Treatment of primary Sjögren's syndrome with ianalumab (VAY736) targeting B cells by BAFF receptor blockade coupled with enhanced, antibody-dependent cellular cytotoxicity.

OBJECTIVES: To evaluate the efficacy and safety of ianalumab (VAY736), a B cell-depleting, B cell activating factor receptor-blocking, monoclonal antibody, in patients with active primary Sjögren's syndrome (pSS) in a double-blind, placebo-controlled, phase II, single-centre study. METHODS: Patients with pSS, EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) ≥6, were randomised to ianalumab single infusion at either 3 mg/kg (n=6), 10 mg/kg (n=12) or placebo (n=9). Outcomes were measured blinded at baseline and weeks 6, 12, 24, and unblinded at end of study (EoS) when B cell numbers had recovered. Clinical outcomes included ESSDAI, EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI), salivary flow rate, ocular staining score, physician global assessment and patient assessments of fatigue and general quality of life. Laboratory-based measures included circulating leucocyte subsets and markers of B cell activity. RESULTS: A similar trend showing positive therapeutic effect by ianalumab was observed across the primary clinical outcome (ESSDAI) and all secondary clinical outcomes (ESSPRI, Multidimensional Fatigue Inventory, Short Form-36, global assessments by physician and patient) versus the placebo-treated group. Rapid and profound B cell depletion of long-lasting duration occurred after a single infusion of ianalumab at either dose. Serum Ig light chains decreased, with return to baseline levels at EoS. Changes in some clinical outcomes persisted through to EoS in the higher dose group. Adverse effects were largely limited to mild to moderate infusion reactions within 24 hours of ianalumab administration. CONCLUSIONS: Overall results in this single-dose study suggest potent and sustained B cell depletion by ianalumab could provide therapeutic benefits in patients with pSS without major side effects.

Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance.

Flow cytometry is increasingly becoming an important technology for biomarkers used in drug discovery and development. Within clinical development flow cytometry is used for the determination of PD biomarkers, disease or efficacy biomarkers or patient stratification biomarkers. Significant differences exist between flow cytometry methodology and other widely used technologies measuring soluble biomarkers including ligand binding and mass spectrometry. These differences include the very heavy reliance on aspects of sample processing techniques as well as sample stabilization to ensure viable samples. These differences also require exploration of new approaches and wider discussion regarding method validation requirements. This paper provides a review of the current challenges, solutions, regulatory environment and recommendations for the application of flow cytometry to measure biomarkers in clinical development.

Comparative expression profiling for human endoplasmic reticulum-resident aminopeptidases 1 and 2 in normal kidney versus distinct renal cell carcinoma subtypes.

Altered expression of the ER-resident aminopeptidases ERAP1 and ERAP2 might play an important role in shaping the MHC class I-presented peptide repertoire, but their function in tumors has not been determined in detail. Thus, the expression of ERAP1, ERAP2 and HLA class I heavy chain (HC) was analysed in various renal tumor types and corresponding kidney parenchyma by immunohistochemistry. Additionally, comparative expression profilings of untreated versus interferon (IFN)-γ-treated RCC cell lines were performed applying qRT-PCR, Western blot and/or flow cytometry. Normal kidney tissues showed strong ERAP1 staining in the proximal tubules of 57.4 % of cases, in the distal tubules of 94.3 % of cases and in the medulla of 88.6 % of cases, whereas high ERAP2 levels were observed in the medulla of 77.1 % of cases and in both, proximal and distal tubules of about 88 % of cases. Imbalanced, downregulated and RCC subtype-specific ERAP1 or ERAP2 expression was detected in 12.7 % or 43.8 % of samples analyzed, respectively. A coordinated downregulation of ERAPs was found in 4.8 %, an upregulation of ERAP1 or ERAP2 in 22.8 % or 2.0 % of RCC lesions. No association exists between ERAP and HLA class I HC expression for any tissue type. A heterogeneous constitutive ERAP expression pattern was also detected in RCC cell lines with lower ERAP2 than ERAP1 expression levels, which was in 11/17 RCC cell lines inducible by IFN-γ. Conclusively, ERAP1 and ERAP2 might be involved in the development of immune escape mechanisms of RCC.

Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma.

Immune surveillance of tumour cells by CD8(+) cytotoxic T cells plays a key role in the establishment and control of an anti-tumour response. This process requires the generation of antigenic peptides, which are largely produced by the proteasome in combination with other proteases located in either the cytoplasm and/or the endoplasmic reticulum (ER). The ER-resident aminopeptidases ERAP1 and ERAP2 trim or even destroy HLA class I-binding peptides thereby shaping the peptide repertoire presented for T cell recognition. So far there exists limited information about the expression pattern of ERAP1 and/or ERAP2 in human tumours of distinct histotypes. Therefore, the expression profiles and modes of regulation of both aminopeptidases were determined in a large series of melanoma cell lines. A heterogeneous expression ranging from high to reduced or even total loss of ERAP1 and/or ERAP2 mRNA and/or protein expression was detected, which often could be induced/upregulated by interferon-gamma treatment. The observed altered ERAP1 and/or ERAP2 expression and activity levels were either mediated by sequence alterations affecting the promoter or enzymatic activities, leading to either transcriptional and/or post-transcriptional downregulation mechanisms or limited or excessive processing activities, which both might have an impact on the antigenic peptide repertoire presented on HLA class I molecules.

Interferon-gamma-induced activation of nitric oxide-mediated antiviral activity of macrophages caused by a recombinant coxsackievirus B3.

Cardiovascular disease is one of the major causes of human death and has been linked to many different risks including viral infections. Coxsackievirus B3 (CVB3) is one of the most important pathogens responsible for virus-induced myocarditis. Cytokines are normally involved in the control of CVB3 replication and pathogenesis. Among them, interferon-gamma (IFN-gamma) in particular is highly protective against CVB3. A novel strategy to circumvent virus-caused heart disease is based on the development of cytokine-expressing recombinant virus vectors. Using in vitro co-culture experiments, the release of IFN-gamma by the recombinant virus variant CVB3/IFN-gamma activates the expression of the inducible nitric oxide synthase (iNOS) in CVB3 non-susceptible murine macrophages and the release of nitric oxide (NO), which reduce coxsackieviral replication directly. In addition, the expression of IFN-gamma by CVB3/IFN-gamma contributes to protect mice from lethal infections by iNOS induction in murine peritoneal macrophages, viral load reduction, and pancreatic tissue protection.