Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21

The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer i mmunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (a a) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immu nogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epit helial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTS APDTRPA with cysteine at the N-terminal end, and used the cysteine for chem ical conjugation to keyhole limpet haemocyanin (KLH), Six breast cancer pat ients immunized with this conjugate plus the immunological adjuvant QS-21 h ave all produced high titre (by ELISA) IgG and IgM antibodies against the 3 0 aa MUC1 peptide, but these sera reacted moderately, or not at all, with M UC1-positive tumour cells. To understand this specificity better, we prepar ed a series of smaller peptides to determine the epitopes recognized by the se immune sera in inhibition assays. Only peptides containing APDTRPA at th e C-terminal end were able to completely inhibit ELISA reactivity for the f ull 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTR PA or any other peptides that did not contain the full APDTRPA epitope. Rem arkably, sera from all six patients recognized this same epitope and were c ompletely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C -terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the beta turn MUC1 assumes in its secondary st ructure may explain the moderate to weak reactivity of these high titer ser a against MUC1-positive tumour cells.