CD8-Targeted, Integrating Viral Vectors Transduce Resting T Cells and Enable Extracorporeal Delivery (ECD) for Rapid CAR T Cell Therapies

Abstract

Introduction: T cell-targeted direct in vivo delivery of a CAR transgene has the potential to provide a fast, "off-the-shelf" option for CAR T cell therapies without the complexity and T cell stress of current autologous manufacturing protocols. We developed a novel, paramyxovirus-based viral vector (VV, termed, fusosome) that can specifically target and transduce "resting" (i.e., non-activated) CD8+ T cells. We evaluated whether extracorporeal delivery (ECD) - the short-term exposure of CD8-targeted fusosomes to resting T cells followed by re-infusion into the patient (apheresis IV) - could generate functional CD19 CAR T cells. Here, we demonstrate that a brief (< 1 hour) exposure of resting leukocytes with CD8-specific CAR T cell fusosomes results in specific transduction of resting CD8+ T cells that can eliminate CD19+ tumors both in vitro and in vivo. Methods: To generate CD8-targeted VV, the paramyxovirus fusogen was mutated to ablate its native tropism and subsequently engineered with a novel single-chain variable fragment (scFv) targeting human CD8α. VV encoding GFP or a CD19 CAR were subsequently pseudotyped with CD8α fusogen to produce CD8-targeted fusosomes. As a control, conventional VSV-G pseudotyped LV was also produced. Transduction efficiency, specificity, and function of CD8-targeted fusosomes and standard LV were measured in resting PBMCs (n=3 in each experiment) and CD3/CD28-activated T cells by flow cytometry, vector copy number (VCN), and functional assays (tumor and B cell killing assays). To assess ECD potential, optimization of short-term incubation assays (30 minutes to 4 hours) at various multiplicity-of-infection (MOI; 0.5 to 3 IU/cell) were determined by assessing CAR transduction and ability to kill CD19+ target cells. In vivo efficacy was measured using immune-deficient NSG mice engrafted with CD19+ Nalm-6 tumors. Freshly thawed PBMCs were incubated with CD8-targeted CD19 CAR VV and subsequently infused via i.v. injection. Tumor bioluminescence (BLI) was measured weekly, and CAR T frequency in peripheral blood was assessed. Results: In contrast to conventional LV, CD8α-targeted fusosomes showed specific transduction of CD8+ activated T cells, and further, demonstrated the ability to quickly bind (within 1 hour) and transduce resting CD8+ T cells (up to 19.9±3.8% CAR+ CD8 T cells at 3 IU/PBMC and VCN<0.5) resulting in functional CD19 CAR T cells that can eliminate CD19+ target cells in coculture assays. Flow cytometry and cell sorting indicate that CD8-targeted fusosomes can target naïve (CD45RA+CCR7+; 10.6±3.1%), memory (CD45RO+; 50.2±0.5%), and effector (CD45RA-CCR7-; 26.9±4.8%) CD8+ T cells. Given the ability of CD8-targeted fusosomes to transduce resting T cells, we explored the potential of ECD, that is, short-term exposure (1 to 4 hours) of apheresis-collected lymphocytes with fusosomes to provide rapid CAR T cell therapy. Transduction of both healthy and patient (DLBCL) PBMCs was vector- and time-dependent, and specific for CD8+ T cells, resulting in the generation of cytotoxic, CAR T cells (at 1 IU/PBMC, healthy donors: 5.0±4.1% CAR+ CD8 T cells vs. DLBCL patients: 5.4±3.2% CAR+ CD8 T cells) capable of lysing CD19+ tumor cells (healthy donors: 74.5±23.5% vs. DLBCL patients: 81.4±17.1% reduction in Nalm-6 representation within co-culture versus cells not exposed to CD8-targeted fusosomes). Short-term exposure of PBMCs, followed by intravenous injection into CD19+ Nalm-6 tumor-bearing animals resulted in CAR expression specifically in CD8 T cells and elimination of tumor (Figure 1). Conclusion: Short-term incubation of healthy or DLBCL patient lymphocytes with CD8-targeted CD19 CAR fusosomes results in the generation of highly functional CD8+ CAR T cells capable of eliminating CD19+ tumor cells both in vitro and in animal models. ECD allows for the optimization of vector and T cell exposure at a given dose to maximize CAR T transduction of freshly isolated lymphocytes (i.e., apheresis). This method has the potential for rapid production of CAR T cells for patients at lower VV doses while also providing important pharmacokinetic and safety data to inform direct intravenous administration of CD8-targeted CD19 CAR T fusosomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal