Improved Antibodies against Carbohydrate Antigens for Cancer Theranostics

Cancer is a leading cause of death worldwide and recently immunotherapy emerged as an important alternative to traditional treatment regimes. Cell surface glycosylation is a common feature of all living cells; however, it is commonly modified on cancer cells. These tumor-associated carbohydrate antigens (TACA) are not normally present on healthy cells and provide important therapeutic targets, some traversing several cancer types. Many of these TACA involve aberrant expression of Sialic acids. Sialic acids (Sia) are 9-carbon backbone acidic sugars found at the tips of various glycoproteins and glycolipids. The two common Sias in most mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated counterpart N-glycolylneuraminic acid (Neu5Gc), which differ by one oxygen atom. Humans are genetically deficient in Neu5Gc synthesis, yet it metabolically incorporates into human cells from dietary sources, especially accumulating on carcinomas. This lead to replacement of Neu5Ac with Neu5Gc at the tips of cell surface carbohydrate chains leading to expression of neo-tumor associated carbohydrate antigens. Antibodies directed towards TACA hold great potential for targeted cancer therapeutics. However, despite availability of monoclonal antibodies to various TACA, some even mediating tumor killing, their therapeutic potential remains extremely limited, largely hampered by their typical low affinity. In this work, we aim to create anti-carbohydrate antibodies that possess high affinity and specificity. We therefore established a scFv expression system using the Yeast surface display method and cloned several known anti-TACA scFv into this system. Anti-SLe^a antibody was then chosen for further alteration by affinity maturation error-prone PCR. We further scanned and isolated five high affinity mutants that can be now examined for their ability to be used as cancer theranostics tools.