We generated a synthetic
epitope, NANA alpha(2-6) GalNAc alpha-O-Crotyl (STn-crotyl), designed to "mimic" the natural O-linked
epitope expressed on human
carcinoma cells, NANA alpha(2-6)GalNAc alpha-O-
Serine (STn-
serine). STn-crotyl was conjugated to the
carrier protein KLH through the crotyl linker arm, and a "
vaccine" containing
STn-KLH plus
DETOX adjuvant was formulated. The immunogenicity of the
vaccine was evaluated preclinically in CAF1 mice and subsequently in patients with metastatic
breast cancer. The specificity and titers of
IgG antibodies were evaluated by kinetic ELISA on synthetic STn-HSA and on ovine submaxillary
mucin (OSM) solid phases. Ovine submaxillary
mucin is a convenient source of repeating, natural O-linked STn-
serine structures. Mice immunized three times with as little as 0.25 micrograms of
STn-KLH produced
IgG titers ranging from 1:10(4) to 1:10(5) when tested on solid phase OSM. Anti-OSM
IgG, both polyclonal and
monoclonal antibodies, generated from these mice were completely inhibited in their binding to solid phase OSM equally well by STn-
serine and STn-crotyl synthetic
haptens but not by several other closely related synthetic
haptens. These
monoclonal antibodies also bound to STn determinants on human
tumor cell surfaces.
Breast cancer patients immunized with 100 micrograms of the same
vaccine produced median peak
IgG titers 1:1280 measured on STn-HSA and 1:160 on OSM.
Hapten inhibition experiments with the human sera demonstrated the specificities of the
IgG antibodies for STn-crotyl and STn-
serine, but not against several other related synthetic
haptens. We found little evidence that the artificial linker arm (crotyl linker) contributed substantially to either the titer or affinity of the
antibodies generated in either mice or human
breast cancer patients. This suggests that the
antibodies recognized the
cancer-associated
disaccharide NANA alpha(2-->6)-GalNAc. Small but not large doses of
STn-KLH immunogen induced anti-STn DTH responses in mice that were inversely proportional to the antibody responses. Evidence of a clinical response was noted in some of the immunized
breast cancer patients, with other patients showing prolonged disease stability.