Frank G.M. Snijdewint

Summary Report on the ISOBM TD-4 Workshop: Analysis of 56 Monoclonal Antibodies against the MUC1 Mucin

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.

Survival in Early Breast Cancer Patients Is Favorably Influenced by a Natural Humoral Immune Response to Polymorphic Epithelial Mucin

PURPOSE Polymorphic epithelial mucin (PEM or MUC1) is being studied as a vaccine substrate for the immunotherapy of patients with adenocarcinoma. The present study analyzes the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relates the presence of these antibodies in pretreatment serum to outcome of disease. MATERIALS AND METHODS We measured immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to MUC1 with an enzyme-linked immunoassay (PEM.CIg), which uses a MUC1 triple-tandem repeat peptide conjugated to bovine serum albumin, 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 greater than the corresponding rounded-up median results obtained in the total breast cancer population. RESULTS 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 significant 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). CONCLUSION 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.

Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and n‐acetylgalactosamine (GalNAc) peptides

Antibodies (Abs) to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. We compared the specificity of natural and induced MUC1 Abs with the objective of defining an effective MUC1 vaccine for active immunotherapy of adenocarcinoma patients. Serum samples, selected out of a screened population of 492 subjects for their high levels of IgG and/or IgM MUC1 Abs, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients immunized 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 (1) overlapping hepta‐ and 20‐mer peptides spanning the MUC1 tandem repeat sequence; (2) two modified 60‐mer peptides with substitutions in the PDTR (PDTA) or in the STAPPA (STAAAA) sequence of each tandem repeat; and (3) four 60‐mer glycopeptides with each 1, 2, 3 and 5 mol N‐acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined, indicating that Abs 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 Abs was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG Abs predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 Abs 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 < 0.0001) in the MUC1 IgG and IgM Abs induced by MUC1 peptide vaccination. Whereas in cancer patients glycans appear to participate in epitope conformation, the epitope(s) recognized by MUC1 Abs induced by peptide vaccination are already masked by minimal glycosylation. Therefore, our results indicate that a MUC1 glycopeptide would be a better vaccine than a naked peptide. Int. J. Cancer 86:702–712, 2000.

Human MUC1 mucin: a multifaceted glycoprotein.

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.

Antibody-dependent cell-mediated cytotoxicity can be induced by MUC1 peptide vaccination of breast cancer patients

Human polymorphic epithelial mucin (PEM, MUC1) is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of glandular epithelium and is over‐expressed and hypo‐glycosylated in adenocarcinomas. The extracellular part of the molecule consists mainly of a variable number of 20 amino acid repeats that contain cryptic epitopes exposed in malignancy. The objective of our study was to determine whether humanized MUC1 MAbs and Abs induced by vaccination of breast cancer patients with MUC1 peptides can effect an antibody‐dependent cell‐mediated cytotoxicity (ADCC). An in vitro assay has been set up in which the breast tumor cell line ZR‐75‐1 is used as target and PBMC of healthy donors as effector cells. Different target and effector cells, as well as various MUC1 MAbs were tested to optimize the efficacy of the in vitro assay. The humanized MAb HuHMFG‐1, which recognizes the PDTR sequence in the MUC1 tandem repeat, induced a strong cell‐mediated cytotoxicity. Nine MUC1‐expressing tumor cell lines, including 3 bone marrow‐derived cell lines, as well as 2 MUC1‐transfected cell lines were susceptible to different extent to MUC1 Ab‐dependent killing. Large variations in the killing capacity of PBMC from healthy donors were found. The NK cells were the essential effector cells for the MUC1 Ab‐dependent killing. Plasma samples with induced high levels of MUC1 Ab were obtained from breast cancer patients repeatedly immunized with a KLH‐conjugated 33‐mer or 106‐mer MUC1 tandem repeat. Pre‐ and post‐vaccinated plasma samples of these patients were compared in the ADCC assay and it could be clearly demonstrated that the induced MUC1 Abs can effect tumor cell killing. MUC1 Ab‐dependent cell‐mediated tumor cell killing may occur in vivo and the ADCC assay can be applied to monitor MUC1 vaccination trials.

An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

Introduction: About one-third of breast and ovarian carcinoma patients have circulating antibodies reactive with polymorphic epithelial mucin (MUC1), either free or bound to immune complexes. While the presence of these immune complexes has prognostic significance in breast cancer patients, the significance of free MUC1 antibodies is less clear. The objective of this study was to develop a reliable assay for the accurate determination of circulating free antibodies to MUC1. Material and Methods: We developed an enzyme-linked immunosorbent assay (ELISA) (PEM.CIg) employing a 60 mer peptide (a triple tandem repeat sequence of the MUC1 peptide core) conjugated to bovine serum albumin and peroxidase-labeled antihuman immunoglobulin G or M antibodies. The assay was standardized and its analytical performance evaluated. A total of 492 serum samples were obtained from 40 healthy men, from 201 healthy women (including 55 women without a history of pregnancy and 45 pregnant women), and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients. MUC1 serum levels were determined with commercial CA 15-3 tests. Results: Circulating antibodies to MUC1 are present both in healthy subjects and in breast cancer patients. The within- and between-assay coefficients of variation 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. The inhibiting effect of modified 60 mer peptides suggests the presence of antibodies directed to more than one epitope. Conclusions: The PEM. CIg assay is a reliable ELISA for measuring free MUC1 antibodies in serum. We are in the process of relating the results obtained in the breast cancer group to disease outcome to evaluate its prognostic significance. In addition, the assay may become a useful tool for vaccine therapy monitoring.

Cellular and humoral immune responses to MUC1 mucin and tandem-repeat peptides in ovarian cancer patients and controls.

Abstract The objective of this study was to demonstrate the presence of proliferative T cell responses to human polymorphic epithelial mucin (MUC1) and its tandem-repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and to correlate these cellular responses to a humoral response to MUC1. PBMC were obtained from 6 healthy women, from 13 women in the third trimester of pregnancy and from 21 ovarian cancer patients. Only 1 of the 6 healthy women showed a weak primary proliferative response (stimulation index, SI<2) 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 ≤ 3.0) and in PBMC from 8/15 ovarian cancer patients (53%) 20-mer and/or 60-mer MUC1 tandem-repeat peptides induced primary responses (SI ≤ 5.4). MUC1 mucin purified from a breast tumor cell line and/or from urine of a healthy donor had a relatively strong stimulating effect (SI ≤ 19) on PBMC from 4 of 16 ovarian cancer patients (25%). In contrast, in PBMC of 9 ovarian cancer patients stimulated by the addition of a Candida albicans extract, MUC1 mucin strongly inhibited proliferation. This inhibition could partially be abrogated by the addition of IL-2. MUC1 (CA 15.3 assay) and free circulating MUC1 IgG and IgM antibodies (PEM.CIg assay) were determined in the plasma of all subjects. 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 Ab 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.

‘Epitope Fingerprinting’ Using Overlapping 20-mer Peptides of the MUC1 Tandem Repeat Sequence

The ISOBM TD-4 Workshop antibodies 122–177 were tested for reactivity with 20 overlapping MUC1 tandem repeat 20-mer peptides by an ELISA, in order to determine the complete amino acid sequences of the epitopes. Of the 56 antibodies studied, 30 showed specific binding and thus the epitopes were characterized. The epitopes appear to be ‘broader’ when compared to those deduced from studies using smaller peptides. Interassay variation is remarkably small, allowing for precise grouping of clusters with very similar epitope patterns. Five groups of antibodies show remarkable similarity: BC3 and VU-4-H5; BC4W154, C595 and Mc5; MF06 and B27.29; VU-11-D1 and VU-11-E2; Ma552, VU-3-C6, 7540MR and BC4E549. We have used the term ‘epitope fingerprinting’ to refer to the ‘fine structure’ of the epitope with all its essential and flanking amino acids. We believe this method is more precise than the usual epitope mapping with short peptides.

Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1

A monoclonal antibody (MAb), VU-2-G7, was generated against a synthetic 60-mer MUC1 triple tandem repeat peptide with N-acetyl-galactosamine (GalNAc) O-linked to the threonine in the PDTR region of each repeat (3M GalNAc). VU-2-G7 and 8 MUC1 MAbs (VU-3-C6, VU-4-H5, 139H2, A76-A/C7, VU-12-E1, BCP9, MF11 and BW835) were tested against various glycosylated and nonglycosylated MUC1 tandem repeat peptides. VU-2-G7 showed strong reactivity with its immunogen, 3M GalNAc, and much lower reactivity with the nonglycosylated 60-mer MUC1 triple tandem repeat peptide. VU-2-G7 showed no reactivity with a 60-mer MUC1 triple tandem repeat peptide modified at the PDTR region or with a 60-mer MUC1 triple tandem repeat peptide with 3 GalNAc per repeat outside the PDTR region (9M GalNAc). In ELISA and flow cytometry, VU-2-G7 ubiquitously reacted with 4 MUC1-expressing breast cancer and 2 ovarian cancer cell lines and with a MUC1-gene-transfected Chinese hamster ovary cell line. The reactivity of VU-2-G7 was always higher than that of VU-4-H5, raised against a nonglycosylated 60-mer MUC1 triple tandem repeat peptide. Immunohistochemical staining of paraffin sections of breast and ovarian tumor tissues showed strong binding of VU-2-G7 predominantly at the cell membrane. The dominant epitope of VU-2-G7 is in the glycosylated PDTR motif of the MUC1 tandem repeat, and this epitope is abundantly present on the surface of tumor cell lines and breast and ovarian tumor tissues. Given the ubiquitously aberrant glycosylation of MUC1 in malignant cells, the production of MAbs against highly purified glycosylated MUC1 tandem repeat peptides may yield MAbs better suited for the immunotherapy of carcinomas than those available at the moment.