Qualification of a homogeneous cell-based neonatal Fc receptor (FcRn) binding assay and its application to studies on Fc functionality of IgG-based therapeutics.

The Fc region of IgG-based molecules plays an important role in determining their in vivo pharmacokinetic profile by its pH-dependent binding to the neonatal Fc receptor (FcRn) which is expressed on the endothelial cells lining blood vessels. By virtue of this pH-specific interaction with IgG-Fc, FcRn mediates IgG homeostasis in human adults by maintaining serum IgG levels, and also transfers maternal IgGs from mother to fetus via the placenta. The Fc-FcRn interaction is also critical for keeping IgG-based therapeutic molecules in circulation thereby enhancing their serum half life. A homogeneous cell-based flow cytometric FcRn binding assay was established to characterize the Fc-FcRn interaction of therapeutic IgG-based molecules. It is a competition-based assay, wherein the IgG-Fc containing test molecule competes with a fixed concentration of fluorescently-labeled IgG-Fc moiety in solution for binding to the cell-expressed FcRn. The cell-bound fluorescence is read on a flow cytometer. Response of the test sample is analyzed relative to the standard sample and the results are reported as % relative binding. The assay is robust and meets the qualification criteria for specificity, method linearity, accuracy and precision over the relative binding range of 60%-160%. This assay was shown to effectively characterize altered Fc-FcRn interactions for photo-stressed, heat-stressed, oxidized, and Fc mutant samples. It was observed that the relative binding of the IgG-Fc to the cell-surface-expressed FcRn in the assay varies across different molecules, even within the same IgG subclass. This indicates that the Fc-FcRn binding can be influenced by the antigen-binding region of the molecules in addition to the IgG subclass. Overall, this assay is reflective of the in vivo mechanism of immunoglobulin binding to membrane-bound FcRn, and can be used as an analytical tool for assessing lot-to-lot consistency and stability testing across different batches of the same molecule. Additionally, the assay can be used as an effective tool for elucidating the amino acids in the IgG-Fc domain that are critical for FcRn binding and also for comparing the binding of different IgG-Fc containing molecules to FcRn.

Management of arrhythmias in heart failure. What a practicing physician should know in the current times.

Arrhythmias play a significant role in the mortality and morbidity as well as hospitalizations of patients who carry a diagnosis of congestive heart failure. With improving survival in a world of novel medications and devices, an understanding of the pathophysiology and management of these arrhythmias is crucial. Majority of the basic heart failure medications such as beta- -blockers, angiotensin converting enzyme inhibitors/aldosterone receptor blockers and aldosterone antagonists play a pivotal role in prevention of sudden cardiac deaths which can be a direct/indirect result of these arrhythmias. Anti-arrhythmic drugs and implantable cardioverter-defibrillators were also beneficial in selected patients. Innovative electrophysiological techniques need to be considered in special situations.

Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity.

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity.

The antibody MAB007 was recently shown to be cysteinylated on an unpaired cysteine residue in the CDR3 variable region. Cysteinylation at this position was not complete and resulted in heterogeneous lots of MAB007 with respect to this posttranslational modification. In this report, a mild redox step was used that effectively removed cysteinylation while keeping native inter and intra-molecular disulfide bonds intact. Biophysical methods were employed to determine what consequences cysteinylation of the variable region had by directly comparing cysteinylated and de-cysteinylated MAB007 antibodies. No differences were detected in secondary structure; however, several pieces of evidence indicated that cysteinylation may result in tertiary or quaternary structural perturbations. These included differences in the cation-exchange chromatography and fluorescence-emission spectra of the cysteinylated and de-cysteinylated antibodies as well as differences in the solvent accessibility of the unpaired cysteine residue determined by labeling experiments. Such structural changes induced by cysteinylation were shown to increase the rate of MAB007 aggregation and to decrease the melting temperature of the Fab region by as much as 6 degrees C. The bioactivity of MAB007 was also shown to be adversely affected by cysteinylation and a direct correlation was made between the percent cysteinylation and biological activity.