title: A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.
abstract: Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.
authors.affiliation: Oncology Drug Discovery, Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Emanuel
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Engle
authors.forename: Ginger
authors.initials: G
authors.lastname: Chao
authors.forename: Rong-Rong
authors.initials: RR
authors.lastname: Zhu
authors.forename: Carolyn
authors.initials: C
authors.lastname: Cao
authors.forename: Zheng
authors.initials: Z
authors.lastname: Lin
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Yamniuk
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hosbach
authors.forename: Jennifer
authors.initials: J
authors.lastname: Brown
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fitzpatrick
authors.forename: Jochem
authors.initials: J
authors.lastname: Gokemeijer
authors.forename: Paul
authors.initials: P
authors.lastname: Morin
authors.forename: Brent A
authors.initials: BA
authors.lastname: Morse
authors.forename: Irvith M
authors.initials: IM
authors.lastname: Carvajal
authors.forename: David
authors.initials: D
authors.lastname: Fabrizio
authors.forename: Martin C
authors.initials: MC
authors.lastname: Wright
authors.forename: Ruchira
authors.initials: R
authors.lastname: Das Gupta
authors.forename: Michael
authors.initials: M
authors.lastname: Gosselin
authors.forename: Daniel
authors.initials: D
authors.lastname: Cataldo
authors.forename: Rolf P
authors.initials: RP
authors.lastname: Ryseck
authors.forename: Michael L
authors.initials: ML
authors.lastname: Doyle
authors.forename: Tai W
authors.initials: TW
authors.lastname: Wong
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Camphausen
authors.forename: Sharon T
authors.initials: ST
authors.lastname: Cload
authors.forename: H Nicholas
authors.initials: HN
authors.lastname: Marsh
authors.forename: Marco M
authors.initials: MM
authors.lastname: Gottardis
authors.forename: Eric S
authors.initials: ES
authors.lastname: Furfine
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D000077544:Panitumumab; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D005353:Fibronectins; D010446:Peptide Fragments; D000077544:Panitumumab; C501466:nimotuzumab; D066246:ErbB Receptors; D017526:Receptor, IGF Type 1