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-FcandPMH3en-sp-Fc)andthentransfectedinto 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.