Hummel, Amber M.

Abstract Therapeutic monoclonal immunoglobulins (mAbs) are used to treat patients with a wide range of disorders including autoimmune diseases. As pharmaceutical companies bring more fully humanized therapeutic mAb drugs to the healthcare market analytical platforms that perform therapeutic drug monitoring (TDM) without relying on mAb specific reagents will be needed. In this study we demonstrate that liquid-chromatography?mass spectrometry (LC?MS) can be used to perform TDM of mAbs in the same manner as smaller nonbiologic drugs. The assay uses commercially available reagents combined with heavy and light chain disulfide bond reduction followed by light chain analysis by microflow-LC?electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). Quantification is performed using the peak areas from multiply charged mAb light chain ions using an in-house developed software package developed for TDM of mAbs. The data presented here demonstrate the ability of an LC?MS assay to quantify a therapeutic mAb in a large cohort of patients in a clinical trial. The ability to quantify any mAb in serum via the reduced light chain without the need for reagents specific for each mAb demonstrates the unique capabilities of LC?MS. This fact, coupled with the ability to phenotype a patient?s polyclonal repertoire in the same analysis further shows the potential of this approach to mAb analysis.

Objective Relapse following remission is common in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), particularly with ANCAs directed at proteinase 3 (PR3). This study was undertaken to evaluate the association of an increase in PR3‐ANCA level with subsequent relapse. Methods Data from the Rituximab versus Cyclophosphamide for ANCA‐Associated Vasculitis (RAVE) trial were used. Starting from the time of achieving complete remission, serial measurements by direct and capture enzyme‐linked immunosorbent assays (ELISAs) were analyzed in 93 patients with PR3‐ANCA, using Cox proportional hazards regression. Results An increase in PR3‐ANCA level was identified in 58 of 93 subjects (62.4%) by direct ELISA and in 59 of 93 (63.4%) by capture ELISA. Relapses occurred in 55 of 93 subjects (59.1%), with 25 and 21 occurring within 1 year after an increase by direct ELISA and capture ELISA, respectively. An increase by direct ELISA was associated with subsequent severe relapses (hazard ratio [HR] 4.57; P < 0.001), particularly in patients presenting with renal involvement (HR 7.94; P < 0.001) and alveolar hemorrhage (HR 24.19; P < 0.001). Both assays identified increased risk for severe relapse in the rituximab group (HR 5.80; P = 0.002 for direct ELISA and HR 4.54; P = 0.007 for capture ELISA) but not the cyclophosphamide/azathioprine group ( P = 0.103 and P = 0.197, respectively). Conclusion The association of an increase in PR3‐ANCA level with the risk of subsequent relapse is partially affected by the PR3‐ANCA detection methodology, disease phenotype, and remission induction treatment. An increase in PR3‐ANCA level during complete remission conveys an increased risk of relapse, particularly severe relapse, among patients with renal involvement or alveolar hemorrhage and those treated with rituximab. Serial measurements of PR3‐ANCA may be informative in this subset of patients, but the risk of relapse must be weighed carefully against the risks associated with therapy.

Summary Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrolment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluoresence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using κ-coefficients. These were &gt; 0·8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were ≥ 0·95 (P &lt; 0·0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.

We present the first evidence that adhesion mediated by a member of the cadherin gene family can be regulated by a G protein-coupled receptor. We show that activating the M3 muscarinic acetylcholine receptor (mAChR) rapidly induces E-cadherin-mediated adhesion in a small cell lung carcinoma (SCLC) cell line. This response is inhibited by E-cadherin antibodies, and does not occur in another SCLC cell line which expresses functional mAChR but reduced levels of E-cadherin. Protein kinase C may be involved, since phorbol 12-myristate 13-acetate also induces E-cadherin-mediated aggregation. Immunofluorescence analyses indicate that mAChR activation does not grossly alter E-cadherin surface expression or localization at areas of cell-cell contact, suggesting mAChR activation may increase E-cadherin binding activity. Our findings suggest that G protein-coupled receptors may regulate processes involving cadherin-mediated adhesion, such as embryonic development, neurogenesis, and cancer metastasis.

SUMMARYANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ-rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With δ-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.