Glycosylated, low antigenicity, low immunogenicity factor VIII

Abstract

The development of inhibitory antibodies to blood coagulation factor VIII (fVIII) results in a severe bleeding tendency. These antibodies arise in patients with hemophilia A (hereditary fVIII deficiency) who have been transfused with fVIII. They also occur in non-hemophiliacs, which produces the condition acquired hemophilia. We describe a method to construct and express novel recombinant fVIII molecules which escape detection by existing inhibitory antibodies (low antigenicity fVIII) and which decrease the likelihood of developing inhibitory antibodies (low immunogenicity fVIII). In this method, fVIII is glycosylated at sites that are known to be antibody recognition sequences (epitopes). This produces the desired properties of low antigenicity fVIII and low immunogenicity fVIII. The mechanism is similar to one used by viruses such as the AIDS virus, which glycosylates its surface proteins to escape detection by the immune system.

1 . A method for preparing a biologically active factor VIII having modified glycosylation comprising the steps of 
 mutating a desired segment of factor VIII DNA to encode -N-X-S/T, where N is asparagine, X is any amino acid, and S/T is serine or threonine, thereby providing mutated factor VIII DNA encoding a post-translational glycosylation site at the desired locus of factor VIII protein, and    expressing the mutated DNA in a host cell capable of post-translational glycosylation, whereby biologically active factor VIII having modified glycosylation is prepared.    
     
     
         2 . The method of  claim 1  wherein said desired segment resides in the A2 domain.  
     
     
         3 . The method of  claim 1  wherein said desired segment resides in the C2 domain.  
     
     
         4 . The method of  claim 3  wherein said desired segment comprises the amino acid residue, glutamine, at position 2189 in the C2 domain.