Identification of high affinity HER2 binding antibodies using CHO Fab surface display.

ABSTRACT: Discovery of monoclonal antibodies is most commonly performed using phage or yeast display but mammalian cells are used for production because of the complex antibody structure, including the multiple disulfide bonds and glycosylation, required for function. As this transition between host organisms is often accompanied by impaired binding, folding or expression, development pipelines include laborious plate-based screening or engineering strategies to adapt an antibody to mammalian expression. To circumvent these problems, we developed a plasmid-based Fab screening platform on Chinese hamster ovary (CHO) cells which allows for antibody selection in the production host and in the presence of the same post-translational modifications as the manufactured product. A hu4D5 variant with low affinity for the human epidermal growth factor receptor (HER2) growth factor receptor was mutagenized and this library of ~10(6) unique clones was screened to identify variants with up to 400-fold enhanced HER2 binding. After two rounds of fluorescence activated cell sorting (FACS), four unique clones exhibited improved antigen binding when expressed on the CHO surface or as purified human IgG. Three of the four clones contained free cysteines in third complementarity determining region of the antibody heavy chain, which did not impair expression or cause aggregation. The improved clones had similar yields and stabilities as hu4D5 and similar sub-nanomolar affinities as measured by equilibrium binding to target cells. The limited size of mammalian libraries restricts the utility of this approach for naïve antibody library screening, but it is a powerful approach for antibody affinity maturation or specificity enhancement and is readily generalizable to engineering other surface receptors, including T-cell receptors and chimeric antigen receptors.

SUBMITTER: Nguyen AW 

PROVIDER: S-EPMC5892863 | biostudies-other | 2018 Mar

REPOSITORIES: biostudies-other