Abstract 7239: Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector
Authors: Krotova Karina , Nenavath Gopal Naik , Packiriswamy Nandakumar , Vandergaast Rianna , McDaniel Yumeng , Moy Melissa , Larson Zachary , Schnebeck Luke , Chin Chia-Hsuan , Gromer Kyle , Peng Kah-Whye , Russell Stephen J. 
Published: March 2024

Abstract
One of the challenges in CAR-T cell therapies is the need for ex vivo processing of autologous T cells. The generation of CAR-T cells directly within the patient has therefore become an attractive alternative. Here, we pseudotyped lentiviral vectors with a VSV-G protein engineered to de-target the LDL receptor and to bind to CD3 via a displayed CD3-specific single-chain antibody (G-CD3), allowing for targeted recognition of T cells. In ex vivo experiments, we demonstrated that engineered G-CD3-LV specifically recognizes and transduces T cells in PBMC and whole blood without apparent off-target transduction of other cells. Furthermore, unlike G-WT-LV, which necessitates T cell activation for transduction, G-CD3-LV does not require pre-activation or the presence of cytokines for successful T cell transduction. The presence of CD3 antibodies on the surface of G-CD3-LV alone was sufficient to activate T cells, as evidenced by CD25 expression and the induction of T cell proliferation. For in vivo evaluation of retargeted LV efficiency NSG mice implanted with NALM6 B-lymphoma were treated with G-CD3 LV encoding αCD19-CAR/GFP (G-CD3/LV-CAR). The tumor was established by tail vein injection of NALM6/F-Luc cells, and 3 or 10 days later animals were humanized via intraperitoneal (IP) injection of human PBMC. Four hours after PBMC transfer, G-CD3/LV-CAR was delivered by IP route. Blood was drawn every week to monitor the generation of CAR-T cells, cytokine levels, and vector copy numbers. Tumor burden was monitored by bioluminescence imaging. Treatment with G-CD3/LV-CAR at both time points resulted in complete tumor eradication. Subsequently, significant levels of CAR-T cells were observed in the blood through Flow analysis, peaking at two weeks after LV injection. Importantly, mice treated on day 10 after tumor establishment reached higher levels of CAR-T cells compared to those treated on day 3 (CAR-T levels were 38±6.5% vs. 10±2.38% of hCD45+ cells, respectively). These results suggest that CAR-T cells proliferated proportionally in response to the level of tumor antigen. With the eradication of the tumor, CAR-T levels in the blood diminished. We also demonstrated that the levels of CAR-T cells correlated well with vector copy numbers. Throughout the treatment, mice did not exhibit any signs of toxicity or body weight loss. Additionally, by measuring cytokines in the blood produced by human cells, we found only a moderate increase in hIFNγ (0.852±0.681 ng/ml) at the peak of CAR-T cell levels in the blood, while the levels of hIL-2 and hTNFα were below the detection limit. In summary, these data highlight the efficiency and potential applicability of fully retargeted, serum stable LV vectors for in vivo CAR-T generation, offering promising prospects to optimize CAR-T cells therapy applications.
Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Yumeng McDaniel, Melissa Moy, Zachary Larson, Luke Schnebeck, Chia-Hsuan Chin, Kyle Gromer, Kah-Whye Peng, Stephen J. Russell. Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7239.