Abstract 4808: Physiological manufacturing conditions enhance in vivo efficacy of CD19 CAR T cells in mice
Authors: Anita Zaitouna, Candy Garcia, Lauren Kucharczyk, Michael Steffey, Abriel Czachorowski, Natalie Czeryba, Ningchun Liu, Sheri Barnes, Yelena Bronevetsky, Shannon Eaker, James Lim, Scott Wise
Journal: Cancer Research (2025)

Abstract Since the first chimeric antigen receptor T-cell therapy was approved in 2017, significant progress has been made in manufacturing to improve patient access to these life-saving therapies. For CD19 CAR T cells, complete response rates of 40-54% have been reported in patients with relapsed or refractory aggressive B cell lymphomas, a staggering result considering the historically poor outcomes associated with this disease. However, roughly half the treated patients did not exhibit the same profound response, owing to the complexity associated with the production of potent autologous cell therapies. In this study, we examined whether manufacturing conditions can impact the in vivo efficacy of CAR T cells in tumor-bearing mice. Utilizing the AVATAR manufacturing platform from Xcellbio, we expanded healthy donor-derived CD19 CAR T cells under physiological conditions mimicking the human vasculature system. We achieved this by tightly regulating oxygen and hyperbaric pressure levels during the expansion process. We then compared tumor-bearing mice treated with AVATAR expanded cells against conventionally manufactured CD19 CAR T cells derived from a conventional CO2 incubator (21% O2 + 0 PSI). CD19 expressing NALM6-Luc-mCh-Puro tumor cells were implanted intravenously in 48 mice, across 6 groups (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories, Bar Harbor, ME), and tumor burden was measured via bioluminescence across 29 days post-implant. We report that CD19 CAR T cells manufactured under conditions mimicking the vasculature environment (15% O2 + 5 PSI) exhibited superior tumor control compared to cells expanded in a conventional CO2 incubator (1.8e8 vs 6.4e6 photons/second, mean BLI signal on day 29). Furthermore, we observed significant increases in plasma IFNg levels in animals treated with AVATAR expanded cells (mean: 111 pg/mL vs 32 pg.mL; day 19 post dose) indicating potent anti-tumor activity compared to animals treated with conventionally manufactured CD19 CAR T cells. Blood analysis revealed similar trends, with AVATAR expanded CD19 CAR+ cells present in higher numbers (21 cells/uL of blood vs. 7 cells/uL; mean, day 28 post dose). Also noteworthy was the increased trafficking of AVATAR-manufactured CD19 CAR+ T cells to the spleen and bone marrow compared to conventionally expanded CAR T cells. In summary, physiological culture conditions mimicking the oxygen and pressure levels of the vasculature system significantly enhanced the potency of CD19 CAR T cells in tumor-bearing mice, as measured by decreased tumor burden, higher effector cytokine production, and increased trafficking of CAR T cells to lymphoid organs. Manufacturing therapeutic cells in the AVATAR system has the promise to boost the efficacy of CD19 CAR T therapy, and may enable solid tumor-targeting cell therapy which has long suffered from poor potency. Citation Format: Anita Zaitouna, Candy Garcia, Lauren Kucharczyk, Michael Steffey, Abriel Czachorowski, Natalie Czeryba, Ningchun Liu, Sheri Barnes, Yelena Bronevetsky, Shannon Eaker, James Lim, Scott Wise. Physiological manufacturing conditions enhance in vivo efficacy of CD19 CAR T cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4808.