Abstract A024: Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo
Authors: Krotova Karina , Nenavath Gopal Naik , Packiriswamy Nandakumar , Vandergaast Rianna , Ziegler Christopher , Moy Melissa , Narjari Riya , Schnebeck Luke , Larson Zachary , Gromer Kyle , Chin Chia-Hsuan , Reiter Samantha , Alía Miguel Muñoz , Peng Kah-Whye , Russell Stephen 
Published: December 2023

Abstract
In recent years, there has been a significant increase in interest regarding the generation of CAR-T cells directly within patients using a simple vector injection containing the CAR transgene. This promising technology has the potential to streamline the procedure and decrease its overall cost. To ensure effective in vivo delivery, these vectors must demonstrate resistance to neutralization by human serum, the ability to specifically target and transduce desired cells, and must support their subsequent expansion. Lentiviral vectors (LV) pseudotyped with VSV G-protein (G-WT) have a broad tropism, targeting various cell types through their affinity for the ubiquitously expressed LDL receptor (LDLR). G-WT LV have proven effective in generating therapeutic CAR-T cells ex vivo, however, requiring proper purification and activation of T cells beforehand. To address challenges related to their in vivo delivery, we utilized rational engineering to redesign the G protein, resulting in a modified version termed G-CD3. This redesigned G protein incorporates a CD3-specific single-chain antibody, allowing for targeted recognition of T cells while avoiding LDLR recognition. Upon testing the newly designed G-CD3 LV, we demonstrated its specificity in targeting and infecting both un-activated and activated T cells in peripheral blood monocytic cell cultures. In addition, the G-CD3 LV powerfully activates T cells, evident from their increased CD25 expression, and the infection is not hindered by the presence of human serum (25%). In further experiments, we successfully demonstrated that G-CD3-LV can infect T cells in fresh whole blood. In vivo evaluation of G-CD3-LV efficiency was performed in humanized NSG mice with more than 40 % of human CD45+ cells by intravenous (IV) or intraperitoneal (IP) LV delivery using LV encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3-LV led to in vivo CAR T cell generation. However, CAR-T blood levels were higher after IP administration, reaching 13-17% of total circulating human CD45+ cells at the peak on day 21. Anti CD19-CAR-T cell generation coincided with the disappearance of endogenous CD19+ B cells, demonstrating functional in vivo CAR-T generation. Tissue analysis at day 39 after LV injection showed the presence of anti-CD19 CAR-T cells accompanied by the absence or dramatic reduction in CD19+ B cells in spleen, bone marrow and liver. In contrast, no CAR-T cell generation was detected at any time point in the mice injected with G-WT-LV, regardless of whether the virus was administered IV or IP. Taken together these data highlight the potential applicability of newly designed retargeted LV for in vivo gene delivery, offering promising prospects for future human gene therapy applications.
Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Christopher Ziegler, Melissa Moy, Riya Narjari, Luke Schnebeck, Zachary Larson, Kyle Gromer, Chia-Hsuan Chin, Samantha Reiter, Miguel Muñoz Alía, Kah-Whye Peng, Stephen Russell. Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A024.