Development of mammalian cell surface binding antibodies by phage display

Abstract

Chinese hamster ovary (CHO) cells are one of the well-established and widely utilized systems for the production of recombinant therapeutic proteins. In contrast to other expression host systems, CHO cells enable complex posttranslational modifications and correct protein folding. However, specific cell productivity is often a limiting factor in large-scale manufacturing. Thus, stable mammalian cell lines with a high expression yield are required for the industrial manufacture of recombinant proteins, including antibodies. This time-consuming and laborious method involves the extensive screening of a large number of cell clones from heterogeneous populations. The aim of this thesis was to discover and enrich antibody single-chain variable fragments (scFv) which recognize and bind to Chinese Hamster Ovary cells. The scFvs could be utilized to develop an efficient screening approach for the isolation of high-producing mammalian cell clones from those with low levels of protein production by capturing the secreted antibodies on the cell surface with a bispecific anchor construct.

Antibody scFvs against the ExpiCHO cells and recombinant CD44 protein, an abundant cell surface receptor, were selected from synthetic antibody phage display libraries by four rounds of cell- and recombinant antigen -based panning. The enrichment of target- specific binders was followed by phage titration and phage immunoreactivity assay and signal to background ratios of 28.4 (CD44) and 2.7 (ExpiCHO) were obtained after the fourth round of panning. The pool of selected clones was screened to identify target- specific single clones. Three selected single clones, and polyclonal scFv gene libraries, were cloned into pHBSC3 vector to create scFv-SpyCatcher003 fusion proteins. The produced fusion proteins were coupled with SpyTag003-Fc, and the covalent isopeptide bond formation between scFv-SpyCatcher003 and SpyTag003-Fc was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The specificity of the scFv-SpyCatcher003+SpyTag003-Fc binders was analyzed with a bead- and cell-based flow cytometry. The proportion of ExpiCHO specific binders increased after each panning round which indicates successful enrichment but only binders from third round were able to recognize CD44. Further optimization is still required but the binders obtained from cell-based panning successfully recognize and bind to CHO cells.