Blockade of Inhibitor Formation by B-Cell Delivered Tolerance to Immunodominant A2 and C2 Domains.

A major impediment in the treatment of hemophilia is the formation of inhibitory antibodies, which occurs in approximately 25–30% of Hemophilia A patients treated with therapeutic Factor VIII (fVIII). We have focused on the development of a gene therapy protocol for tolerance induction, with an emphasis on the elimination of inhibitor production. Our lab has demonstrated that LPS-stimulated B-cell blasts, transfected with a retrovirus encoding an IgG-peptide fusion protein, such as fVIII domains, are tolerogenic in both normal and primed recipients. Last year, we reported (http://www.abstracts-on-line.com/abstracts/hemphiladelphia03; Scott and Lei 2003) that specific tolerance to the immunodominant epitopes in the C2 domain of fVIII (a major target of inhibitors) could be induced by our protocol. However, the immune response to full length fVIII was only modestly affected. Most inhibitory antibodies are reactive with conformational epitopes on the exposed surfaces of the A2, as well as the C2, domain of fVIII. Therefore, in this study, we inserted residues S2173-Y2332 of the C2 domain and S373-R740 of the A2 domain onto the IgG heavy chain backbone, respectively, to induce tolerance in hemophilic mice. Specific tolerance to each domain was induced by this protocol. Importantly, a combination of A2-IgG and C2-IgG expressing B cells induced tolerance to the full length fVIII molecule, a result which supports the dominance of these domains in the immune response to fVIII. Tolerance was manifested in terms of ELISA, T-cell proliferation and especially Bethesda Unit titers (95% reduction). Similar results were obtained even when treatment was initiated after priming injections of fVIII. In conclusion, this protocol offers great promise for prevention and potential reversal of this serious complication of fVIII replacement therapy. (Supported by HL61883 and a Laboratory Grant from the National Hemophilia Foundation).