title: A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).
abstract: Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Chao
authors.initials: C
authors.lastname: Tu
authors.affiliation: Global Biotherapeutics Technologies, Pfizer R&D, Grange Castle Business Park, Dublin D22, Ireland.
authors.forename: Virginie
authors.initials: V
authors.lastname: Terraube
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Amy Sze Pui
authors.initials: AS
authors.lastname: Tam
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Wayne
authors.initials: W
authors.lastname: Stochaj
authors.affiliation: Global Biotherapeutics Technologies, Pfizer R&D, Grange Castle Business Park, Dublin D22, Ireland.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Laura
authors.initials: L
authors.lastname: Lin
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Mark
authors.initials: M
authors.lastname: Stahl
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Edward R
authors.initials: ER
authors.lastname: LaVallie
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.affiliation: Global Biotherapeutics Technologies, Pfizer R&D, Grange Castle Business Park, Dublin D22, Ireland.
authors.forename: William J J
authors.initials: WJ
authors.lastname: Finlay
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and.
authors.forename: Lydia
authors.initials: L
authors.lastname: Mosyak
authors.affiliation: From Global Biotherapeutics Technologies, Pfizer R&D, Cambridge, Massachusetts 02140 and joel.bard@pfizer.com.
authors.forename: Joel
authors.initials: J
authors.lastname: Bard
authors.affiliation: Global Biotherapeutics Technologies, Pfizer R&D, Grange Castle Business Park, Dublin D22, Ireland orla.cunningham@pfizer.com.
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham
mesh_terms: D019943:Amino Acid Substitution; D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000378:metabolism; D001666:Binding Sites, Antibody; D001688:Biological Products / Q000737:chemistry* / Q000378:metabolism; D054382:Chemokine CXCL13 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D066329:Protein Aggregates; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014961:X-Ray Diffraction
chemical_list: D000936:Antigen-Antibody Complex; D001688:Biological Products; C110663:CXCL13 protein, human; D054382:Chemokine CXCL13; D022801:Complementarity Determining Regions; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: antibody engineering; chemokine; crystal structure; mutagenesis in vitro; protein stability