A Limited Number of Anti-Idiotypic Antibodies Is Required To Maintain FVIII Function in Presence of Polyclonal Anti-FVIII Antibodies of Haemophilia A Patients with Inhibitors.

Abstract

Replacement therapy using either plasma-derived or recombinant FVIII elicits FVIII-specific Abs in about 25% of the patients. The majority of such antibodies are directed towards specific FVIII regions on which major epitopes have been identified. Most of these epitopes are located in the carboxy-terminal end of the C2 domain of the FVIII light chain and in the amino-terminal end of the A2 domain of the heavy chain, whereas, antibodies directed towards the C1 domain have been described in cases of mild/moderate haemophilia A. From a functional point of view, epitopes have been mapped to the phospholipid and von Willebrand binding sites in the C1 and C2 domains and to the FIX binding site in A2.We have derived human mabs (mabRhd5, mabBo2C11 and mabBoIIB2) that react with high affinity to the FVIII C1, C2 and A2 domains, respectively, and are representative of most of the specific inhibitors observed in haemophilia A patients.Recently, we have generated mouse anti-Id mab (mab14C12) against mabBo2C11 and provided the first demonstration that an anti-Id mab directed towards a C2-specific inhibitor restored FVIII activity both in vitro and in vivo in the presence of the inhibitor. Futher more, we demonstrated that FVIII inhibition obtained by a mixture of two anti-FVIII mabs (mabBo2C11 (anti-C2) and mabBoIIB2 (anti-A2)) was neutralized up to 100% when a mixture of the corresponding anti-Ids (mab14C12 and mab30D1) was added to the assay establishing the proof of concept that combination of anti-Id mabs will be able to neutralize the anti-FVIII immune response of haemophilia A patients. Based on this observation, We have also generated an anti-idiotypic antibody (mab18B6) towards an anti-C1 domain inhibitor (mabRHD5). In ELISA, mab18B6 specifically prevents, in a dose-dependent manner, mabRHD5 binding to C1 and fully neutralized mabRHD5 inhibitory properties in a coagulation assay. With a mixture of anti-Ids (mab14C12, mab30D1 and mab18B6) we were able to completely neutralized the cumulative anti-FVIII activity obtained in functional assay by a mixture of the corresponding monoclonal anti-FVIII antibodies. We next evaluated whether a combination of anti-Ids (anti-anti-A2,-C1,-C2) had the ability to neutralize the inhibitory properties of polyclonal antibodies obtained from haemophilia A patients. To this end, eight patients with severe haemophilia A and inhibitor were tested. An average of 70% neutralization of the inhibition was obtained in 6 out of 8 cases. These data allow to conclude that potent polyclonal high-affinity FVIII inhibitors can be neutralized with corresponding anti-idiotypic antibodies and that only a limited number of anti-idiotypic antibodies is required for inhibitor neutralization in a majority of patients.