Impact of Ori-TTV particles on in vivo CAR-T cell engineering for the treatment of hematologic malignancies.

Abstract

6558 Background: CAR-T cell therapy has revolutionized hematological malignancy treatment, yet traditional autologous therapies face substantial logistical and financial challenges. An efficient in vivo CAR-T cell generation platform represents a transformative approach to make immunotherapies more accessible. Here, we report the design and validation of Ori-TTV, a T cell-specific lentiviral vector system capable of directly generating functional CAR-T cells within the body. Methods: Ori-TTV was engineered with five synergistic components: (1) an Ori Multiple Targeting Domain (Ori-MTD) for selective delivery to T lymphocytes; (2) a de-targeted fusogen; (3) a T cell-specific synthetic promoter; (4) phagocytosis-resistant modifications; and (5) serum-resistant surface engineering. Ori-TTV encodes a dual-target CAR recognizing CD19 and BCMA, enabling treatment of B-cell malignancies (e.g., non-Hodgkin lymphoma) and plasma cell–derived cancers (e.g., multiple myeloma) with potential extension to autoimmune diseases. Results: Ori-TTV particles are produced at high functional titers comparable to conventional broad-tropism lentiviruses and demonstrate significant resistance to macrophage phagocytosis (>50% reduction). The T cell-specific synthetic promoter drives CAR/GFP expression comparable to the EF-1α promoter in primary T cells while maintaining minimal off-target expression in non-T cells, including HEK293T producer cells and various tumor lines. Ori-TTV efficiently transduces resting T cells, achieving >80% CAR expression in PBMCs in vitro . The resulting CAR-T cells exhibit a predominantly memory phenotype with increased TSCM cells compared to Kymriah-like ex vivo CAR-T cells. Serial killing assays demonstrated potent, multi-round cytotoxicity against CD19+ and BCMA+ target cell lines with varying antigen densities. In vivo efficacy was evaluated in humanized mouse models of NHL and MM. The PBMC humanized mice were challenged with tumors and administered Ori-TTV. Bioluminescence imaging demonstrated robust tumor regression, with complete remissions in the majority of treated animals. Pharmacodynamics revealed efficient in vivo transduction, with CAR-T expansion kinetics showing peak levels at day 13 (20–30% of circulating T cells). Expanded CAR-T cells maintained a memory-enriched phenotype with significantly increased TSCM cells. Long-term monitoring confirmed durable responses without toxicity or vector-related adverse events. Furthermore, in CD34+ humanized mice, single-injection Ori-TTV caused durable depletion of peripheral B cells. Conclusions: These findings establish Ori-TTV as a next-generation lentiviral vector system enabling safe, efficient, and selective in vivo CAR-T cell generation. It has the potential to redefine CAR-T therapy by eliminating manufacturing barriers, reducing costs, and expanding patient access.