P-030: crizanlizumab reduces endothelial adhesion of red blood cells from scd patients in standardized microfluidic platform: in vitro technology assessment

Purpose: Crizanlizumab (ADAKVEO®, Novartis, Switzerland) a monoclonal P-selectin blocking antibody designed to disrupt the blood cells and endothelium interactions in sickle cell disease (SCD) patients, which leads to reduction of vaso-occlusive crises, the major manifestation of the disease. We report the novel microfluidic platform (endothelium-on-a-chip), to test the effects of Crizanlizumab on the adhesion of red blood cells (RBCs) to perfusion-cultured, acutely and chronically activated human endothelial cells (ECs). Materials and methods: Whole blood samples collected from SCD subjects (n=21), 20 HbSS and 1 HbSC in EDTA and sodium citrate. RBCs were isolated via centrifugation from whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were cultured within the microfluidic platform channels at 15 dyne/cm2 for at least 48 hours prior to experiments. To mimic chronic pre-activation in SCD HUVECs were pretreated for 4 hours with heme (40 µM), TNFα (20 ng/ml) or 50% plasma of SCD patients in basal media, +/- 100 µg/ml Crizanlizumab followed by injection of blood samples through the microfluidic channels. For acute EC activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml Crizanlizumab and injected through the microfluidic channels for 15 minutes. Thereafter, non-adherent RBCs were rinsed via washing solution with or without Crizanlizumab and the remaining RBCs were quantified based on previous published methods. Paired t-test was used to calculate statistical significance. Results: The inhibitory effects of Crizanlizumab on blood cell adhesion to ECs was linked to the type and duration of EC activation (Fig. 1). Crizanlizumab slightly reduced RBC adhesion to 4-hours heme activated ECs (1170±413vs1671±522 p>0.05). Reduction of RBC adhesion due to Crizanlizumab treatment was significant in 15-minutes heme activated ECs (135±40 vs 1513±617, p≤0.05). The effect of Crizanlizumab on decreasing RBC adhesion was significant when ECs were pre-activated by TNF-α (4404±1393 vs 2016±609, p≤0.05) or subjects’ autologous plasma for 4 hours followed by a 15-minute heme activation (5876±2579 vs 2397±1381p≤0.05). Conclusion: In accordance with the label of Crizanlizumab to reduce VOC, application of microfluidic platform associated with Crizanlizumab use displayed a reduced RBC adhesion to acutely heme-activated EC. Effects were preserved in presence or absence of chronic TNFα or autologous plasma pre-activation. Endothelium-on-a-chip microfluidic platform has been shown as reliable in monitoring patient response to anti-adhesive therapies in SCD.Effects of Crizanlizumab on RBC adhesion levels to HUVECs activated for (A) 4-hours with heme (B) 15-minutes with heme (C) 4-hours with TNF-? and 15-minutes with heme, (D) 4-hours with plasma and 15-minutes with heme. Error bars represent the standard err J. ZAK declares a conflict of interest: Research support/Scientific studies: I am the Administrative Officer on several federal grants that support the development of the BioChip Labs assay platform: NIH phase I & II grant NSF phase I grant State of Ohio TVSF grant Stock shareholder: I am the founder of BioChip Labs Inc. and own a majority stake in the company Other: I hold the administrative title of CEO at BioChip Labs Inc. U. GURKAN declares a conflict of interest: Consultancy, Expert: Hemex Health Inc, BioChip Labs Inc Patent or product inventor: Hemex Health Inc, BioChip Labs Inc, Xatek Inc, DxNow Inc Research support/Scientific studies: Hemex Health Inc, BioChip Labs Inc, Stock shareholder: Hemex Health Inc, BioChip Labs Inc, Xatek Inc, DxNow Inc