Affinity improvement of the fully human anti‑TSLP recombinant antibody

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 antiTSLPsinglechain antibody variable fragment (antiTSLPscFv) 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 antiTSLPscFv84. Specific primers were designed and mutated DNA sequences of antiTSLPscFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinityenhanced antiTSLPscFvM4 was screened using ELISA. However, in general the scFvs have low stability and short halflives in vivo. Therefore, scFv84 and scFvM4 were inserted into eukaryotic expression vectors (pcDNA3.1spFc and PMH3ENspFc) and then transfected into 293F cells to express antiTSLPscFvFc. ELISA and western blotting results indicated the size of the antiTSLPscFvFc to be ~50 kDa. Binding of antiTSLPscFvFcM4 to TSLP was enhanced compared with the premutated scFvFc84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10fold greater than the premutated antibody.