Details

Autor(en) / Beteiligte

• Fennell, Brian J
• McDonnell, Barry
• Tam, Amy Sze Pui
• Chang, Lijun
• Steven, John
• Broadbent, Ian D
• Gao, Huilan
• Kieras, Elizabeth
• Alley, Jennifer
• Luxenberg, Deborah
• Edmonds, Jason
• Fitz, Lori J
• Miao, Wenyan
• Whitters, Matthew J
• Medley, Quintus G
• Guo, Yongjing J
• Darmanin-Sheehan, Alfredo
• Autin, Bénédicte
• Shúilleabháin, Deirdre Ní
• Cummins, Emma
• King, Amy
• Krebs, Mark RH
• Grace, Christopher
• Hickling, Timothy P
• Boisvert, Angela
• Zhong, Xiaotian
• McKenna, Matthew
• Francis, Christopher
• Olland, Stephane
• Bloom, Laird
• Paulsen, Janet
• Somers, Will
• Jensen, Allan
• Lin, Laura
• Finlay, William JJ
• Cunningham, Orla

Titel

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery

Ist Teil von

• mAbs, 2013-11, Vol.5 (6), p.882-895

Ort / Verlag

United States: Taylor & Francis

Erscheinungsjahr

2013

Link zum Volltext

Quelle

Taylor & Francis Journals Auto-Holdings Collection

Beschreibungen/Notizen

• 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 (A 4 T) 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 V L -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.