ALPHA-GALACTOSYL (GA1-ALPHA-1-3GAL-BETA-1-4GLCNAC-R) EPITOPES ON HUMAN-CELLS - SYNTHESIS OF THE EPITOPE ON HUMAN RED-CELLS BY RECOMBINANT PRIMATE ALPHA-1,3GALACTOSYLTRANSFERASE EXPRESSED IN ESCHERICHIA-COLI

Developing methods for in vitro synthesis of the carbohydrate structur e Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epito pe) on human tumour cells may be of potential clinical significance in cancer immunotherapy, Tumour vaccines with this epitope would be opso nized in vivo by the natural anti-Gal antibody, which is present in la rge amounts in humans, and which interacts specifically with a-galacto syl epitopes. Binding of anti-Gal to alpha-galactosyl epitopes on tumo ur cell membranes is likely to increase uptake of the cell membranes b y antigen-presenting cells, such as macrophages, via the adhesion of t he Fc portion of anti-Gal to Fc receptors on these cells, This, in tur n, may increase processing and presentation of tumour-associated antig ens by antigen-presenting cells, and induce an effective immune respon se against tumour cells with these antigens, The present study describ es a method for the synthesis of alpha-galactosyl epitopes on human ce lls (red cells used as a model) by recombinant alpha-1,3galactosyltran sferase (rec. alpha 1,3GT) expressed in bacteria, Escherichia coil was transformed with cDNA of the luminal portion of New World monkey rec, alpha 1,3GT 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. alpha 1 ,3GT produced in COS cells, Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine accepters, then subjected to rec, alpha 1,3GT activity, This enzyme synthesized at least 4 x 10(4) alpha -galactosyl epitopes/red cell. These epitopes were found to be accessi ble for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4 l ectin, It is argued that the method presented can be used for the synt hesis of alpha-galactosyl epitopes on membranes of autologous tumour v accines in humans.