Targeting pathogenic alloantibody generation using a chimeric HPA-1a-containing fc fusion protein: A novel treatment for fetal and neonatal alloimmune thrombocytopenia (FNAIT) Free

Background: Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) is a rare, pregnancy-associated bleeding disorder caused by maternal alloantibodies targeting paternally inherited platelet antigens, most commonly HPA-1a on integrin β3, which complexes with both aIIb or aV integrin alpha-chain subunits. These alloantibodies cross the placenta and attack fetal and neonatal platelets, and can lead to severe thrombocytopenia and life-threatening bleeding complications such as intracranial hemorrhage (ICH). Current treatments, such as intravenous immunoglobulin (IVIG) or FcRn-blocking antibodies, are non-specific and either broadly suppress maternal immunity or indiscriminately inhibit antibody transfer to the fetus. While alleviating symptoms, these treatments fail to selectively target the root cause - pathogenic alloantibodies - and can result in adverse side effects due to global immune suppression. Aim: To develop a targeted immunotherapy for FNAIT using an alloantigen-specific chimeric fusion protein designed to selectively lower the titer of circulating maternal anti-HPA-1a alloantibodies by (1) neutralizing and clearing existing antibodies from circulation and (2) depleting HPA-1a-specific antibody-producing B cells. Methods: We designed, expressed, and purified an HPA-1a-containing Fc fusion protein comprised of an α-subunit-independent murine single-chain chimeric β3 integrin fragment (cβ3-E2) containing five humanized amino acids (A₃₀P₃₂L₃₃D₃₉Q₄₇₀) that directed expression of the human HPA-1a epitope, fused to the Fc region of mouse IgG2a. Wild-type (WT) murine cβ3-E2-Fc fusion proteins were generated as controls. The binding specificity and stoichiometry of APLDQ-cβ3-E2 Fc for anti-HPA-1a antibodies was confirmed using ELISA. Results: APLDQ-cβ3-E2-Fc, but not WT cβ3-E2-Fc, bound specifically to four different human HPA-1a monoclonal antibodies, as well as to a polyclonal antiserum that had been generated in WT mice immunized with APLDQ (HPA-1a)-positive murine platelets. Introduction of APLDQ-cβ3-E2-Fc into APLDQ-immunized mice was effective in lowering the circulating concentration of HPA-1a-specific alloantibodies. Finally, B and T cells harvested from the spleens of APLDQ-immunized mice were adoptively transferred into irradiated Rag1-deficient BALB/c mice. Upon alloantigen challenge, these mice produced a robust HPA-1a-specific antibody response. Co-incubation of the B/T cell mixture with APLDQ-cβ3-E2-Fc prior to their adoptive transfer, however, significantly attenuated in vivo HPA-1a antibody production, consistent with its ability to target and deplete B cells involved in the alloantibody response. Conclusions: We have successfully developed an HPA-1a-containing Fc fusion protein that selectively targets pathogenic HPA-1a antibodies and their antibody-producing B cells - the root cause of FNAIT. This approach provides an antigen-specific, targeted alternative to traditional immunosuppressive therapies for FNAIT patients, with the potential for curative treatment. Future studies will focus on optimizing the therapeutic dose and timing, as well as evaluating the long-term efficacy in FNAIT mouse models.