Engineering of stable bispecific antibodies targeting IL-17A and IL-23.
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.
Mabry
Robert
R
ZymoGenetics, Inc., Seattle, WA 98102, USA. mabryr@zgi.com
Lewis
Katherine E
KE
Moore
Margaret
M
McKernan
Patricia A
PA
Bukowski
Thomas R
TR
Bontadelli
Kristen
K
Brender
Ty
T
Okada
Shannon
S
Lum
Karen
K
West
James
J
Kuijper
Joseph L
JL
Ardourel
Dan
D
Franke
Secil
S
Lockwood
Luann
L
Vu
Tuyen
T
Frank
Amanda
A
Appleby
Mark W
MW
Wolf
Anitra
A
Reardon
Brian
B
Hamacher
Nels B
NB
Stevens
Brenda
B
Lewis
Patsy
P
Lewis
Kenneth B
KB
Gilbertson
Debra G
DG
Lantry
Megan
M
Julien
Susan H
SH
Ostrander
Craig
C
Chan
Chung
C
Byrnes-Blake
Kelly
K
Brody
Jennifer
J
Presnell
Scott
S
Meengs
Brent
B
Levin
Steven D
SD
Snavely
Mark
M
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
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D020381:Interleukin-17; D053759:Interleukin-23; D057127:Single-Chain Antibodies