Recombinant HPA-1a antibody therapy for treatment of fetomaternal alloimmune thrombocytopenia: proof of principle in human volunteers

Abstract

Fetomaternal alloimmune thrombocytopenia, caused by the maternal generation of antibodies against fetal human platelet antigen-1a (HPA-1a), can result in intracranial hemorrhage and intrauterine death. We have developed a therapeutic human recombinant high-affinity HPA-1a antibody (B2G1Δnab) that competes for binding to the HPA-1a epitope but carries a modified constant region that does not bind to Fcγ receptors. In vitro studies with a range of clinical anti-HPA-1a sera have shown that B2G1Δnab blocks monocyte chemiluminescence by >75%. In this first-in-man study, we demonstrate that HPA-1a1b autologous platelets (matching fetal phenotype) sensitized with B2G1Δnab have the same intravascular survival as unsensitized platelets (190 hours), while platelets sensitized with a destructive immunoglobulin G1 version of the antibody (B2G1) are cleared from the circulation in 2 hours. Mimicking the situation in fetuses receiving B2G1Δnab as therapy, we show that platelets sensitized with a combination of B2G1 (representing destructive HPA-1a antibody) and B2G1Δnab survive 3 times as long in circulation compared with platelets sensitized with B2G1 alone. This confirms the therapeutic potential of B2G1Δnab. The efficient clearance of platelets sensitized with B2G1 also opens up the opportunity to carry out studies of prophylaxis to prevent alloimmunization in HPA-1a-negative mothers.

Figure 1

Platelet recovery (%) after re-injection. The graph shows the percentage of platelets remaining in circulation post-reinjection after normalizing at 100% for the first sample (5 minutes, see “Results”) for each condition: unsensitized platelets (n = 12) and platelets sensitized with either B2G1Δnab (n = 7), B2G1 (n = 8), or a combination of 75% B2G1Δnab/25% B2G1 (n = 3) or 90% B2G1Δnab/10% B2G1 (n = 5). Error bars represent the SD.

Figure 2

In vivo platelet tracking using flow cytometry. (A) Flow cytometric data showing sensitized platelets in peripheral blood. Sensitized platelets can be detected with an FITC-labeled anti-human IgG antibody in the peripheral blood after re-injection. Platelet sensitized with B2G1Δnab (top panel) are detected up to 1 hour post-reinjection but after 24 hours the antibody has redistributed to the whole platelet population as evidenced by the shoulder on the histogram and increased MFI for the whole platelet population. In contrast, when platelets are sensitized with a combination of 75% B2G1Δnab/25% B2G1, the majority of platelets are rapidly cleared from circulation after 60 minutes. (B) Correlation between flow cytometry and radiolabeling studies data in 4 volunteers. The percentage of platelets (normalized at 100% at the 5-minute time point) remaining in circulation in the first hour, measured either by flow cytometry or radiolabeling, is consistent between the 2 methods. Over 75% of platelets sensitized with B2G1Δnab only remain in circulation (n = 1) while platelets sensitized with 75% B2G1Δnab/25% B2G1 show a sharp decrease in the first hour down to below 25%.