title: Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4.
abstract: Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.
authors.affiliation: Early Development PKPD, Genentech, Inc., South San Francisco, CA, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bumbaca
authors.forename: Anne
authors.initials: A
authors.lastname: Wong
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Drake
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Reyes
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Lin
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Stephan
authors.forename: Luc
authors.initials: L
authors.lastname: Desnoyers
authors.forename: Ben-Quan
authors.initials: BQ
authors.lastname: Shen
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dennis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism / Q000493:pharmacokinetics; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism / Q000493:pharmacokinetics; D000915:Antibody Affinity; D000970:Antineoplastic Agents / Q000737:chemistry / Q000276:immunology* / Q000493:pharmacokinetics / Q000627:therapeutic use; D006528:Carcinoma, Hepatocellular / Q000628:therapy; D045744:Cell Line, Tumor; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008819:Mice, Nude; D008969:Molecular Sequence Data; D051499:Receptor, Fibroblast Growth Factor, Type 4 / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D003176:Complement C3; D051499:Receptor, Fibroblast Growth Factor, Type 4