Early breast cancer patients with a natural humoral response to MUC1 have a higher probability of freedom from distant failure and a better disease-specific survival. MUC1 antibodies may control hematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1-expressing tumor cells. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides in an adjuvant setting may favorably influence the outcome of disease.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.

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Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells. MUC1 is overexpressed and aberrantly glycosylated in carcinoma cells. The role MUC1 plays in cancer progression represents two sides of one coin: on the one hand, loss of polarity and overexpression of MUC1 in cancer cells interferes with cell adhesion and shields the tumor cell from immune recognition by the cellular arm of the immune system, thus favoring metastases; on the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses. Tumor-associated MUC1 has short carbohydrate sidechains and exposed epitopes on its peptide core; it gains access to the circulation and comes into contact with the immune system provoking humoral and cellular immune responses. Natural antibodies to MUC1 present in the circulation of cancer patients may be beneficial to the patient by restricting tumor growth and dissemination: early stage breast cancer patients with a humoral response to MUC1 have a better disease-specific survival. Several MUC1 peptide vaccines, differing in vectors, carrier proteins and adjuvants, have been tested in phase I clinical trials. They are capable of inducing predominantly humoral responses to the antigen, but evidence that these immune responses may be effective against the tumor in humans is still scarce.

The epithelial mucin 1 (MUC1) is an accepted serum tumor marker and cellular tumor antigen. We discuss recent views on the difference(s) between normal and tumor MUC1, and its implication for the development of cancer vaccines and antibody therapies, with special emphasis on the role of glycosylation.

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Key words: MUC1; breast cancer; ADCC; NK cells; vaccination; immunotherapyMUC1 is a transmembrane O-linked glycoprotein present on the apical surface of normal secretory epithelial cells. 1 The extracellular domain of MUC1 consists mainly of a variable number of tandem repeats 2 and it has a cytoplasmatic tail of 69 amino acids. 3 In the vast majority of human adenocarcinomas this protein is over-expressed and hypo-glycosylated 4 exposing an immunodominant repetitive amino acid sequence. The overproduction and secretion of MUC1 is correlated with the progression of breast, 5 ovarian 6,7 and colon 8 carcinoma. In breast cancer patients MUC1 serum levels are used to monitor therapy and for early detection of recurrences. 9,10 Proliferative responses to MUC1 and its tandem-repeat peptides have been demonstrated with peripheral blood mononuclear cells (PBMC) of patients with ovarian adenocarcinoma 11 and MUC1 specific cytotoxic T lymphocytes (CTL) have been isolated from tumor-draining lymph nodes of breast 12 and ovarian 13 cancer patients. In patients with ovarian, breast and pancreatic adenocarcinomas cytotoxic T cells can be induced that are specific for the MUC1 tandem repeat. 14,15 Recognition of MUC1 by T lymphocytes may be due to exposure of the immunogenic PDTRP region of the tandem repeat that is normally not exposed due to extensive O-glycosylation of the peptide core. The cytotoxic action of these T cells has also been described to be MHC-nonrestricted, due to crosslinkage to poorly glycosylated MUC1 tandem repeats on tumor cells. 16 Not only cellular but also humoral responses to MUC1 are found in breast, ovarian, colon and pancreatic carcinomas. [17][18][19] Moreover, the presence of immune-complexed MUC1 in breast cancer patients is related to a favorable disease outcome 20 and survival in early breast cancer patients is favorably influenced by a natural humoral immune response to MUC1. 21 These findings point to MUC1 and its repeat peptide as a target for immunotherapy of carcinomas. One possible approach is the use of MAbs against tumor-associated antigens, 22,23 another the use of tumorassociated antigens such as MUC1 as a vaccine and target for cellular and humoral immune responses.MUC1 MAbs have already been used in a number of vaccination trials as carrier molecule of radioactive elements 24,25 and recently a clinical trial has been set up with unlabeled humanized (Hu)HMFG-1 MAb for adjuvant therapy of breast cancer patients (Dr. D.W. Miles, Guy's Hospital, London). Also active vaccination trials with MUC1 tandem-repeat peptides 26 have been initiated. Recently, in 3 phase I vaccination trials executed by Dr. P.O. Livingston in the Memorial Sloan-Kettering Cancer Center, NY, breast cancer patients with no evidence of disease were repeatedly injected with a 30-mer 27,28 a 33-mer or a 106-mer MUC1 tandem repeat peptide conjugated to KLH plus adjuvant QS-21. In these patients high MUC1 IgG and IgM could be induced. 21 MAbs specific for tumor-associated antigens (TAA) can induce a complement-depende...