Synthesis of alpha-gal epitopes (Ga1 alpha 1-3Gal beta 1-4GlcNAc-R) on human tumor cells by recombinant alpha 1,3galactosyltransferase produced in Pichia pastoris

This study describes the processing of human tumor cells or cell membranes to express a-gal epitopes (Gal alpha1-3Gal-beta1-4GlcNAc-R) by the use of N ew World monkey (marmoset) recombinant alpha1,3galactosyltransferase (r alp ha1,3GT), produced in the yeast Pichia pastoris, Such tumor cells and membr anes may serve, in cancer patients, as autologous tumor vaccines that are t argeted in vivo to antigen-presenting cells by the anti-Gal antibody. This r alpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solu bility without detergent. It is effectively produced in P, pastoris under c onstitutive expression of the P,,, promoter and is secreted into the cultur e medium in a soluble, truncated form fused to a (His), tag. This tag enabl es the simple affinity purification of r alpha1,3GT on a nickel-Sepharose c olumn and elution with imidazole, The purified enzyme appears in SDS-PAGE a s two bands with the size of 40 and 41 kDa and displays the same acceptor s pecificity as the mammalian native enzyme. r alpha1,3GT is very effective i n synthesizing a-gal epitopes on membrane-bound carbohydrate chains and dis plays a specific activity of 1.2 nM membrane bound alpha -gal epitopes/min/ mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9) cells) with neuraminidase, r alpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6) alpha -gal epitopes per cell. Eff ective synthesis of a-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcino ma. These data imply that r alpha1,3GT produced in P, pastoris is suitable for the synthesis of a-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.