INTRACELLULAR EXPRESSION IN PIG CELLS OF ANTI-??1,3GALACTOSYLTRANSFERASE SINGLE-CHAIN FV ANTIBODIES REDUCES GAL??1,3GAL EXPRESSION AND INHIBITS CYTOTOXICITY MEDIATED BY ANTI-GAL XENOANTIBODIES1

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Abstract
Background. The carbohydrate structure Galα1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Galα1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of α1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. Methods. As a novel strategy to inhibit α1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig α1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Galα1,3Gal expression. Results. After in vitro transfection in pig cells, the scFv antibody anti-pig α1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Galα1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in 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-α1,3-galactosyltransferase in pig cells significantly decreases the amount of Galα1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.

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