Different membrane cofactor protein (CD46) isoforms protect transfected cells against antibody and complement mediated lysis

Abstract

The need for organ transplantation, especially of kidneys, exceeds the availability of human donors and the possibility of xenotransplantation from suitable animals is now being addressed. The immediate barrier to success is hyperacute graft rejection, resulting from naturally occurring xenoreactive antibodies and the activation of complement. It is proposed that the intensity of the hyperacute response can be reduced by providing additional regulatory molecules to limit activation of the complement cascade, initially as transfected gene products in cultured cells as an in vitro model and eventually as a transgene in potential donor animals, such as pigs. Limiting the activity of C3b reduces the production of the C3a, C4a and CSa anaphylotoxins, thus curtailing not only the immediate C3b-mediated lytic pathway but also the later effects of a cellular inflammatory response including endothelial and platelet cell activation. To develop and assess the first part of this strategy, we have transfected several cDNA's encoding isoforms of CD46 (membrane cofactor protein). At least four different CD46 isoforms are commonly expressed in almost all human cells, and we have compared two of these and a third form to determine if they mediate different functions. After transfection, CD46-expressing CHO-K1 cells were selected with methionine sulphoximine and identified using monoclonal antibodies. Transfectants with suitable CD46 expression were assayed for primary CD46 function using a lysis assay dependent on the reaction of antibody and complement. In this in vitro model of hyperacute rejection, normal human sera containing natural xenoreactive antibodies were shown to lyse CHO cells, but only in the presence of complement. CD46 expression was sufficient to provide significant protection against higher titered rabbit-anti-CHO antibodies and the more potent rabbit complement. In fact, complement from rat, rabbit, human and guinea pig sera was lytic to CHO cells but not to CD46-transfected cells. Complement regulation in these cells was proved to be specifically CD46 mediated because Fab fragments of the M177 monoclonal antibody, which blocks the interaction between CD46 and C3b, abrogated protection, whereas Fab of E4.3 antibody, which reacts with CD46 without inhibiting C3b binding, did not affect protection. The CHO cells can be used as a model for studies of natural human antibodies reactive with pig cells, since absorptions of human sera with pig erythrocytes or CHO cells effectively removed most of the antibody reaction for CHO or pig cells respectively. Thus, CD46 is an appropriate human complement-regulatory molecule to provide one of the defensive mechanisms for xenogeneic transplants. Each of the three isoforms provided similar density-dependent protection, and we have concluded that CD46 isoforms do not differ in their initial interaction with complement proteins.