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

Bong, Ji-Hong; Thömmes, Sarah; Kim, Hyun-Ok; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

Zusammenfassung

Bacterial surface display (Autodisplay) enables efficient selection of antibody fragments, but conventional phage display cannot express variable heavy (VH) and light (VL) chains separately, limiting combinatorial diversity and direct affinity-based screening. This study aimed to develop a combinatorial twin-chain Fv (tcFv) library enabling co-autodisplay of VH and VL chains on the surface of E. coli, and to enhance binding affinity through structural refolding. The Fv 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 × 10⁷ 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 VH and Alexa 546–labeled VL. 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 (KD = 49.2 nM, n = 3). FRET confirmed structural assembly of VH.K18 and VL.23 within < 10 nm proximity. Optimization of the VH:VL mixing ratio to 44%:56% maximized binding activity (n = 3, p < 0.05). As expected, refolding further improved affinity by approximately ninefold (KD = 5.6 nM, n = 3). The developed combinatorial Fv (tcFv) library enables functional co-autodisplay and refolding of VH and VL 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.