This project will establish a protein engineering platform for evolving monoclonal antibody binding affinity and specificity to solve the notorious challenge of developing clinical mAbs against tumor associated carbohydrate antigens (TACAs). Our central hypothesis is that the merger of Qβ carrier protein-elicited mAb discovery and rationally-guided directed evolution will outpace existing methodologies for discovering powerful antibodies against challenging TACA glycosylated biomarkers. TACAs are unique biomarkers to multiple tumor types, yet they have been underutilized for molecular imaging and diagnostics because of challenges in developing selective, potent binders. Distinct glycosylation patterns of tumor cell surfaces are hallmark features that arise during oncogenesis through changes in expression levels of glyco-processing enzymes. Problematically, these aberrant tumorigenic features are usually undetected by the immune system and rarely identified as non-self. Even when recognized as an antigen, weak binding against monovalent glycans leads to an insufficient immune response. To address this need, we will apply our directed evolution methodology to develop lead candidate mAbs against TACAs selective to cancer with in vivo binding of KD<10nM and specificity >100-fold binding above control cells. This will be accomplished by first generating a diverse panel of TACA-specific antibodies via immunization of transgenic mice with multivalent Qβ vaccines. Dominant antibodies will be isolated and characterized for paratope diversity and the ability to selectively bind the glyco-targets. Next, we use rationally- guided directed evolution to achieve mAb binding affinity and specificity. Multiple TACA-specific mAbs obtained through immunization will undergo high-throughput yeast display directed evolution with site-wise diversification based on structural, stabilizing, and phylogenetic factors to overcome the routinely low affinity of anti-carbohydrate binders. Specificity and affinity will be evaluated against multiple human tumor cell lines. This project will: 1) establish a platform that drastically reduces initial discovery time for translatable molecular imaging and diagnostic tools against carbohydrate antigens; 2) significantly advance understanding of tumorigenic cell glycosylation patterns; and 3) mark a major step towards improving sensitivity and specificity of biomarker-based diagnosis of cancers including ovarian, breast, and pancreatic cancers.