SELECTION OF HEREGULIN VARIANTS HAVING HIGHER AFFINITY FOR THE ERBB3 RECEPTOR BY MONOVALENT PHAGE DISPLAY

Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of t he ErbB receptor family, Although HRG can bind to ErbB3 and ErbB4 homo dimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRG beta egf doma in was displayed on the surface of M13 phage to facilitate mutagenic a nalysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion, Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids . These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservati on. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scannin g mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgre n, J. A. Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sli wkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674), Some variants from several libraries showed significant improvements in binding affi nity to the ErbB3-IgG, These optimized segments were combined in vario us ways in the same molecule to generate variants (containing up to 16 mutations) that had > 50-fold higher affinity than wild-type HRG beta , The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wildtype. This indicates wild-type affinity is o ptimized for potency and that factors other than affinity for ErbB3 ar e limiting, These variants showed enhanced affinity toward the ErbB4 h omodimer, suggesting these receptors use very similar binding determin ants despite them having 65% sequence identity.