Generation of Affinity Purified Antibody Reagents for Specific Determination of Efruxifermin in Biological Matrices

Abstract

Efruxifermin (EFX) is a novel Fc-fusion analog of human fibroblast growth factor 21 (FGF21), currently in clinical development as a potential treatment for non-alcoholic steatohepatitis (NASH). Each molecule of EFX consists of two modified FGF21 molecules, each attached at their N-termini to a human IgG1 Fc domain by a short polyglycine-serine linker. The FGF21 moiety of EFX incorporates three amino acid substitutions (L98R, P171G, and A180E relative to native FGF21). Two of these are proximal to the C-terminus (P171G and A180E), and reduce cleavage and inactivation by an endogenous protease, fibroblast activation protein (FAP), thereby prolonging its half-life. Fusion to human IgG1 Fc domain further extends circulating half-life, enabling once-weekly subcutaneous dosing. Accordingly, to support on-going clinical development of EFX, a specific assay is needed to distinguish intact EFX from both endogenous FGF21 and any in vivo biotransformation products of EFX that display reduced pharmacology. To maximize the antigenicity of EFX, FGF21 amino acid sequences were compared across species. Based on this, an antibody generation campaign was initiated in both rabbits and chickens. Comparison of titer responses against EFX and human FGF21 suggested that antisera from chickens was superior to rabbit antisera. Following a scaled-up, 12-week antibody campaign, antisera were purified by a combination of batch and column chromatographic procedures. By exploiting differences in structure and amino acid sequence of EFX relative to human FGF21, a purification strategy was designed to isolate chicken antibodies with increased specificity for EFX unique sequences. This reagent is being used as a capture antibody in the development of a noncompetitive ECLIA employing chemiluminescence detection. Presently, a number of different antibodies are being evaluated for potential pairing with the specific capture. We conclude that application of affinity purified chicken anti-EFX IgY will enable sensitive and specific determination of EFX in biological matrices with decreased cross-reactivity from endogenous hFGF21 and EFX metabolites.