined the antigen presentation capacity of bone marrow dendritic cells (BMDCs) by co-culturing them with supernatant from TM@eOA-treated B16OVA cells. Our results demonstrated that TM@eOA treatment enhanced the antigen presentation capacity of BMDCs, as shown in Figure 5c, with a 15-fold increase in the level of SIINFEKL-MHC-I complexes compared to the control group (due to OVA antigen presentation). Furthermore, TM@eOA treatment was found to enhance the effector function of carrier T cells (Figures 5d, e and 6d), as evidenced by significant T cell proliferation, production of effector cytokines (IFNγ and GZMB), and increased eOA concentrations in the supernatant of B16OVA cells treated with TM@eOA (Figure 6e). When BMDCs were co-cultured with supernatant from TM@eOA-treated B16OVA cells and cultured with naive OT-1 CD8+ T cells, we observed enhanced T cell proliferation and effector function, suggesting a potent effect of antigen cross-presentation (Figure 5f). These results indicate that genome-editing-capable oncolytic viruses on oncolytic virus-T cell chimeras can activate ICDs, enhance the effector function of ONCOTECH, and enhance DC-mediated T cell immune activation.

[0071] To further mimic the in vivo tumor immune microenvironment, we constructed 3D multicellular spheroids consisting of mouse bone marrow-derived macrophages (BMDMs), BMDCs, T cells, and B16OVA-mCherry cells, which were treated with TM@eOA (Figure 5g). Overall, TM@eOA treatment increased the ability of BMDCs to present the OVA peptide SIINFEKL, increased the proportion of M1 macrophages, and reduced the level of immunosuppressive regulatory T cells (Figure 5h). In addition, TM@eOA treatment led to a significant reduction in regulatory T cells (Tregs) in 3D multicellular spheroids (Figure 5h). In addition, we observed that endogenous CD8 + T cells and exogenous vectors (OT-1CD8 + )T cell killing function (IFNγ + ) were significantly improved (Figure 5i). Overall, ONCOTECH reprogrammed the immunosuppressive TME by enhancing the effector function of endogenous T cells and exogenous vector T cells carrying oncolytic viruses, thereby inhibiting the growth of 3D tumor spheres (Figure 5j).

[0072] 4. ONCOTECH improves the immunosuppressive TME to study the distribution of TM@eOA in vivo

[0073] We first stained T cells and eOA with DiR and Cy7, respectively, and then injected fluorescently labeled TM@eOA intravenously into B16OVA tumor-bearing mice (Figure 7a). Flow cytometry results showed that TM@eOA could maintain its in vivo stability after systemic administration, and there was no obvious shedding of oncolytic virus on the carrier T cells (Figure 8a). In vivo fluorescence imaging showed that 24 hours after TM@eOA injection, the Cy7 signal in the tumor area was the strongest, while the M@eOA or T+M@eOA groups showed moderate fluorescence intensity. In addition, most of the carrier T cells in the TM@eOA group were also located in the tumor area, and their ability to infiltrate the tumor was improved compared with the native T cells or T+M@eOA groups (Figure 7b). It is worth noting that compared with the T+M@eOA group, the distribution of TM@eOA-treated mice in other organs was significantly reduced and was mainly enriched in the tumor, indicating that the fluorescence intensity of both Cy7 and DiD was enhanced (Figure 7c and Figure 8b, c). In addition, TM@eOA-mediated eOA expression in tumor tissues was more robust than that mediated by M@eOA or T+M@eOA (Figures 7d and 8d). These results suggest that the simultaneous delivery of oncolytic viruses and T cells into the body in an integrated mode (TM@eOA), rather than a physical mixture of the two (T+M@eOA), may synergistically enhance the targeting ability of oncolytic viruses and increase the infiltration of vector T cells. M@eOA delivered by vector T cells targets tumors. In vivo delivery of TM@eOA induced a 20.6% gene mutation frequency in the PDL1 genomic locus (Figure 8e), which was confirmed by spectral flow cytometry and RNA-Seq by reducing the expression of PDL1 in tumor cells and immune cells (Figures 7e, f and Figures 8f, g). Importantly, we did not observe off-target editing in other organs, including the heart, liver, spleen, lung, and kidney (Figure 8e), and TM@eOA-treated B16OVA tumor-bearing mice had moderate levels of cellular and humoral antiviral responses (Figure 9a-c), indicating the tumor specificity and safety of TM@eOA.

[0074] In addition, we found that the oncolytic virus in TM@eOA induced the overexpression of CRT and HSP70 (Figure 7g), as well as increased levels of ATP and HMGB1 released by tumor cells (Figure 7h). These events also coincided with increased levels of cleaved caspase-3, caspase-1, and cleaved GSDMD (Figure 10a), indicating that TM@eOA strongly induced ICD effects in vivo. In addition, we investigated whether TM@eOA cells could promote DC antigen cross-presentation to T cells in lymph nodes (LN). After in vivo delivery of TM@eOA, the proportion of mature DCs increased significantly (Figure 10b), and the expression of DCs presenting OVA SIINFEKL also increased significantly (Figure 7i and Figure 10c), which further promoted the cross-presentation of SIINFEKL-MHC-I complexes to naive T cells (Figure 7j and Figure 10d). In addition, M1 TAMs and endogenous cytotoxic CD8 + The levels of T cells were also significantly increased (Fig. 7k, l and Fig. 10e, f), while the levels of Treg cells in tumors were significantly decreased (Fig. 7m and Fig. 10g).

[0075] Principal component analysis (PCA) of these results showed that TM@eOA occupied a region distinct from that of the other treatments (Figure 7n). We also evaluated the killing effector function of the carrier T cells in TM@eOA. Five days after TM@eOA treatment, we observed that OT-1CD8 + T cells (carrier T cells) infiltrated into the tumor (Figure 7o), with the highest level of cell division (Figure 7p), and endogenous CD8 + T cells and exogenous vector T cells also increased cytokine production, including IFNγ and GZMB (Figure 7q and Figure 10h,i), indicating that the increased infiltration, proliferation and cytokine release of vector T cells contributes to the reshaping of the tumor microenvironment. To further elucidate the mechanism of therapeutic efficiency of TM@eOA, we performed intratumoral RNA sequencing (Figure 10j). The Venn diagram showed that TM@eOA treatment altered 230 significantly differentially expressed transcripts, 199 of which were different from those treated with T cells or M@eOA (Figure 10k). In addition, gene set enrichment analysis (GSEA) revealed the important role of major activation pathways, including the interaction of viral proteins with cytokines and cytokine receptors, as well as antigen processing and presentation (Figure 7r). Taken together, these transcriptome profiles further confirm that ONCOTECH treatment can reverse immunosuppressive components in the tumor microenvironment through multiple pathways while stimulating the systemic immune system.

[0076] 5. ONCOTECH for solid tumors

[0077] To evaluate the anti-tumor efficacy of ONCOTECH in vivo, B16OVA cells were subcutaneously inoculated into C57BL / 6 mice on day -7, and autologous lymphocytes were removed by X-ray on day -1 before treatment with TM@eOA (Figure 11a). After M@eOA treatment, tumor growth in mice was slightly delayed, while TM@eOA treatment resulted in the slowest tumor growth over a 30-day period (Figure 11b). Consistent with the tumor growth curve, the survival rate was 80% on day 70 after TM@eOA treatment, which was much higher than that of the T (intravenous injection) + eOA (intratumoral injection) group (Figure 11c and Figure 13a). In addition, we found that the anti-tumor efficacy of TM@eOA became moderate when CD4+ T cells were depleted (Figures 12a-c, eh). To investigate the long-term immune memory effect induced by TM@eOA treatment, we found that CD4+ T cells increased significantly in responders but not in non-responders after treatment (Figure 11d), and memory T cells in the spleen, among which we found effector memory T cells (Tem, CD44 + CD62L - ) and central memory T cells (Tcm, CD44 + CD62L + ) were all increased compared to healthy mice or saline-treated groups, indicating that TM@eOA triggered a strong immune memory response (Figure 12d). We also re-challenged 9 of the 12 mice in the TM@eOA group with tumors and found that after re-challenging the B16OVA tumor, 7 of the 9 mice completely suppressed tumor growth (Figure 11e), maintaining a long-term complete remission state for more than 90 days. In addition, we used anti-CD4, CD8, and NK1.1 antibodies to deplete CD4 in mice that were re-challenged with tumors after TM@eOA treatment. + T, CD8 + Comparative analysis of isotype control groups showed that tumor growth was significantly increased after administration of anti-CD8 or anti-CD4 antibodies. The results indicate that long-term tumor protection is mainly dependent on CD4 + T and CD8 + Furthermore, we observed that the body temperature of mice remained stable over 12 days after TM@eOA treatment (Figure 13b), and no systemic cytokine release (IL-6 and IFNγ) or liver or kidney damage was detected (Figure 13c, d), indicating the safety of ONCOTECH treatment.

[0078] To enhance the therapeutic efficacy of ONCOTECH in treating melanoma tumors, we investigated the antitumor efficacy of TM@eOA in combination with anti-CTLA4 antibodies. Our results demonstrated that the addition of anti-CTLA4 significantly enhanced the therapeutic potential of TM@eOA, resulting in sustained slowing of tumor growth and prolonged survival (Figures 11g-i). Furthermore, we generated oncolytic adenoviruses (OAs) expressing single-chain variable fragments (scFv) targeting PDL1 (termed OA-aPDL1) and compared their efficacy with that of eOA expressing Cas9. Both types of OAs demonstrated comparable efficiency in inhibiting tumor growth and improving survival outcomes (Figures 13e, f). To evaluate the potential of ONCOTECH in treating diffuse tumors, we established a diffuse tumor model in mice by intravenously injecting luciferase-expressing B16OVA cells. Following lymphodepletion, tumor-bearing mice were treated with intravenously administered TM@eOA (Figure 11j). Our results showed that after TM@eOA treatment, PDL1 gene mutations and PDL1 protein expression were most significantly downregulated in lung tumor nodules (Figure 11k). In addition, we found that after TM@eOA treatment, infiltrating T cells were more proliferative than those treated with T cells alone. In addition, carrier T cells secreted cytokines (IFNγ and GZMB) more efficiently (Figure 13g, h). Mice treated with TM@eOA had the fewest lung and other metastatic tumors compared to mice receiving single treatment (T cells or M@eOA) or combined treatment of T cells and M@eOA (T+M@eOA) (Figure 11l and Figure 13i). In addition, 80% of mice treated with TM@eOA survived for at least 50 days, while no mice in the other treatment groups survived for more than 50 days (Figure 11m). To simulate spontaneous metastasis, we also constructed a 4T1-OVA triple-negative breast cancer model (at day -12) that was able to spontaneously metastasize to the lungs. After lymph node removal and TM@eOA treatment, we found that TM@eOA could induce 43.2% PDL1 gene mutations in primary 4T1-OVA tumors (Figure 11n,o), inhibit primary tumor growth, and reduce the risk of lung metastasis after 40 days of observation (Figure 11p-r). These results demonstrate that ONCOTECH treatment has a potent anti-metastatic effect in treating systemic metastatic tumors.

[0079] We then expanded our studies to an orthotopic mouse model with LN-229 human glioblastoma cells that overexpress the disulfide ganglioside GD-2. We generated anti-GD-2 CAR-T cells and constructed a GD2-CAR-TM@eOA chimera with a CAR-scFv that interacts with the GD-2 protein (Figure 14a). After in vitro evaluation of human antiviral responses (Figures 9d-g), we investigated the in vivo antitumor effects of CAR-TM@eOA treatment in orthotopic LN-229 tumor-bearing NSG mice with a humanized tumor microenvironment generated by transplanting human HLA-A2+CD45+ peripheral blood mononuclear cells (PBMCs) into NSG mice (Figures 14b-e). After treating LN-229 tumor-bearing mice with the indicated formulations (Figures 15a-d), we found that mice treated with CAR-TM@eOA also disrupted the PDL1 gene and significantly reduced Treg infiltration within brain tumor nests, thereby promoting local secretion of anti-tumor cytokines (including human TNFα, IL12p70, IL-2, and IFNγ) (Figures 15e-h). Importantly, mice treated with CAR-TM@eOA produced increased IFNγ and GZMB in tumors (Figure 15i), which significantly contributed to the suppression of tumor growth and prolonged survival (Figures 15j, k). To further investigate the therapeutic potential of CAR-TM@eOA in human systemic metastatic tumor models, we established a lung metastatic A549 tumor model in humanized NSG mice and developed EphA2-CAR-T cells specifically targeting A549 cells. We then combined these cells with A549-M@eOA to generate CAR-TM@eOA for the treatment of diffuse tumors (Figures 14a, 10l). Similar to our previous experiments, we observed that CAR-TM@eOA treatment was most effective in disrupting the PDL1 gene and downregulating PDL1 protein expression levels (Figure 15m, n). In addition, this treatment promoted the secretion of anti-tumor effector molecules (including human TNFα, IL12p70, IL-2, IFNγ, and GZMB, Figure 15o, p), demonstrating its potent therapeutic effect against A549 human lung metastatic tumors (Figure 15q, r).

[0080] In addition to CAR-T and TCR-T cells, we also investigated whether eOAs could be loaded into tumor-infiltrating lymphocytes (TILs) derived from human PDAC tumors. As shown in Figure 16a, the isolation process of autologous TILs from resected human PDAC tumor tissue also allowed the isolation of syngeneic tumor cells, which could be further processed to obtain cell membranes paired with the TCRs of TILs. We showed that eOAs could be successfully loaded onto TILs using membranes obtained from isolated syngeneic pancreatic tumor cells (Figure 16b, c) and demonstrated the powerful ability of TIL-M@eOA to disrupt the PDL1 gene and efficiently kill tumor cells (Figure 16d-f).

[0081] 6. Cell-produced viral vesicles MV@eOA form chimeras on the surface of CAR-T cells to increase therapeutic applications.

[0082] Due to the limited availability of patient tumor cell membranes, we used lentiviral transduction to generate PSCA (prostate stem cell antigen) in the membrane of 293T cells. This tumor antigen can be specifically recognized by PSCA-specific CAR. We then constructed microcapsules (MVs) by infecting 293T cells expressing PSCA and using cytochalasin B (CB) induction and lipid extrapolation to isolate MV-containing eOA (Figure 17a). Transmission electron microscopy (TEM) and dot blotting confirmed that MVs encapsulated eOA. We found that each microgram of MV contained 4×10 7 VP eOA (Figure 19a, b). These virus-containing MVs were then loaded onto CAR-T cells through PSCA-CAR interaction. Confocal imaging showed that MV@eOA could attach to the surface of PSCA-CAR-T cells (Figure 17b and Figure 19c), with an average of 25 viral particles loaded per CAR-T cell. This engineering strategy did not affect the ability of carrier CAR-T cells to recognize additional recombinant PSCA (Figure 19d), nor did it affect their function (Figure 17c, d) or the infection and genome editing capacity of MV@eOA (Figure 17e, f). Notably, CAR-T-MV@eOA remained stable in serum-containing buffer, indicating their in vivo stability (Figure 19e).

[0083] We then established a patient-derived pancreatic cancer organoid (PDO) derived from a PSCA-expressing PDAC sample (Figure 19f) to evaluate the anti-tumor ability of PSCA-CAR-T-MV@eOA (Figure 17g). CLSM images showed that treatment with CAR-T-MV@eOA induced a large amount of PDO cell death (Figure 17h, i). Flow cytometry results showed that PDO cells were efficiently infected by eOA, resulting in significant downregulation of PDL1 in PDO cells (Figure 17j, k). After treatment, vector CAR-T cells proliferated well in co-culture with PDO (Figure 17l), releasing various effector molecules, including IFNγ, GZMB, and TNFα (Figure 17m). These results indicate that CAR-T-MV@eOA effectively inhibited the in vitro growth of patient-derived tumor organoids.

[0084] 7. Treatment of humanized PDX models with cell-derived CAR-T-MV@eOA

[0085] To further explore the potential of CAR-T-MV@eOA therapy in clinically relevant animal models, we established a humanized PDX model in humanized NSG mice and injected CAR-T-MV@eOA intravenously (Figure 18a). Since prostate stem cell antigen (PSCA) is highly expressed in PDX samples, we selected HLA-A2 +Vector CAR-T cells targeting PSCA were constructed in healthy donors (Figures 14a, 11f). 40,41 Following CAR-T-MV@eOA treatment, we observed accumulation of eOA (labeled by Cy7) and vector CAR-T cells (labeled by DiD) in the tumor region (Figures 18b, c), with higher levels of adenovirus expression observed compared to MV@eOA or CAR-T+MV@eOA treatment (Figure 18d). We also observed significant PDL1 gene mutation frequency and downregulation of PDL1 protein expression in PDX tumor tissues (Figures 18e, f). Compared to MV@eOA or CAR-T+MV@eOA treatment, we found higher expression of CRT and HSP70, and highest levels of ATP and HMGB1 in PDX tissues following CAR-T-MV@eOA treatment, indicating a strong ICD effect (Figures 18f and 19g). We also observed that after CAR-T-MV@eOA treatment, the number of Treg cells decreased (Figure 18g and Figure 19h), the proliferation of vector T cells increased (Figure 18h), and the levels of effector molecules (GZMB, TNFα and IFNγ) were enhanced (Figure 18i and Figure 19i-k). Therefore, CAR-T-MV@eOA treatment strongly inhibited the growth of PDX tumors within 20 days and effectively prolonged the survival time of tumor-bearing mice, with a survival rate of 80% within 30 days after treatment (Figure 18j). No significant changes in body temperature were observed after treatment (Figure 19l). Finally, to evaluate the long-term effects of CAR-T-MV@eOA treatment, we used humanized mice transplanted with hematopoietic stem cells (HSC CD34) and intravenously injected CAR-T-MV@eOA (Figure 18k). Compared with the saline control group, we observed that CAR-T-MV@eOA could significantly disrupt the PDL1 gene in PDX tumor tissues (Figure 18l) and strongly inhibited the growth of PDX tumors within 40 days. Importantly, this treatment regimen effectively prolonged the survival of tumor-bearing mice, with a survival rate of 40% within 60 days after treatment ( FIG. 18m ).

[0086] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An oncolytic virus-T cell chimera, characterized in that, The oncolytic virus-T cell chimera is obtained by immobilizing oncolytic virus OVs on the outer surface of carrier T cells through methods such as engineering biological membranes or vesicles carrying T cell-specific antigens.

2. The oncolytic virus-T cell chimera according to claim 1, wherein The T cells include one or more of TCR-T cells, CAR-T cells, and tumor-infiltrating lymphocytes TILs derived from human PDAC pancreatic cancer tumors.

3. The oncolytic virus-T cell chimera according to claim 1, characterized in that, The tumor-targeted oncolytic virus delivery system is obtained by attaching oncolytic virus to T cells through the biophysical interaction between the T cell receptor TCR and pMHC-I, or the chimeric antigen receptor CAR and tumor cell surface antigens.

4. The oncolytic virus-T cell chimera according to claim 1, characterized in that, The oncolytic virus includes one or more of oncolytic adenovirus and oncolytic herpes simplex virus HSV-1.

5. The oncolytic virus-T cell chimera according to claim 1, wherein The specific preparation method of the oncolytic virus-T cell chimera includes the following steps: Step 1, eOA is encapsulated with B16OVA cell membranes expressing H-2Kb-SIINFEKL through liposome extrusion technology to form M@eOA; Step 2, M@eOA is anchored to OT-1 CD8+ T through the biophysical interaction between TCR and MHC-I-OVA 257-264 ; Or the specific preparation method of the oncolytic virus-T cell chimera includes the following steps: Step 1, eOA is encapsulated with tumor cell membranes overexpressing specific tumor antigens (EphA2, PSCA, GD2) through liposome extrusion technology to form M@eOA; Step 2, M@eOA is anchored on CAR-T cells through the biophysical interaction between CAR and specific tumor antigens (EphA2, PSCA, GD2); Or the specific preparation method of the oncolytic virus-T cell chimera includes the following steps: Step 1, after eOA infects 293T cells overexpressing PSCA, through relaxin B treatment, vesicles carrying eOA in the supernatant are collected, and nanovesicles MV@eOA carrying eOA are obtained through liposome extrusion technology; Step 2, MV@eOA is anchored on CAR-T cells through the biophysical interaction between CAR and tumor antigen PSCA; Or the specific preparation method of the oncolytic virus-T cell chimera includes the following steps: Step 1, eOA is encapsulated with autologous tumor cell membranes from patients through liposome extrusion technology to form M@eOA; Step 2, M@eOA is anchored on TIL cells through the biophysical interaction between TCR and pMHC-I of autologous tumors.

6. The oncolytic virus-T cell chimera according to claim 4, wherein The oncolytic adenovirus includes engineered oncolytic adenovirus, and the engineered oncolytic adenovirus is obtained by cloning Cas9, sgRNA targeting the target gene, and EGFP into oncolytic adenovirus.

7. The oncolytic virus-T cell chimera according to claim 6, wherein, The cloning is to clone Cas9, sgRNA targeting the target gene, and EGFP into the shuttle plasmid of OA, and then transform the shuttle plasmid into oncolytic adenovirus to obtain it.

8. The oncolytic virus-T cell chimera according to claim 7, wherein, The shuttle plasmid includes pDC315-hTERT-E1A.

9. A pharmaceutical composition for treating cancer, characterized in that, The pharmaceutical composition contains the oncolytic virus-T cell chimera according to any one of claims 1-8.

10. The pharmaceutical composition according to claim 9, wherein The pharmaceutical composition further includes anti-CTLA4 antibody.

11. Use of the oncolytic virus-T cell chimera according to any one of claims 1-8 in the preparation of a medicament for cancer treatment.

12. The application according to claim 11, characterized in that, The tumor includes a primary or disseminated tumor expressing an antigen.

13. The application according to claim 12, wherein The tumor includes melanoma, lung cancer, pancreatic cancer, or glioblastoma.

14. Use of the oncolytic virus-T cell chimera according to any one of claims 1-8 in combination with an anti-CTLA4 antibody in the preparation of a cancer therapeutic agent.

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