anti-MUC1 Antibodies Research Articles

Mumps and ovarian cancer: modern interpretation of an historic association

Epidemiologic studies found childhood mumps might protect against ovarian cancer. To explain this association, we investigated whether mumps might engender immunity to ovarian cancer through antibodies against the cancer-associated antigen MUC1 abnormally expressed in the inflamed parotid gland. Through various health agencies, we obtained sera from 161 cases with mumps parotitis. Sera were obtained from 194 healthy controls. We used an ELISA to measure anti-MUC1 antibodies and electro-chemiluminescence assays to measure MUC1 and CA 125. Log-transformed measurements were analyzed by t-tests, generalized linear models, and Pearson or Spearman correlations. We also conducted a meta-analysis of all published studies regarding mumps and ovarian cancer. Adjusting for assay batch, age, and sex, the level of anti-MUC1 antibodies was significantly higher in mumps cases compared to controls (p = 0.002). Free circulating levels of CA 125, but not MUC1, were also higher in cases (p = 0.02). From the meta-analysis, the pooled odds ratio estimate (and 95% CI) for the mumps and ovarian cancer association was 0.81 (0.68-0.96) (p = 0.01). Mumps parotitis may lead to expression and immune recognition of a tumor-associated form of MUC1 and create effective immune surveillance of ovarian cancer cells that express this form of MUC1.

Anti-MUC1 Antibodies and Ovarian Cancer Risk: Prospective Data from the Nurses' Health Studies

The surface epithelial glycoprotein MUC1 becomes overexpressed and hypoglycosylated in adenocarcinomas; similar changes occur during nonmalignant inflammatory events. Antibodies developed against tumor-like MUC1 in response to such events could be one way through which ovarian cancer risk factors operate. We evaluated the association between anti-MUC1 antibodies and risk of ovarian cancer in a prospective nested case-control study in the Nurses' Health Studies. We used an ELISA to measure plasma anti-MUC1 antibodies in 117 ovarian cancer cases collected at least 3 years before diagnosis and 339 matched controls. In controls, younger women (P-trend = 0.03), those with a tubal ligation (P = 0.03), and those with fewer ovulatory cycles (P-trend = 0.04) had higher antibody levels. In cases, women with late-stage disease (P = 0.04) and those whose specimen was >11 years remote from diagnosis (P = 0.01) had higher antibody levels. Overall, increasing anti-MUC1 antibody levels were associated with a nonsignificant trend for lower risk for ovarian cancer, but there was highly significant heterogeneity by age (P-heterogeneity = 0.005). In women <64 years, the antibody level in quartiles 2 to 4 versus quartile 1 were associated with reduced risk (relative risk = 0.53; 95% confidence interval, 0.31-0.93; P-trend = 0.03), whereas in women > or = 64 years, the corresponding relative risk was 2.11 (95% confidence interval, 0.73-6.04); P-trend = 0.05). Anti-MUC1 antibodies evaluated several years before diagnosis may be associated with lower risk of subsequent ovarian cancer in women <64 years old at assessment. Key elements of an "immune model" to explain ovarian cancer risk factors are confirmed and should be evaluated in larger prospective studies.

Abstract 2444: Targeting the cell-bound MUC1-SEA module with monoclonal antibodies and peptides to ablate MUC1+ malignancies

Abstract The MUC1 oncoprotein is overexpressed in a variety of high-incidence malignancies including breast, colon, and pancreatic carcinoma and by the malignant plasma cells of myeloma. It is a heterodimer composed of a large extracellular α chain that is noncovalently bound to a membrane-bound β subunit. The α-chain's ‘on-again-off-again’ interaction with the β-chain results in intracellular signaling. A variety of anti-MUC1 antibodies have been described to date, almost all of which recognize the immunogenic tandem-repeat-array of the MUC1 α-subunit which is shed into the peripheral circulation. Because it circulates freely, the α subunit actively sequesters anti-α chain Abs, precluding their even reaching and adequately targeting MUC1+ malignant cells. To circumvent this problem we identified the MUC1 SEA module that comprises the junction of the extracellular α-subunit with the cell-bound β-subunit as a valuable cancer cell target since it invariably remains attached to the cell surface. We generated a series of anti-MUC1 α/β junction IgGγ1 monoclonal Abs, denominated DMB mAbs, which bind with exceptionally high affinity to MUC1-expressing human breast cancer cells. The antibodies were non-reactive when used as probes in Western blot analyses of the MUC1 protein resolved on denaturing SDS gels, suggesting that the epitopes recognized by the DMB mAbs are configurational. The novel mAbs bound well to MUC1-expressing cancer cells such as T47D and ZR-75 and to the extracellular domain of the MUC1/X protein which comprises only the SEA module that is fused C-terminally to the thirty N-terminal amino acids of the MUC1 protein. The DMB mAbs were much less reactive with recombinant SEA module alone, which corresponds to the MUC1/X extracellular domain that lacks the 30 N-terminal amino acids. Unexpectedly, all DMB mAbs showed high reactivity with mutant ‘SEA module 4G’ protein that comprises an additional 4 glycine residues upstream to the GSVVV cleavage site resulting in a non-cleaved protein. From these results we conclude that the native structure of the cleaved SEA module associated with the MUC1/X protein is different to that of the SEA module that lacks the N-terminal 30 amino acids, and more similar to mutant ‘SEA module 4G’ protein. To reconcile these seemingly contradictory findings, we hypothesized that the N-terminal portion of the MUC1 protein binds to the SEA module, thereby forming epitopes recognized by the DMB mAbs. Based on these structural findings, small peptides were derived from the MUC1 N-terminus and appear to selectively bind the MUC1 SEA module. We conclude that (1)The anti-MUC1 α/β junction Abs and peptides described here bind to specific configurationally-determined MUC1 α/β junction epitopes, and (2)Abs and peptides targeting those epitopes are likely to prove biologically-significant and clinically useful in selectively targeting and killing MUC1+ malignant cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2444.

Aberrant Expression of MUC1 Mucin in Pediatric Inflammatory Bowel Disease

Mucin 1 (MUC1) glycoprotein is normally expressed at low levels on the luminal side of healthy colonic epithelial cells. In colon cancer and other epithelial tumors, MUC1 is overexpressed and hypoglycosylated. Antibodies specific for this "tumor form" of MUC1 are found in cancer patients. We hypothesized that MUC1 expression might be altered in chronic inflammation, such as in inflammatory bowel disease (IBD). Furthermore, we hypothesized that these alterations might induce antibody responses. The aims of this study were to characterize MUC1 expression in IBD and to examine whether pediatric patients with IBD have an MUC1-specific antibody. Colon biopsies were examined for MUC1 expression by immunochemistry using anti-MUC1 antibodies that detect normal or abnormal forms of MUC1. Sera were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) for evidence of anti-MUC1 antibody. We found marked overexpression of MUC1 in IBD, most of which was hypoglycosylated. On colon specimens from healthy age-matched controls, we found low levels of luminal MUC1 and no alteration in its glycosylation. We detected antibody to MUC1 in sera of IBD patients as well as controls, and in a limited number of IBD samples examined longitudinally, we could correlate the rise and fall of antibody levels with clinical disease severity. MUC1 is overexpressed and hypoglycosylated in pediatric IBD and may play an important role in the pathogenesis of IBD, and thus warrants further study as a potential therapeutic target. Similarly, antibodies to MUC1 may influence IBD and should be explored as potential diagnostic or prognostic markers.

A conditional mouse model for human MUC1-positive endometriosis shows the presence of anti-MUC1 antibodies and Foxp3+ regulatory T cells

Endometriosis is defined by the presence of tissue implants resembling endometrial glands outside of the uterus, at ectopic sites, frequently on the ovarian surface. The ectopic lesions are often invasive, resistant to therapy, and may predispose to endometrioid and clear cell ovarian tumors. The complex mechanisms leading to chronic endometriosis are mediated partly by impaired immune surveillance in the host. Although innate immunity has been addressed previously, the response of adaptive immune effectors to specific antigens has not been characterized, mostly because very few endometriosis antigens have been defined to date. We postulated that the mucin 1 (MUC1) glycoprotein, which is normally present on eutopic human endometrial glands and overexpressed in endometrioid and clear cell ovarian tumors, is also present in ectopic lesions of ovarian endometriosis. Furthermore, changes in MUC1 expression in endometriosis could promote adaptive anti-MUC1 immunity that might play a role in the malignant progression. To test our hypothesis, we crossed MUC1 transgenic mice, which express human MUC1 under the endogenous promoter, with the loxP-Stop-loxP-Kras(G12D/+) (Kras) mice, in which endometriosis can be induced through Cre-loxP recombination. The double transgenic MUC1Kras mice develop benign, MUC1-positive ovarian lesions, closely resembling human endometriosis. Subsequent to disease induction, the mice generate high titers of IgM and IgG antibodies that are specific for MUC1. Antibodies appear early in disease and the predominance of the IgG1 subclass suggests Th2-driven immunity. Immune phenotyping revealed an accumulation of Foxp3+ CD4 regulatory T cells (Tregs) in the draining lymph nodes at late-stage disease. Furthermore, our observations in human endometriosis showed a similar recruitment of FOXP3+ CD4 T cells. Overall, our results reveal a Th2/Treg-dominant natural immunity in endometriosis with potential implications for cancer progression.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

BackgroundTumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.ResultsBased on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli.ConclusionBi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.

Humoral immune response to abnormal MUC1 in subjects with colorectal adenoma and cancer

Colorectal cancer (CRC) expresses a hypoglycosylated (abnormal) form of MUC1 different than MUC1 expressed in normal colon, which elicits antibodies in patients with CRC. This form of MUC1 is expressed in other abnormal but non-malignant lesions in the colon, such as adenomatous polyps, precursors to CRC. Estimates of the prevalence of anti-MUC1 antibodies in subjects with these lesions are lacking. We evaluated IgM and IgG anti-MUC1 antibodies in 148 subjects with non-advanced adenomas (NAA), advanced adenomas (AA), colorectal cancer, hyperplastic polyps (HPP), and normal controls. We hypothesized that the prevalence of anti-MUC1 antibodies would increase along the adenoma-carcinoma sequence as more dysplastic tissues express more abnormal MUC1. Anti-MUC1 IgM was found in 5/47 (10.6%) of normals, 5/45 (11.1%) of NAA, 7/47 (14.9%) of AA, and 4/20 (20.0%) of CRC ( p = 0.70). The prevalence of anti-MUC1 IgG was 8/47 (17.0%) of normals, 14/45 (31.1%) of NAA, 14/47 (29.8%) of AA, and 6/20 (30.0%) of CRC ( p = 0.36). We found no significant differences in the prevalence of anti-MUC1 antibodies between subjects along the adenoma-carcinoma sequence. However, in an exploratory analysis, the median normalized anti-MUC1 IgG OD level of the combined abnormal groups (NAA, AA, CRC) was significantly higher than the normals (0.045 OD vs. 0.030 OD; p = 0.017). Our data support further studies into the potential role of anti-MUC1 immunity in preventing progression of premalignant colon lesions to colon cancer.

The MUC1 oncoprotein as a functional target: Immunotoxin binding to α/β junction mediates cell killing

MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.

Generation of antibodies that target and kill MUC1 expressing cancer cells via a novel cell-tethered MUC1 α/β junction epitope

3034 Background: MUC1 protein has generated considerable interest as a target for tumor killing. However, a serious shortcoming of anti-MUC1 antibodies generated to date is that they recognize epitopes within the strongly immunogenic tandem-repeat-array of the MUC1 a- chain which is released from the cell into the circulation. Soluble shed MUC1 a-chain sequesters anti-tandem-repeat-array antibodies, severely limiting their ability to reach MUC1-expressing malignant cells. Rather than target freely circulating MUC1 a-chain, we identified the junction of the MUC1 a-subunit to cell-membrane-bound MUC1 β-subunit as a preferable cell-tethered MUC1 epitope; since the site is not shed, antibodies recognizing it are more effective in targeting MUC1 expressing cells. Methods: To circumvent the immunogenicity of the a-chain tandem repeat array we formulated a novel antibody generating protocol utilizing immunization with both MUC1 cDNA and with the alternatively-spliced MUC1/X protein isoform from which the tandem repeat array is deleted. After immunization and hybridoma formation, anti- MUC1 a/β junction antibodies were selected. Results: DMC209 monoclonal antibodies uniquely specific for the MUC1 a/β junction were generated. The antibodies specifically bind the MUC1 a/β junction on full-length MUC1 expressed by breast and ovarian cancer cells, and on MUC1-positive malignant plasma cells of multiple myeloma. To demonstrate that anti-MUC1 a/β junction antibodies kill malignant cells, immunotoxin conjugates were formed with anti-MUC1 a/β junction polyclonal antibodies generated in our cDNA/protein immunization protocol and PE38, a powerful pseudomonas exotoxin. The antibody-exotoxin conjugates were potently cytocidal to MUC1-expressing malignant cells. Significantly, cell killing was abrogated by addition of soluble MUC1/X protein, highlighting that the cell-killing immunotoxin gains cell entry via the MUC1 a/β junction. Conclusions: The MUC1 a/β junction has been identified as an important cell-tethered MUC1 epitope against which highly specific antibodies can be generated capable of killing MUC1-expressing cells. These studies point to effective anti-MUC1-based immuno-therapeutic strategies. No significant financial relationships to disclose.

Expression of Tumor-Associated Differentiation Antigens, MUC1 Glycoforms and CEA, in Human Thymic Epithelial Cells: Implications for Self-Tolerance and Tumor Therapy

Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancer-associated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials.

Incessant Ovulation, Mucin 1 Immunity, and Risk for Ovarian Cancer

Risk for ovarian cancer correlates directly with "ovulatory years or cycles" estimated from time not pregnant, breast-feeding, or using oral contraceptives. Recently, we reported that several factors known to reduce ovarian cancer risk may operate by inducing antibodies against mucin 1 (MUC1), a glycoprotein overexpressed in ovarian cancer. Conversely, other events might increase risk by interfering with the development of protective immunity. In this study, we examined whether the total number of ovulatory cycles decreases the likelihood of anti-MUC1 antibodies and provides an immune basis for the association between "incessant ovulation" and ovarian cancer risk. From 1998 to 2003, we enrolled 668 epithelial ovarian cancer cases and 721 controls residing in eastern Massachusetts or New Hampshire, collected information on menstrual and reproductive events, and obtained blood samples from controls to measure anti-MUC1 antibodies. Using logistic regression, we calculated odds ratios to evaluate the influence of reproductive factors, including the estimated lifetime number of ovulatory cycles on ovarian cancer risk and on the presence of MUC1 antibodies in controls. Overall, we observed that early age at first birth, cycle lengths >or=30 days, and oral contraceptive use increased the likelihood of having anti-MUC1 antibodies. Estimated ovulatory cycles were correlated positively with ovarian cancer risk and inversely with the presence of anti-MUC1 antibodies among controls ages 46 to 60 years. These data suggest that suppression of MUC1-specific immunity should be considered as an additional explanation for the observation that ovarian cancer risk increases with the lifetime number of ovulatory cycles.

MUC1 expression and anti-MUC1 serum immune response in head and neck squamous cell carcinoma (HNSCC): a multivariate analysis

BackgroundHNSCC progression to adjacent tissue and nodes may be mediated by altered glycoproteins and glycolipids such as MUC1 mucin. This report constitutes a detailed statistical study about MUC1 expression and anti-MUC1 immune responses in relation to different clinical and pathological parameters which may be useful to develop new anti HNSCC therapeutic strategies.Patients and methodsFifty three pre treatment HNSCC patients were included: 26 (49.1%) bearing oral cavity tumors, 17 (32.1%) localized in the larynx and 10 (18.8%) in the pharynx. Three patients (5.7%) were at stage I, 5 (9.4%) stage II, 15 (28.3%) stage III and 30 (56.6%) at stage IV. MUC1 tumor expression was studied by immunohistochemistry employing two anti-MUC1 antibodies: CT33, anti cytoplasmic tail MUC1 polyclonal antibody (Ab) and C595 anti-peptidic core MUC1 monoclonal antibody. Serum levels of MUC1 and free anti-MUC1 antibodies were detected by ELISA and circulating immune complexes (CIC) by precipitation in polyethylene glycol (PEG) 3.5%; MUC1 isolation from circulating immune complexes was performed by protein A-sepharose CL-4B affinity chromatography followed by SDS-PAGE and Western blot. Statistical analysis consisted in Multivariate Principal Component Analysis (PCA); ANOVA test (Tukey's test) was employed to find differences among groups; nonparametrical correlations (Kendall's Tau) were applied when necessary. Statistical significance was set to p < 0.05 in all cases.ResultsMUC1 cytoplasmic tail was detected in 40/50 (80%) and MUC1 protein core in 9/50 (18%) samples while serum MUC1 levels were elevated in 8/53 (15%) patients. A significant statistical correlation was found between MUC1 serum levels and anti-MUC1 IgG free antibodies, while a negative correlation between MUC1 serum levels and anti-MUC1 IgM free antibodies was found. Circulating immune complexes were elevated in 16/53 (30%) samples and were also statistically associated with advanced tumor stage. MUC1 was identified as an antigenic component of IgG circulating immune complexes. Moreover, poorly differentiated tumors were inversely correlated with tumor and serum MUC1 detection and positively correlated with node involvement and tumor mass.ConclusionPossibly, tumor cells produce MUC1 mucin which is liberated to the circulation and captured by IgG antibodies forming MUC1-IgG-CIC. Another interesting conclusion is that poorly differentiated tumors are inversely correlated with tumor and serum MUC1 detection.

Cancer specific Mucin‐1 glycoforms are expressed on multiple myeloma

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules

MUC1 glycoprotein that is overexpressed in aberrant forms in epithelial cancers has been used for diagnosis, staging and therapy. As normal prostate and prostate cancer tissues express MUC1, it represents a potential target, but MUC1 epitopes specific to prostate cancer have not been well characterized. In order to assess MUC1 epitopes in prostate cancer, and their correlation with Gleason grades, binding of 7 well-characterized anti-MUC1 monoclonal antibodies (MAbs) (BrE-3, SM3, BC2, EMA, B27.29, HMFG-1 and NCL MUC1 core), were studied on a prostate tissue microarray. This microarray contained 197 prostate tissue cores representing: i) normal/benign prostate; ii) prostatic intraepithelial neoplasia and Gleason grades 1 and 2; and iii) Gleason grades 3-5. These MAbs bind the MUC1 extracellular domain, but have variable sensitivity to MUC1 glycosylation. To further characterize the effect of glycosylation on their binding, MAb reactivities with unglycosylated MUC1 core peptide and breast and prostate cancer cell lysates were compared. These studies demonstrated strong binding of BrE-3, BC2 and EMA to the peptide core and recognition by BrE-3, SM3, BC2 and EMA of hypoglycosylated MUC1. The results for the microarray indicated that higher Gleason grades were associated with markedly increased cellular staining by MAbs that preferentially recognize less glycosylated MUC1 (BrE-3, p<0.001; SM3, p<0.004; EMA, p=0.009; and BC2, p<0.001). Staining by MAbs that bind preferentially to hyperglycosylated MUC1 (B27.29, p=0.33; HMFG-1, p=0.89; and NCL MUC1 core, p=0.96) did not correlate with Gleason grade. These results demonstrated that hypoglycosylated MUC1 expression increased with Gleason grade, thus supporting the targeting of hypoglycosylated MUC1 epitopes in prostate cancer for more specific imaging and therapy applications.

Synthesis and Structural Model of an α(2,6)‐Sialyl‐T Glycosylated MUC1 Eicosapeptide under Physiological Conditions

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, a 20-residue peptide (GSTAPPAHGVTSAPDTRPAP) representing the full length tandem repeat sequence of the human mucin MUC1 and its analogue glycosylated with the (2,6)-sialyl-T antigen on Thr11, were prepared and investigated by NMR and molecular modeling. The peptides contain both the GVTSAP sequence, which is an effective substrate for GalNAc transferases, and the PDTRP fragment, a known epitope recognized by several anti-MUC1 monoclonal antibodies. It has been shown that glycosylation of threonine in the GVTSAP sequence is a prerequisite for subsequent glycosylation of the serine at GVTSAP. Furthermore, carbohydrates serve as additional epitopes for MUC1 antibodies. Investigation of the solution structure of the sialyl-T glycoeicosapeptide in a H(2)O/D(2)O mixture (9:1) under physiological conditions (25 degrees C and pH 6.5) revealed that the attachment of the saccharide side-chain affects the conformational equilibrium of the peptide backbone near the glycosylated Thr11 residue. For the GVTSA region, an extended, rod-like secondary structure was found by restrained molecular dynamics simulation. The APDTR region formed a turn structure which is more flexibly organized. Taken together, the joined sequence GVTSAPDTR represents the largest structural model of MUC1 derived glycopeptides analyzed so far.

Talc and Anti-MUC1 Antibodies

To the Editors: Cramer et al. ([1][1]) present very intriguing findings on anti-MUC1 antibodies and their relationship to ovarian cancer risk factors in healthy women, raising new theories on the role of immunity in ovarian cancer and opening up avenues for new research in this area. The analysis is

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli

In contrast to the murine and human VHs, camels’ single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a + vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS–PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Conditions Associated with Antibodies Against the Tumor-Associated Antigen MUC1 and Their Relationship to Risk for Ovarian Cancer

Many cancers, including ovarian, overexpress epithelial mucin (MUC1) and promote anti-MUC1 antibodies that may correlate with more favorable prognosis. By extension, risk for ovarian cancer might be reduced by preexisting MUC1-specific immunity. We measured anti-MUC1 antibodies in 705 control women, identified events predicting antibodies, and estimated ovarian cancer risk by comparing profiles of events generating antibodies in controls with those in 668 ovarian cancer cases. Factors predicting antibodies included oral contraceptive use, breast mastitis, bone fracture or osteoporosis, pelvic surgeries, nonuse of talc in genital hygiene, and to a lesser extent intrauterine device use and current smoking. There was a significant increase in the likelihood of having anti-MUC1 antibodies from 24.2% in women with 0 or 1 condition, to 51.4% in those with five or more conditions. By the same index of events, the risk for ovarian cancer was inversely associated with number of conditions predisposing to anti-MUC1 antibodies. Compared with having experienced 0 or 1 event, the adjusted risk for ovarian cancer decreased progressively with relative risks (and 95% confidence limits) of 0.69 (0.52-0.92), 0.64 (0.47-0.88), 0.49 (0.34-0.72), and 0.31 (0.16-0.61), respectively for women with two, three, four, and five or more events related to the presence of antibodies (P(trend) < 0.0001.) We conclude that several traditional and new risk factors for ovarian cancer may be explained by their ability to induce MUC1 immunity through exposure of MUC1 to immune recognition in the context of inflammatory or hormonal processes in various MUC1-positive tissues.

Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.

Phase I trial of a murine antibody to MUC1 in patients with metastatic cancer: evidence for the activation of humoral and cellular antitumor immunity

BrevaRex mAb-AR20.5 is a murine anti-MUC1 monoclonal antibody generated to induce MUC1 antigen-specific immune responses through the formation of immune complexes with circulating MUC1 and/or MUC1-expressing tumor cells that may target these immune complexes (IC) to receptors on dendritic cells (DCs). A phase I study focusing on safety and immunology evaluated 1, 2 and 4-mg doses. Seventeen patients with MUC1-positive cancers received intravenous infusions of the antibody over 30 min on weeks 1, 3, 5, 9, 13 and 17 of treatment. mAb-AR20.5 was well-tolerated, not associated with dose-limiting toxicity, and did not induce hypersensitivity reactions. Overall, five of 15 evaluable patients developed human anti-mouse antibodies (HAMA), five developed anti-idiotypic antibodies (Ab2) and seven developed anti-MUC1 antibodies. Immune responses were most prominent in the 2-mg dose cohort for all parameters tested, and treatment-emergent MUC1-specific T-cell responses were detected in five of 10 evaluable patients treated with mAb-AR20.5. The injection of a murine antibody to MUC1 induces MUC1-specific immune responses in advanced cancer patients. Anti-MUC1 antibody increases correlated with decrease or stabilization of CA15.3 levels (P=0.03). The 2-mg dose of mAb-AR20.5 showed strongest biological activity, and will be evaluated in future efficacy trials.