S107: P-SELECTIN INHIBITOR INCLACUMAB REDUCES CELL ADHESION IN AN IN-VITRO ASSAYS SHOWING POTENTIAL FOR PREVENTION OF VASO-OCCLUSION EVENTS IN SICKLE CELL DISEASE (2022)

Introduction: Sickle cell disease (SCD) is a genetic disorder that leads to serious clinical complications including vaso-occlusive events (VOE). Development of VOEs had been linked to translocation of endothelial P-Selectin from Weibel–Palade bodies to the cell surface, a process promoting sickle Red Blood Cell (SS RBC) adhesion to vessel walls. Up-regulation of P-selectin contributes to cell–cell interactions, as activated platelets bind to neutrophils to form aggregates in a P-selectin–dependent manner. Previous studies have shown that anti-P-selectin agents decrease SS RBC adhesion in vivo, increase microvascular flow, and reduce adhesion of the leukocyte to endothelium, showing the potential of P-selectin as a therapeutic target. In November 2019, the FDA approved the first P-selectin inhibitor, crizanlizumab-tmca (Adakveo, Novartis), to reduce the frequency of vaso-occlusive crises (VOCs) in adults and pediatric patients with SCD. Different types of P-selectin inhibitors can offer physicians and patients new treatment options enhancing the arsenal of drugs to combat SCD. Inclacumab (Global Blood Therapeutics) is a fully human monoclonal antibody designed to bind to and selectively inhibit P-selectin. Differences in static cell adhesion to P-selectin substrate due to inclucumab were shown previously. The present study aimed to validate the dose-dependentability of inclacumab in blocking P-selectin cell adhesion in samples from SCD patients using in-vitro Whole Blood (WB) and Isolated White Blood Cell (I-WBC) assays. Methods: Five deidentified samples from SCD patients undergoing phlebotomy prior to blood exchange had been collected at the Children’s Hospital of Michigan with Flow Adhesion on P-selectin measured using proprietary assays in both Whole Blood (WB) and in Isolated WhiteBlood Cells (I-WBC), with and without Inclacumab after 5 minutes incubation at room temperature. Microfluidic channels were coated with P-selectin, and I-WBC (5x106/mL) suspension or WB sample diluted 1:1 with phosphate buffer was passed through the channel for 6 (I-WBC) or 10 (WB) minutes at a shear rate of 1 dyne/cm and pulsatility of 1.67 Hz. The channel was washed for 5 minutes with the same buffer, and the number of cells adherring to the channel surface was quantified. Results: Incubation of either WB or I-WBC with inclacumab significantly reduced flow adhesion to P-Selectin with the overall inhibition being stronger when assessed on isolated white cells (ab. 35% inhibition on WB vs. ab. 55% on I-WBC after incubation with 40 mM Inclacumab as compared to baseline Flow Adhesion values, Fig. 1). Adhesion on P-selectin showed a pronounced dose response that followed logarithmic response curve, with changes in adhesion with I-WBC better correlated with inclacumab dose, than those measured using WB. Decrease in adhesion on P-selectin was reliably detected at 2 μg/mL treatment dose for 3 out of 5 samples, with dose-depemdent decrease in adhesion in all samples at 10 and 40 μg/mL treatment doses (for I-WBC; and similar for WB). For flow adhesion with both WB and I-WBC elevated adhesion at lower doses was observed in some samples. Conclusions: Both WB and I-WBC adhesion to P-Sselectin show a dose dependent inhibition by inclacumab, with clinically relevant doses likely demonstrating greater inhibition. P-selectin adhesion assays described in this study may serve as potential surrogate biomarkers of clinical response tp p-selectin inhibitors, like inclacumab.References 1. Schmitt et al, J Cardiovasc Pharmacol 2015; 65:611