EXPRESSION OF FACTOR I-RESISTANT MUTANTS OF THE HUMAN-COMPLEMENT COMPONENT C3 IN HETEROLOGOUS SYSTEMS

Complement plays a major role in hyperacute rejection of xenografts. I n order to overcome this, we are developing, by minimal mutagenesis, a modified C3 molecule that, like cobra venom factor (CVF), escapes nor mal complement regulatory processes and inhibits complement-mediated r esponses by systemic depletion of C3. Unlike CVF, this protein should have little or no immunogenicity and be suitable for repeat administra tions. As an initial step in this process, we have modified human C3 t o make it resistant to inactivation by factor I. The factor I resistan t C3 is capable of forming an active C3 convertase. Preincubation with normal human serum abrogated subsequent complement-mediated cytolysis by both the classical and alternative pathways, while wild-type (wt) C3 was inactive. The modified human C3 also blocked complement activit y of guinea-pig serum. For economical and rapid production, we have de veloped expression of recombinant C3 wt and mutant proteins in the Bac ulovirus system. Large quantities are also being produced from stably transfected CHO cell lines. In addition, we have developed a fast C3 p urification method by engineering a 6XHIS tag into the C3a portion of the molecule, thereby avoiding the need for subsequent separation of t he tag from active C3b molecules.