Combined analytical assays for the characterization of drugs binding to human IgE: Applicability to omalizumab-bearing biosimilar candidates assessment.

Analytical and functional comparison is key for substantiating the level of convergence (essential sameness) or divergence between versions or variants of a given biological medicine. Accordingly, an overlapping biological activity between products meant to be equal probably reflects a highly similar structure and anticipates a comparable pharmacodynamic behavior. We developed an orthogonal approach to compare the human IgE binding features of different lots and versions of Xolair® (omalizumab), an anti-human IgE monoclonal antibody. The IgE binding affinity and kinetics were measured by surface plasmon resonance. Ability to prevent mast cell activity was assessed in vitro and in vivo in mast cell-based models. The variability of monoclonal antibodies with identical amino acid sequences produced either in Chinese hamster ovarian cells or in human HEK293 cells, was compared. Monoclonal antibodies from the two sources exhibited slightly different human IgE binding and neutralizing features. A known variant exhibiting a three amino acid replacement in the Fab region had lower IgE binding affinity than the original omalizumab. The lower binding affinity translated into reduced IgE neutralizing capacity and, in turn, a difference in the ability to prevent mast cell activation in vitro and in vivo. The proposed set of analytical and functional assays was sensitive enough to detect Fab-linked differences between anti-IgE antibody versions exhibiting an identical aminoacid sequence. In addition to add value to the comparative assessment of biosimilar candidates bearing omalizumab, these methods can aid pre-assessments of new anti-IgE agents that aim to improve therapeutic performance.

Preclinical Characterization of Pharmacologic NAD+ Boosting as a Promising Therapeutic Approach in Rheumatoid Arthritis.

OBJECTIVE: We analyzed NAD+ metabolism in patients with rheumatoid arthritis (RA), its association with disease activity and clinical outcomes of RA, and the therapeutic potential of pharmacologic NAD+ boosting. METHODS: Our study included 253 participants. In the first cohort, comprising 153 RA patients and 56 healthy donors, we assessed NAD+ levels and NAD+ -related gene pathways. We analyzed 92 inflammatory molecules by proximity extension assay. In the second cohort, comprising 44 RA patients starting anti-tumor necrosis factor (anti-TNF) drugs, we evaluated changes in NAD+ levels and their association with clinical response after 3 months. Mechanistic studies were performed ex vivo on peripheral blood mononuclear cells (PBMCs) from patients with RA to test the beneficial effects of NAD+ boosters, such as nicotinamide and nicotinamide riboside. RESULTS: Reduced NAD+ levels were found in RA samples, in line with altered activity and expression of genes involved in NAD+ consumption (sirtuins, poly[ADP-ribose] polymerase, CD38), transport (connexin 43), and biosynthesis (NAMPT, NMNATs). Unsupervised clustering analysis identified a group of RA patients with the highest inflammatory profile, the lowest NAD+ levels, and the highest disease activity (as shown by the Disease Activity Score in 28 joints). NAD+ levels were modulated by anti-TNF therapy in parallel with the clinical response. In vitro studies using PBMCs from RA patients showed that nicotinamide riboside and nicotinamide increased NAD+ levels via NAMPT and NMNAT and reduced their prooxidative, proapoptotic, and proinflammatory status. CONCLUSION: RA patients display altered NAD+ metabolism, directly linked to their inflammatory and disease activity status, which was reverted by anti-TNF therapy. The preclinical beneficial effects of NAD+ boosters, as shown in leukocytes from RA patients, along with their proven clinical safety, might pave the way for the development of clinical trials using these compounds.

In Vitro and In Vivo Validation of EP2-Receptor Agonism to Selectively Achieve Inhibition of Mast Cell Activity.

PURPOSE: Agonism of the prostaglandin E2 receptor, E-prostanoid receptor 2 (EP2), may represent an alternative protective mechanism in mast cell (MC)-mediated diseases. Previous studies have suggested that activation of the MC EP2 receptor prevents pathological changes in the murine models of allergic asthma. This work aimed to analytically validate the EP2 receptor on MCs as a therapeutic target. METHODS: Murine MC lines and primary cultures, and MCs bearing the human immunoglobulin E (IgE) receptor were subjected to IgE-mediated activation subsequent to incubation with selective EP2 agonists. Two molecularly unrelated agonists, butaprost and CP-533536, were tested either in vitro or in 2 in vivo models of allergy. RESULTS: The diverse range of MC populations was consistently inhibited through selective EP2 agonism in spite of exhibiting a heterogeneous phenotype. Such inhibition occurred in both mouse and human IgE (hIgE)-mediated activation. The use of molecularly unrelated selective EP2 agonists allowed for the confirmation of the specificity of this protective mechanism. This effect was further demonstrated in 2 in vivo murine models of allergy where MCs are a key to pathological changes: cutaneous anaphylaxis in a transgenic mouse model expressing the hIgE receptor and aeroallergen-induced murine model of asthma. CONCLUSIONS: Selective EP2 agonism is a powerful pharmacological strategy to prevent MCs from being activated through IgE-mediated mechanisms and from causing deleterious effects. The MC EP2 receptor may be an effective pharmacological target in allergic and other MC-mediated conditions.