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.

SUBMITTER: Chen Q 

PROVIDER: S-EPMC6947841 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature