Stability engineering of scFvs for the development of bispecific and multivalent antibodies.
Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.
Miller
Brian R
BR
Biogen Idec, Inc., 5200 Research Place, San Diego, CA 92122, USA. brian.miller@biogenidec.com
Demarest
Stephen J
SJ
Lugovskoy
Alexey
A
Huang
Flora
F
Wu
Xiufeng
X
Snyder
William B
WB
Croner
Lisa J
LJ
Wang
Norman
N
Amatucci
Aldo
A
Michaelson
Jennifer S
JS
Glaser
Scott M
SM
D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053259:Lymphotoxin beta Receptor / Q000235:genetics; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D053259:Lymphotoxin beta Receptor; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D057127:Single-Chain Antibodies