PROJECT SUMMARY/ABSTRACT The overarching objective of TR&D 3 is to design and develop a novel category of immunomodulatory proteins, both as tools to study the immune response and as targeted disease therapeutics. Cytokines control all aspects of immune homeostasis and are thus of great interest as potential interventions to either stimulate immune activity for treatment of cancer and infectious diseases or to suppress activity response for treatment of autoimmune disorders and to promote transplant tolerance. Unfortunately, natural cytokines have several intrinsic properties that limit their therapeutic application, including: (1) Pleiotropy, which hinders specificity, safety, and efficacy; (2) Instability, which leads to challenges in formulation and manufacturability; and (3) Short in vivo half-life, which limits durability of disease control. To overcome inherent limitations of natural cytokines, emerging molecular engineering technologies aim to modify native cytokines in order to improve their suitability as drugs. Our team has established a unique approach to empower cytokines as therapeutics: fusion of natural cytokines to anti-cytokine antibodies, creating intramolecularly assembled immunocytokines (ICs) that selectively direct the activities of cytokines to particular immune cell subsets while also extending circulation half-life. Using this approach, we generated a complementary pair of interleukin-2 (IL-2)-based ICs that mount either immune effector cell-biased (immunostimulatory) or regulatory T cell (Treg)-biased (immunosuppressive) responses. Our promising preliminary work in developing ICs illustrates the potential for designing off-the-shelf molecular therapeutics that selectively manipulate immune activity. Here, we aim to advance clinical translation of our technology by evaluating IL-2 ICs in animal models of cancer, inflammatory bowel disease, and type 1 diabetes. In parallel, we will extend our versatile cytokine/antibody fusion protein concept to the IL-7 system to promote T cell survival. We will also harness our team’s recent discovery of a particularly suppressive subset of tumor-infiltrating Tregs to specifically target an antagonistic IC to the tumor microenvironment that will promote anti-cancer immunity. Our interdisciplinary team of protein engineers and immunologists is uniquely equipped to carry out the proposal aims, which promise to help realize the potential of cytokine therapeutics. The IC deliverables resulting from this project will be integrated with other platforms in the Center, installing these targeted proteins on artificial antigen- presenting cells from TR&D 1 to promote antigen-specific immune activation, and coating ICs on nanoimmunomaterials in TR&D 2 to selectively direct gene delivery. Moreover, ICs will be iteratively developed through Collaborative Projects 1, 8, 9, and 10 and deployed in Service Projects 8, 10, and 12. Collectively, the proposed work will demonstrate the translatability, modul...