Characterization of a Human Platelet Antigen-1a–Specific Monoclonal Antibody Derived from a B Cell from a Woman Alloimmunized in Pregnancy

Abstract

Human platelet Ag (HPA)-1a, located on integrin β3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.4, was derived from an immortalized B cell from an alloimmunized woman who had an infant affected by FNAIT. It is the only HPA-1a–specific human mAb with naturally paired H and L chains. Specific binding of mAb 26.4, both native and recombinant forms, to platelets and to purified integrins αIIbβ3 (from platelets) and αVβ3 (from trophoblasts) from HPA-1a+ donors was demonstrated by flow cytometry and surface plasmon resonance technology, respectively. No binding to HPA-1a− platelets or integrins was detected. Moreover, the Ab binds with higher affinity to integrin αVβ3 compared with a second HPA-1a–specific human mAb, B2G1. Further in vitro experimentation demonstrated that mAb 26.4 can opsonize HPA-1a+ platelets for enhanced phagocytosis by monocytes, inhibit binding of maternal polyclonal anti–HPA-1a Abs, and weakly inhibit aggregation of HPA-1a–heterozygous platelets, the latter with no predicted clinical relevance. Thus, mAb 26.4 is highly specific for HPA-1a and could potentially be explored for use as a prophylactic or therapeutic reagent for FNAIT intervention and as a phenotyping reagent to identify women at risk for immunization.

Current perspectives on fetal and neonatal alloimmune thrombocytopenia – increasing clinical concerns and new treatment opportunities

“…HPA-1 antigens are expressed on the surface of fetal invading trophoblasts as part of the vitronectin receptor, and we know that anti-HPA-1a antibodies can bind the HPA-1a antigen when present on the vitronectin receptor 90,91. The vitronectin receptor promotes cell adhesion and migration of primary cytotrophoblasts 92,93.…”

Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model

“…In this study, we used a human anti-HPA-1a mAb, clone26.4, generated from a B cell derived from a woman HPA-1a immunized in connection with pregnancy, who had two children affected by FNAIT. mAb 26.4 was expressed recombinantly and found to be highly specific for HPA-1a, bound strongly to HPA-1a epitopes on αIIbβ3 from platelets as well as αVβ3 from trophoblasts [16]. Thus, the HTR8/SVneo cell line with mAb 26.4 could be a useful model to study possible effect of anti-HPA-1a antibodies on EVT.…”

“…A recently developed human recombinant anti-HPA-1a IgG1 mAb (clone 26.4) [16] was used to explore the effect on invasive trophoblast cells. Murine anti-human αVβ3 mAb, clone LM609 (Millipore, Billerica, MA) was used as positive control for cell functional studies.…”

“…It has therefore been speculated that anti-HPA-1a antibodies may affect placental development [4]. Anti-HPA-1a antibodies can bind HPA-1a on αVβ3 expressed on trophoblast cells [9, 16], and we hypothesize that this binding may affect EVT invasion, spiral artery remodeling, and in turn lead to reduced placental function.…”

“…For functional experiments we used an experimental in vitro model with human recombinant anti-HPA-1a monoclonal antibody (mAb), clone 26.4 [16], and a first trimester human EVT-derived cell line, HTR8/SVneo [17]. …”