Specific peptide conjugation to a therapeutic antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics.

Antibody-drug conjugates are powerful tools for combatting a wide array of cancers. Drug conjugation to a therapeutic antibody often alters molecular characteristics, such as hydrophobicity and effector function, resulting in quality deterioration. To develop a drug conjugation methodology that maintains the molecular characteristics of the antibody, we engineered a specific peptide for conjugation to the Fc region. We used trastuzumab and the chelator (DOTA) as model antibody and payload, respectively. Interestingly, peptide/DOTA-conjugated trastuzumab exhibited enhanced antibody-dependent cellular cytotoxicity (ADCC) and increased thermal stability. Detailed structural and thermodynamic analysis clarified that the conjugated peptide blocks the Fc dynamics like a "wedge." We revealed that (1) decreased molecular entropy results in enhanced ADCC, and (2) blockade of Fc denaturation results in increased thermal stability. Thus, we believe that our methodology is superior not only for drug conjugation but also as for reinforcing therapeutic antibodies to enhance ADCC and thermal stability.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using FcγRIIIa ligand.

Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N-glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG-Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant FcγRIIIa. Expi293 cell-derived monoclonal antibodies had higher affinity for the mutant FcγRIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the FcγRIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.

Chemoselective epoxidation of cholesterol derivatives on a surface-designed molecularly imprinted Ru-porphyrin catalyst.

A new molecularly imprinted Ru-porphyrin complex catalyst on a SiO2 support was designed, prepared, and characterized in a step-by-step manner for the C5[double bond, length as m-dash]C6 epoxidation of cholesterol derivatives. High chemoselectivity for the C5[double bond, length as m-dash]C6 epoxidation of cholesterol derivatives without protecting the 3-position OH group and other oxidizable functional groups was achieved on the molecularly imprinted catalyst.

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Purification and characterization of arylmalonate decarboxylase from Achromobacter sp. KU1311.

We have isolated, purified and characterized arylmalonate decarboxylase (AMDase; EC 4.1.1.76). This is an unique enzyme that gives optically pure arylpropionates from the corresponding arylmalonates. Recently, we have screened similar enzyme producers from soil samples and succeeded in isolating Achromobacter sp. KU1311. The gene encoding the enzyme was cloned and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240 amino acid protein with a relative molecular mass of 24,735. This enzyme was purified and its characteristics were compared with those of the hitherto known enzyme from Alcaligenes bronchisepticus KU1201.

Introduction of single mutation changes arylmalonate decarboxylase to racemase.

The introduction of only one mutation based on the estimated reaction mechanism endowed arylmalonate decarboxylase with a racemase activity, which catalyses racemisation of alpha-arylpropionates.

Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism.

The introduction of two mutations (G74C/C188S) based on the estimated reaction mechanism resulted in the inversion of enantioselectivity of arylmalonate decarboxylase, which catalyses the asymmetric decarboxylation of arylmethylmalonate to give optically active arylpropionate.