A Tetravalent PD-L1/VEGF Bispecific Antibody Blocks Tumor Growth In Vivo

The VEGF/VEGFR and PD-L1/PD-1 pathways are often activated in tumors to promote angiogenesis and suppress cytotoxic T cells, respectively. We used phage-displayed libraries of human antigen-binding fragments (Fabs) with a common light chain to develop anti-PD-L1 and anti-VEGF Fabs that inhibit their respective pathways with high potency and efficacy. We then used these Fabs to construct a bispecific tetravalent antibody with anti-PD-L1 and anti-VEGF Fabs fused to the N- and C-termini of an Fc domain, respectively. This Fab-Fc-Fab format was produced and purified at high yields with standard methods used for IgG production, and proved to be highly stable. Importantly, the tetravalent antibody was more effective at activating T cell-mediated killing and inhibiting tumor growth than the clinically approved anti-PD-L1 antibody Atezolizumab, both in vitro and in vivo . Moreover, the C-terminal anti-VEGF Fabs acted as a trap and made the tetravalent antibody more potent than Bevacizumab for inhibition of VEGF-mediated signalling in vitro. A structure of the anti-PD-L1 Fab in complex with the PD-L1 IgV domain revealed that the antibody inhibited binding to PD-1 through direct steric hinderance. Collectively, these results establish the Fab-Fc-Fab with a common light chain as a viable format for producing stable bispecific tetravalent antibodies, and the anti-PD-L1/VEGF tetravalent antibody as a promising lead candidate for the development of a single agent that can inhibit both pathways for cancer treatment.