Multivalent nanobodies with rationally optimized linker and valency for intravitreal VEGF neutralization

Intravitreal VEGF neutralization is the cornerstone of current wet AMD therapy. To achieve longer duration of action, anti-VEGF proteins with high affinity and low molecule weight are highly desirable. Besides binding affinity maturation, multivalency antibody connected with optimal linkers could also achieve high binding affinity. In this study, we systematically investigated the effects of linkers on the structure and binding affinity of multivalent antibodies, and further elucidated the mechanism of binding affinity enhancement through structure prediction by AlphaFold 2 and molecular dynamics (MD) simulation. We observed that flexible linkers improved the binding affinity of bivalent nanobodies independent of linker length, while an optimal length was required when rigid linkers were used. With linker analysis and optimization, we successfully constructed tetravalent nanobodies for efficient VEGF neutralization, with an ∼ 134 times enhancement of binding affinity and ∼ 6 times increase of VEGF-VEGFR pathway inhibition in vitro, when compared to the monovalent nanobody.