Fibroblast growth factor 21 (FGF21) is a recently discovered metabolic regulator. Exogenous FGF21 produces beneficial metabolic effects in animal models; however, the translation of these observations to humans has not been tested. Here, we studied the effects of LY2405319 (LY), a variant of FGF21, in a randomized, placebo-controlled, double-blind proof-of-concept trial in patients with obesity and type 2 diabetes. Patients received placebo or 3, 10, or 20 mg of LY daily for 28 days. LY treatment produced significant improvements in dyslipidemia, including decreases in low-density lipoprotein cholesterol and triglycerides and increases in high-density lipoprotein cholesterol and a shift to a potentially less atherogenic apolipoprotein concentration profile. Favorable effects on body weight, fasting insulin, and adiponectin were also detected. However, only a trend toward glucose lowering was observed. These results indicate that FGF21 is bioactive in humans and suggest that FGF21-based therapies may be effective for the treatment of selected metabolic disorders.

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.

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

A monoclonal antibody, 9.2.27, with a high specificity for human melanoma cell surfaces has been utilized for biosynthetic studies in M21 human melanoma cells to define a unique antigenic complex consisting ofa 250kdilodalton N-linked glycoprotein and a high molecular weight proteoglycan component larger than 400 kilodaltons. The 250-kilodalton glycoprotein has endoglycosidase H-sensitive precursors and shows a lower apparent molecular weight after treatment with neuraminidase. The biosynthesis of the proteoglycan component is inhibited by exposure of M21 cells to the monovalent ionophore monensin; this component can be labeled biosynthetically with 35SO4, is sensitive to 3-elimination in dilute base, and is degraded by both chondroitinase AC and ABC lyases, suggesting that it is a chondroitin sulfate proteoglycan. These data demonstrate that the antigenic determinant recognized by monoclonal antibody 9.2.27 is located on a glycoprotein-proteoglycan complex which may have unique implications for the interaction of glycoconjugates at the. human melanoma tumor cell surface.The development ofmonoclonal antibodies to tumor cell surface components has provided specific probes to establish the molecular nature of these antigens. Antigenic determinants with unique biochemical properties defined by monoclonal antibodies specific for colorectal carcinoma have recently been identified as glycolipids (1). Several additional monoclonal antibody reagents have defined components that appear to be either cellsurface or secreted glycoproteins of human tumors (2-4). The high specificity of these monoclonal .antibody probes and their ability to immunoprecipitate antigens selectively from complex soluble tumor extracts permits the biochemical analysis ofthese potentially tumor-associated gene.products.This laboratory has recently developed a. monoclonal antibody, 9.2.27, that is directed against an antigen highly associated with the surface of all melanoma cell lines and frozen sections of fresh surgical melanoma specimens thus far examined (4).In this study, we utilized the 9.2.27 monoclonal antibody in a combination of biosynthetic and enzymatic. digestion studies to define a unique cell surface antigen that consists of a glycoprotein-proteoglycan complex expressed on the surface of human melanoma cells.MATERIALS AND METHODS Cells. The M21 human melanoma cell line, originally derived from a metastatic melanoma lesion (5), was maintained in longterm tissue culture in RPMI-1640 medium-supplemented with 10% fetal calf serum (GIBCO), 2 mM L-glutamine, and 50 ,ug of gentamycin sulfate per ml at 37°C in a humidified 5% COJ/ 95% air atmosphere. Cells used for all experiments exceeded 90% viability by trypan blue exclusion.Antisera. Monoclonal antibody 9.2.27 was developed in this laboratory from a fusion of the nonsecreting 653 variant of the murine P3-X63-Ag8 myeloma cell line with spleens of BALB/ c mice previously primed with 4 M urea extracts of M21 melanoma cells. The specificity ofthis reagent is reported elsewhere (4). ...