Introduction: Direct in vivo delivery of lentiviral vectors (LV) to generate CD19 CAR+ cells without the need for ex vivo preparation represents a promising approach to transform autologous CAR therapy into an off-the-shelf treatment. In these studies, direct administration of a new LV encoding a CD19 CAR into humanized NOD SCID gamma (NSG) mice expressing human IL-3, GM-CSF, and SCF (NSG-SGM3) resulted in a dose-dependent elimination of B cells in the peripheral blood, peritoneal fluid, bone marrow, and tissue of treated mice. Methods: CD3-directed LV encoding a CD19 CAR with a novel synthetic driver element was manufactured utilizing a 25L clinical scale suspension-based process. NSG-SGM3 mice transplanted with human CD34+ cells from cord blood were injected with LV doses (1E6 IU, 1E7 IU, or 5E7 IU) intraperitoneally (IP) or 1E7 IU intravenously (IV). Quantification of CD19 CAR+ cells and CD20+ B cells in peripheral blood, peritoneal fluid, and bone marrow was assessed by flow cytometry. Additionally, immunohistochemical analysis was performed to evaluate the tissue-resident human B cells and for any other histopathological observations following test article administration. Results: All CD34+ humanized NSG-SGM3 mice were confirmed to exhibit efficient human hematopoietic engraftment by flow cytometry prior to test article administration (peripheral blood hCD45: 68.9% ± 4.93%; hCD19+ B cells: 53.7% ± 5.11%). Direct LV administration at the 1E7 IU and 5E7 IU doses demonstrated a dose-dependent reduction in circulating human B cells compared to the control and 1E6 IU dose (p < 0.05). The synthetic driver elements co-expressed with the CAR led to the formation of unique CD3+ CD8+ CD56+ T and NK-like (TaNK) CD19 CAR+ cells in circulation. The 1E7 IU IP dose demonstrated ablation of B cells (total cells/uL) in peripheral blood (control: 9.67 ± 3.72 vs. LV treated: 0.157 ± 0.117), intraperitoneal fluid (control: 0.322 ± 0.244 vs. LV treated: 0.038 ± 0.029), bone marrow (control: 9.16± 1.83 vs. LV treated: 0.734 ± 0.864), and splenic tissue. Both IP and IV routes of administration showed significant B cell depletion at the 1E7 IU dose. However, complete B cell elimination in splenic tissue was only observed at the 5E7 IU dose. Non-treated CD34+ humanized NSG-SGM3 mice exhibited hepatic portal inflammation and moderate graft-versus-host disease (GVHD) in the colon. Interestingly, mice treated with LV encoding CD19 CAR exhibited decreased inflammatory pathology, suggesting potential B cell involvement in the inflammatory response in this model. Conclusion: In this study, direct in vivo delivery of LV encoding CD19 CAR resulted in the generation of functionally active CD19 CAR TaNK cells capable of eliminating target B cells in peripheral blood, peritoneal fluid, bone marrow, and tissue. Citation Format: Frederic Vigant, Ani Kundu, Ramya Yarlagadda, Cody Gowan, Michael Betts, Jonathan Kato, Renata Soares, Alan Ponce, Lintao Liu, Junyi Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Suraj Kachgal, Gregory Schreiber, Wei Zhang, Gregory Wade, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice [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 2918.

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 (>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.