title: CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.
abstract: While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Barry
authors.initials: B
authors.lastname: McDonnell
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Amy Sze Pui
authors.initials: AS
authors.lastname: Tam
authors.affiliation: Pfizer; Global Biotherapeutics Technologies, Foresterhill; Aberdeen, UK.
authors.forename: Lijun
authors.initials: L
authors.lastname: Chang
authors.affiliation: Pfizer; Global Biotherapeutics Technologies, Foresterhill; Aberdeen, UK.
authors.forename: John
authors.initials: J
authors.lastname: Steven
authors.affiliation: Pfizer; Global Biotherapeutics Technologies, Foresterhill; Aberdeen, UK.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Broadbent
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Huilan
authors.initials: H
authors.lastname: Gao
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kieras
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Alley
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Luxenberg
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Edmonds
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Lori J
authors.initials: LJ
authors.lastname: Fitz
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Miao
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Whitters
authors.affiliation: Pfizer; Immunoscience; Cambridge, MA USA.
authors.forename: Quintus G
authors.initials: QG
authors.lastname: Medley
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Yongjing J
authors.initials: YJ
authors.lastname: Guo
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Darmanin-Sheehan
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Autin
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Deirdre Ní
authors.initials: DN
authors.lastname: Shúilleabháin
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Emma
authors.initials: E
authors.lastname: Cummins
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Amy
authors.initials: A
authors.lastname: King
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Mark R H
authors.initials: MR
authors.lastname: Krebs
authors.affiliation: Pfizer; Pharmacokinetics, Dynamics, and Metabolism; Sandwich, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Grace
authors.affiliation: Pfizer; Pharmacokinetics, Dynamics, and Metabolism; Sandwich, UK.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Hickling
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Boisvert
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: McKenna
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Francis
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Stephane
authors.initials: S
authors.lastname: Olland
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Laird
authors.initials: L
authors.lastname: Bloom
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Paulsen
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Allan
authors.initials: A
authors.lastname: Jensen
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Cambridge, MA USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Lin
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: William J J
authors.initials: WJ
authors.lastname: Finlay
authors.affiliation: Pfizer; Global Biotherapeutics Technologies; Dublin, Ireland.
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics; D022801:Complementarity Determining Regions / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007279:Injections, Subcutaneous; D019151:Peptide Library; D015202:Protein Engineering; D055550:Protein Stability; D051381:Rats; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D018033:Antibodies, Bispecific; D022801:Complementarity Determining Regions; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: affinity optimization; bispecific scFv-Fc-scFv; high concentration formulation; phage display; thermal stability