The Wilms Tumor 1 (WT1) protein is widely expressed across a variety of cancer types, but is rarely expressed in adult tissues. WT1 is involved in the oncogenic process and in cancer stem cells. However, because WT1 is an intracellular transcription factor, therapies that target the WT1 protein have remained elusive. Our lab has discovered and developed a T-cell receptor (TCR)-like monoclonal antibody (mAb) that specifically binds to a WT1-derived peptide in the context of the major histocompatibility complex (MHC) class I haplotype HLA-A02 expressed on cancer cell surfaces. This mAb, ESK1, is a human IgG1 that has high affinity for this epitope and has demonstrated significant anti-cancer potency in vivo. The efficacy of ESK1 has prompted its movement from the laboratory to development for clinical use. Therefore, it is important for us to understand the mechanism of this antibody in vivo in order to select patients that will benefit most from this therapy, to determine ways to amplify therapeutic efficacy, and to predict mechanisms of resistance and/or toxicities. Studies in vitro and in vivo have demonstrated the dominant role of antibody-dependent cellular cytotoxicity (ADCC) mediated by Fcγ receptors (FcγRs) in ESK1 activity and have ruled out Fc-independent mechanisms. While commercially available mAbs target tens of thousands to hundreds of thousands of epitopes on a cancer cell surface, ESK1 only targets hundreds to a few thousand epitopes. Despite these low numbers, ESK1 exhibits strikingly potent clearance of tumor burden in vivo, which led us to believe that ADCC is more powerful than previously thought. Therefore, in the first aim of this project, we will quantify theminimum number of epitopes required for ESK1 to bind in order to promote the recruitment and activation of effector cells for ADCC. Identifying this binding site number, which we predict to beless than one hundred per cell, will not only inform us on the potential impact of epitope site number in patient selection and in predicting toxicity, but will also support the further development of therapeutic mAbs against other low density cancer specific epitopes. NK cells are the main mediators of ADCC in humans, yet murine NK cells express few low-affinity activating FcγRs, making these cells virtually dispensable for successful antibody therapy in a mouse. As mAb therapies are validated in mouse models before moving to human trials, this may lead to large discrepancies in the observed efficacy, toxicities, and mechanisms of therapy identified. Therefore, this project will also develop a novel mouse model for appropriately evaluating human mAbs for the first time: an immunodeficient, human FcγR-expressing mouse. This mouse model will be used to validate the therapeutic activity of ESK1, as well as to determine which human FcγRs and immune effector cells are involved in its anti-tumor activity. This information will be essential to determine ways to best engineer the Fc region of ESK1 to promote the most effective anti-tumor response, as well as to properly select patients (e.g. based on their immune cell content post therapy or according to disease type) for increasing the chance of success in clinical trials.