Abstract 5372: CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells

Introduction: Adoptive cell transfer (ACT) of T cells has emerged in recent years as a powerful immunotherapy against cancer. Currently, T cells are harvested from the patient or donor and are genetically modified ex vivo to enhance their cancer-fighting capabilities. However, this process is costly and more complex than administering off-the-shelf therapies such as small molecule drugs or monoclonal antibodies. The development of methods to transfect T cells in vivo remains an important endeavor for immunotherapies. Ionizable lipid nanoparticles (LNPs) have recently played a key role as carrier vehicles for mRNA in the Pfizer-BioNTech and Moderna mRNA vaccines. While ionizable LNPs delivering mRNA have now been FDA-approved for non-targeted immune cell delivery, their potential for targeting specific immune cell subtypes has yet to be fully realized. Delivering therapeutic genes that could reprogram T cells in vivo to recognize disease-relevant antigens would be of great clinical significance in reducing the lead time and cost of current ACT therapies. Methods: Ionizable LNPs were synthesized to encapsulate mCherry or Firefly Luciferase (FLuc) reporter gene mRNA with a fluorescent Cy7-labeled lipid on the LNP surface. CD3 monoclonal antibody, F(ab’)2 CD3e fragment, or CD8 diabody was conjugated to the LNP surface. In vitro, the aCD3- or aCD8-LNPs were incubated with Jurkat or TK-1 cell lines, respectively. LNP uptake was imaged using fluorescent confocal microscopy, and mCherry expression was quantified via flow cytometry. For in vivo delivery, the ionizable LNPs were injected i.v., and reporter gene expression and cell activation were analyzed via flow cytometry and bioluminescence imaging. Results: Confocal microscopy of ionizable LNP uptake in vitro highlighted differences in the internalization of the aCD3- and aCD8-LNPs in their respective cell lines, and transfection was observed with each targeting ligand. In murine models, aCD3-LNPs led to FLuc expression in the spleen and liver with increased accumulation in lymph nodes. aCD3-LNPs also transfected mCherry mRNA and activated splenic and circulating T cells. Cytokine concentrations were elevated in blood 5 and 24 h after aCD3-LNP injection. The impact of LNP lipid composition on transfection will be detailed in the presentation. Conclusions: We successfully packaged mRNAs of mCherry and FLuc reporter genes within ionizable LNPs and targeted them to T cells via the CD3 or CD8 receptor. The ionizable LNPs were capable of transfecting T cells in vitro and in vivo while causing transient activation, depletion, migration, cytokine release, and phenotypic shifts. Working towards the goal of receptor-mediated T cell targeting in vivo, we elucidate the circulation time, transfection efficiency, and activating potential of ionizable LNPs targeted to T cells in the spleen and blood via two potential targets, the CD3 and CD8 receptors. Citation Format: Elise R. Robinson, Aris J. Kare, Azadeh Kheirolomoom, Mohammed Inayathullah, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Jai W. Seo, Felix B. Salazar, Ramasamy Paulmurugan, Anna M. Wu, Katherine W. Ferrara. CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells [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 5372.