Developing methods for in vitro synthesis of the
carbohydrate structure
Galalpha1-3Galbeta1-4GlcNAc-R (termed the
alpha-galactosyl epitope) on human tumour cells may be of potential clinical significance in
cancer immunotherapy. Tumour
vaccines with this
epitope would be opsonized in vivo by the natural anti-Gal antibody, which is present in large amounts in humans, and which interacts specifically with alpha-galactosyl
epitopes. Binding of anti-Gal to alpha-galactosyl
epitopes on tumour cell membranes is likely to increase uptake of the cell membranes by antigen-presenting cells, such as macrophages, via the adhesion of the Fc portion of anti-Gal to
Fc receptors on these cells. This, in turn, may increase processing and presentation of tumour-associated
antigens by antigen-presenting cells, and induce an effective immune response against tumour cells with these
antigens. The present study describes a method for the synthesis of alpha-galactosyl
epitopes on human cells (red cells used as a model) by recombinant alpha1,3galactosyltransferase (rec. alpha1,3GT) expressed in bacteria. Escherichia coli was transformed with
cDNA of the
luminal portion of New World monkey rec. alpha1,3GT linked to six histidines (
His)6 at the N-terminus. The
enzyme produced by the bacteria was isolated from
bacterial lysates on a
nickel-
Sepharose column and eluted with
imidazole. This recombinant
enzyme displayed acceptor specificity similar to that of rec. alpha1,3GT produced in COS cells. Red cells were pre-treated with
sialidase for exposure of
N-acetyllactosamine acceptors, then subjected to rec. alpha1,3GT activity. This
enzyme synthesized at least 4 x 10(4) alpha-galactosyl
epitopes/red cell. These
epitopes were found to be accessible for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4
lectin. It is argued that the method presented can be used for the synthesis of alpha-galactosyl
epitopes on membranes of autologous tumour
vaccines in humans.