Project Summary For decades, the treatment of cancer has relied on surgical resection, chemotherapy and/or radiotherapy. Recently, a number of immune based therapies have provided promising new approaches for cancer treatment. These include checkpoint inhibitors that block T cell suppression (eg anti-PD-1), bi-specific antibodies that cross-link T cells to cancer cells (eg Bi-specific T cell engagers – BiTE) and T cells engineered to express antigen receptors specific to cancer cells (eg Chimeric Antigen Receptor T cells or CAR T). All three approaches induce T cell mediated killing of cancer cells. However, widespread development of bi-specific antibodies and CAR T cells is limited by the small number of known cell-surface proteins that are sufficiently specific to cancer to safely allow targeting by antibodies. This is particularly true for solid cancers, where unlike hematopoietic malignancies, loss of healthy cells cannot be readily replenished by stem cell progenitors. A solution to this issue is to target cancer specific glycan antigens rather than protein antigens. Indeed, altered glycosylation is a near universal feature of cancer and represent the most abundant and widely expressed cell surface cancer antigens, while also having limited or no expression in normal tissue. However, generation of monoclonal antibodies specific to complex carbohydrates has proven to be very challenging, greatly limiting their usefulness as targets for cancer immunotherapy. Here we propose to address these issues and develop a novel class of immunotherapeutics that target an N-linked carbohydrate antigen common to the vast majority of solid and hematopoietic cancers. We have termed these molecules as Glycan- dependent T cell Recruiter (GlyTR) technology. Critically, GlyTR technology does not utilize antibodies to target carbohydrate cancer antigens. Preliminary data demonstrates that the GlyTR bi-specific protein 1) specifically bound to both human CD3 and its N-glycan target, 2) robustly activated T cells only in the presence of cancer cells, 3) induced T cell dependent killing of cancer cells with an EC50 as low as 5pM, 4) inhibited in vivo growth of established colon cancer xenografts in humanized NSG mice and 5) did not trigger production of antibodies in mice. To further expand on this approach, the following Aims are proposed. Aim 1 will characterize human and mouse reactive GlyTR bi-specific proteins targeting N-glycans. Aim 2 will examine the efficacy and safety of GlyTR proteins targeting N-glycans using humanized NSG mice. Aim 3 will examine the efficacy and safety of GlyTR proteins targeting N-glycans using mice with a normal immune system. If successful, these experiments will provide proof of principal data for an entire new class of cancer killing immunotherapeutic's capable of targeting multiple solid and hematopoietic cancers with minimal toxicity.