Use of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in preparation of drugs for inhibiting tumor growth

Abstract

The application of an oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs to inhibit tumor growth was demonstrated through in vitro and in vivo experiments. The virus significantly enhanced the migration ability, tumor accumulation level, and anti-tumor effect of HER2 CAR-T cells expressing CCR7 in an ovarian cancer model. By combining a locally delivering chemokine-based oncolytic adenovirus with systemic adoptive T-cell therapy, this strategy effectively overcomes the major bottleneck in CAR-T cell therapy for solid tumors—insufficient T-cell infiltration—providing a new approach and strategy for immunotherapy of solid tumors. The combination of oncolytic adenovirus carrying CCL19 and CAR-T cell therapy significantly increased the infiltration level of CAR-T cells within tumors, effectively delaying tumor growth.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs to inhibit tumor growth. Background Technology

[0002] Despite continuous advancements in tumor diagnostic technologies and treatments, the survival rate for patients with metastatic cancer remains concerning. CAR-T cell therapy has achieved landmark efficacy in hematologic malignancies, and the U.S. Food and Drug Administration (FDA) has approved several CAR-T products, including lecithin, acrifenex, toripalix, and lecithin, for clinical treatment.

[0003] In stark contrast to its remarkable efficacy in hematologic malignancies, CAR-T cell therapy has demonstrated limited clinical activity in early clinical trials for solid tumors. Objective response rates (ORRs) are generally low, and progression-free survival (PFS) is relatively short in unselected study cohorts. A meta-analysis of 22 early clinical trials involving 262 patients showed an overall ORR of only about 9% for CAR-T cell therapy in solid tumors, highlighting the numerous challenges that need to be addressed in its treatment of solid tumors. Among these challenges, the unique properties of the tumor microenvironment (TME) play a crucial role in limiting CAR-T cell infiltration into tumor tissue and exerting anti-tumor efficacy.

[0004] The tumor microenvironment exhibits significant immunosuppressive properties, coupled with an unfavorable chemokine gradient distribution, posing a significant obstacle to the effective migration of CAR-T cells to the tumor site. Multiple chemokines, including CXCL9, CXCL10, CXCL11, and CCL5, play indispensable roles in recruiting T cells to the tumor site. Previous studies have shown that the expression levels of these chemokines are closely related to increased T cell infiltration and significantly improved survival rates in patients with various cancers. High expression of the chemokine CCL19 is closely associated with enhanced anti-tumor immunity and improved clinical prognosis in various tumors; in cohorts of patients with solid tumors such as breast cancer, high expression of CCL19 is significantly associated with increased T cell infiltration and improved patient survival rates, suggesting that CCL19 plays a crucial role in regulating immune cell migration and influencing tumor clinical prognosis.

[0005] Numerous preclinical studies and early clinical trials have confirmed that CAR-T cells carrying the chemokine CCL19 exhibit significantly enhanced migration to solid tumors, prolonged survival time within the tumor, and effective initiation of endogenous immune synergy, thereby significantly improving anti-tumor efficacy. This strategy holds promise as a core universal module for next-generation cell therapy for solid tumors. Oncolytic viruses (OVs) are a promising class of drugs for treating solid tumors in recent years. These viruses can selectively infect and lyse tumor cells without damaging normal tissues, providing new ideas and directions for tumor treatment. In addition to their inherent oncolytic activity, oncolytic viruses can also be genetically engineered to express exogenous genes, potentially reconstructing the tumor microenvironment and enhancing the efficacy of immunotherapy. Utilizing oncolytic viruses as vectors to deliver chemokines that improve T cell migration may effectively overcome the barriers of the tumor microenvironment, significantly enhancing the efficacy of CAR-T cell therapy for malignant tumors. Summary of the Invention

[0006] To address the technical deficiencies of existing technologies, this invention provides an application of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs to inhibit tumor growth. This significantly increases the infiltration level of CAR-T cells within tumors, effectively delaying tumor growth. The combined treatment regimen is well-tolerated, and the observed adverse reactions are not significantly different from those in the control group. The research results confirm that the combination of oncolytic adenovirus carrying CCL19 and CAR-T cell therapy is both safe and effective, laying a solid experimental foundation for its potential clinical application.

[0007] The technical solution adopted in this invention is the application of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs that inhibit tumor growth.

[0008] The oncolytic adenovirus is a conditionally replicating human adenovirus type 5 that lacks the E1B-55 kDa gene.

[0009] The CAR-T cells mentioned are human epidermal growth factor receptor 2 (HER2) CAR-T cells.

[0010] The dosage of the oncolytic adenovirus expressing human CCL19 in the aforementioned tumor growth inhibitory drug is 2 × 10⁻⁶. 9 PFU.

[0011] The number of CAR-T cells expressing CCR7 in the tumor growth inhibitory drug is 1×10⁻⁶. 6 indivual.

[0012] The oncolytic adenovirus expressing human CCL19 is an oncolytic adenovirus with the CCL19 gene inserted (oAd-CCL19).

[0013] The tumor growth inhibitor mentioned is an intratumoral injection drug.

[0014] The tumor in question is an ovarian tumor.

[0015] The beneficial effects of this invention are as follows: This invention provides an application of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs to inhibit tumor growth. In vitro and in vivo experiments have confirmed that this virus can significantly enhance the migration ability, tumor enrichment level, and anti-tumor effect of HER2 CAR-T cells expressing CCR7 in an ovarian cancer model. By combining oncolytic adenovirus that delivers chemokines locally with systemic adoptive T cell therapy, this strategy effectively overcomes the main bottleneck of CAR-T cell therapy for solid tumors—insufficient T cell infiltration, providing a new idea and strategy for immunotherapy of solid tumors. The use of oncolytic adenovirus carrying CCL19 combined with CAR-T cell therapy significantly increases the infiltration level of CAR-T cells in tumors and effectively delays tumor growth. Attached Figure Description

[0016] Figure 1 shows the in vitro expansion of stem cell-like memory CAR-T cells with high CCR7 expression; A and B: flow cytometry analysis of CCR7 expression levels in freshly isolated PBMCs and T cells after 5 days of in vitro activation; C: flow cytometry analysis of the phenotype of the prepared CAR-T cells (initial T cells Tn: CCR7). + CD45RA + CD45RO - Central memory T cells (Tcm): CCR7 + CD45RA - CD45RO + Stem cell-like memory T cells (Tscm): CCR7 + CD45RA + CD45RO + D, E: Transwell assay to detect the migration ability of CAR-T cells to different concentrations of recombinant human CCL19 (rhCCL19); Scale bar: 100 μm.

[0017] Figure 2 shows the identification of recombinant oncolytic adenovirus carrying CCL19; where A: schematic diagram of the gene structure of wild-type adenovirus, control oncolytic adenovirus lacking E1B-55 kDa, and recombinant oncolytic adenovirus expressing CCL19; B: SKOV3 cells were infected with oAd or oAd-CCL19 at MOI=5, and the expression of E1A protein was detected by Western blotting at 24, 48, and 72 h; C and D: A549, SK-BR-3, and SKOV3 cells were infected with different titers of oAd-CCL19 or control adenovirus oAd, and cell viability was detected at different time points at MOI=20, or at different MOIs 72 h after infection; E: SKOV3 ovarian cancer cells were infected with physiological saline, oAd, or oAd-CCL19 at MOI=10 for 72 h, and annexin V was used as the immunoblotting result. Positive cells are apoptotic cells, used to detect cell apoptosis; F: apoptosis rate statistics for each treatment group; statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test, and data are expressed as mean ± standard deviation. P<0.05, P<0.001, ns: no statistical difference.

[0018] Figure 3 shows the functional validation of CCL19 expression in recombinant oncolytic adenovirus; where A: qPCR detection of CCL19 mRNA expression level in SKOV3 cells infected with oAd or oAd-CCL19; B: ELISA detection of secretory CCL19 protein content in the culture supernatant of infected SKOV3 cells; C: Transwell assay diagram: conditioned medium of oAd or oAd-CCL19 infected SKOV3 cells for 48 h added to the lower chamber, and 2×10⁻⁶ cells added to the upper chamber. 5 A: HER2 CAR-T cells; D: Representative image of HER2 CAR-T cells migrating to conditioned medium; E: Statistical result of the number of migrating cells in image D; Scale bar: 100 μm; Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Data are expressed as mean ± standard deviation. P<0.05, P<0.001, ns: no statistical difference.

[0019] Figure 4 shows the in vitro validation of the antitumor efficacy of oAd-CCL19 combined with HER2 CAR-T cells; where A: Schematic diagram of chemotaxis experiment: detecting the infiltration ability of HER2 CAR-T cells into tumor cells infected with oncolytic virus; CFSE-labeled SKOV3 cells were infected with oAd or oAd-CCL19 at MOI=1 for 24 h, and 2×10⁻⁶ cells were added to the upper chamber. 5 A: PE-labeled HER2 CAR-T cells; B: Representative images of CAR-T cell migration taken under a fluorescence microscope after 5 h of culture; C: Statistical results of the number of migrating HER2 CAR-T cells; D, E: In vitro modified co-culture experiments based on Transwell assay to verify the anti-tumor effect of single or combination therapy, using crystal violet staining to detect SKOV3 cell viability and performing statistical analysis; F, G: ELISA detection of IL-2 and IFN-γ concentrations in cell culture supernatant; H: Viability assessment of HER2 CAR-T cells infected / uninfected with oAd-CCL19; Statistical analysis used one-way ANOVA combined with Tukey multiple comparison test, scale bar: 100 μm, data are expressed as mean ± standard deviation. P<0.001, P<0.0001, ns: no statistical difference.

[0020] Figure 5 shows the in vivo efficacy and safety validation of oncolytic virus combined with CAR-T cell therapy; where A: experimental timeline: 4×10 6 SKOV3-Luc cells were subcutaneously injected into NSG mice to establish a human ovarian cancer mouse model; approximately day 10, the mice were given two intratumoral injections, each at a dose of 1×10⁻⁶. 9 PFU oAd, oAd-CCL19, or solvent control; 3 days after the last viral injection, intravenous infusion of 1×10 6A: HER2 CAR-T cells or solvent control, with a second infusion performed 3 days later; B: Tumor growth curves of mice in different treatment groups; C: Tumor size and weight statistics of mice in different treatment groups after 42 days; D, E: qPCR detection of CCL19 and HER2-CAR mRNA expression levels in tumor tissues of mice in different treatment groups; F: Changes in body weight of mice in each treatment group, recorded every 2 days; G, H: ELISA detection of serum ALT and BUN levels in peripheral blood of mice in each treatment group; Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test, and data are expressed as mean ± standard deviation. P<0.001, P<0.0001, ns: no statistical difference. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] cell lines

[0023] The 293A, 293T, A549, SK-BR-3, and SKOV3 cell lines were all cryopreserved in our laboratory. The firefly luciferase gene was transfected into SKOV3 cells using a lentiviral vector to construct the SKOV3-Luc cell line. All cells were cultured using standard cell culture protocols in a constant temperature incubator at 37°C and 5% CO2.

[0024] laboratory animals Female NSG immunodeficient mice aged 6–8 weeks were purchased from Shanghai Model Biotechnology Co., Ltd. The mice were housed in a specific pathogen-free (SPF) grade animal facility under constant temperature and humidity conditions, using a standard 12-hour light / 12-hour dark cycle, with free access to food and water.

[0025] CAR vector construction and human T cell transduction The HER2-targeting CAR vector consists of a CD8α signal peptide, a HER2-targeting single-chain variable region antibody (ScFv), a hinge region, a CD28 transmembrane domain, a human CD28 co-stimulatory domain, and a CD3ζ signaling domain. Based on published sequences, a human HER2-targeting ScFv was synthesized artificially. The CAR vector and two packaging plasmids (pMD.2G and psPAX2) were co-transfected into HEK293T cells using polyethyleneimine (PEI). 48 h after transfection, the cell supernatant was collected, filtered through a 0.45 μm filter (Millipore), and concentrated by ultracentrifugation at 100,000 × g for 120 min at 4 °C. The concentrated lentivirus was resuspended in a suitable buffer, aliquoted, and stored at -80 °C. The lentivirus titer was determined using the half-maximal tissue culture infectious dose (TCID50) method.

[0026] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy donors using density gradient centrifugation (800×g, 20 min). The isolated PBMCs were seeded into complete culture medium containing 15% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin, and cultured at 37°C in a 5% CO2 incubator. T cells were stimulated with human T cell activation beads CD3 / CD28 (Thermo Fisher Scientific), supplemented with 300 U / mL human interleukin-2 (IL-2) and 10 ng / mL human interleukin-15 (IL-15). After 24 h of stimulation, lentivirus was transduced into activated T lymphocytes at a multiplicity of infection (MOI) of 5, with IL-2 and IL-15 supplemented. T cells were collected after 12 h and cultured for approximately 8 days with continued supplementation of IL-2 and IL-15 to obtain HER2CAR-T cells. Control T cells without lentivirus transduction were prepared using the same method. Cells. Written informed consent was obtained for all PBMC samples from healthy donors.

[0027] Preparation of oncolytic adenovirus The blank oncolytic adenovirus (oAd) used in this invention is a conditionally replicating human adenovirus type 5 lacking the E1B-55 kDa gene; oAd-EGFP and oAd-CCL19 are its derived vectors, with the open reading frames of enhanced green fluorescent protein (EGFP) and human CCL19 inserted into the E1 region of the virus, respectively. All oncolytic adenoviruses were amplified in HEK293 cells, purified by cesium chloride (CsCl) density gradient equilibration centrifugation, dialyzed against 5% sucrose solution, aliquoted, and stored at -80°C; the viral infection titer was determined on HEK293 monolayer cells using the TCID50 method and converted to plaque-forming units (PFU); the viral working solution was diluted to 2 × 10¹ with sterile Duchenne phosphate-buffered saline (D-PBS). 0 PFU / mL was administered via intratumoral injection, with 50 μL injected per mouse, and a single dose of 1×10⁻⁶. 9 PFU.

[0028] Oncolytic activity assay The oncolytic activity of oncolytic adenovirus was detected in vitro using the Cell Counting Kit-8 (CCK-8, Elairet). Tumor cells were seeded at a density of 3000 cells / well in 96-well culture plates and cultured for 8 h until cell adhesion was achieved. Different concentrations of oncolytic adenovirus were then added. Cell viability in each well was measured using the CCK-8 assay at 24, 48, 72, and 96 h after virus addition. The relative survival rate of cells in the virus-infected group was calculated using uninfected cells as a blank control.

[0029] Western blot assay for proteins SKOV3 cells were seeded in 6-well culture plates. After cell adhesion, the cells were infected with oAd and oAd-CCL19 at MOI=5. Two hours after infection, the infection medium was discarded and replaced with fresh complete culture medium. Cells were collected at different time points after infection, total protein was extracted, and the expression level of the early viral gene E1A was detected.

[0030] Protein samples were thoroughly lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime) containing 1% phosphatase inhibitor and inactivated by heating at 100°C for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Thermo Fisher Scientific). The membranes were blocked for 1 h with Tris buffered saline containing 5% skim milk (containing 1% Tween 20, TBST), and E1A monoclonal antibody (Santa Cruz Biotechnology, 1:1000 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Bio-Raybone, 1:5000 dilution) were added and incubated overnight at 4°C. Horseradish peroxidase (HRP)-labeled secondary antibody (Bio-Raybone, 1:1000 dilution) was then added and incubated at room temperature for 1 h. Chemiluminescence imaging was performed using a chemiluminescence imaging system (Bio-Raybone, ChemiDoc). 1200) Digital analysis of the protein immunoblotting results was performed, with GAPDH as an internal reference.

[0031] Real-time quantitative polymerase chain reaction (qPCR) Total RNA was extracted from tumor tissues or cultured cells using the GeneJET RNA purification kit (Thermo Fisher Scientific); the extracted RNA was reverse transcribed into cDNA using the PrimeScript RT kit (containing gRNA inhibitor, Takara Bio); and the expression level of the target gene was detected by qPCR experiments using the SYBR Green quantitative PCR kit (Takara Bio) on a 7500 real-time quantitative PCR instrument (Applied Biosystems).

[0032] The primer sequences used for RT-PCR are as follows: CCL19 upstream primer: 5'-AACTCTGAGTGGCACCAATG-3'CCL19 Downstream primer: 5'-TGCAGCCATCCTTGATGAGA-3'HER2-CAR Upstream primer: 5'-GCCGGATTTCACTTTCACCA-3'HER2-CAR Downstream primer: 5'-CCGAACGTGAATGGAGTACG-3'GAPDH Upstream primer: 5'-GTGGTCTCCTCTGACTTCAACA-3'GAPDH Downstream primer: 5'-CTCTTCCTCTTGTGCTCTTGCT-3' Using GAPDH as an internal reference gene, the relative expression levels of CCL19 and HER2-CAR genes were calculated using the 2^(-ΔΔCt) method.

[0033] Flow cytometry Flow cytometry was used to detect the expression levels of CCR7 and HER2 on the cell surface. Cultured cells were collected, washed three times with PBS, and then blocked with Fc receptor blocking solution for 30 min. The positivity rate of CAR-T cells was detected using phycoerythrin (PE)-Texas Red channel. The expression level of HER2 in SKOV3 cells was detected using PE-labeled HER2-specific antibody (Bio-Rad Laboratories). All flow cytometry experiments were performed using a CytoFLEX flow cytometer (Beckman), and data were processed using appropriate analysis software.

[0034] Apoptosis analysis SKOV3 cells were infected with oncolytic adenovirus at MOI=10. Two hours after infection, the infection medium was discarded and replaced with fresh complete culture medium. Seventy-two hours after infection, all cells were collected and stained with Annexin V-PI apoptosis detection kit. After incubation at room temperature in the dark for 20 minutes, apoptosis was immediately detected using a CytoFLEX flow cytometer (Beckman). The experiment was set up in triplicate, and the proportion of apoptotic cells was analyzed using appropriate software.

[0035] Crystal violet staining experiment Tumor cells were divided into 5×10 4 HER2 CAR-T cells were seeded at a density of 10 cells / well in 24-well culture plates and cultured in complete culture medium. After cell adhesion, cells were infected with oAd and oAd-CCL19 at an MOI of 10. After 48 h of infection, HER2 CAR-T cells were added to the wells with or without the addition of CAR-T cells for co-culture for 24 h. After co-culture, the culture plate was washed once with PBS, and the cells were fixed with 4% paraformaldehyde for 10 min. The fixative was discarded, and the fixed cells were stained with 2% crystal violet staining solution for 30 min. After washing three times with PBS, the cells were photographed and recorded. The crystal violet precipitate was then dissolved in 33% acetic acid solution, and the absorbance was measured using a microplate reader. The cell coverage was calculated based on the absorbance value.

[0036] Enzyme-linked immunosorbent assay (ELISA) 1×10 5Tumor cells were seeded into 24-well culture plates. After cell adhesion, the cells were infected with oAd and oAd-CCL19 at MOI=1. After 48 h of culture, the cell supernatant was collected from each group, and the concentration of CCL19 protein in the supernatant was detected using a CCL19 ELISA kit (Lianke Biotechnology). The secretion levels of IL-2 and interferon-gamma (IFN-γ) in the cell supernatant were detected using the same method. All experiments were performed in triplicate.

[0037] Chemotaxis experiment The migration ability of CAR-T cells was detected using a 24-well Transwell chamber (Corning) containing a 5 μm pore size filter membrane; 5 × 10⁵ cells were infected with oncolytic virus (oAd-EGFP or oAd-CCL19) at MOI=1 for 48 h. 4 Add tumor cells, or culture medium containing different concentrations of recombinant CCL19 (0, 1, 10, 100 nM), to the lower chamber of the Transwell; add 2 × 10⁶ tumor cells. 5 CAR-T cells were added to the upper chamber and cultured in a 37°C, 5% CO2 incubator. After 5 h of culture, the unmigrated cells on the filter membrane of the upper chamber were wiped off, and the migrating cells in the lower chamber were counted to compare the differences in the migration ability of CAR-T cells among different groups.

[0038] In vivo experiments 4×10 6 SKOV3-Luc cells were subcutaneously injected into the right back of NSG mice to establish a human ovarian cancer xenograft model. Approximately 10 days later, when the tumor volume reached about 100 mm³, treatment was initiated. Mice received a single intratumoral injection of 2 × 10⁻⁶ cells. 9 PFU containing oAd-EGFP or oAd-CCL19; 48 h later, 1×10⁻⁶ PFU was administered to the tail vein of mice in two divided doses. 6 One CAR-T cell was administered, with two infusions 3 days apart. The long diameter (L) and short diameter (S) of the tumor were measured every 2 days using a digital caliper to dynamically monitor tumor growth. The tumor volume was calculated using the formula V=1 / 2×(L×S²).

[0039] Safety assessment The potential treatment-related toxicities of the combination therapy were assessed in tumor-bearing mice. Peripheral blood was collected from the mice before their sacrifice at the experimental endpoint, and serum was separated by centrifugation. Serum biochemical indicators, including alanine aminotransferase (ALT) and blood urea nitrogen (BUN), were measured to evaluate the effects of the treatment on the liver and kidney function of the mice. Meanwhile, changes in body weight of the mice were recorded weekly throughout the treatment period to observe their general condition and toxic reactions.

[0040] Statistical analysis All statistical analyses were performed using GraphPad Prism 9 software (GraphPad, San Diego, USA). Quantitative data are expressed as mean ± standard deviation (x ± s). Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA combined with Tukey's multiple comparison test was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0041] result Activated T cells highly express CCR7 and CCL19, which are ideal chemokines for recruiting CAR-T cells. Both naive T cells (Tn) and central memory T cells (Tcm) express the CCR7 receptor. To clarify the changes in CCR7 expression before and after T cell activation, this study isolated peripheral blood cells (PBMCs) from four healthy donors and used flow cytometry to detect CCR7 expression levels. The results showed that after T cell activation in vitro, CCR7 expression levels increased. + The proportion of cells increased significantly (Figures 1A and 1B). This study aimed to verify whether the cell expansion protocol could effectively enrich CCR7. + CAR-T cells: This study constructed CAR-T cells targeting HER2. Flow cytometry analysis showed that the final CAR-T cells contained CAR... + The cells are mainly CCR7. + The phenotype was more than 97% stem cell-like memory T cell (Tscm) subset (Figure 1C).

[0042] Further in vitro culture experiments revealed a significant increase in CCR7 expression levels in activated CAR-T cells, which remained elevated for at least 6 days under continuous culture conditions (Figure S1). Transwell assays were used to assess the chemotactic migration ability of CAR-T cells to CCL19. The results showed that, compared to the control group without chemokines, the addition of recombinant CCL19 (rCCL19) significantly promoted CAR-T cell migration; however, further increasing the concentration of rCCL19 did not enhance migration ability (Figures 1D and 1E). In summary, the amplification protocol established in this invention effectively preserves CCR7. + Stem cell-like memory CAR-T cells, which exhibit a strong response to CCL19-mediated directed migration, suggest that CCL19 is an ideal chemokine for recruiting this type of CAR-T cells.

[0043] Construction and in vitro identification of oncolytic adenovirus carrying CCL19 This invention successfully constructed an oncolytic adenovirus expressing human CCL19 (oAd-CCL19) and a matched control virus not expressing CCL19 (oAd). Simultaneously, schematic diagrams of the gene structures of wild-type adenovirus type 5 (Ad5 WT) and the genetically engineered oncolytic adenovirus were drawn (Figure 2A). To clarify whether the insertion of CCL19 affects the replication ability of the oncolytic adenovirus, this invention first compared the expression levels of the early viral gene E1A after infecting SKOV3 cells with oAd-CCL19 and the control virus. Western blot results showed no significant difference in E1A protein expression levels between the oAd and oAd-CCL19 groups at each detection time point (Figure 2B), suggesting that the insertion of the CCL19 gene does not affect the viral replication ability.

[0044] This study then verified whether the insertion of CCL19 altered the inherent oncolytic activity of adenovirus. In three tumor cell lines (A549, SK-BR-3, and SKOV3), cells were infected with oAd-CCL19 and control viruses at different MOIs, and cell viability was assessed at different time points. CCK-8 assays showed that both viruses induced tumor cell lysis in a dose-dependent manner, and their cytotoxic characteristics were almost identical (Figures 2C and 2D). Flow cytometry analysis of the proportion of annexin V-positive cells showed no significant difference in apoptosis levels between oAd and oAd-CCL19-infected SKOV3 cells (Figures 2E and 2F). In conclusion, the insertion of the CCL19 gene into oncolytic adenovirus does not affect viral replication capacity or weaken the inherent oncolytic activity of adenovirus.

[0045] oAd-CCL19 can efficiently express and secrete biologically active CCL19, promoting CAR-T cell migration. To verify whether oAd-CCL19 can effectively express and secrete the chemokine CCL19, SKOV3 cells were infected with oAd and oAd-CCL19, respectively, with an uninfected blank control group. Cells and cell supernatant were collected 48 h after infection. RT-PCR results showed that only the oAd-CCL19 group showed highly efficient transcription of CCL19 mRNA (Figure 3A). ELISA results showed that high concentrations of secreted CCL19 protein were detectable in the conditioned medium of the oAd-CCL19 group. Figure 3 (B) This confirms that oAd-CCL19 can be efficiently expressed and secreted in tumor cells.

[0046] Finally, this invention verified whether virus-expressed CCL19 could enhance the recruitment ability of CAR-T cells. Transwell assay results showed that, compared with the conditioned medium of oAd-infected SKOV3 cells, the conditioned medium after oAd-CCL19 infection significantly promoted the migration of HER2 CAR-T cells to oAd (Figures 3C, 3D, 3E). These results confirm that oAd-CCL19 can secrete biologically active CCL19, effectively recruiting CAR-T cells to migrate to tumor cells.

[0047] Oncolytic adenovirus carrying CCL19, combined with HER2-targeting CAR-T cells, exhibits synergistic antitumor activity in vitro. This invention further verifies whether CCL19 expressed by oAd-CCL19 can enhance the migration ability of HER2 CAR-T cells, thereby improving their anti-tumor activity. A two-compartment Transwell migration assay was used (Figure 4A). CFSE-labeled SKOV3 cells were seeded in the lower compartment and infected with oAd or oAd-CCL19 at MOI=1 for 24 h. PE-labeled HER2 CAR-T cells were then added to the upper compartment. The migrating CAR-T cells were observed and quantitatively analyzed using fluorescence microscopy (Figure 4B). The results showed that the number of CAR-T cells recruited in the oAd-CCL19 infection group was 3.8 times that of the control virus oAd infection group (Figure 4C), confirming that the virus-secreted CCL19 is sufficient to drive the efficient migration of CAR-T cells to tumor cells.

[0048] Based on the above results, this invention hypothesizes that the enhanced recruitment capacity of CAR-T cells can be translated into increased cytotoxicity. To verify this hypothesis, SKOV3 cells were pretreated with oAd, oAd-CCL19, or saline for 48 h, respectively, and then HER2 CAR-T cells or blank control T cells were added to the upper chamber. After co-culturing for 24 h, crystal violet staining results showed that compared with the oAd combined with HER2 CAR-T group, the oAd-CCL19 combined with HER2 CAR-T group significantly enhanced the lysis effect on tumor cells (Figures 4D and 4E). ELISA results were consistent with this; compared with the control group, the secretion levels of IL-2 and IFN-γ in the cell supernatant of the oAd-CCL19 combined with HER2 CAR-T group were significantly increased (Figures 4F and 4G), suggesting that this combination regimen can significantly enhance the activation and effector function of CAR-T cells.

[0049] This invention further evaluated whether oAd-CCL19 would adversely affect the viability of CAR-T cells. Cell viability assays revealed no significant difference in viability between oAd-CCL19-infected and uninfected HER2 CAR-T cells. Figure 4 (H), confirming that oAd-CCL19 does not affect the survival of CAR-T cells, and the two have good in vitro compatibility.

[0050] Oncolytic adenovirus carrying CCL19 can enhance the inhibitory effect of CAR-T cells on transplanted tumors without significant toxicity. To translate the above in vitro research results into in vivo evaluation, this invention first subcutaneously inoculated SKOV3 cells into NSG mice to construct a human ovarian cancer xenograft model (Figure 5A); after the tumor became palpable, the mice were given two intratumoral injections, respectively oAd, oAd-CCL19, or a solvent control; two days after the last viral injection, HER2CAR-T cells or a solvent control were intravenously infused; throughout the treatment period, tumor growth was dynamically monitored, and changes in mouse body weight and systemic toxicity were assessed.

[0051] The results showed that, compared with all other treatment groups, the oAd-CCL19 combined with HER2 CAR-T cell therapy significantly and persistently inhibited tumor growth (Figure 5B); at the experimental endpoint, the tumor weight of mice in this group was significantly lower than that in other groups (Figure 5C), confirming that the combination of the two has a significant synergistic anti-tumor effect in vivo. To verify the in vivo recruitment effect of oAd-CCL19 on HER2 CAR-T cells, tumor tissue was collected 6 days after the last HER2 CAR-T cell infusion for subsequent analysis. RT-PCR detection of tumor tissue showed that CCL19 mRNA was highly expressed in the tumor tissue of mice treated with oAd-CCL19. In the tumor tissue of mice that received HER2 CAR-T cells alone, the expression level of HER2 CAR mRNA was low, while the expression level of HER2 CAR mRNA in the oAd-CCL19 combined treatment group was significantly increased (Figures 5D and 5E), suggesting that CCL19 expressed in the tumor can effectively recruit adoptive CAR-T cells and increase their accumulation in the tumor.

[0052] Although the combined treatment significantly enhanced oncolytic activity, no obvious toxic reactions were observed in this invention: the body weight changes of mice in all experimental groups showed a consistent trend with no significant differences (Figure 5E); the serum ALT and BUN levels of mice in each treatment group were within the normal range and showed no significant differences compared with the control group (Figures 5F and 5G). In summary, oAd-CCL19 can safely enhance the accumulation of HER2 CAR-T cells in tumors and the anti-tumor efficacy in vivo, and the combination of the two has a good safety profile.

[0053] in conclusion: This invention successfully constructed an oncolytic adenovirus carrying CCL19, and in vitro and in vivo experiments confirmed that this virus significantly enhanced the migration ability, tumor accumulation level, and anti-tumor effect of HER2 CAR-T cells expressing CCR7 in an ovarian cancer model. By combining oncolytic adenovirus delivering chemokines locally with systemic adoptive T cell therapy, this strategy effectively overcomes the major bottleneck of CAR-T cell therapy for solid tumors—insufficient T cell infiltration—providing a new approach and strategy for immunotherapy of solid tumors.

[0054] From a mechanistic perspective, the significant enhanced antitumor effect observed in this invention may depend on the regulatory role of the CCR7-CCL19 axis—a central axis that plays a crucial role in T cell homing and intratumoral retention. This study confirms that activated HER2 CAR-T cells sustainably overexpress the CCR7 receptor, enabling them to strongly respond to virally expressed CCL19. In vitro experiments show that CCL19 expressed by oAd-CCL19 significantly promotes CAR-T cell migration to tumor cells (Figures 4B and 4C) and significantly upregulates the secretion of effector cytokines such as IL-2 and IFN-γ (Figures 4F and 4G), thereby achieving more efficient tumor lysis. Notably, the CAR-T cell preparation protocol established in this invention can stably obtain a high proportion of CCR7. + CD45RO + CD45RA - Stem cell-like memory CAR-T cells (Fig. 1B). Since stem cell-like memory T cells (TSCM) have significantly better survival and anti-tumor activity than central memory T cells (TCM)

[38] , the enrichment of this cell subpopulation may be a key factor in the stronger therapeutic effect of HER2 CAR-T cells in this invention.

[0055] The findings of this invention provide strong preclinical evidence for the clinical translation of oncolytic adenovirus carrying CCL19 combined with CAR-T cell therapy. This strategy is modular and widely applicable, effectively addressing the key issue of insufficient CAR-T cell infiltration in solid tumors. It holds promise as a universal treatment to overcome T cell migration barriers in solid tumors, offering a new direction for the development of immunotherapy for solid tumors.

[0056] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of patent protection of this patent.

[0057] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Application of oncolytic adenovirus expressing human CCL19 and CAR-T cells expressing CCR7 in the preparation of drugs to inhibit tumor growth.

2. The application according to claim 1, characterized in that, The oncolytic adenovirus is a conditionally replicating human adenovirus type 5 that lacks the E1B-55 kDa gene.

3. The application according to claim 1, characterized in that, The CAR-T cells mentioned are human epidermal growth factor receptor 2 (HER2) CAR-T cells.

4. The application according to claim 1, characterized in that, The dosage of the oncolytic adenovirus expressing human CCL19 in the aforementioned tumor growth inhibitory drug is 2 × 10⁻⁶. 9 PFU.

5. The application according to claim 1, characterized in that, The number of CAR-T cells expressing CCR7 in the tumor growth inhibitory drug is 1×10⁻⁶. 6 indivual.

6. The application according to claim 1, characterized in that, The oncolytic adenovirus expressing human CCL19 is an oncolytic adenovirus with the CCL19 gene inserted (oAd-CCL19).

7. The application according to claim 1, characterized in that, The tumor growth inhibitor mentioned is an intratumoral injection drug.

8. The application according to claim 1, characterized in that, The tumor in question is an ovarian tumor.

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