PROJECT SUMMARY Altered cell-surface glycosylation is recognized as a hallmark of cancer. Two frequently observed cancer- associated glyco-phenotypes are hypersialylation and mucin overexpression. These cancer glycosylation patterns strongly correlate with disease aggressiveness and poor patient outcomes, but until recently their functional significance was unclear. In previous funding periods, we discovered that hypersialylation allows cancer cells to evade immune surveillance through engagement of immune inhibitory Siglec receptors. We speculate that poor responses of cancer patients to current immune therapies, including monoclonal antibodies, T cell checkpoint receptor blockade, bispecific T cell engagers and targeted cell therapies, are due to immune suppression via the Siglec/sialoglycan axis. Accordingly, we developed a new class of immune therapies comprising antibody- sialidase conjugates that degrade Siglec ligands on targeted cancer cells. In collaborative work, we found that mucin glycoproteins promote focal adhesion formation and enhance cancer cell survival and proliferation in the metastatic niche by virtue of their bulk physical properties. We also discovered that certain cancer-associated mucin glycoforms engage Siglec receptors thereby mediating immune suppression. These dual functions of cancer mucins make them attractive targets for cancer therapy. Accordingly, we made targeted mucin degraders comprising mucin-specific proteases fused to cancer antigen- binding nanobodies. Nanobody-mucinase fusions had anti-metastatic and immune potentiating activities in mouse cancer models. In the next funding period, we will build on these discoveries with three Specific Aims. In Aim 1, we will develop targeted mucin degraders based on engineered human proteases, an important step toward clinical translation. In Aim 2, we will explore a new approach to precision targeting of mucin degraders to shed and secreted mucins that are immune suppressive in circulation. Finally, in Aim 3 we will develop a new cancer immune therapy modality comprising antibody-lectin chimeras (AbLecs). These bispecific molecules simultaneously bind cancer antigens, block immune suppressive glycan ligands, and mediate immune effector functions via their Fc domains. Overall, the proposed work will set the stage for preclinical development of two new therapeutic strategies targeting the cancer glycocalyx.