232 Non-viral engineered next generation CD19 UltraCAR-T with membrane bound IL-15 (mbIL15) and PD-1 blockade preserves stem cell memory/naïve phenotype and enhances anti-tumor efficacy

<h3>Background</h3> Despite great advances in CD19 CAR-T therapy for B-cell malignancies, challenges of durability, manufacturing complexity, high treatment cost, and treatment-related toxicities remain. The NextGen (Next Generation) UltraCAR-T platform is designed to address these challenges, with decentralized overnight manufacturing of cells that express CD19 CAR, membrane-bound IL-15 (mbIL15) for enhanced persistence and maintenance of stem cell memory/naïve (Tscm) phenotype, a miRNA-based PD-1 blockade without complex and expensive gene editing techniques to avoid exhaustion, and a safety/kill switch, all from a single non-viral transposon to ensure a safe, homogenous cell product. <h3>Methods</h3> <i>In vitro</i> CAR-T polyfunctionality, a trait linked to improved clinical outcomes, was assessed in CAR-T cells co-cultured with CD19⁺ tumor cells using a single-cell proteomics assay. The sustained cytotoxic potential of CAR-T cells was evaluated with a repetitive antigen challenge in stimulated culture conditions that mimic the tumor microenvironment. For <i>in vivo</i> testing, mice with established tumors (NALM-6 and Jeko-1) were treated with CAR-T cells and monitored for tumor growth by IVIS. CAR-T cell expansion, persistence, and phenotype were assessed by flow cytometry in peripheral blood collected at regular intervals. <h3>Results</h3> NextGen UltraCAR-T cells showed enhanced T cell polyfunctionality, and durable tumor killing after repeated antigen challenges compared to conventional CAR-T cells lacking mbIL15 or PD-1 blockade. In <i>in vivo</i> tumor models, a single administration of NextGen CD19 UltraCAR-T manufactured overnight, without activation or <i>ex vivo</i> expansion, showed enhanced <i>in vivo</i> expansion and persistence, robust antitumor efficacy with a complete tumor clearance, and significantly longer survival compared to the conventional CD19 CAR-T cells. Furthermore, long-term antitumor immunity was observed when these tumor-free mice were rechallenged 76 days later with tumors for a second time (to simulate tumor relapse), which led to significant efficacy and overall survival without additional CD19 UltraCAR-T treatment. In contrast to conventional CD19 CAR-T cells, the NextGen CD19 UltraCAR-T treatment resulted in a significant enrichment of Tscm/naïve cell population that are associated with durable T cell memory and prevention of tumor relapse. <h3>Conclusions</h3> NextGen CD19 UltraCAR-T cells are engineered to address limitations of conventional CAR-T, including efficacy, durability, safety, and manufacturing cost and complexity. The inclusion of mbIL15 and intrinsic PD-1 blockade potentiates CAR-T functions in tumor models and provides a phenotype associated with improved clinical effectiveness. These results demonstrate that the NextGen CD19 UltraCAR-T has potential to be the best-in-class treatment. <h3>Ethics Approval</h3> NSG or NSG MHCI/II DKO mice, which lack an adaptive immune system, were obtained from Jackson Laboratories under a protocol approved by the Institutional Animal Care and Use Committees (IACUC) of Precigen.