The Innovative Factor VIII Molecules HAT and HAT RI Overcome Impairments of Hemophilia A Therapy

Objective Current Factor VIII (FVIII) substitution therapies are limited by a short plasma half-life, the lack of subcutaneous bioavailability, and a high immunogenic potential resulting in the development of inhibitory antibodies. Thus, we aimed for novel recombinant FVIII molecules addressing all major limitations of current FVIII substitution therapies to provide a safe and convenient treatment option raising the patient’s quality of life.

Material and Methods For the generation of the Hemophilia A Therapeutic (HAT) molecule, four albumin-binding domains were incorporated into a B-domain deleted, single chain FVIII sequence. Additionally, 19 deimmunizing amino acid substitutions were integrated into HAT’s FVIII core sequences resulting in HAT RI (Reduced Immunogenicity). Both molecules were produced in a human cell line, purified and extensively tested in vitro. Albumin-mediated half-life extending effects were investigated after i. v. injection in hemophilia A mice, Göttingen minipigs and albumin-deficient mice expressing the human FcRn a-chain. Pharmacokinetic properties and bioavailability for subcutaneous administration were evaluated in hemophilia A mice and in Göttingen minipigs. Pharmacodynamic investigations were performed using the tail vein transection assay in hemophilia A mice to assess in vivo efficacy. As FVIII-bound albumin may shield HAT and HAT RI from present FVIII inhibitors, the bypassing activity of both molecules was determined by a modified Bethesda assay. Further immunological aspects were investigated by LC-MS to identify HLA-presented peptides of HAT RI on dendritic cells.

Results Both FVIII molecules HAT and HAT RI demonstrated full in vitro and in vivo coagulating activity with a great efficacy. Pharmacokinetic assessment revealed a 4-fold half-life extension compared to Moroctocog alfa in the albumin-deficient mouse model. Bioavailability of 50 % after subcutaneous administration was observed in the Göttingen minipig model. A high activity despite the presence of inhibitors was observed in combination with reduced HLA-presentation showing improved immunological compatibility.

Conclusion HAT and HAT RI address the major challenges of current FVIII substitution therapy as both molecules provide a superior half-life, show a high subcutaneous bioavailability in mice and pig models, and reduce FVIII immunogenicity according to utilized in vitro models.

Publication History

Article published online:
18 June 2021