In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift

Kheirolomoom, Azadeh; Kare, Aris J.; Ingham, Elizabeth S.; Paulmurugan, Ramasamy; Robinson, Elise R.; Baikoghli, Mo; Inayathullah, Mohammed; Seo, Jai W.; Wang, James; Fite, Brett Z.; Wu, Bo; Tumbale, Spencer K.; Raie, Marina N.; Cheng, R. Holland; Nichols

Abstract

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nano-particles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25(+), OX40(+), and CD69(+) T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a(+) T cells from the white-pulp to red-pulp, and differentiation from naive to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Keywords: T-cell transfection; T-cell activation; mRNA; Reporter gene; Lipid nanoparticle

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Transfection

In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles represents a cutting-edge approach in immunotherapy, aiming to enhance the body's natural ability to fight diseases like cancer and viral infections. T-cells are a crucial component of the immune system, responsible for identifying and attacking infected or malignant cells. However, in many cases, T-cells need to be genetically modified to improve their efficacy against specific targets, a process known as transfection. Traditional methods of T-cell transfection often involve ex vivo manipulation, where T-cells are extracted from the body, genetically modified in the lab, and then reintroduced into the patient. This process is complex, costly, and time-consuming. In situ transfection offers a promising alternative by delivering genetic material directly into T-cells within the body. Anti-CD3-conjugated lipid nanoparticles are at the heart of this innovation. These nanoparticles are designed to specifically target T-cells by binding to the CD3 receptor, a molecule present on the surface of T-cells. The lipid nanoparticles encapsulate the desired genetic material, protecting it from degradation and facilitating its entry into the T-cells upon binding to CD3. This targeted delivery system ensures that the genetic material reaches the T-cells efficiently, leading to effective transfection.