PROJECT SUMMARY/ABSTRACT Background: Immunotherapy has emerged as one of the most promising approaches for the treatment of cancer. Unfortunately, a large number of patients and common tumor types do not respond to existing immunotherapies. Tumor-associated macrophages (TAMs), which accumulate in many cancers and suppress anti-tumor immunity, likely contribute to this problem. Hypothesis and Objective: Based on encouraging preliminary data, we hypothesize that engagement of Dectin-2, a pattern recognition receptor that is highly expressed by TAMs in certain tumors, can reprogram TAMs into potent antigen-presenting cells capable of inducing immunity against a wide range of cancers. Our objective is to validate this hypothesis by analyzing the immunological and anti-tumor effects of natural and synthetic Dectin-2 agonists in mouse models of cancer. Specific Aims: Aim 1: Analyze the factors that regulate Dectin-2 expression and the mechanisms by which Dectin-2 agonists reprogram TAMs and induce anti-tumor immunity. Aim 2: Assess the anti-tumor effects of natural Dectin-2 agonists, alone and in combination with other agents, on a range of aggressive cancers. Aim 3: Synthesize glycopeptide agonists of Dectin-2 and assess their anti-tumor activity alone and in combination with other agents. Aim 4: Construct tumor-targeted antibody-glycopeptide conjugates and evaluate their functional and therapeutic effects. Study Design and Methods: Since TAMs in some tumors express little or no Dectin-2, we will analyze the effects of natural Dectin-2 agonists on TAMs in the presence of GM-CSF, which induces Dectin-2 expression. The anti-tumor effects of the agonists will be studied in mouse models of pancreas, breast, colon, lung, and skin cancer with a spectrum of Dectin-2 expression, both as monotherapies and in combination with GM-CSF and other agents shown to enhance the efficacy of Dectin-2 agonists in our mouse models. Mass cytometry and recently developed informatics tools will be utilized to analyze the effects of Dectin-2 ligands on the anti- tumor immune response in multiple tissues. To generate more effective and clinically applicable therapies, a novel synthetic approach will be used to produce compositionally defined Dectin-2 ligands that are optimized for TAM activation. The most efficacious of these synthetic Dectin-2 ligands will be conjugated to tumor- targeted antibodies for purposes of enhanced tumor delivery and TAM-mediated tumor cell killing, and their safety and efficacy assessed. Expected Results and Impact: We expect these experiments to demonstrate that engaging Dectin-2 on TAMs induces immunity against a wide range of tumors, including tumors resistant to checkpoint blockade, and that Dectin-2 agonists used alone or in combination with other anti-tumor agents can induce durable tumor regression. The most promising of these agonists will be candidates for clinical development.