Abstract LB311: Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells

Abstract

Abstract Introduction: Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate CD19-directed chimeric antigen receptor (CAR) T cells in patients after direct intravenous delivery or extracorporeal delivery (ECD), i.e., a short-term exposure of apheresis product to fusosome before re-infusion into the patient. Here, we demonstrate that an improved manufacturing process resulted in higher fusosome titer and quality, increasing the potency of CAR T generation from resting CD8 T cells. This potency is also observed when fusosomes are evaluated in a clinically relevant mock ECD approach. Methods: Fusosome titer was quantified by ddPCR directly for physical titer (vector genomes, GQA) or after infections of SupT1 cells for functional titer (IU/mL). Transduction of CD8 T cells was measured by flow cytometry and vector copy number analysis after short-term (1-4 hour) incubation of PBMCs with fusosome followed by wash and culture in resting or CD3/CD28-activated conditions. To assess CD19CAR T cell generation at a mock clinical setting and scale, a healthy donor was apheresed to collect PBMCs, fusosome was administered into the apheresis bag and PBMCs were sampled from the bag over a 2.5-hour time course for further analysis. In vivo anti-tumor efficacy of the fusosome was measured in immune-deficient NSG mice engrafted intravenously with Nalm6, then PBMCs followed by fusosome one day later. Tumor growth was monitored by bioluminescence imaging. Results: Fusosomes produced by the improved process had both a higher functional concentration (>14-fold higher IU/mL) and better infectivity (>10-fold higher IU/GQA) in comparison to fusosomes produced by the original process. Exposure of PBMCs to fusosomes produced by the improved process led to increased fusosome binding to CD8 T cells at high IU/PBMC doses and higher transduction efficiency in CD8 T cells at the same calculated dose per cell compared to fusosomes produced by the original process (>3-fold higher mean CAR+ percentage after 2-hour exposure to 2 IU/PBMC, n=8). Furthermore, fusosomes produced by the improved process generated higher frequencies of CAR+ CD8 T cells and resulted in increased ability to control tumor growth at lower doses in vivo in a Nalm6 model. Importantly, the ECD approach translated well to a mock clinical setting, resulting in successful generation of CAR T cells (12.6% of CD8 T cells after a 2.5-hour incubation). Conclusion: CD8-targeted fusosome encoding a CD19CAR transgene represents a potential novel therapeutic approach for cancer. This study demonstrates that improvements in the manufacturing process can significantly increase the potency of the fusosome in T cells, and we intend to move forward to clinical trials using this improved process. Citation Format: Jesse Green, Brian Granger, Ouwen Liang, Jesus Moreno, Andrew Tucker, Aesha Vakil, Reuben Burch, Nicholas Mozdzierz, Matthew Scott-Skandera, Stephanie Riofrio, Mike DeCosta, Amey Gaikwad, Sanket Phadke, Kaely Gallagher, Adam Charlton, Sarah Miller, Hosein Kouros-Mehr, Misha Shamashkin, Neal Van Hoeven, Kutlu Elpek, Terry Fry. Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB311.