Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization.

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (kD) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.

Long-acting MIC-1/GDF15 molecules to treat obesity: Evidence from mice to monkeys.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion: analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion (huLCAT-Fc) is a chimeric protein produced by fusing human Fc to the C-terminus of the human enzyme via a linker sequence. The huLCAT-Fc homodimer contains five N-linked glycosylation sites per monomer. The heterogeneity and site-specific distribution of the various glycans were examined using enzymatic digestion and LC-MS/MS, followed by automatic processing. Almost all of the N-linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N-linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N-linked site exclusively contain typical asialobiantennary structures. HuLCAT-Fc was also confirmed to have mucin-type glycans attached at T407 and S409 . When LCAT-Fc fusions were constructed using a G-S-G-G-G-G linker, an unexpected +632 Da xylose-based glycosaminoglycan (GAG) tetrasaccharide core of Xyl-Gal-Gal-GlcA was attached to S418 . Several minor intermediate species including Xyl, Xyl-Gal, Xyl-Gal-Gal, and a phosphorylated GAG core were also present. The mucin-type O-linked glycans can be effectively released by sialidase and O-glycanase; however, the GAG could only be removed and localized using chemical alkaline ß-elimination and targeted LC-MS/MS. E416 (the C-terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O-xylosyltransferase. HuLCAT-Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPPE416 GS418 GGGGDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.

Treating diabetes and obesity with an FGF21-mimetic antibody activating the ßKlotho/FGFR1c receptor complex.

Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to ßKlotho with high affinity and specifically activates signaling from the ßKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on ßKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.