Deglycosylated Human Monoclonal Antibody Against HPA-1a Prevents Anti-HPA-1a Alloantibodies Mediated Endothelial Apoptosis
Authors: Wihadmadyatami Hevi , Burg-Roederfeld Monika , Werth Silke , Ruan Changgeng , Wei Mingwang , Bein Gregor , Sachs Ulrich J. , Santoso Sentot 
Published: October 2019

Abstract
Foetal/neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of fetal platelets by maternal platelet alloantibodies which crossed through the placenta during pregnancy. Alloantibodies (aabs) against Human Platelet Antigen-1a (HPA-1a) residing on the b-subunit of the fibrinogen receptor (alphaIIbbeta3 integrin) on platelets are responsible for intracranial hemorrhage (ICH) in severe FNAIT cases. Recently, we could demonstrate that the deglycosylated mouse mab SZ21 against HPA-1a is able to cross through placenta, and prevents the destruction of platelets in mouse model (Bakchoul et al, Blood 2013). Meanwhile, this mab is humanized as chimeric antibody (mab 813). It is known that the beta3-subunit is abundantly expressed as vitronectin receptor (alphavbeta3 integrin) on endothelial surface. In this study, we sought to investigate the effect of deglycosylated mab 813 (deg-813) towards endothelial function.
Deg-813 was prepared and characterized as previously described (Bakchoul et al, Blood 2013). Flow cytometry analysis showed similar binding activity of intact 813 and deg-813 with platelets and endothelial cells. This result could be confirmed by surface plasmon resonance technology; native and deg-813 interacted equally with purified alphavbeta3 immobilized on the sensor chip (Kd 7.43x10-11 and 6.67x10-11, respectively). To study the influence of mab 813 on endothelial function, apoptosis experiment with HUVEC was performed using Tunel and Caspase-3/7 assays. In both assays, endothelial apoptosis was not observed with intact 813 and deg-813. In contrast, inhibitory mabs against alphavbeta3 receptor (clones 23C6, LM609) as well as cyclic Arg-Gly-Asp peptide caused significant apoptosis of these cells. Similar results were obtained with brain endothelial cells (hCMEC/D3). Accordingly, deg-813 did not inhibit the adhesion of HUVEC on vitronectin matrix when compared to alphavbeta3 specific mabs.
Furthermore, we asked the question whether maternal anti-HPA-1a aabs may induce endothelial apoptosis. Anti-HPA-1a aabs derived from FNAIT mothers giving birth of babies with ICH (n = 3) were purified by absorption/elution method using HUVEC. Analysis of the eluted material by antigen capture assay showed specific binding to alphavbeta3, but not to alphaIIbbeta3. This result could be confirmed by immunoprecipitation analysis. When alphavbeta3-reactive anti-HPA-1a antibodies were tested with endothelial cells, significant apoptosis was observed. In the control experiment, anti-alphaIIbbeta3 complex specific antibodies did not cause endothelial apoptosis. Finally, incubation of HUVEC with deg-813 prevented cell death mediated by anti-HPA-1a antibodies.
Taking together, our observation indicates that deg-813 can protect not only anti-HPA-1a aabs mediated platelet destruction but also endothelial disturbance without altering endothelial function. These results suggest that deg-813 may represent a novel drug to prevent ICH in severe FNAIT during pregnancy.

Disclosures
No relevant conflicts of interest to declare.