Screening of Anti-Erythropoietin Antibody from Autodisplayed Combinatorial Twin-Chain Fv Libraries

Abstract

Bacterial surface display (Autodisplay) enables efficient selection of antibody fragments, but conventional phage display cannot express variable heavy (V-H) and light (V-L) chains separately, limiting combinatorial diversity and direct affinity-based screening. This study aimed to develop a combinatorial twin-chain F-v (tcFv) library enabling co-autodisplay of V-H and V-L chains on the surface of E. coli, and to enhance binding affinity through structural refolding. The F-v fragments derived from a human anti-thyroid peroxidase antibody were expressed independently using the autodisplay system. Randomized CDR3 regions of both chains were used to construct combinatorial libraries (genetic diversity: 1.52 & times; 10(7) cfu). Screening was performed with Alexa 488-labeled erythropoietin (EPO) by flow cytometry and fluorescence-activated cell sorting. tcFv formation was confirmed by fluorescence resonance energy transfer (FRET) between Alexa 488-labeled V-H and Alexa 546-labeled V-L. Refolding was optimized using an oxido-shuffling reagent containing reduced and oxidized glutathione (GSH/GSSG, 10:1 ratio). A variant, tcFv.K18.23, exhibited high affinity toward EPO (K-D = 49.2 nM, n = 3). FRET confirmed structural assembly of VH.K18 and VL.23 within < 10 nm proximity. Optimization of the V-H:V-L mixing ratio to 44%:56% maximized binding activity (n = 3, p < 0.05). As expected, refolding further improved affinity by approximately ninefold (K-D = 5.6 nM, n = 3). The developed combinatorial F-v (tcFv) library enables functional co-autodisplay and refolding of V-H and V-L chains on E. coli, allowing rapid selection of high-affinity antibody fragments. This system provides a simple, cost-effective platform for antibody discovery and engineering.