Delivery of cytokines for cancer immunotherapy using nanolayer-controlled trafficking of liposomal nanoparticles

An immunosuppressive or immune excluded tumor microenvironment (TME) plays a key role in limiting the response of many tumor types to immunotherapy. One attractive strategy to accomplish increased lymphocyte infiltration in tumors is the use of cytokines, which can directly impact multiple immune pathways and reprogram the TME to enable a robust immune response against cancer cells. Unfortunately, despite this obvious potential, many cytokines have been limited clinically due to toxicity concerns. Rational drug delivery strategies that can rescue the therapeutic potential of cytokines could act as an important step in our ability to carefully manipulate the anti-tumor immune response in the TME and open the door for more effective immunotherapies. The layer- by-layer (LbL) approach allows us to modify NP surface properties via electrostatically absorbed polymeric coatings that promote specific cancer cell association and influence intracellular trafficking. In the first funding period of this grant, we employed this strategy to target proinflammatory cytokines such as interleukin-12 (IL-12) directly to OC cell surfaces. This therapeutic approach elicited robust CD8+ T-cell and NK cell infiltration into the TME, and when coupled with combination checkpoint therapies (CTLA-4 and PD-1), this treatment strategy yielded complete remission in a metastatic ovarian tumor model and promoted effective immune memory. The successful improvement of IL-12 localization and retention within the TME demonstrated the potential of IL-12 as a monotherapy; however, in many cases, IL-12 alone is insufficient to promote curative responses and often a combination treatment is needed. Messenger RNA (mRNA) provides a viable alternative approach to deliver combinations of cytokines and engineered immunomodulatory therapeutics, as mRNA readily enables combinations due to facile exchange of different nucleic acids and potentially lower systemic exposure of high bolus doses due to prolonged and targeted expression. The rapid development of mRNA constructs and their facile encapsulation into lipid nanoparticles (LNPs) allows for the rapid screening of therapeutic constructs in a high throughput manner, whereas in contrast the use of an engineered protein therapy requires extensive protein production optimization and purification steps. This mRNA-based approach will thus allow us to facilitate clinical translation of cytokine therapies while introducing targeted mRNA production as a means of addressing systemic toxicity. In this renewal, we seek to maintain or improve the extended duration of cytokine bioavailability gained in current work, while facilitating combination cytokine therapies by developing layered lipid nanoparticles (LLNPs) for the targeted delivery of cytokine-encoding mRNA to OC tissues. We will develop, optimize, characterize and test LLNPs encoding for IL-12 cytokine, IL-12 fused to an anti-CD45 Ab for targeting leukocytes in the TME, and (3) IL-12 fused to a GPI anchor. We will test these treatments in a range of highly metastatic ovarian cancer models and leverage the platform to incorporate combination cytokines IL12 and IL15, as well as combinations with checkpoint inhibitors in orthotopic syngeneic animal models. 1