Fc gamma receptor IIIb binding of individual antibody proteoforms resolved by affinity chromatography–mass spectrometry

The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb contributes to neutrophil activation and is involved in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These processes present important mechanisms-of-actions of therapeutic antibodies. The very low affinity of IgG toward FcɣRIIIb (KD ~ 10 µM) is a technical challenge for interaction studies. Additionally, the interaction is strongly dependent on IgG glycosylation, a major contributor to proteoform heterogeneity. We developed an affinity chromatography–mass spectrometry (AC-MS) assay for analyzing IgG-FcɣRIIIb interactions in a proteoform-resolved manner. This proved to be well suited to study low-affinity interactions. The applicability and selectivity of the method were demonstrated on a panel of nine different IgG monoclonal antibodies (mAbs), including no-affinity, low-affinity and high-affinity Fc-engineered or glycoengineered mAbs. Thereby, we could reproduce reported affinity rankings of different IgG glycosylation features and IgG subclasses. Additional post-translational modifications (IgG1 Met252 oxidation, IgG3 hinge-region O-glycosylation) showed no effect on FcɣRIIIb binding. Interestingly, we observed indications of an effect of the variable domain sequence on the Fc-binding that deserves further attention. Our new AC-MS method is a powerful tool for expanding knowledge on structure–function relationships of the IgG-FcɣRIIIb interaction. Hence, this assay may substantially improve the efficiency of assessing critical quality attributes of therapeutic mAbs with respect to an important aspect of neutrophil activation

The contribution of comorbidities to mortality in hospitalized patients with heart failure

BACKGROUND: Heart failure (HF) with reduced ejection fraction (HFrEF) has a worse prognosis than HF with preserved EF (HFpEF). The study aimed to evaluate whether different comorbidity profiles of HFrEF- and HFpEF-patients or HF-specific mechanisms contribute to a greater extent to this difference. METHODS: We linked data from two health insurances to data from a cardiology clinic hospital information system. Patients with a hospitalization with HF in 2005–2011, categorized as HFrEF (EF < 45%) or HFpEF (EF ≥ 45%), were propensity score (PS) matched to controls without HF on comorbidites and medication to assure similar comorbidity profiles of patients and their respective controls. The balance of the covariates in patients and controls was compared via the standardized difference (SDiff). Age-standardized 1-year mortality rates (MR) with 95% confidence intervals (CI) were calculated. RESULTS: 777 HFrEF-patients (1135 HFpEF-patients) were PS-matched to 3446 (4832) controls. Balance between patients and controls was largely achieved with a SDiff < 0.1 on most variables considered. The age-standardized 1-year MRs per 1000 persons in HFrEF-patients and controls were 267.8 (95% CI 175.9–359.8) and 86.1 (95% CI 70.0–102.3). MRs in HFpEF-patients and controls were 166.2 (95% CI 101.5–230.9) and 61.5 (95% CI 52.9–70.1). Thus, differences in MRs between patients and their controls were higher for HFrEF (181.7) than for HFpEF (104.7). CONCLUSIONS: Given the similar comorbidity profiles between HF-patients and controls, the higher difference in mortality rates between HFrEF-patients and controls points more to HF-specific mechanisms for these patients, whereas for HFpEF-patients a higher contribution of comorbidity is suggested by our results

YjbH-Enhanced Proteolysis of Spx by ClpXP in Bacillus subtilis Is Inhibited by the Small Protein YirB (YuzO)▿†

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein