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UNDERSTANDING THE RELATIONSHIP BETWEEN BISPECIFIC ANTIBODY DESIGN AND FUNCTION: HOW VALENCY AND SPATIAL CONFIGURATION CAN IMPROVE ANTI-TUMOR RESPONSES

T-cell bispecific antibodies (BsAb) couple cytotoxic T-lymphocytes to tumor cells, thereby inducing their destruction. Although there are over 60 classes of BsAb in development, the relative significance of parameters such as valency or spatial configuration are largely unknown. During my graduate school work, I have sought to identify design parameters that significantly enhance to BsAb potency in vitro and in vivo. The studies presented here support the roles of affinity, valency and spatial configuration in driving strong BsAb-mediated T-cell responses against antigen-expressing tumors. To begin, we found that improving tumor binding affinity 13-fold improved in vivo cytotoxicity substantially and in vitro cytotoxicity up to 5,000-fold. Next, we compared a dual bivalent IgG-[L]-scFv design to two monovalent FDA-approved designs and found bivalency as a critical parameter in improving T-cell cytotoxicity in vitro and in vivo . Following this, we dissected the IgG-[L]-scFv platform and revealed that positioning tumor and T-cell binding domains on the same side (cis-configuration) of a BsAb elicited significantly stronger anti-tumor activity, in vitro and in vivo , compared to positioning them on opposite sides (trans-configuration) and using two such cis-modules in the same BsAb improved cytotoxicity even more (up to 2,000-fold). Finally, we showed how spacing these domains using a single Ig domain (C L ) was superior to smaller (G 4 S) or larger (C H1 -C H2 -C H3 ) domains. These findings provide critical guidelines for improving BsAb function and highlight the importance of affinity, spatial configuration and dual-bivalency as development parameters.