Project Summary Cancer cells undergo changes that the immune system can recognize, and this can be leveraged for therapy. Direct-targeting antibodies that recognize such changes can operate via multiple mechanisms, including blocking oncogenic signaling, serving as Trojan horses carrying toxic compounds, coordinating innate immune engagement for cytotoxicity through cellular and non-cellular means, and promoting antigen uptake to jumpstart adaptive immunity that can provide durable protection. We recently generated a dozen high-affinity fully human monoclonal antibodies (mAbs) that recognize a tumor-specific hypoglycosylated form of Mucin-1 (MUC1), a form that is present and overexpressed in >80% of all cancers. Our anti-MUC1 mAbs are different and unique from other humanized murine anti-MUC1 mAbs, having been isolated from a healthy human individual receiving the MUC1-100mer peptide vaccine. They have undergone selection and affinity maturation in a healthy immune environment. Importantly, in all vaccinated individuals that generated an antibody response to MUC1, there have been no adverse events in 8 years, increasing the likelihood that these agents will be safe. Our new preliminary data show these mAbs can mediate immune effector functions in vitro. Our objective is to provide preclinical data on the safety, efficacy and the mechanism of action of our fully-human MUC1 antibodies. We hypothesize they can safely cause tumor regression in vivo and will reveal fundamental aspects of antibody-antigen interactions that will inform future therapeutic antibody design. In the first aim, we plan to test the safety, efficacy and mechanism(s) of action of our anti-MUC1 antibodies against huMUC1- transgenic murine tumors in immunocompetent mice expressing both the human MUC1 gene and human antibody receptor genes, and against MUC1+ human tumor xenografts in immunodeficient huMUC1-Tg mice. We will monitor weight, food intake, temperature, and compare histological tumor sections to those of other tissues to assess safety and cross-reactivity with normal tissues. We will compare our MUC1 antibodies against well-characterized FDA-approved mAbs such as rituximab (anti-CD20) and trastuzumab (anti-Her2) for their ability to control tumors co-expressing huMUC1 together with huCD20 or huHer2. We will assess whether the MUC1 antibodies mediate effector functions through innate or adaptive immune mechanisms. In the second aim, we will determine the requirements of MUC1/ anti-MUC1 interactions that mediate immune effector functions. Using our panel of 12 anti-huMUC1 mAbs and four MUC1 constructs, we will evaluate first in vitro the requirements of antibody affinity, level of antigen expression, epitope proximity to the cell membrane, and membrane location on antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody internalization. Key findings from in vitro studies will be validated in vivo. Together these studies will increase our understanding of antibody-antigen interactions and may identify one or more candidates to develop for testing in a future clinical trial.