Abstract 2734: Modulation of resting T cell status to enhance transduction and CAR T expansion following exposure to CD8-targeted fusosomes
Authors: Dolinski Brian , Chaturvedi Vandana , Humes Daryl , Proano Christina , Ott Mariliis , Moreno Jesus , Zipp Garrett , Lampano Aaron , Aamer Hadega , Liang Ouwen , van Hoeven Neal , Elpek Kutlu G. , Foster Aaron E. , Fry Terry J. 
Published: June 2023

Abstract
Introduction: Fusosomes are novel viral vectors pseudotyped with a modified paramyxovirus envelope targeting specific cell types. We have generated a CD8-targeted fusosome to deliver a CD19-directed chimeric antigen receptor (CAR) transgene to CD8+ T cells in vivo following intravenous (i.v.) injection. Although fusosomes are capable of genetically modifying resting T cells, modulation with chemical or biological agents may increase transduction and facilitate CAR T expansion. Here, we demonstrate that rapamycin and IL-7 enhance resting T cell transduction, and IL-7 supports the expansion of in vivo generated CAR T cells to increase anti-tumor efficacy.
Methods: CD8a-targeted fusosome encoding GFP or CD19CAR transgene were generated. To measure the effect on transduction, T cells were pre-treated with IL-7 (15 ng/ml) or rapamycin (30 μM) prior to fusosome exposure. Transduction was assessed by flow cytometry and vector copy number after 7-10 days in culture. CAR T cell function and expansion were assessed in coculture assays with CD19+ Nalm6 tumor cells using live cell imaging. To measure anti-tumor efficacy with a combined fusosome and IL-7 approach, immunodeficient NSG mice were challenged i.v. with Nalm6 tumors expressing firefly luciferase (5E5 cells/mouse) on day -4, PBMC (1E7 cells/mouse) on day -1 followed by CD8/CD19CAR fusosome (1E7 IU/mouse) on day 0. IL-7 (1-5 μg/mouse) was administered subcutaneously twice weekly. Tumor growth was monitored by bioluminescence imaging.
Results: CD8/CD19CAR fusosome were effective at transducing resting T cells. Pre-treatment with IL-7 alone resulted in a moderate increase in transduction efficiency (1.5x). In contrast, pre-treatment with both IL-7 and rapamycin further increased transduction efficiency (&gt;5x). As IL-7 could also be used to support CAR T expansion, we tested whether IL-7 post-fusosome exposure could increase CAR T cell numbers and anti-tumor efficacy. In the absence of tumor targets, IL-7 promoted the expansion of CD8+ CAR T cells as well as non-transduced T cells. However, in the presence of tumor antigen, IL-7 treatment resulted in selective expansion of CAR T cells over bystander T cells (40x vs 10x, respectively) in vitro. Importantly, systemic IL-7 treatment following CD8/CD19CAR fusosome administration increased efficacy in Nalm6 tumor bearing NSG mice compared to those treated with fusosome only.
Conclusion: T cell-targeted fusosomes encoding a CAR transgene potentially represent a novel, off-the-shelf therapeutic approach for cancer. This study demonstrates that modulation of the resting T cell state can increase susceptibility to fusosome-mediated transduction. The potency of CD8/CD19CAR fusosomes can be increased by pre-treatment of resting T cells with IL-7 and rapamycin to increase transduction or post-treatment with IL-7 to expand CAR T cells to increase anti-tumor efficacy
Citation Format: Brian Dolinski, Vandana Chaturvedi, Daryl Humes, Christina Proano, Mariliis Ott, Jesus Moreno, Garrett Zipp, Aaron Lampano, Hadega Aamer, Ouwen Liang, Neal van Hoeven, Kutlu G. Elpek, Aaron E. Foster, Terry J. Fry. Modulation of resting T cell status to enhance transduction and CAR T expansion following exposure to CD8-targeted fusosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2734.