Raman spectroscopy characterization of antibody phases in serum.

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm(-1), which are typical of mAb ß-sheets, and lacked bands at 1270 and 1655 cm(-1), which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm(-1) and lacked those at 1240 and 1670 cm(-1). Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,(-1) demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.

Effect of sequence and stereochemistry reversal on p53 peptide mimicry.

Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15-29) using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15-29) result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.

The dimerization of glucagon-like peptide-2 MIMETIBODY™ is linked to leucine-17 in the glucagon-like peptide-2 region.

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY™ refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY™ scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY™ in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY™, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.

Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.