Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv

Single chain antibody fragments (scFvs) are favored in diagnostic and therapeutic fields thanks to their small size and the availability of various engineering approaches. Linker between variable heavy (V(H)) and light (V(L)) chains of scFv covalently links these domains and it can affect scFv’s bio-physical/chemical properties and in vivo activity. Thus, scFv linker design is important for a successful scFv construction, and flexible linkers are preferred for a proper pairing of V(H)–V(L). The flexibility of the linker is determined by length and sequence content and glycine-serine (GS) linkers are commonly preferred for scFvs based on their highly flexible profiles. Despite the advantage of this provided flexibility, GS linkers carry repeated sequences which can cause problems for PCR-based engineering approaches and immunogenicity. Here, two different linkers, a repetitive GS linker and an alternative non-repetitive linker with similar flexibility but lower immunogenicity are employed to generate anti-Vascular Endothelial Growth Factor scFvs derived from bevacizumab. Our findings highlight a better in vitro profile of the non-repetitive linker such as a higher monomer ratio, higher thermal stability while there was no significant difference in in vivo efficacy in a zebrafish embryonic angiogenesis model. This is the first study to compare in vivo efficacy of scFvs with different linkers in a zebrafish model.