Abstract LB091: T cell targeted lentiviral gene delivery using the PACK-IT Platform generates CAR-T cells with superior potency compared to conventional lentivirus and enables in vivo generation of CD19-CAR T cells capable of controlling leukemia in preclinical models

Chimeric Antigen Receptor (CAR)-T cell therapy has revolutionized outcomes for patients with B cell and plasma cell malignancies; however, a sizable fraction of CAR-T cell recipients fail to experience long term disease control. In some cases, therapeutic failure may be attributed to inadequate T cell potency induced by prolonged activation and ex vivo proliferation during the traditional multiday CAR-T cell manufacturing process. Moreover, only a small fraction of eligible patients receive commercial CAR-T cell therapies due to access barriers such as cost and difficulty meeting supply demand equilibrium. With the goal of enhancing anti-tumor efficacy and patient access, we developed the Programmable Antibody-mediated Cellular Knock-In of T cells (PACK-IT) Platform. To direct genetic integration specifically into T cells, the PACK-IT Platform incorporates a mutated form of the lentiviral viral envelope protein, Vesicular Stomatitis Virus glycoprotein (VSV-G), that ablates binding to the natural cognate receptor. Mutant VSV-G is coupled with envelope expression of a T cell targeting scFv, leading to PACK-IT Platform cargo delivery specifically in T cells. Using an optimized anti-CD3.PACK-IT to deliver a CD19.28.z-CAR, we generated human CAR-T cells ex vivo via a rapid 4 day manufacturing process that eliminates the need for a T cell purification or activation step. When tested against Nalm-6 leukemia in NSG mice, ex vivo generated anti-CD3.PACK-IT.CD19.28.z-CAR T cells outperformed CD19.28.z-CAR T cells produced with a 4 day conventional lentiviral engineering process. The PACK-IT platform also enabled an ultra-rapid 4 hour manufacturing process, eliminating the need for T cell purification, activation, and ex-vivo CAR-T cell expansion. Notably, intravenous administration of anti-CD3.PACK-IT.CD19.28.z-CAR lentiviral particles (3.92e9 lentiviral particles/ mouse) to immunodeficient NSG mice inoculated with human T cells (5e6/mouse) generated CD19.28z CAR-T cells in vivo that mediated significant antitumor effects against Nalm-6 leukemia. These findings demonstrate the feasibility of using anti-CD3.PACK-IT, an envelope engineered lentivirus, to enable ultra-rapid ex vivo manufacturing of functionally superior CAR-T cells and to produce CAR-T cells in vivo capable of tumor control. This platform offers the potential to improve access by reducing costs and delays associated with CAR-T cell manufacturing and enhance outcomes by delivering products of greater potency. Citation Format: Kylie A. Burdsall, Peng Xu, Daniela Castro-Martinez, Louai Labanieh, Katie Ho, Quanming Shi, Bingfei Yu, Elena Sotillo, Howard Y. Chang, Crystal L. Mackall. T cell targeted lentiviral gene delivery using the PACK-IT Platform generates CAR-T cells with superior potency compared to conventional lentivirus and enables in vivo generation of CD19-CAR T cells capable of controlling leukemia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited s); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2): nr LB091.