title: Engineering of stable bispecific antibodies targeting IL-17A and IL-23.
abstract: Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
authors.affiliation: ZymoGenetics, Inc., Seattle, WA 98102, USA. mabryr@zgi.com
authors.forename: Robert
authors.initials: R
authors.lastname: Mabry
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Lewis
authors.forename: Margaret
authors.initials: M
authors.lastname: Moore
authors.forename: Patricia A
authors.initials: PA
authors.lastname: McKernan
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Bukowski
authors.forename: Kristen
authors.initials: K
authors.lastname: Bontadelli
authors.forename: Ty
authors.initials: T
authors.lastname: Brender
authors.forename: Shannon
authors.initials: S
authors.lastname: Okada
authors.forename: Karen
authors.initials: K
authors.lastname: Lum
authors.forename: James
authors.initials: J
authors.lastname: West
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Kuijper
authors.forename: Dan
authors.initials: D
authors.lastname: Ardourel
authors.forename: Secil
authors.initials: S
authors.lastname: Franke
authors.forename: Luann
authors.initials: L
authors.lastname: Lockwood
authors.forename: Tuyen
authors.initials: T
authors.lastname: Vu
authors.forename: Amanda
authors.initials: A
authors.lastname: Frank
authors.forename: Mark W
authors.initials: MW
authors.lastname: Appleby
authors.forename: Anitra
authors.initials: A
authors.lastname: Wolf
authors.forename: Brian
authors.initials: B
authors.lastname: Reardon
authors.forename: Nels B
authors.initials: NB
authors.lastname: Hamacher
authors.forename: Brenda
authors.initials: B
authors.lastname: Stevens
authors.forename: Patsy
authors.initials: P
authors.lastname: Lewis
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Lewis
authors.forename: Debra G
authors.initials: DG
authors.lastname: Gilbertson
authors.forename: Megan
authors.initials: M
authors.lastname: Lantry
authors.forename: Susan H
authors.initials: SH
authors.lastname: Julien
authors.forename: Craig
authors.initials: C
authors.lastname: Ostrander
authors.forename: Chung
authors.initials: C
authors.lastname: Chan
authors.forename: Kelly
authors.initials: K
authors.lastname: Byrnes-Blake
authors.forename: Jennifer
authors.initials: J
authors.lastname: Brody
authors.forename: Scott
authors.initials: S
authors.lastname: Presnell
authors.forename: Brent
authors.initials: B
authors.lastname: Meengs
authors.forename: Steven D
authors.initials: SD
authors.lastname: Levin
authors.forename: Mark
authors.initials: M
authors.lastname: Snavely
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000493:pharmacokinetics; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000915:Antibody Affinity; D030562:Databases, Protein; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006207:Half-Life; D006801:Humans; D020381:Interleukin-17 / Q000276:immunology*; D053759:Interleukin-23 / Q000276:immunology*; D007700:Kinetics; D051379:Mice; D015202:Protein Engineering; D055550:Protein Stability; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D020381:Interleukin-17; D053759:Interleukin-23; D057127:Single-Chain Antibodies