Search

MUC1 is a potential target in breast cancer immunotherapy as MUC1 is overexpressed in breast cancer, and is absent or expressed in low level in normal mammary gland. In addition, MUC1 is mostly aberrantly underglycosylated in cancer and the antigens on the cancer surface are different from normal cell. Therefore targeting MUC1 for cancer immunotherapy can exploit the difference between cancer and normal cells, and eliminating the cancerous cells while leaving the normal mammary cells unharmed. This review will focus on the recent advance of MUC1 breast cancer immunotherapy currently being investigated.

Introduction: Cripto is a founding member of the EGF-CFC family, and plays an important role in tumourigenesis, tumour cell proliferation and migration. We aimed to determine the significance of Cripto expression on the survival of patients with breast cancer., Methods: Immunohistochemical detection of Cripto was performed by using mAb C13 on 120 formalin-fixed paraffin-embedded breast tumour specimens in tissue microarrays. This cohort comprises a series of 120 patients with primary operable breast cancer diagnosed between 1989 and 1995, retrieved from the Concord Repatriation General Hospital breast carcinoma database., Results: Using a cutoff value of 80%, Cripto overexpressed in 57 of the 120 (47.5%) patients. We found significant associations between overexpression of Cripto and the Nottingham Prognostic Index (NPI, p<0.01), histological grade (p<0.01), pathological tumour type (p=0.04), PR (p=0.02) as well as Ki-67 (p=0.02). Univariate analysis reveals that there is a significant correlation between overexpression of Cripto and survival (p=0.0003). Cox regression analysis indicates that the overexpression of Cripto is an independent prognostic factor in breast cancer (HR 2.79, 95%CI 1.20-6.50)., Conclusion: The unique epitope recognized by mAb C13 is overexpressed on breast tumour tissues. In this series of invasive breast cancers, overexpression of Cripto was more often found in high grade and poor prognosis tumours compared to low grade and good prognosis breast cancers. Moreover, overexpression of Cripto was significantly associated with decreased patient survival.

Mucin 1 (MUC1) is a large complex glycoprotein that is highly expressed in breast cancer, and as such could be a target for immunotherapy. In mice, human MUC1 is highly immunogenic, particularly when conjugated to mannan, where a high frequency of CD8(+) MHC-restricted cytotoxic T lymphocytes is induced, accompanied by tumor protection. On this basis, a clinical trial was performed in which 25 patients with advanced metastatic carcinoma of breast, colon, stomach, or rectum received mannan-MUC1 in increasing doses. After 4 to 8 injections, large amounts of IgG1 anti-MUC1 antibodies were produced in 13 out of 25 patients (with antibody titers by ELISA of 1/320-1/20,480). Most of the antibodies reacted to the epitopes STAPPAHG and PAPGSTAP. In addition, T cell proliferation was found in 4 out of 15 patients, and CTL responses were seen in 2 out of 10 patients. Mannan-MUC1 can immunize patients, particularly for antibody formation, and to a lesser extent, cellular responses. It remains to be seen whether such responses have antitumor activity.

Mucin 1 (MUC1) is highly expressed in breast cancer, has an ubiquitous distribution and, due to altered glycosylation, peptides within the VNTR are exposed. These peptides are the target for anti-MUC1 antibodies, which give a differential reaction on cancer compared with normal tissue. The amino acids, APDTR or adjacent amino acids, are highly immunogenic in mice for antibody production (after immunisation with either breast cancer cells, human milk fat globule (HMFG) or the VNTR peptide). In addition, human studies show that this region of the MUC1 VNTR functions as target epitopes for cytotoxic T cells. We have performed preclinical and clinical studies to examine the immune responses to MUC1 in mice and humans: (a) MUC1+ 3T3 or P815+ 3T3 cells in syngeneic mice are rejected, with the generation of both cytotoxic T lymphocyte (CTL) and DTH responses and a weak antibody response and a weak antibody responses; this type of immunity gives rise to total resistance to re-challenge with high doses of these tumors; (b) immunisation with peptides (VNTR x 2), a fusion protein (VNTR x 5), or HMFG leads to no CTLs, DTH, good antibody production and weak tumour protection (to 10(6) cells, but not 5 x 10(6) cells) (possibly a TH2 type response); (c) immunisation with mannan-fusion protein (MFP) gives rise to good protection (resistance to 50 x 10(6) cells), CTL and DTH responses and weak antibody responses (possibly a TH1 type response, similar in magnitude to that obtained after tumor rejection); (d) established tumors can be rapidly rejected by delayed treatment of MFP; (e) the CTL responses are MHC restricted (in contrast to the human studies); (f) APDTR appears not to be the T cell reactive epitope in mice. On the basis of these findings, two clinical trials are in progress: (a) VNTR x 2 (diphtheria toxoid) which gives rise to some T cell proliferation, DTH and antibody responses in some patients and (b) an MFP trial. The ability to alter the immune response towards cellular immunity with mannan or to humoral immunity with peptides, allows the immune response to be selectively manipulated.

Breast cancer is considered as the major cause of mortality by cancer for women. Even if chemotherapy, radiotherapy and surgery have improved the life expectancy of patients bearing tumours, breast cancer is responsible for the death of 42,000 women per year in USA and 25,000 women in France. In this context, cancer vaccines may add an attractive alternative therapeutic strategy to the current existing treatments. We describe here the construction of recombinant vaccinia viruses co-expressing a tumour associated antigen (MUC 1) and an "adjuvant" cytokine, which have potential applications in the active immunotherapy of breast cancer. Indeed, recombinant vaccinia viruses have been extensively used during the past decade to induce a protective response against a whole variety of pathogens, and has proven to be of great value in the elicitation of a cellular immune response leading to the rejection of tumour grafts in mouse models.

Two murine Mabs VA1(IgG1) and VA2(IgG1) were produced against a bacterial fusion protein comprising glutathione S-transferase and five tandem repeats of the MUC1 protein. Using the immunoperoxidase staining technique, VA1 detected 46/53 and VA2 detected 48/53 breast cancers and both also reacted with a range of other human epithelial carcinomas. In addition VA1 gave weak reactions with normal breast tissues whereas VA2 was non-reactive and could be a relatively tumour specific antibody for breast cancer. The antibodies were also tested by ELISA-VA1 reacted weakly with glycosylated HMFG but strongly with deglycosylated HMFG, whereas VA2 reacted strongly with both forms of HMFG. The reactivities of the two Mabs with synthetic peptides of the MUC1 tandem repeat were used to map the epitopes recognised by VA1 (amino acids RPAPGS) and VA2 (amino acids DTRPA). The use of fusion proteins provides another means of immunisation to produce anti-tumour antibodies.

Anti-breast cancer antibodies (BC2, HMPV and 4B6) and an anti-ovarian cancer antibody (OM1) were found to react with mucins--indeed with the protein core encoded by the MUC1 gene. This gene contains a VNTR (variable number of tandem repeats) encoding a 60 bp (= 20 amino acids) repeat sequence and within this amino acid sequence SAPDTRPAP was predicted, by hydrophilicity analysis, to be the immunogenic peptide sequence. The four antibodies were shown to react with MUC1 VNTR encoded peptides in direct binding and inhibition studies. The precise reactivity of the 4 mAbs was mapped using ELISA in both solid and liquid phase, and demonstrated the epitopes to be: APDTR (BC2 and HMPV), PDTR (4B6) and DTRPA (OM1). By using the pepscan method, the epitopes were shorter (PDTR, DTR and DTRP). However when these short peptides (except DTR) were synthesized they did not react; flanking amino acids are needed for the epitopes. Clearly several different methods should be used to define the reactive epitope. Within (S)APDTR, major amino acid substitutions could be made--even of three to four amino acids without altering antibody binding, provided that P and R were not substituted. It was of interest that an anti-ovarian cancer antibody gave similar anti-peptide reactions to the anti-breast cancer antibodies; apparently MUC1 peptides in ovarian cancer are the same as in breast cancer.

Monoclonal anti-mucine antibodies BC1, BC2 and BC3 produced using human milk fat globule membrane react with a synthetic peptide p1-24 (PDTRPAPGSTAPPAHGVTSAPDTR) representing the repeating amino acid sequence of the mucin core protein. The minimum epitope recognized by these three monoclonal antibodies (mAb) in p1-24 was contained in the five amino acids APDTR. To analyse the variation of position of the epitope, various modifications of the APDTR sequence were made by synthesizing peptides and testing by direct binding and inhibition enzyme-linked immunosorbent assays. Firstly, peptides p13-32 and C-p13-32, in which the epitope APDTR was placed in the middle instead of the C-terminal as in p1-24, were examined. These peptides had a greater reaction with mAb BC1, BC2 and BC3 compared with the reaction with p1-24. Secondly, A-p1-24 and TSA-p1-24 were made wherein two APDTR epitopes were present--these peptides were shown to bind two IgG antibody molecules. Finally, the contribution of each amino acid in the APDTR epitope was studied using the pepscan polyethylene rods, making all 20 of the amino acid substitutions in each position for SAPDTR (the minimum epitope APDTR with an adjacent amino acid S). In the 120 peptides examined there were some 'permissible' substitutions in A, D and T but not in P or R for BC1 and BC2; there were more 'permissible' substitutions for BC3; different substitution patterns were found with each antibody and some substitutions gave an increased reaction compared with the native peptide SAPDTR. The studies are of value in analysing the reaction of antibodies with epitopes expressed in breast cancer and in determining the antigenicity of synthetic peptides.

This study reports the characterization of a breast cancer-associated antigen identified by murine monoclonal antibody (MoAb) RCC-1 (formerly called 24-17.1). Immunoperoxidase staining indicated that RCC-1 recognized an antigen highly expressed in malignant tumours of breast origin, and no reactivity was noted with connective tissue, muscle or lymph nodes, which is an important consideration in its successful use in immunolymphoscintigraphy. The RCC-1 was shown to consist of 94,000 dalton disulfide-bonded dimers which were shown to be different from the transferrin receptor. In addition, the antibody RCC-1 did not react with components of human milk or with an antigenic peptide derived from the core protein of a mammary mucin. Chemical treatment and enzymatic digestion suggested that the epitope recognized by antibody RCC-1 was protein as it was resistant to neuraminidase and periodate treatment but was sensitive to trypsin. The RCC-1-defined antigen detects a novel breast cancer associated antigen.

Mucins are heavily glycosylated proteins that are produced in excessive amounts in breast cancers and other adenocarcinomas. These proteins are potent immunogens; indeed, most monoclonal antibodies raised against extracts of breast cancer cells or tumors are directed toward a single family of mucins. The anti-mucin antibodies currently available are valuable diagnostic aids for detecting advanced stages of breast cancer. To improve the sensitivity of these antibodies so that they might be suitable for use in screening and early diagnosis, tumor imaging, and therapy of breast cancer, recent studies have focused on identifying the precise epitopes they recognize.

The monoclonal antibody (Mab) OM-1, which defines the ovarian carcinoma-associated sebaceous gland antigen (SGA), and Mab F36/22, which defines the ductal carcinoma antigen (DCA), were tested for reactivity against a synthetic peptide representing the repeat twenty amino acid sequence of the human polymorphic epithelial mucin core protein plus four amino acids of the adjacent sequence (p1-24). OM-1 bound strongly to the peptide by direct dot blot assay and ELISA and the minimum epitope for OM-1 was shown to be APDTRP(A) by inhibition assay. F36/22 reacted weakly with the peptide under the same conditions and its affinity for peptide in solution was relatively very low. Mab OC125, which defines the ovarian cancer-associated antigen CA125, did not react with the p1-24 peptide. Five other anti-mucin Mabs (HMFG1, HMFG2, BC1, BC2, BC3), previously shown to bind to the p1-24 peptide, reacted strongly with SGA by direct binding and in a sandwich assay with OM-1 as the capture Mab. F36/22 was weakly positive under the same conditions suggesting that both peptide and SGA do not express the optimal epitope for F36/22 binding. These results indicate that SGA and possibly DCA have the repeat sequence core protein of the breast carcinoma-associated human polymorphic epithelial mucin.

Three murine monoclonal antibodies (BC1, BC2 and BC3) were developed against human milk fat globule membrane (HMFGM). By immunoperoxidase staining, it was found that the antigenic determinants had a predominant distribution in breast cancer tissue. In addition, the antibodies reacted preferentially with mucin derived from human milk rather than that derived from the breast cancer cell line ZR75; they also recognized polymorphic high molecular weight components (MW greater than or equal to 230,000) in serum and in human milk fat globule membrane. Thus the antibodies appear to react with a component of the family of mucins found in breast cancer and human milk and it appears likely that at least part of each epitope is protein in nature. Antibodies BC1, BC2 and BC3 recognized related but not identical epitopes, and they appear to be co-expressed on the same molecules as 3E1.2-defined antigen (mammary serum antigen, MSA) which is also a member of the family of breast cancer-related mucin. However, the 3E1.2 epitope is distinct and non-cross-reactive with those described for BC1, BC2 and BC3. The BC2 and BC3 defined epitopes were examined for their value in serum assays. Immunoassay was developed with a combination of two antibodies, using antibody BC3 for antigen capture and antibody BC2 or 3E1.2 for antigen detection and gave reasonable sensitivity (approximately 85%) and specificity (approximately 95%) in such serum tests for breast cancer. In a limited study, these tests appeared to complement the MSA test in the detection of breast cancer.

A member of the high molecular weight glycoproteins of human milk and breast cancer was isolated from the sera, ascites and breast carcinoma tissue of patients with breast cancer using monoclonal antibody 3E1.2. The 3E1.2 defined antigen, termed mammary serum antigen (MSA) was obtained by immunoaffinity chromatography and a solid phase immuno-precipitation technique (SPIT) from serum of patients with metastatic breast cancer. MSA was found to be a high molecular weight glycoprotein with a Mr greater than 300,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a native Mr approximately 1 x 10(6) by gel filtration chromatography; in accord with the published Mr of other high molecular weight glycoproteins obtained from human milk and breast cancer. A high degree of glycosylation of MSA molecule was shown by its poor staining with Coomassie blue but good staining in a PAS-silver stain. In addition, MSA contained N-acetyl neuraminic acid and N-acetyl glucosamine as indicated by its binding to wheat-germ agglutinin. The epitope defined by antibody 3E1.2 is sensitive to treatment by sodium periodate and neuraminidase, implying that both carbohydrate and sialic acid are required for binding of antibody 3E1.2. Sandwich immunoassays demonstrated that MSA+ molecules are likely to express repeated 3E1.2 defined epitopes. Furthermore, MSA was susceptible to degradation by pronase, subtilisin and proteinase K and gave a different peptide profile from that of the PAS-O glycoprotein of human milk. MSA+ molecules were found to carry epitopes for a number of other monoclonal antibodies which were reactive with the PAS-O glycoprotein. It is suggested that MSA has the same core protein as is recognised by antibody DF3 which has been used to clone the same cDNA as was cloned with antibodies HMFG-1, HMFG-2 and SM-3. However, the epitope detected by the 3E1.2 antibody is either absent or weakly expressed on human milk, human milk-fat globule membrane (HMFGM) or deglycosylated HMFGM--all of which react strongly with various anti-HMFG antibodies. The antibody 3E1.2 thus recognises a unique epitope of the high molecular weight glycoproteins of human milk and breast cancer, being found in cancer tissue, serum and ascitic fluid of patients with breast cancer but weakly expressed or absent in human milk.