Characterization of antibody binding to the tumor marker MUC1

MUC1 is an important marker in adenocarcinomas and represents an important target for cancer drug development. The N-terminal domain of MUC1 consists of a repeating amino acid sequence called the VNTR that in healthy cells is highly glycosylated. However, in tumor cells the heavy glycosylation is reduced, exposing the protein backbone. Tumor-associated changes in glycosylation make MUC1 a possible target for antibody immunotherapy. The present study tested the effects of cancer specific glycosylation and the number of VNTR repeats on antibody affinity. Circular dichroism revealed that MUC1 is a conformational epitope. Glycosylation of MUC1stabilized an extended form, which promoted antibody affinity. The affinity of three MUC1 antibodies against different numbers of VNTR repeats was examined using ELISA. In contrast to previous reports, there was no effect on affinity associated with increasing VNTR length. This observation suggested that changes in VNTR repeat number do not alter the conformation of MUC1. This hypothesis was further supported by ELISA experiments using the scFv fragment C595, which displayed higher affinity for increased VNTR number. We propose a model where antibody binding is modulated by glycosylation through conformational effects, and by multivalency associated with increased VNTR repeat number.