Figure 1.
(a) Design and format of the GUCY2C T-BsAb. (A) Schematic of individual chains. For the GUCY2C T-BsAb, we applied the diabody Fc format. Heterodimerization is driven using knobs-into-holes. The Fc-knob (containing Y349C and T366W, numbered according to the EU index) and Fc-hole (containing S354C, T366S, L368A, and Y407V) are derived from human IgG126,27 and contain mutations L234A, L235A, and G237A in the lower hinge to reduce Fcγ receptor binding, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.28–30 EFN = effector function null. (b) Schematic of paired domains. (c) High-throughput anti-GUCY2C x anti-CD3 bispecific discovery workflow. To support bispecific discovery efforts for the GUCY2C T-BsAb program, automated reformatting, production and screening tools were implemented. This allows for parallel discovery and optimization activities where hundreds-to-thousands of hits can be cloned, produced and characterized in a very short time frame. Multiple formats were used to test for improvement of specific characteristics during optimization. This allows for rapid triaging and lead selection for scale-up as the final bispecific