Abstract 482: Development of a CD3-targeted lentiviral vector compatible with M. <i>nemestrina</i> to evaluate in vivo CAR-T gene therapy

Abstract While LV gene transfer into human lymphocytes is well-characterized, most non-human primate species carry TRIM5α restriction factors that limit the sensitivity for in vivo biodistribution and RCL safety assessments. While LV targeting T cells through the TCR complex can both provide activation signals in the process of endocytosis, envelopes with broad tropism may also cause unintended transduction of other cell types. Additionally, immune complement mediated inactivation of envelope proteins may influence biodistribution. In this study, CD3-targeted LV’s were redesigned for reactivity with both human and M. nemestrina. A modified pH-dependent fusogen was also incorporated into the LV envelope for endosomal escape following CD3-mediated endocytosis. The impact of envelope design on serum resistance, cell tropism, T cell activation, and CAR-T expansion using in vitro cultures of isolated NHP PBMC compared to human PBMC. 3rd generation SIN vectors with codon modified GAGPOL sequences were utilized to mitigate risks of vector remobilization. Methods: PBMC from M. nemestrina or human donors were used fresh or cryopreserved. SIN LV variants encoding a primatized α -CD20 CAR transgene +/- a synthetic lymphoproliferative element with EGFR cell removal epitope were manufactured in a 5-plasmid system including transmembrane or GPI anchored NHP/human cross-reactive CD3 targeting moieties and pH dependent fusogens. LV titers, specific activities, CD3 signaling, and LV endocytosis were characterized for each vector. Immunophenotype, CAR-T expansion and functionality of LV-treated cells were analyzed by flow cytometry and cytokine analysis. Results: Several α-CD3 scFv targeting elements presented on the LV surface showed weak CD3 stimulating activity but could be stabilized through incorporation of additional protein domains. One α-CD3 scFv (NCD3) was identified with strong CD3 stimulation of both human and M. nemestrina T cells in transmembrane or GPI configuration. LV constructs with human-only reactive CD3 targeting moieties showed no transduction or stimulation of M. nemestrina PBMC while effectively modifying human PBMC as expected. NCD3 targeting LV constructs were shown to effectively transduce both M. nemestrina and human T cells, generating CAR-T cells capable of expansion and response to antigen re-challenge. No transduction was observed using a pH dependent fusogen on CD3 negative cells compared to VSV-G envelope controls. Finally, LV constructs expressing the NCD3 targeting moiety and fusogen retained greater than 50% activity across multiple serum donors, demonstrating resistance to complement mediated inactivation. Conclusion: A CD3 targeting LV construct capable of activating and transducing both human and NHP PBMC in a CD3 specific fashion was successfully engineered as a product suitable for testing biodistribution, potency and RCL in an NHP model. Citation Format: Anirban Kundu, Frederic Vigant, Cody Gowan, Michael Betts, Ewa Jaruga-Killeen, Gregory Schreiber, Michelle Andraza, Gregory I. Frost. Development of a CD3-targeted lentiviral vector compatible with M. nemestrina to evaluate in vivo CAR-T gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 482.