title: Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge.
abstract: Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.
authors.affiliation: a Department of Antibody Engineering; Genentech Inc. ; South San Francisco , CA USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Devin
authors.initials: D
authors.lastname: Tesar
authors.forename: C Andrew
authors.initials: CA
authors.lastname: Boswell
authors.forename: Hendry S
authors.initials: HS
authors.lastname: Cahaya
authors.forename: Anne
authors.initials: A
authors.lastname: Wong
authors.forename: Jianhuan
authors.initials: J
authors.lastname: Zhang
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Homer
authors.initials: H
authors.lastname: Pantua
authors.forename: Jinyu
authors.initials: J
authors.lastname: Diao
authors.forename: Sharookh B
authors.initials: SB
authors.lastname: Kapadia
authors.forename: Rong
authors.initials: R
authors.lastname: Deng
authors.forename: Robert F
authors.initials: RF
authors.lastname: Kelley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics / Q000276:immunology* / Q000493:pharmacokinetics*; D019540:Area Under Curve; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008252:Macaca fascicularis; D008657:Metabolic Clearance Rate; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D017207:Rats, Sprague-Dawley; D011961:Receptors, Fc / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc
keywords: FcRn recycling; antibody catabolism; antibody pharmacokinetics; humanized; isoelectric point