ENGINEERING OF RECOMBINANT SOLUBLE CD46 - AN INHIBITOR OF COMPLEMENT ACTIVATION

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that F unctions to protect autologous cells from complement-mediated damage b y binding C3b and C4b for their factor I-mediated cleavage. We now des cribe the production and function of recombinant soluble CD46 (rsCD46) , which was produced as a truncated form by mutagenesis using the spli ce overlap extension polymerase chain reaction, by inserting a transla tional stop codon into the CD46 cDNA at the junction of the transmembr ane and extracellular domains. After transfection of an expression con struct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay u sing monoclonal antibodies. Following a single-step immunoaffinity pur ification, the protein resolved as a single band of similar to 56 000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE). The purified rsCD46 (51 mu g/ml) protected Chinese hamster ovar y (CHO) cells from lysis initiated by a high titre rabbit anti-CHO ant ibody and complement from rabbit or human. The protection was specific ally mediated by rsCD46 because the monoclonal antibody M177, which bl ocks interaction between CD46 and C3b/C4b, abrogated the protection. T he results demonstrate that rsCD46 is effective as a fluid-phase regul ator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Adminstration of a bolus injection of rsCD46 was effective at delaying hyperacute g raft rejection. These data suggest that rsCD46 may have a role as a th erapeutic agent.