Citation

  • Authors: Robertson N. et al.
  • Year: 2020
  • Journal: J Biol Chem
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK 293 EBNA
    Description: Transformed with adenovirus 5 DNA Increased recombinant protein yield with EBV oriP vector
    Known as: 293 c18 ; 293 EBNA ; ATCC® CRL-10852™

Abstract

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labelled enabling enrichment and isolation by magnetic sorting and Förster resonance energy transfer (FRET)-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a CHO cell system. We validate this method by screening a synthetic naïve human scFv library against CHO cells expressing the oncogenic target epithelial cell adhesion molecule (EpCAM) and identify a panel of three novel binders to this membrane protein, one with dissociation constant (KD) as low as 0.8nM. We further demonstrate that the identified antibodies have utility for killing EpCAM positive cells when used as a targeting domain on CAR-T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

Pubmed