Advances of MUC1 as a target for breast cancer immunotherapy.

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.

Anti-Cripto Mab inhibit tumour growth and overcome MDR in a human leukaemia MDR cell line by inhibition of Akt and activation of JNK/SAPK and bad death pathways.

Doxorubicin (DOX) selection of CCRF-CEM leukaemia cell line resulted in multidrug resistance (MDR) CEM/A7R cell line, which overexpresses MDR, 1 coded P-glycoprotein (Pgp). Here, we report for the first time that oncoprotein Cripto, a founding member of epidermal growth factor-Cripto-FRL, 1-Criptic family is overexpressed in the CEM/A7R cells, and anti-Cripto monoclonal antibodies (Mab) inhibited CEM/A7R cell growth both in vitro and in an established xenograft tumour in severe combined immunodeficiency mice. Cripto Mab synergistically enhanced sensitivity of the MDR cells to Pgp substrates epirubicin (EPI), daunorubicin (DAU) and non-Pgp substrates nucleoside analogue cytosine arabinoside (AraC). In particular, the combination of anti-Cripto Mab at less than 50% of inhibition concentrations with noncytotoxic concentrations of EPI or DAU inhibited more than 90% of CEM/A7R cell growth. Cripto Mab slightly inhibited Pgp expression, and had little effect on Pgp function, indicating that a mechanism independent of Pgp was involved in overcoming MDR. We demonstrated that anti-Cripto Mab-induced CEM/A7R cell apoptosis, which was associated with an enhanced activity of the c-Jun N-terminal kinase/stress-activated protein kinase and inhibition of Akt phosphorylation, resulting in an activation of mitochondrial apoptosis pathway as evidenced by dephosphorylation of Bad at Ser136, Bcl-2 at Ser70 and a cleaved caspase-9.

Overexpression of Cripto and its prognostic significance in breast cancer: a study with long-term survival.

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.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Anti-mucin monoclonal antibodies.

Mucins are of major interest in cell biology, not only are they highly over-expressed in many adenocarcinomas (up to 40-fold increase), but also have important physiological function, and probably more to be determined (1-3). There is much information available on mucins - doubtless because of their unusual structure being heavily glycosylated, but also containing a repeat region rich in the amino acids serine, threonine and proline. This repeat region confers high immunogenicity of the mucins, and as a result, many antibodies (Abs) have been made to mucins of different species (4). Furthermore, the production of Abs led to the cloning of the cDNAs and armed with these reagents (antibodies, cDNA and genomic structures), advances in the knowledge of the structure and function of mucins has been rapid, together with the development of transgenic and gene knockout animals for biological studies (1-9). Here we describe monoclonal antibodies (Mabs) made to the different mucins, including Mucins 1-4, concentrating on human Mucin 1 (MUC1), to variants of MUC1, to regions outside the VNTR of MUC1, mouse Mucin1 (muc1), unusual features and cross reactions of anti-MUC1 Mabs and Abs made by patients in clinical trials. We will especially describe the Mabs produced in our laboratory.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

BACKGROUND: Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. METHODS: Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. RESULTS: The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. CONCLUSIONS: These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Glycoconjugate metabolism in a cystic fibrosis knockout mouse model.

Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.

Breast cancer in mice: effect of murine MUC-1 immunization on tumor incidence in C3H/HeOuj mice.

Mucin-1 (MUC-1), which is overexpressed in more than 90% of human breast cancers, is a potential target for immunotherapy. To develop a mouse model appropriate for the immunotherapy of human cancer, mouse mucin-1 (muc-1) fusion protein, containing ten tandem repeats, was made and used to immunize C3H/HeOuj mice, which supposedly have a high incidence of breast cancer. C3H/HeOuj mice were injected eight times with 5 microg oxidized mannan muc-1-glutathione-S-transferase (MMFP) with or without cyclophosphamide, which is used to increase cellular immunity. At 80 age weeks, only 12.1% (4 of 33) mice of the untreated C3H/HeOuj mice had mammary tumors. The reason for the low incidence of breast cancer in these mice is not known, but all the mammary tumors were MUC-1+ breast adenocarcinomas and were transplantable to C3H/HeOuj mice. The incidence was 11.4% (4 of 35) in mice injected with MMFP: 38.2% (13 of 34) in mice given cyclophosphamide; and 14.3% (2 of 14) in mice treated with glutathione-S-transferase. That is, cyclophosphamide increased the incidence of mammary tumors, and metastases were found in only these mice. Fewer tumors (6 of 34 or 17.6% compared with 13 of 34 or 38.2% with cyclophosphamide only) occurred in the group immunized with MMFP and cyclophosphamide. Mice immunized with MMFP had high levels of muc-1 antibodies and cellular immune responses (the frequency of the precursor of the cytotoxic Tlymphocyte cell was 1 of 40,000 to 1 of 100,000), which were not found in control groups. The occurrence of muc-1 immunity, particularly the presence of large amounts of anti-mucin-1 antibodies, had no effect on tumor incidence. Thus, the immunization with murine muc-1 reduced the tumor incidence in only cyclophosphamide-treated mice and led to strong muc-1 antibody production and to cellular responses. These findings have implications for human tumor immunotherapy in which strong antibody and weak cellular responses are to be expected and, indeed, have been found.

Development of a fecal occult blood test using a monoclonal antibody to haptoglobin.

Monoclonal antibodies (mAbs) were produced to human haptoglobin by immunising with fecal extracts from patients with colon cancer. An enzyme-linked immunosorbent assay was developed with one of the mAbs (FE14.1), and its ability to diagnose colorectal carcinoma evaluated. Patients with colorectal cancer were positive (43/46 = 93.5%) compared to normal individuals (4/211 = 1.9%). The assay has a specificity 93.5% and sensitivity 98.1% and has several advantages over current fecal occult blood tests. The test is potentially useful for bowel cancer diagnosis and to quantitate the level of haptoglobin in other body fluids such as urine and in effusions.

Construction and characterization of a chimeric receptor containing the cytoplasmic domain of MUC1 mucin.

MUC1 mucin is a transmembrane glycoprotein that is highly expressed in various cancer cell lines and is also present in most of the glandular epithelial cells including the airway. Although the presence of numerous phosphorylation sites in its cytoplasmic domain suggests its potential role as a receptor, the unavailability of a ligand for MUC1 mucin has limited our understanding of its function. In this paper, we tried to circumvent this problem by constructing a chimeric receptor containing the cytoplasmic domain of MUC1 mucin, which can be phosphorylated on activation. To this end, we constructed a chimeric plasmid vector (pCD8/MUC1) by replacing the extracellular and transmembrane domains of human MUC1 mucin with those of human CD8. Transient transfection of the vector into COS-7 cells resulted in expression of the chimeric receptor on the surface of the COS-7 cells as judged by immunologic assays with various antibodies as well as by fluorescence-activated cell-sorting analysis. Treatment of the transfected COS-7 cells with an anti-CD8 antibody resulted in a significant increase in phosphorylation of tyrosine moieties of the chimeric receptor. This chimeric receptor will serve as a powerful tool in elucidating the signaling mechanism as well as the functional role of MUC1 mucin in the airway.

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Expression of mucin 1 (MUC1) in esophageal squamous-cell carcinoma: its relationship with prognosis.

Using 2 anti-mucin 1 (MUC1) monoclonal antibodies (MAbs), DF3 and BCP8, we examined MUC1 expression immunohistochemically in 192 esophageal squamous-cell carcinomas (SCCs). In normal squamous epithelium of the esophagus, DF3 was not expressed, but BCP8 was expressed on the cell membrane, mainly in the surface layer. In esophageal SCCs, DF3 and BCP8 were expressed mainly on the cell membrane of SCC cells, but also in the cytoplasm in several cases. To analyze the correlation of MUC1 expression and the prognosis of the patients, the 192 cases were divided into 2 groups: high-expression group (HEG, > 50% of the neoplastic cells stained) and low-expression group (LEG, < 50% of neoplastic cells stained). DF3-HEG (24 patients) showed a significantly poorer survival rate than DF3-LEG (168 patients), whereas there was no significant difference in survival between BCP8-HEG (43 patients) and BCP8-LEG (149 patients). Also, in the analysis of 162 patients with advanced stage (submucosal or deeper invasion) to exclude the influence of low expression of DF3 and BCP8 in 30 patients with early stage (up to the level of muscularis mucosae), DF3-HEG (24 patients) showed significantly poorer survival than DF3-LEG (138 patients), whereas there was no significant difference in survival between BCP8-HEG (42 patients) and BCP8-LEG (120 patients). The results of our study on esophageal SCC suggest that the expression of sialyl oligosaccharides detected by DF3 is related to poor prognosis.

Expression of MUC2 gene in gastric regenerative, metaplastic, and neoplastic epithelia.

It has been reported that MUC2 mucin is expressed in goblet cells of gastric intestinal metaplasia, but not in its normal epithelium. To confirm this finding, we have examined the expression of the MUC2 gene by reverse transcriptase-polymerase chain reaction and immunohistochemical methods in gastric tissues obtained by routine upper gastrointestinal tract endoscopy and compared the results with pathological findings based on hematoxylin and eosin (H&E) staining. In 16.7% of the tissue specimens tested, MUC2 mRNA was detected in spite of the absence of intestinal metaplasia in HE specimens. A possible explanation for this was the identification by immunohistochemistry of MUC2 protein in regenerative gastric mucosal cells in biopsies that did not contain intestinal metaplasia. Sialyl-Le(x) epitope, which is suggested to be located on MUC2 mucin core protein (MUC2 protein), was also immunohistochemically detected in both goblet cells of intestinal metaplasia and regenerative epithelium. With regard to carcinoma, MUC2 protein was predominantly expressed in intestinal-type adenocarcinoma. These data indicate that MUC2 mucin is expressed in gastric regenerative epithelium in addition to intestinal metaplasia and intestinal type adenocarcinoma.

Oxidised mannan antigen conjugates preferentially stimulate T1 type immune responses.

It is desirable to be able to produce either T1 or T2 responses and we have found that, in mice, mannose--coupled antigens stimulated T2 type responses antibodies and CTLs, whereas if oxidized, mannose--coupled antigens stimulated T1 responses little antibody and a potent CTL response. In addition, the cytokine profiles support the T1rT2 differentiation with these immunizations, in that oxidized mannan antigen gives IFNg, IL-2 and IL-12 production, whereas in the absence of oxidization, IL-4 and not the other cytokines is produced. A number of antigens have been examined--particularly Mucin 1 and the delivery method using mannose may be applicable to the other antigens.

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules.

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.

Mouse mucin 1 (MUC1) defined by monoclonal antibodies.

Mucins are highly expressed in many different human cancers and numerous murine monoclonal antibodies (MAbs) to human mucins, particularly Mucin 1 (MUC1), have been produced. However, no such antibodies to murine mucin 1 (muc1) have been described and we now describe 6 different antibodies produced to murine muc1 and to human MUC1 cytoplasmic tail, either by immunising rats, or muc1 o/o mice with synthetic peptides or a fusion protein composed of glutathione-s-transferase (GST) linked to the tandem repeat region of muc1. The antibodies to both the extracellular tandem repeat region and to the cytoplasmic tail were found to react with mucin-containing murine tissues such as breast, stomach, colon, ovary, kidney and pancreas, and the staining patterns were similar to those found in humans. The reagents reacted specifically with muc1 peptides and tissues; however, some cross reactivity with other mucin-derived peptides was noted, particularly those containing the amino acid sequence TSS. Three different epitopes (TSS, TAVLSGTS and LSGTSSP) of the M30, M70 and MFP25 MAbs were detected. Of interest was the finding that some of the antibodies reacted with murine lymphocytes; it was not clear whether these reactions were due to mucin 1 on mouse lymphocytes (MUC1 was considered to be absent from human lymphocyte), or due to cross reaction with a sialic adhesion molecule on lymphocytes. The antibodies should prove valuable reagents when studying differentiation and expression in murine glandular tissues and the ontogeny of mucin-secreting tumours.

Immunohistochemical detection of MUC2 mucin core protein in ulcerative colitis.

MUC2 mucin is predominantly expressed in the colon and is considered to play an important role in the protection of that organ. Recent findings suggested that MUC2 protein levels are significantly decreased in active ulcerative colitis (UC). We therefore performed an immunohistochemical study to reveal if the expression of MUC2 protein is altered in UC. Seventy-nine biopsy tissue specimens from 31 UC patients, along with normal colon tissues, were immunostained with anti-MUC2 mucin core protein monoclonal antibody (MoAb) CCP58 (IgG1). UC tissue specimens were divided into two groups based on the histological severity of inflammation, i.e., 64 with active inflammation (grades 2-5) and 15 without (grade 1). In the former group, 52 out of 64 (81.3%) tissue specimens contained sections of glands with a few cells positive for MoAb CCP58. These glands were small and consisted of MUC2 negative-short cells and a few positive cells without apparent mucus formation, considered to be immature regenerative glands. In contrast, the staining pattern was almost the same as that of the normal colon and no immature glands were seen in the group without active inflammation. The sections of immature regenerative glands with a few MUC2-positive cells were exclusively found in the UC tissues with active inflammation, but not in those without it, suggesting that the expression of MUC2 protein may be decreased in active UC.

Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogeneous tumor-associated aberrations in glycosylation.

In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, alpha-fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans.

Correlative histochemical study providing evidence for the dual nature of human colorectal cancer mucin.

Luminal secretions within colorectal cancers have been assumed to be the counterpart of normal goblet cell mucin. The aim of this study was to establish whether secretory material within colorectal cancers may in fact be traced to different lineages: goblet cells and columnar cells. The distribution of the apomucins MUC1 and MUC2, non-O-acetylated sialic acid and the carbohydrate structures sialosyl Tn, Tn, Lewis(x), sialosyl Lewis(x) and Lewis(y) was studied in normal colorectal mucosa and colorectal cancer specimens using standard histochemical techniques. Unmasking of MUC1 and MUC2 was achieved using periodic acid and saponification-neuraminidase-periodic acid pretreatment respectively. Within normal and malignant epithelium, correlations and/or co-localization could be demonstrated for goblet cells with MUC2, non-O-acetylated sialic acid, sialosyl Tn, Tn (Golgi region) and sialosyl Lewis(x), and for columnar cells with MUC1, Lewis(x), sialosyl Le(x), Tn (cytoplasm) and Lewis(y) (UEA-1). The goblet cell spectrum was associated with mucin-like (type I) luminal secretions within cancers, whereas the columnar cell spectrum characterized non-mucin-like (type II) secretions and intracytoplasmic lumina. These data indicate that colorectal cancer mucin can be broadly separated into two types: secretory mucin linked to cells of goblet lineage and up-regulated membrane-associated mucin of presumed columnar cell origin.