Targeting Selectins Mediated Biological Activities With Multivalent Probes

Selectins are type-I transmembrane glycoproteins that are ubiquitously expressed on activated platelets, endothelial cells, and leukocytes. They bind to cell surface glycoproteins and extracellular matrix ligands, regulate the rolling of leukocytes in the blood capillaries, and recruit them to inflammatory sites. Hence, they are potential markers for the early detection and inhibition of inflammatory diseases, thrombosis, cardiovascular disorders, and tumor metastasis. Fucosylated and sialylated glycans, such as sialyl Lewisx, its isoform sialyl Lewisa, and heparan sulfate, are primary selectin ligands. Functionalization of these selectin-binding ligands on multivalent probes, such as nanoparticles, liposomes, and polymers, not only inhibits selectin-mediated biological activity but is also involved in direct imaging of the inflammation site. This review briefly summarizes the selectin-mediated various diseases such as thrombosis, cancer and recent progress in the different types of multivalent probes used to target selectins.

Characterization of Two Anti-P-Selectin Monoclonal Antibodies (mAbs): Crizanlizumab Shows Comparable or Stronger Effects Versus Inclacumab across Cell Adhesion Assays in Vitro

Background: Vaso-occlusive crises (VOCs) are the hallmark of sickle cell disease (SCD) and are associated with significant morbidity and mortality. Crizanlizumab, a first-in-class humanized anti-P-selectin IgG2 mAb, is approved in >40 countries to reduce/prevent VOCs in SCD patients aged ≥16 yrs. Inclacumab, a fully human anti-P-selectin IgG4 mAb, is in clinical development for SCD. In vitro data suggest that inclacumab may show stronger affinity to P-selectin and greater maximal inhibition of cell-cell interactions vs crizanlizumab (Geng et al ASH 2020). Aim: We investigated in blinded experiments whether crizanlizumab and inclacumab can be differentiated in terms of P-selectin binding and inhibition of P-selectin-mediated cell-cell interactions, via 5 different in vitro assays, including testing of blood samples from healthy volunteers and SCD patients. Methods: For both mAbs, we assessed: P-selectin affinity (surface plasmon resonance [SPR]/Biacore assay); inhibition of adhesion of P-selectin-expressing cells to P-selectin glycoprotein ligand-1 (PSGL-1; cell adhesion bioassay); inhibition of platelet aggregation in native blood from healthy volunteers (whole blood impedance aggregometry [WBA] and flow cytometry-based platelet-leukocyte aggregate [PLA] assays); and inhibition of adhesion of SCD patient whole blood or isolated leukocytes to P-selectin-coated substrate under physiologically relevant shear rates in microfluidic systems (flow adhesion bioassays). Results: The SPR/Biacore assay indicated that both mAbs recognize P-selectin with binding affinities of the same order of magnitude, although there was a trend towards higher binding affinity with inclacumab (mean equilibrium dissociation constant [K D ± standard deviation]: 12.4 ± 1.0 nM [crizanlizumab] vs 6.7 ± 0.7 nM [inclacumab]). However, the function-related cell adhesion bioassay revealed a significant difference between the mAbs in their ability to inhibit adhesion of P-selectin-expressing cells to PSGL-1. Crizanlizumab showed stronger inhibition than inclacumab (potency vs crizanlizumab reference standard: 98.0% for the crizanlizumab control sample [95% CI 85.2‒112.7%] and 26.2% for the inclacumab sample [95% CI 20.7%‒32.6%]). Native whole blood samples for WBA and PLA assays were provided by 12 and 10 healthy volunteers, respectively. Blocking of platelet (WBA assay) or platelet-leukocyte aggregation (PLA assay) in whole blood was comparable for crizanlizumab and inclacumab, with a clear trend towards stronger inhibition by crizanlizumab. In the WBA assay, the half maximal inhibitory concentration (IC 50) was twice as high with inclacumab (10.36 μg/mL) as crizanlizumab (5.11 μg/mL); similar data were seen in the PLA assay (Figure 1; IC 50: 4.11 vs 2.81 μg/mL for inclacumab vs crizanlizumab). Blood samples were provided by 9 SCD patients for the flow adhesion bioassays. Dose-dependent inhibition of adhesion of whole blood and isolated leukocytes to P-selectin was seen with both crizanlizumab and inclacumab, with no difference observed between them (Figure 2). A mAb concentration of 10 μg/mL resulted in statistically significant inhibition of adhesion by 60‒75%; further increasing the mAb concentration did not result in stronger inhibition of adhesion beyond this threshold. In relation to the clinically approved crizanlizumab dose (5.0 mg/kg), a concentration of 100 μg/mL corresponds with the maximum plasma concentration (C max) and ~10-15 μg/mL corresponds with the trough concentration (C trough). Conclusions: While the SPR/Biacore data suggested higher P-selectin binding affinity for inclacumab compared with crizanlizumab, there was a stronger inhibition of P-selectin-mediated cell adhesion with crizanlizumab vs inclacumab in other well-characterized functional in vitro assays. In microfluidic flow adhesion bioassays, blockage of cell adhesion from SCD whole blood and leukocytes was comparable for both mAbs. In summary, these data indicate that comparable or stronger blockage of cell adhesion with crizanlizumab vs inclacumab does not require superior P-selectin binding affinity. The data from healthy volunteers will be complemented by data from SCD patients for the WBA and PLA assays. Ultimately, clinical data are required to evaluate potential differences in the profiles and efficacy of crizanlizumab and inclacumab as treatments for SCD patients. Figure 1 Figure 1. Disclosures Rubic-Schneider: Novartis Pharma AG: Current Employment. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ledieu: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Garcia: Novartis Pharma AG: Current Employment. Hehlen: Novartis Pharma AG: Current Employment. Burnet-Merlin: Light Chain Bioscience - Novimmune SA: Current Employment; Novartis Pharma AG: Ended employment in the past 24 months. Cochin de Billy: Novartis Pharma AG: Current Employment. Greutmann: Novartis Pharma AG: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Krӧner: Novartis Pharma AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: WO2021087050A1. Verneret: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Bruederle: Novartis Pharma AG: Current Employment. Gao: Functional Fluidics: Current Employment. Dajee: Novartis: Current Employment.

The kinetics of E-selectin- and P-selectin-induced intermediate activation of integrin αLβ2 on neutrophils

ABSTRACT Selectins and integrins are key players in the adhesion and signaling cascade that recruits leukocytes to inflamed tissues. Selectin binding induces β2 integrin binding to slow leukocyte rolling. Here, a micropipette was used to characterize neutrophil adhesion to E-selectin and intercellular adhesion molecule-1 (ICAM-1) at room temperature. The time-dependent adhesion frequency displayed two-stage kinetics, with an E-selectin-mediated fast increase to a low plateau followed by a slow increase to a high plateau mediated by intermediate-affinity binding of integrin αLβ2 to ICAM-1. The αLβ2 activation required more than 5 s contact to E-selectin and spleen tyrosine kinase (Syk) activity. A multi-zone channel was used to analyze αLβ2 activation by P-selectin in separate zones of receptors or antibodies, finding an inverse relationship between the rolling velocity on ICAM-1 and P-selectin dose, and a P-selectin dose-dependent change from bent to extended conformations with a closed headpiece that was faster at 37°C than at room temperature. Activation of αLβ2 exhibited different levels of cooperativity and persistent times depending on the strength and duration of selectin stimulation. These results define the precise timing and kinetics of intermediate activation of αLβ2 by E- and P-selectins.

Glycocalyx Impairment in Vascular Disease: Focus on Inflammation

The glycocalyx is a complex polysaccharide-protein layer lining the lumen of vascular endothelial cells. Changes in the structure and function of the glycocalyx promote an inflammatory response in blood vessels and play an important role in the pathogenesis of many vascular diseases (e.g., diabetes, atherosclerosis, and sepsis). Vascular endothelial dysfunction is a hallmark of inflammation-related diseases. Endothelial dysfunction can lead to tissue swelling, chronic inflammation, and thrombosis. Therefore, elimination of endothelial inflammation could be a potential target for the treatment of vascular diseases. This review summarizes the key role of the glycocalyx in the inflammatory process and the possible mechanism by which it alleviates this process by interrupting the cycle of endothelial dysfunction and inflammation. Especially, we highlight the roles of different components of the glycocalyx in modulating the inflammatory process, including components that regulate leukocyte rolling, L-selectin binding, inflammasome activation and the signaling interactions between the glycocalyx components and the vascular cells. We discuss how the glycocalyx interferes with the development of inflammation and the importance of preventing glycocalyx impairment. Finally, drawing on current understanding of the role of the glycocalyx in inflammation, we consider a potential strategy for the treatment of vascular diseases.

A combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P‑selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface

Background Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. Results This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. Conclusions An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Exofucosylation of Adipose Mesenchymal Stromal Cells Alters Their Secretome Profile

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.