Manufacturing CAR-T Cells in a Closed-System, End-to-End Manufacturing Workflow Enhances CAR-T Cell Function 2303378

Abstract

Abstract Introduction The Tumor Microenvironment (TME)–characterized by hypoxia and altered biophysical stress–is a primary driver of resistance in Chimeric Antigen Receptor (CAR) T cell therapy. Current manufacturing protocols fail to prepare CAR-T cells for hostile in vivo environments, leading to reduced potency and decreased persistence against cancer cells. This study presents a novel, closed-system, end-to-end CAR-T manufacturing workflow that incorporates automated liquid handling and cell harvest capabilities to reduce manufacturing time, complexity and cost. The core innovation lies in the platform’s ability to accurately model key TME stressors through advanced environmental controls, including sustained hypoxia and hyperbaric pressure to produce highly resilient therapeutic T cells. Methods The manufacturing process is fully automated and contained within a single, modular consumable, providing a high-throughput and robust workflow designed to operate in GMP environments. Key steps–including media changes, transduction, and cell harvesting–are performed without operator intervention. Results Expanding anti-CD19 CAR-T cells under hypoxia (5-10% O2) and hyperbaric pressure (5 PSI), conditions analogous to the bone marrow niche, significantly enhanced their potency and persistence. Following tumor target engagement, these cells demonstrated a 3- to 5-fold improvement in efficacy and persistence compared to CAR-T cells cultured under standard conditions (21% O2). In vitro data confirm improved function and increased resistance to TME-like stress. This enhanced therapeutic profile correlates with the enrichment of a stem-like memory T cell (Tscm) subpopulation (characterized by CD45RA+, CD62L+, CCR7+, and CD95+) and the upregulation of the metabolic regulator GLUT1, facilitating metabolic adaptation to the low-oxygen environment. Conclusion This workflow validates the AVATAR Foundry system as a robust, standardized platform for commercial-scale production of complex, physiologically relevant cell therapies. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)