Affinity improvement of the fully human anti‑TSLP recombinant antibody.

Abstract

Thymic stromal lymphopoietin (TSLP) is a potentially important target for the treatment of asthma and malignancies. However, a fully human antibody reactive with TSLP is currently unavailable for clinical use. In a previous study, a human anti-TSLP-single-chain antibody variable fragment (anti-TSLP-scFv) 84 was selected by phage display from a constructed human scFv library. In the present study, a computer simulation method was developed using Discovery Studio 4.5 software, to increase the affinity of anti-TSLP-scFv-84. Specific primers were designed and mutated DNA sequences of anti-TSLP-scFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinity-enhanced anti-TSLP-scFv-M4 was screened using ELISA. However, in general the scFvs have low stability and short half-lives in vivo. Therefore, scFv-84 and scFv-M4 were inserted into eukaryotic expression vectors (pcDNA3.1-sp-Fc and PMH3EN-sp-Fc) and then transfected into 293F cells to express anti-TSLP-scFv-Fc. ELISA and western blotting results indicated the size of the anti-TSLP-scFv-Fc to be ~50 kDa. Binding of anti-TSLP-scFv-Fc-M4 to TSLP was enhanced compared with the pre-mutated scFv-Fc-84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10-fold greater than the pre-mutated antibody.