title: Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.
abstract: The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.
authors.affiliation: Zyngenia, Inc., Gaithersburg, MD, USA. dlafleur@zyngenia.com
authors.forename: David W
authors.initials: DW
authors.lastname: LaFleur
authors.forename: Donara
authors.initials: D
authors.lastname: Abramyan
authors.forename: Palanisamy
authors.initials: P
authors.lastname: Kanakaraj
authors.forename: Rodger G
authors.initials: RG
authors.lastname: Smith
authors.forename: Rutul R
authors.initials: RR
authors.lastname: Shah
authors.forename: Geping
authors.initials: G
authors.lastname: Wang
authors.forename: Xiao-Tao
authors.initials: XT
authors.lastname: Yao
authors.forename: Spandana
authors.initials: S
authors.lastname: Kankanala
authors.forename: Ernie
authors.initials: E
authors.lastname: Boyd
authors.forename: Liubov
authors.initials: L
authors.lastname: Zaritskaya
authors.forename: Viktoriya
authors.initials: V
authors.lastname: Nam
authors.forename: Bridget A
authors.initials: BA
authors.lastname: Puffer
authors.forename: Pete
authors.initials: P
authors.lastname: Buasen
authors.forename: Shashi
authors.initials: S
authors.lastname: Kaithamana
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Burnette
authors.forename: Rajesh
authors.initials: R
authors.lastname: Krishnamurthy
authors.forename: Dimki
authors.initials: D
authors.lastname: Patel
authors.forename: Viktor V
authors.initials: VV
authors.lastname: Roschke
authors.forename: Peter A
authors.initials: PA
authors.lastname: Kiener
authors.forename: David M
authors.initials: DM
authors.lastname: Hilbert
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000595:Amino Acid Sequence; D042702:Angiopoietin-2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000627:therapeutic use; D000918:Antibody Specificity; D049109:Cell Proliferation / Q000187:drug effects; D000068818:Cetuximab; D005260:Female; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000628:therapy*; D009389:Neovascularization, Pathologic; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D015202:Protein Engineering / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D015398:Signal Transduction; D000068878:Trastuzumab; D016896:Treatment Outcome; D023041:Xenograft Model Antitumor Assays
chemical_list: D042702:Angiopoietin-2; D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000068818:Cetuximab; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D000068878:Trastuzumab
keywords: antibody engineering; multi-specific antibody