Poster O11 - CD45 exclusion-driven drug-free cellular backpacking to improve adoptive T-cell transfer therapy for solid tumors.

Introduction: Adoptive cell transfer therapies utilizing cytotoxic T cells have been remarkably successful in liquid tumors. However, their clinical efficacy in solid tumors has been very modest due to the rapid loss of their functional activity post-infusion. Current strategies that safely reinforce T cell functionality exhibit limited success because of the inability to sustain stimulatory effects on T cells while navigating through the tumor microenvironment. Here, we address these challenges with a purely material-based cellular backpacking approach by hitchhiking CD45-excluding drug-free micropatches onto the surface of T cells. We develop a mathematical model based on kinetic segregation theory and apply findings from it to design our CD45-excluding cellular backpacks. Such cellular backpacks, in contrast to artificial antigen-presenting cell (aAPC) inspired Dynabeads®, offer an effective way to provide supporting stimulation post-infusion to cytotoxic T cells without affecting their migration. After adoptive transfer in mice bearing aggressive B16F10 melanoma, backpack-bound T cells migrate similarly but persist in spleen and tumor tissues longer than cells without backpacks. They elicit superior anti-tumor benefits, resulting in ~53% longer median survival. Altogether, the material-based CD45-exclusion-driven cellular backpacking presents a simple, drug-free avenue to improve solid tumor control with adoptive T cell transfer.

Materials and

Methods: We first generated a mathematical model to understand T-cell stimulation dynamics based on phosphatase-kinase polarization. This model provided a basis for selecting micropatch-shaped particles as a potent CD45-excluding cellular backpack. Hence, we fabricated the micropatches comprised of PLGA polymer using soft photolithography and microcontact printing techniques. These polymeric micropatches were functionalized with antibodies to mediate firm binding with T cells. We performed flow cytometry to quantify the percentage of cells bound with backpacks and screened for antibodies that gave optimal binding efficiency. We then stained for CD45 molecules on backpack-bound T cells and imaged using confocal microscopy to identify CD45 distribution across the T cell surface. We also identified stimulatory effects from backpack binding by performing intracellular calcium staining in T cells. Finally, we identified migration hallmarks of backpack-bound cells compared to unmodified cells using transwell and time-lapse microscopy. After the preliminary in vitro findings supporting our hypothesis, we intravenously injected backpack-bound antigen-specific T cells in the B16F10 subcutaneous melanoma-bearing mouse model and assessed their biodistribution compared to unmodified cells. We further performed a therapeutic study in the same mouse model with backpack-bound T cells and monitored the tumor size and animal survival over time. The immunophenotyping study mapping changes in the tumor microenvironment and lymphoid system after the adoptive transfer of backpack-bound T cells in this tumor model is underway.

Results, Conclusions, and Discussions: Our mathematical model indicated that single micron-sized contact is effective in T cell stimulation with CD45 exclusion compared to 1000 discrete nano-sized contacts. It further informed that micropatch capable of forming micron-sized contact with T cells are the suitable choice of backpacks to stimulate T cells even with partial CD45 exclusion. We fabricated micropatches at scale and bound them firmly to the T cell surface with antibody functionalization. Confocal imaging of CD45-stained cellular backpack samples revealed micron-sized CD45 exclusion at the T-cell backpack contacts. In accordance with the mathematical model, we observed a two-fold increase in intracellular calcium ions after backpack binding, while nanoparticle binding showed no change. Interestingly, the backpack binding preserved migration hallmarks of T cells, while aAPC-based particles significantly inhibited T cell migration. After adoptive transfer in tumor-bearing mice, backpacks attached to transferred T cells migrated into tumor and spleen tissues without affecting cellular biodistribution and supported cells to persist longer than cells without backpacks. The adoptive transfer of backpack-bound T cells was well-tolerated and significantly delayed subcutaneous B16F10 melanoma tumor growth to prolong median survival by ~53%. To our knowledge, this is the first instance where a purely material-based strategy without any active biological modality improves adoptive T cell transfer therapy for solid tumors. This strategy advances previous work of cellular backpacking for T cells of tethering drug-releasing nanoparticles in a new direction with a simple drug-free CD45 exclusion-driven approach. Altogether, the purely material-based approach described here presents a simple platform to sustain stimulatory support post-adoptive transfer of T cells for improving solid tumor management.

Acknowledgements (Optional): We acknowledge the support from the Wyss Institute and John A. Paulson School of Engineering & Applied Sciences at Harvard University. Suyog Shaha thanks the Simmons Award for funding the imaging at the Harvard Center for Biological Imaging (HCBI) facility.