title: Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies.
abstract: Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Sue
authors.initials: S
authors.lastname: Townsend
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Cambridge, MA 02139;
authors.forename: James R
authors.initials: JR
authors.lastname: Apgar
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Matthew
authors.initials: M
authors.lastname: Lambert
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Barry
authors.initials: B
authors.lastname: McDonnell
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Joanne
authors.initials: J
authors.lastname: Grant
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Jason
authors.initials: J
authors.lastname: Wade
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Edward
authors.initials: E
authors.lastname: Franklin
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Niall
authors.initials: N
authors.lastname: Foy
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Deirdre
authors.initials: D
authors.lastname: Ní Shúilleabháin
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Conor
authors.initials: C
authors.lastname: Fields
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Alfredo
authors.initials: A
authors.lastname: Darmanin-Sheehan
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Cambridge, MA 02139;
authors.forename: Amy
authors.initials: A
authors.lastname: King
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Cambridge, MA 02139;
authors.forename: Janet E
authors.initials: JE
authors.lastname: Paulsen
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Biotherapeutics R&D, Andover, MA 01810.
authors.forename: Timothy P
authors.identifier: http://orcid.org/0000-0002-8918-2140
authors.initials: TP
authors.lastname: Hickling
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Cambridge, MA 02139;
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Tchistiakova
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland;
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Biotherapeutics R&D, Dublin D22, Ireland; william.finlay@pfizer.com.
authors.forename: William J J
authors.initials: WJ
authors.lastname: Finlay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use*; D000918:Antibody Specificity / Q000276:immunology; D002999:Clone Cells; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000276:immunology*; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005854:Germ Cells / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D016875:tau Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D018984:Epitopes, T-Lymphocyte; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D016875:tau Proteins
keywords: antibody; humanization; immunogenicity; paratope; plasticity