Radhakrishnan Rathnachalam

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate, LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

Leptin is a four-helix bundle: secondary structure by NMR

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N ‐labeled) have been assigned and its secondary structure reveals it to be a four‐helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short‐helix cytokine family. A three‐dimensional model was built verifying the mechanical consistency of the identified elements with a short‐helix cytokine core.

Determination of the Mechanism of Action of Anti-FasL Antibody by Epitope Mapping and Homology Modeling

Fas ligand (FasL) is a 40-kDa type II transmembrane protein belonging to the tumor necrosis factor (TNF) family of proteins and binds to its specific receptor, Fas, a member of the TNF receptor family. Membrane-bound FasL can be processed into a soluble form by a metalloprotease similar to that which cleaves TNFalpha. Elevated levels of FasL have been implicated in a wide variety of diseases ranging from cancer to inflammatory abnormalities, which could be targeted by antibody therapy. We generated a fully human high-affinity antibody against FasL that binds to and neutralizes the activity of both soluble and membrane-associated human FasL. In order to elucidate the mechanism of function of this antibody, we have mapped the region and critical residues involved in the recognition of FasL using a combination of homology modeling, immunoprecipitation, hydrogen-deuterium exchange mass spectrometry (H/DXMS), and alanine scanning site-directed mutagenesis. These studies have revealed the antibody binding site on human FasL. Furthermore, through molecular homology modeling, we have proposed a mechanism for the neutralizing activity of this antibody that involves interference with the docking of the ligand to its receptor by the antibody.