Engineering of an anti‐epidermal growth factor receptor antibody to single chain format and labeling by sortase A‐mediated protein ligation

Abstract

Sortase‐mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three‐component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo‐glycine acceptor molecule. We describe cloning of the single‐chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG‐biotin onto sc528. EGFR is an important cancer target and is over‐expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA‐biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen‐specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site‐specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody–receptor interaction. Biotechnol. Bioeng. 2012; 109:1461–1470. © 2011 Wiley Periodicals, Inc.

Sortase A enzyme (SrtA) from Staphylococcus aureus site‐specifically conjugates poly‐glycine constructs to LPETG‐tagged acceptor molecules. Here, an anti‐Epidermal Growth Factor Receptor scFv antibody fragment is functionalized with biotin or fluorescein, and used to specifically label an A431 EGFR over‐expressing cell line with Streptavidin Alexa 488. This technology has further application in directed immobilization of antibody fragments to biosensor surfaces for rapid generation of high quality kinetic data sets.