CAR-TTS-2021

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in glioblastoma (GBM) due to tumor antigen heterogeneity and the immunosuppressive microenvironment. To address these barriers, we developed a novel combinatorial approach: engineering CAR-T cells with third-generation oncolytic adenoviruses (OAd) to enable targeted viral delivery and sustained immune activation. Unlike conventional OAd administration, this strategy leverages CAR-T cells as tumor-tropic vectors for localized oncolysis and cytokine modulation.

CD70-specific CAR-T cells were transduced with two third-generation OAds (E1B19K/E3-deleted, replication-selective): OAd-GFP (control) or OAd-IL15 (TS-2021), generating CAR-TOAd−GFP and CAR-TTS−2021. Viral replication kinetics and CAR-T expansion were assessed in vitro. OAd delivery efficiency was quantified by co-culturing CAR-TOAd cells with GBM cells. Flow cytometry was used to analyze IL15-mediated effects on stem-like markers (CCR7, CD45RA) and exhaustion markers (PD-1, TIM-3, and LAG-3) after repeated antigen stimulation. Antitumor activity was evaluated in vitro using cytotoxicity assays and in NCG mice bearing orthotopic GBM xenografts. Mechanistic studies were conducted using RNA-seq and Western blotting.

In this study, we found that genetically engineered OAd-GFP can specifically replicate within CAR-T cells and be precisely delivered to GBM through an antigen-specific mechanism. Prolonged antigen stimulation induced T-cell exhaustion, limiting the efficacy of CAR-T therapy. TS-2021-infected CAR-T cells exhibited enhanced expansion and persistence in vitro, with reduced expression of exhaustion markers under sustained antigen stimulation. IL15 autocrine signaling activated JAK-STAT and MAPK-ERK pathways. This process repaired the DNA damage induced by OAd in CAR-T cells and maintained their expansion and persistence. By combining OAd-mediated oncolysis with IL15-driven CAR-T persistence, CAR-TTS−2021 cells demonstrated potent antitumor efficacy against GBM both in vitro and in vivo.

By integrating IL15-armed OAd into CAR-T cells, we demonstrate a synergistic strategy that simultaneously enhances viral oncolysis, sustains T-cell persistence, and counteracts GBM immunosuppression. This approach addresses both antigenic heterogeneity and microenvironment-driven resistance, providing a translatable paradigm for solid tumor immunotherapy.