Intracellular expression in pig cells of anti-alpha 1,3galactosyltransferase single-chain Fv antibodies reduces Gal alpha 1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies

Background The carbohydrate structure Gal alpha 1,3Gal expressed on pig cel ls is the major antigen recognized by xenoreactive natural antibodies in th e higher primates. In xenotransplantation, natural antibodies binding to th at structure initiate hyperacute rejection, and the anti-Gal alpha 1,3Gal a ntibodies that are elicited probably take part in later phases of vasculari zed graft rejection. This epitope also appears to be involved in innate cel lular responses. Inactivation of alpha 1,3 galactosyltransferase in transge nic pigs would certainly lead to the success of xenotransplantation, but ge ne knockout in pigs is not feasible yet. Methods. As a novel strategy to inhibit alpha 1,3 galactosylation, we gener ated recombinant single-chain Fv (ScFv) antibodies directed against pig alp ha 1,3-galactosyltransferase and evaluated the effect of their intracellula r expression on enzyme activity and Gal alpha 1,3Gal expression. Results. After in vitro transfection in pig cells, the scFv antibody anti-p ig alpha 1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-as sociated activity. Consequently, Gal alpha 1,3Gal on cell membranes was red uced to the same extent. This led to a profound (more than 90%) reduction i n the cytotoxicity involving anti-Gal antibodies and complement. Conclusion, Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti -alpha 1,3-galactosyltransferase in pig cells significantly decreases the a mount of Gal alpha 1,3Gal and could be important to protect cells from elic ited antibodies as well as from innate effectors.