Characterization of a Novel Disulfide-Stabilized single-chain variable fragment (scFv) Library

Abstract

Single-chain variable fragments (scFvs), consisting of variable light and heavy chain domains, are small antibody fragments capable of antigen binding. While easily produced in E. coli and suited for phage display, scFvs are less stable than larger antibody constructs. To improve stability, a disulfide bond was introduced between CDR-L1 and CDR-H3. However, one intradomain disulfide bond had to be removed for phage display compatibility. A scFv library was constructed with varying CDR- H3 loop lengths (13–19 amino acids) using this stabilized framework. This study aimed to characterize this library by isolating unique binders against HER2, Gremlin1, hTROP2, and Protein L and analyzing their performance. Key methods included a single selection round against Protein L to remove misfolded variants and frameshifted clones, followed by three panning rounds against HER2, Gremlin1, and hTROP2. Post- selection, scFvs were cloned into the pLK04 vector, purified via Ni-NTA and SEC, and analyzed for affinity using Octet and thermostability through differential scanning fluorimetry. Protein L selection increased in-frame sequences from 23% to 76%. Effective enrichment was achieved for HER2 and Gremlin1, identifying six unique HER2 binders and four for Gremlin1, though no unique hTROP2 binders were found. Dissociation constants were 15.61 nM for HER2 and 4.81 nM for Gremlin1, with Gremlin1 exhibiting a thermal stability (Tm) of +65°C, while HER2 stability results were inconclusive. This ds-scFv library shows potential as a source of superstable scFvs with promising binding characteristics. Future work will assess the effects of restoring intradomain disulfide bonds on binder performance.