Immune Responses to Human MUC1 Mucin: Relevance for Cancer Immunotherapy

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Summary

Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells [Chapter I]. 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 favouring metastases. On the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses to it. Tumor-associated MUC1 has short carbohydrate side-chains and exposed epitopes on its peptide core; it gains access to the circulation and comes in contact with the immune system provoking humoral and cellular immune responses to it. Natural antibodies to MUC1 are present in the circulation of cancer patients, both as free antibodies [see Chapter IV] and as immune complexes, bound to MUC1 [see Chapter II]. MUC1 is now being tested in various laboratories as a target antigen for the immunotherapy of carcinomas, but evidence that these immune responses may be effective against the tumor in humans is still scarce.

Chapter II describes the development of an enzyme-linked immunosorbent assay, based on a murine monoclonal antibody (139H2) directed to the PDTR sequence of the MUC1 tandem repeat, for the detection of immune complexes containing MUC1 (PEM.CIC) in the bloodstream. Immunoglobulins bound to the mucin were detected with horseradish peroxidase-conjugated goat anti-human Ig (G + M + A) immunoglobulins. Circulating immune complexes (ClCs) containing MUC1 were found in sera of 24.5% of 151 primary breast carcinoma patients and in 18 and 21.4%, respectively, of patients with advanced breast (n = 61) and ovarian (n= 56) carcinomas, and in 37% of patients with benign breast tumours, but in only 2.1 % of 96 healthy individuals.

The study contained in Chapter III investigates the clinical significance of the presence of circulating immune complexes containing MUC1 in pretreatment serum samples obtained from 140 breast cancer patients. Measurements in 96 healthy women defined an upper level of normal, excluding 98 % of the healthy population, of 120 Optical Density Units (O.D.) x 10³. In breast cancer patients an inverse correlation between positivity for PEM.CIC and extent of disease at diagnosis was present: the highest frequency of positivity for PEM.CIC was found in patients with a carcinoma in situ while all patients with distant metastasis at first diagnosis were PEM.CIC negative. Only one out of 15 patients with tumor in more than 5 axillary lymph nodes had positive PEM.CIC levels in pretreatment serum. Twenty-three out of the 28 patients (82%) that relapsed during the observation period were PEM.CIC negative. Patients with pretreatment elevation of CA 15.3 and a concomitant elevation of PEM.CIC suffered no relapses during the observation period. These preliminary results in a small cohort of patients suggested that humoral immune response to MUC1 protects against disease progression.

In Chapter IV we describe the development of an enzyme-linked immunosorbent assay for the measurement of circulating free antibodies to MUC1 that has become an useful tool for the detection of natural humoral responses to MUC1 and for MUC1 vaccine therapy monitoring. The assay is based on a triple tandem repeat MUC1 peptide conjugated to BSA. This length of peptide was chosen because studies by other groups demonstrated that the immunodominant region for the mouse, PDTRP, attains its native conformation in synthetic peptides with more than two repeats. We tested a total of 492 serum samples, obtained from 40 healthy men, 201 healthy women and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients, for the presence of IgG or IgM bound to the peptide (as catcher) with horseradish peroxidase-labelled rabbit anti-human IgG or IgM immunoglobulins (as tracer). Circulating antibodies to MUC1 are present both in healthy subjects and in patients with breast cancer. The assay was standardised and its analytical performance was assessed.  The within- and between-assay coefficients of variation (CV) were, respectively, 2% and 12% for the IgG determinations and 1.2% and 3% for the IgM determinations. Correlation coefficients for serially diluted serum samples ranged from 0.9998 to 0.9920 for IgG and from 0.9996 to 0.9818 for IgM determinations. The reactivity of serum samples was partially blocked by the addition of various MUC1 peptides and by MUC1 mucin. Results of inhibition tests with modified triple tandem repeat peptides suggest the presence of an­tibodies directed to more than one epitope.

In Chapter V we analyse the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relate their presence in pretreatment serum to outcome of disease, in addition shedding light on the utility of MUC1 as a vaccine substrate for the immunotherapy of adenocarcinoma patients. IgG and IgM antibodies to MUC1 were measured with the assay described in Chapter IV in pretreatment serum samples obtained from 154 breast cancer patients (52 with stage I disease and 102 with stage II) and 302 controls. The median disease-specific survival time of breast cancer patients was 74 months (range, 15 to 118 months). A positive test result was defined as MUC1 IgG or IgM antibody levels equal to or higher than the corresponding median results obtained in the total breast cancer population. A positive test result for both MUC1 IgG and IgM antibodies in pretreatment serum was associated with a significant benefit in disease-specific survival in stage I and II (P = .0116) breast cancer patients. Positive IgG and IgM MUC1 antibody levels had additional prognostic value to stage (P = .0437) in multivariate analysis. Disease-free survival probability did not differ significantly. However, stage II patients who tested positive for MUC1 IgG and IgM antibody and who relapsed had predominantly local recurrences or contralateral disease, as opposed to recurrences at distant sites in the patients with a negative humoral response (P = .026). The results of this retrospective study indicate that 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. The possible role of MUC1 antibodies in controlling haematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1 expressing tumor cells will be further elucidated in a prospective study in breast cancer patients.

Antibodies to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. The study in Chapter VI compares the specificity of natural and induced MUC1 antibodies with the objective of defining an effective MUC1 vaccine for active immunotherapy of patients with adenocarcinoma. Serum samples, selected out of a screened population of 492 subjects (see Chapter IV) for their high levels of IgG and/or IgM antibodies to MUC1, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients taking part in two phase I vaccination trials (Memorial Sloan-Kettering Cancer Center, NY) with MUC1 peptides (coupled to keyhole limpet hemocyanin (KLH) and mixed with QS-21). The samples were tested with enzyme-linked immunoassays for reactivity with (a) overlapping hepta- and 20-mer peptides spanning the MUC1 tandem repeat sequence; (b) two modified 60-mer peptides with substitutions, one in the PDTR (PDTA) and the other in the STAPPA  (STAAAA) sequence of each tandem repeat and (c) four 60-mer glycopeptides with each one, two, three or five molecules N-acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined in 13/31 MUC1 IgG and 5/18 MUC1 IgM positive samples, indicating that antibodies directed to more than one region of the MUC1 peptide core can coexist in one and the same subject. The most frequent minimal epitopic sequence of natural MUC1 IgG and IgM antibodies was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG antibodies predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 antibodies from breast cancer patients reacted more strongly with the N-acetylgalactosamine (GalNAc) peptides than with the naked 60-mer peptide, while reactivity with the GalNAc-peptides was significantly reduced (2-tailed P < .0001) in the MUC1 IgG and IgM antibodies induced by MUC1 peptide vaccination. The results of this study indicate that, whereas in cancer patients glycans appear to participate in epitope conformation, the epitopes recognized by MUC1 antibodies induced by peptide vaccination are already masked by minimal glycosylation. As glycosylation, albeit aberrant, is still present in the tumor-associated mucin, we conclude that the optimal MUC1 vaccine is not a peptide but a glycopeptide, possibly a 33-mer peptide ending with RPAPGS at the C-terminal end and with GalNAcs attached to the threonine in the PDTR sequence and to the serine and threonine in the STAPPA sequence of the repeat.

Chapter VII explores the presence of proliferative T cell responses to MUC1 and its tandem repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and correlates these cellular responses to humoral response to MUC1. Only 1 of 6 healthy women showed a weak primary proliferative (stimulation index, SI < 2) response to a 20-mer MUC1 tandem repeat peptide in the presence of interleukin-2 (IL-2). In PBMC from 5/13 pregnant women (38%) a weak response could be induced by the 20-mer and/or 60-mer tandem repeat peptides (SI £ 3). Primary responses to the 20-mer and/or 60-mer tandem repeat peptides (SI £ 5.4) could be induced in PBMC from 8/15 ovarian cancer patients (53%). Purified MUC1 mucin had a relatively strong proliferative effect (SI £ 19) on PBMC from 4/16 ovarian cancer patients (25%). In contrast, MUC1 mucin strongly inhibited proliferation in PBMC of 9 ovarian cancer patients stimulated by the addition of Candida albicans extract. This inhibition could partially be abrogated by the addition of IL-2. The MUC1 and the free circulating MUC1 IgG antibody plasma levels were significantly higher in the ovarian cancer patients than in the healthy women. Although no significant correlations were found between MUC1 mucin, MUC1 antibody plasma levels and the individual proliferative responses to the MUC1 antigens, an association may exist between them, since all three are significantly higher in the ovarian cancer patients than in the healthy women.

In Chapter VIII the results of the studies presented in this thesis are discussed in conjunction with some forms of immunotherapy of cancer.