Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry.

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli.

Background: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don't require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E.

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands.

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions.

Enhancement of DNA uptake in FUT8-deleted CHO cells for transient production of afucosylated antibodies.

Removal of the core alpha1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody-dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins.

Superior in vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer.

The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcgammaRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function-enhanced antibodies for solid tumor therapy.

Cigarette smoke synergistically enhances respiratory mucin induction by proinflammatory stimuli.

Pathogenic factors associated with chronic obstructive pulmonary disease (COPD), such as cigarette smoke, proinflammatory cytokines, and bacterial infections, can individually induce respiratory mucins in vitro and in vivo. Since co-presence of these factors is common in lungs of patients with COPD, we hypothesized that cigarette smoke can amplify mucin induction by bacterial exoproducts and proinflammatory cytokines, resulting in mucin hyperproduction. We demonstrated that cigarette smoke extract (CSE) synergistically increased gene expression and protein production of MUC5AC mucin induced by LPS or TNF-alpha in human airway epithelial NCI-H292 cells.