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

Abstract

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 (VH) and light (VL) 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 VH–VL. 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.

Semiconductive and Biocompatible Nanofibrils from the Self-Assembly of Amyloid π-Conjugated Peptides

“…We have previously reported that the I 10 sequence (SSNNFGAILSS) has a very robust capacity to self-assemble into polymorphic twisted unbranched fibrils characterized by a cross-β-sheet quaternary structure, which tolerates functionalization of 18 to 22-mer peptide antigens. , A flexible tetrapeptide linker (GSGS) was added at the N-terminus of the amyloid core followed by two charged lysine (KK) residues. The KK motif was added to promote solubility in aqueous solution, whereas the GSGS linker, which is commonly used in protein engineering, was incorporated in order that the PDI does not interfere with the self-assembly process driven by the amyloid segment. The PDI unit (PDI-[L] 2 ) previously functionalized by leucine at both imide positions (Figure S1) was attached to the peptidyl-resin by means of a short aliphatic 6-amino hexanoic acid (Ahx) linker to provide low steric hindrance between the PDI core and the self-recognition sequence.…”

Natural Killer Cell Engagers (NKCEs): a new frontier in cancer immunotherapy

“…Several linker options are available to bridge the V regions, such as (G 4 S) n /(G 3 S) n linker ( 185 ), 218s linker (GSTSGSGKPGSGEGSTKG) ( 186 ), or HMA linker (PSGQAGAAASESLFVSNHAY) ( 49 ) ( Table 3 ). The (G 4 S) n linker is most commonly used ( 187 , 188 ), due to its high flexibility, low immunogenicity, and serine residues’ contribution to solubility. Moreover, the 218s linker is reported to be more proteolytic stable and has reduced aggregation ( 186 ), while the HMA linker exhibits lower immunogenicity ( 189 ).…”