title: Rational optimization of a bispecific ligand trap targeting EGF receptor family ligands.
abstract: The human epidermal growth factor (EGF) receptor (HER) family members cooperate in malignancy. Of this family, HER2 does not bind growth factors and HER3 does not encode an active tyrosine kinase. This diversity creates difficulty in creating pan-specific therapeutic HER family inhibitors. We have identified single amino acid changes in epidermal growth factor receptor (EGFR) and HER3 which create high affinity sequestration of the cognate ligands, and may be used as receptor decoys to downregulate aberrant HER family activity. In silico modeling and high throughput mutagenesis were utilized to identify receptor mutants with very high ligand binding activity. A single mutation (T15S; EGFR subdomain I) enhanced affinity for EGF (two-fold), TGF-alpha (twenty-six-fold), and heparin-binding (HB)-EGF (six-fold). This indicates that T15 is an important, previously undescribed, negative regulatory amino acid for EGFR ligand binding. Another mutation (Y246A; HER 3 subdomain II) enhanced neuregulin (NRG)1-beta binding eight-fold, probably by interfering with subdomain II-IV interactions. Further work revealed that the HER3 subunit of an EGFR:HER3 heterodimer suppresses EGFR ligand binding. Optimization required reversing this suppression by mutation of the EGFR tether domain (G564A; subdomain IV). This mutation resulted in enhanced ligand binding (EGF, ten-fold; TGF-alpha, thirty-four-fold; HB-EGF, seventeen-fold; NRG1-beta, thirty-one-fold). This increased ligand binding was reflected in improved inhibition of in vitro tumor cell proliferation and tumor suppression in a human non-small cell lung cancer xenograft model. In conclusion, amino acid substitutions were identified in the EGFR and HER3 ECDs that enhance ligand affinity, potentially enabling a pan-specific therapeutic approach for downregulating the HER family in cancer.
authors.affiliation: Receptor BioLogix Inc., Palo Alto, California, United States of America.
authors.forename: Pei
authors.initials: P
authors.lastname: Jin
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Beryt
authors.forename: Lisa
authors.initials: L
authors.lastname: Turin
authors.forename: Cathleen
authors.initials: C
authors.lastname: Brdlik
authors.forename: Ying
authors.initials: Y
authors.lastname: Feng
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bai
authors.forename: Jim
authors.initials: J
authors.lastname: Liu
authors.forename: Brett
authors.initials: B
authors.lastname: Jorgensen
authors.forename: H Michael
authors.initials: HM
authors.lastname: Shepard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002616:Chemical Engineering / Q000379:methods*; D019281:Dimerization; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
chemical_list: D008024:Ligands; D066246:ErbB Receptors