title: Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.
abstract: The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Nilvebrant
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Åstrand
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Georgieva-Kotseva
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Björnmalm
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
mesh_terms: D000418:Albumins / Q000378:metabolism*; D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D044366:Transition Temperature; D000068878:Trastuzumab
chemical_list: D000418:Albumins; D018033:Antibodies, Bispecific; D061067:Antibodies, Monoclonal, Humanized; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab
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