Recombinant Human HPA-1A Antibodies for Treatment of Fetomaternal Alloimmune Thrombocytopenia (FMAIT): Proof of Principle in An in Vivo Murine Model and Human Volunteer Studies

Abstract

Abstract Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A single nucleotide polymorphism (SNP) in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a–specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a–specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor–dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a+ platelets at micromolar concentration and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less (<15%) than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. We set out to prove the principle of our strategy in vivo using a murine model. Beta3-deficient mice (WT) were transplanted with littermate bone marrow transduced with a lentiviral vector containing the human ITGB3 encoding either Leucine-33 or Proline-33. B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia whilst WT mice and HPA-1b+ mice remained unaffected. Given the differences between murine and human Fcγ receptors, F(ab)2 B2G1 was used as a non-destructive proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies. These results provide rationale for human clinical studies, which are now underway. Eight HPA-1a1b heterozygotes volunteers have been recruited and from each of them we generate 2 pools of autologous platelets. Each pool is sensitized with either B2G1 or B2G1Δnab and radiolabeled with either Cr-51 or In-111. After re-injection into the volunteer, platelet survival is followed by the means of the radiolabel in order to show the differential effect on platelet destruction of the G1Δnab constant region when compared to the reference IgG1 destructive antibody. Full data of the volunteer studies will be presented in support of further clinical trials.