Thomas Wang

Versatile technologies that can deliver both RNA and protein payloads could streamline development, simplify manufacturing and expand the capabilities of combination therapies. Here we demonstrate an efficient approach to forming ca. 100 nm polymer vesicles (polymersomes) capable of rapid self-assembly without organic solvents, avoiding the need for post-encapsulation purification. Block copolymers are designed with a lower critical solution temperature that renders them soluble in aqueous medium under standard refrigeration, but they spontaneously assemble at room temperature into large batches of nanoparticles with predictable size and morphology. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity and in vivo targeting, while both siRNA and proteins can be encapsulated during warming at >75% loading efficiencies. Formulations can be stored in a dry state for greater hydrolytic stability under standard refrigeration and can be diluted directly from the vial, bypassing the need for purification required for high scalability. We use our system for in vivo delivery in protein subunit vaccination, immune tolerance induction and siRNA interference therapy in cancer.

Current asthma treatments manage disease symptoms but fail to address the underlying cause of allergic disease. Allergen immunotherapy holds the promise for durable disease control by establishing allergen-specific tolerance through repeated introduction of native allergens; however, its efficacy can be limited by long interventions, reactions upon administration, and poor patient compliance. Here, we developed a rapid, safe, and effective liver-targeted allergen immunotherapy (LIT) to provide long-term disease control. We developed LIT tolerogens from native respiratory allergens, which induced antigen-specific regulatory T (T _reg ) cells in vivo with only two interventions. Synthetic mannosylation of native allergens prevented antibody-mediated recognition and subsequent life-threatening anaphylaxis upon administration. Protein engineering prevented sensitization events that occurred because of the proteolytic activity of native respiratory allergens, which limit the effectiveness of allergen immunotherapy. In preclinical models of allergic asthma, LIT ameliorated clinical, pathological, and serological features, and protection was dependent on antigen-specific T _reg cells. In sensitized mice, LIT provided a year-long control of allergic asthma symptoms in the absence of additional intervention. Last, LIT induced antigen-specific T _reg cells against Der p 1, a major protein in the clinically relevant house dust mite (HDM) respiratory allergen. In mice with established HDM allergy, LIT was well tolerated and provided allergic symptom relief. Together, our data provide a proof of concept that LIT with synthetically mannosylated tolerogens provides a rapid, safe, and effective approach to allergen immunotherapy and holds promise for durable control of allergic asthma.

Although great advances in siRNA and mRNA delivery have been made (e.g. with lipid nanoparticles), there remains a pressing need for a versatile platform that can deliver both RNA and protein payloads with convenient processing and storage. Here, we demonstrate a highly efficient approach to forming ca. 100 nm vesicular polymer nanoparticles (polymersomes) that requires no organic solvents by utilizing thermoresponsive block copolymers capable of self-assembling in aqueous medium as the solution is raised to room temperature. This is achieved using block copolymers with a domain that has a lower critical solution temperature (LCST) such that they are soluble in aqueous medium under standard refrigeration (4-7°C) temperature but assemble upon warming to room temperature, resulting in large batches of nanoparticles with predictable size and morphology as dictated by polymer structure. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity as well as in-vivo targeting, respectively. Both siRNA and protein payloads can be incorporated during warming at higher than 75% loading efficiencies. Payload-polymer suspensions can also be lyophilized into a dry state, allowing for greater hydrolytic stability under mild (4-7°C) refrigeration conditions. This greatly reduces processing and storage requirements, as the powder can simply be reconstituted in cold aqueous medium, then used as described. Here, we selected biomedical applications that did not require removal of unencapsulated payload, bypassing further purification processes and demonstrating a highly scalable approach for formulating nanoparticle-based treatments. Finally, we demonstrate that our system is capable of in-vivo delivery in the contexts of protein subunit vaccination, prophylactic immune tolerance induction, and siRNA interference therapy in cancer. We believe this polymersome platform addresses several challenges to broad translation of nanoparticle delivery of biologics to the clinic, such as scalability, loading efficiency, quality control, and dry storage.

Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)–resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.