Abstract After several decades of substantial investment in antibody drug conjugates (ADCs), the field has dramatically expanded with the FDA approval of seven new agents in the past four years including five ADCs against solid tumors. This recent success has reignited interest in the field, yet despite these approvals, fundamental questions as to how these agents work in the clinic remain. One of the most important unanswered questions is the role of the immune system in therapeutic responses to ADCs. This is a complex question given the multiple ways in which ADCs can interact with the immune system. First, ADC payloads can drive immunogenic cell death (ICD), which can stimulate immune cells via damage associated molecular patterns (DAMPs). Second, the payloads can have direct effects on immune cells following bystander uptake of payloads or Fc-gamma receptor mediated uptake. Finally, current ADCs in solid tumors use an IgG1 framework, where the antibodies themselves are capable of Fc-effector functions that may also contribute to efficacy. The relative magnitude of these effects is unknown, and until this question is answered, the most effective design of ADCs for cancer and other diseases remains unclear. For example, these results determine what protein format will be most effective and how different protein domains can be engineered to enhance efficacy. It also highlights what therapies will synergize with ADCs to enhance a durable immune response. The long-term goal of this research is to understand the fundamental properties of these complex drugs in sufficient detail to rationally combine the antibody, linker, and payload with a particular target (in a select patient population) for maximum clinical efficacy in both cancer and non-oncologic applications. The goal for this proposal is to quantify the relative contributions of direct payload cell killing, indirect immune stimulation from ICD, and Fc-gamma receptor mediated signaling and payload uptake in driving a strong immune response to ADC therapy in immunocompetent mouse models. By using antibody ‘carrier’ doses in combination with ADCs and manipulating which species can bind Fc-gamma receptors, these studies will dissect the relative contributions from each mechanism. Quantitative single-cell pharmacokinetic measurements are also used to independently measure payload efficacy in cancer and immune cell populations. The results are combined with hybrid agent-based computational models to rapidly simulate thousands of permutations for predictive insight into the role of each mechanism in animal models and scaling to the clinic. A graphical user interface can allow other scientists to make falsifiable predictions about their own ADCs to guide the development of ADC therapeutics against any target of interest. These targets/payloads include non-oncological applications, such as immunosuppressive ADCs and depletion of select cell populations in the body. The successful completion of this w...