Abstract 3294: In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion
Authors: Vigant Frederic , Kundu Ani , Zhang Dongming , Zhang Wei , Jaruga-Killeen Ewa , Andraza Michelle , Schreiber Gregory , Kerner Alissa , Zhang Junyi , Henkelman John , Soares Renata , Yarlagadda Ramya , Frost Gregory I. , Kerkar Sid P. 
Published: June 2022

Abstract
Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies.
Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers.
Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (&gt;50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood.
Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies.
Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3294.