Abstract
Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. FGF21 functions through a receptor complex that consists of an FGF receptor (FGFR) and a co-receptor β-Klotho. Here, we identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against β-Klotho that mimics FGF21 function. The co-crystal structure of β-Klotho KL1 domain in complex with 39F7 Fab revealed that the recognition of 39F7 is centered on Trp-295 of β-Klotho in a FGF21 noncompetitive manner. KL1 adopts a (β/α)8 TIM barrel fold which resembles that of β-glycosylceramidase, but lacks molecular features for enzymatic activity, suggesting that KL1 functions as a scaffold protein instead. In vitro characterization demonstrated that, although 39F7 does not compete with FGF21, it is specific for β-Klotho/FGFR1c activation. Furthermore, the agonistic activity of 39F7 required the full IgG molecule to be bivalent, suggesting that 39F7 functions by promoting receptor/co-receptor dimerization. Supported by negative stain EM analysis of full-length β-Klotho, we propose a molecular model wherein the agonistic antibody 39F7 acts in a β-Klotho– and FGFR1c-dependent manner, mimicking FGF21 activity. More importantly, 39F7 offers promising therapeutic potential in the axis of FGF21 signaling as an antibody therapy alternative to FGF21 analogs for treatment of metabolic diseases.
Highlights
Fibroblast growth factor 21 (FGF21), an endocrine hormone in the Fibroblast growth factors (FGFs) family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus
We previously described an immunization campaign in XenoMouse that resulted in the identification of “mimAb1” that activates the -Klotho/FGFR1c receptor complex [22]
We identified another highaffinity antibody, 39F7, with unique properties that are distinctive from mimAb1. 39F7 binds human -Klotho with a KD value of 86 picomolar as determined by surface plasmon resonance (SPR) analysis (Fig. 1A) and activates FGF receptor activity in a FGF21-responsive reporter cell assay (Fig. 1B)
Summary
Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. We identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against -Klotho that mimics FGF21 function. Paracrine FGFs utilize HS as a cofactor for high-affinity interaction with FGFRs and to activate receptor signaling. It is believed that these two co-receptors, ␣-Klotho and -Klotho, serve primarily as docking sites for the endocrine FGFs to facilitate their interactions with FGFRs and subsequent signaling [9, 10]. Both ␣- and -Klotho are single-pass membrane proteins with very short intracellular domains. Because of the divergence among the active site residues, it is
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