anti-MUC1 Antibody Research Articles

Comparative Immunohistochemical Study of MUC1 and Carbohydrate Antigens in Breast Benign Disease and Normal Mammary Gland

The aim was to compare the expression of MUC1 and carbohydrate antigens in 124 tissue samples; 42 fibroadenoma (FA), 23 nonproliferative benign diseases (NPF), 25 usual epithelial hyperplasia (UEH), 7 atypical ductal hyperplasia (ADH), and 27 breast normal tissues. An immunohistochemical approach was adopted, using the following antibodies: reactive with MUC1 variable number of tandem repeats (C595, HMFG2, and SM3 monoclonal antibodies), anti-MUC1-cytoplasmic tail polyclonal antibody (CT33), and anti-carbohydrate antigens (sialyl Lewis x, Lewis x, Lewis y, Tn, and Thomsen-Friedenreich epitopes). Positive area of reaction, intensity, and pattern of expression were considered. A reactivity index was calculated as intensity (I) x 100+percentage of positive area (A). Statistical analysis comprised frequency analysis, P < 0.05, analysis of variance, and multiple correlation with principal component analysis. All samples expressed MUC1, detected by at least one anti-MUC1 antibody whereas Lewis x was the carbohydrate antigen most frequently found in all groups whereas variable number of tandem repeats MUC1 and Lewis x showed the highest correlation: 93% of normal samples, 62.5% of NPF, 87% of FA, 85% of UEH, and finally 80% of ADH. Although principal component analysis using reactivity indexes explained only 39% of data variability, normal samples appeared grouped and separated from benign breast diseases, which remained spread. Thomsen-Friedenreich was the only antigen that showed an increased tendency for positive expression and intensity from NPF through FA, UEH to ADH, whereas it was not detected in normals. With respect to the pattern of expression, an apical pattern was predominantly found in all the groups.

Abstract B128: Affinity optimization of an anti-MUC1 antibody, (HuHMFG1) also enhances ADCC activity

Abstract HuHMFG1 (AS1402) is a humanized antibody which binds to MUC1, a cell surface glycoprotein which is over-expressed on tumor cells. A phase II clinical trial of huHMFG1 in metastatic breast cancer was recently terminated prior to completion as it was thought unlikely to give sufficiently positive efficacy findings. HuHMFG1 contains a potential glycosylation site within the variable region; this is partially glycosylated in the drug product. The presence of the glycan reduces the affinity of the antibody for MUC1, and enzymatic deglycosylation of huHMFG1 increases affinity 3-fold. The asparagine residue which undergoes partial glycosylation (N56) was mutated to all other amino acids and binding affinities to antigen determined: several mutants exhibited enhanced binding to MUC1. The mutation to glycine (N56G) demonstrated approximately 10-fold increased affinity as assessed by antigen binding ELISA and cell binding ELISA against a variety of MUC1-expressing cell lines; BIAcore analysis further supported these observations. The primary mechanism of action by which huHMFG1 is proposed to function is antibody dependent cell mediated cytotoxicity (ADCC). The N56G mutant exhibited 10 to 20-fold increased ADCC activity compared to huHMFG1 (5-10% of the huHMFG1 dose was required to elicit maximum cell kill); in addition the percentage of target cells killed also increased. This second generation huHMFG1 exhibits increased antigen affinity and ADCC activity and furthermore has the potential to offer improved in vivo activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B128.

Functional Neoglycopeptides: Synthesis and Characterization of a New Class of MUC1 Glycoprotein Models Having Core 2-Based O-Glycan and Complex-Type N-Glycan Chains

An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-Pro-Asp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of thhe structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.

Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer

IntroductionMUC1 is a cell-surface glycoprotein that establishes a molecular barrier at the epithelial surface and engages in morphogenetic signal transduction. Alterations in MUC1 glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion, and immune surveillance. A 20-amino-acid tandem repeat that forms the core protein of MUC1 is overexpressed and aberrantly glycosylated in the majority of epithelial tumors. AS1402 (formerly R1550) is a humanized IgG1k monoclonal antibody that binds to PDTR sequences within this tandem repeat that are not exposed in normal cells. AS1402 is a potent inducer of antibody-dependent cellular cytotoxicity (ADCC), specifically against MUC1-expressing tumor cells. The objective of this study was to determine the safety, tolerability, and pharmacokinetic (PK) characteristics of AS1402 monotherapy in patients with locally advanced or metastatic MUC1-positive breast cancer that had progressed after anthracyclines- and taxane-based therapy.MethodsPatients received AS1402 over a 1- to 3-hour intravenous (i.v.) infusion at doses between 1 and 16 mg/kg, with repeated dosing every 1 to 3 weeks (based on patient-individualized PK assessment) until disease progression. Serum AS1402 levels were measured at multiple times after i.v. administration. Human anti-human antibody (HAHA) responses were measured to determine the immunogenicity of AS1402. Noncompartmental pharmacokinetic parameters were determined and were used to assess dose dependency across the dose range studied.ResultsTwenty-six patients were treated. AS1402 was generally well tolerated. Two grade 3/4 drug-related adverse events were reported, both at the 3-mg/kg dose. Neither was observed in expanded or subsequent dosing cohorts. No anti-human antibodies were detected. Plasma concentrations of AS1402 appeared to be proportional to dose within the 1- to 16-mg/kg dose range assessed, with a mean terminal half-life of 115.4 ± 37.1 hours.ConclusionsRepeated iv administration of AS1402 was well tolerated, with a maximum tolerated dose (MTD) exceeding 16 mg/kg, the highest dose administered in this study. The half-life and exposure of AS1402 were such that weekly dosing could achieve plasma concentrations corresponding to the maximal ADCC activity observed in vitro. A phase II study is ongoing to evaluate the clinical activity of AS1402 in patients with advanced breast cancer.Trial registrationClinicalTrials.gov Identifier: NCT00096057.

Internalization of MUC1 by anti-MUC1 antibody from cell membrane through the macropinocytotic pathway

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. It is thought to serve as a physical barrier from the extracellular environment and also as a receptor for various extracellular molecules. However, little is known about the fate of MUC1 during and after the interaction with these molecules. In the present study, we used anti-MUC1 antibody as an interacting molecule and investigated the cellular trafficking of MUC1. Our results showed that: (1) anti-MUC1 antibody was internalized only in MUC1 expressing cells and triggered internalization and down-regulation of MUC1; (2) the internalization of MUC1 by anti-MUC1 antibody required the cytoplasmic tail of MUC1 and was suppressed by inhibitors of Na +/H + exchanger, and caveola/raft-dependent internalization, but not by an inhibitor of clathrin-dependent internalization. We conclude that antibody-induced internalization of MUC1 involves the macropinocytotic pathway.

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of alpha-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.

Anti-MUC1 antibody responses in endometriosis and ovarian cancer in humans and preclinical MUC1 transgenic models (88.29)

Abstract Ovarian cancer is the deadliest gynecologic malignancy and endometriosis is the only currently described precursor lesion. MUC1 mucin is an ovarian epithelial tumor-associated antigen and a vaccine candidate for ovarian cancer immunotherapy. Natural and vaccine-induced MUC1 immunity, often studied in ovarian cancer is much less understood in endometriosis. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models expressing the human antigen are needed. We generated two novel preclinical models for MUC1 positive endometriosis and MUC1-expressing ovarian tumors, respectively. The MUC1KrasG12D mice develop MUC1-positive ovarian endometriosis after AdCre injection under the ovarian bursa. MUC1KrasG12D transgenic mice injected with MUC1-expressing immortalized ovarian surface epithelial cells (OSE) develop tumors throughout the peritoneal cavity and serve as a transplantable in vivo tumor model. Our results show that MUC1 is immunogenic in humans and mice with either malignant or premalignant lesions and triggers MUC1-specific IgM and Th2-driven IgG antibodies. Furthermore, the presence of IgG in cancer patients correlates inversely with survival. In additions, mice and humans show several differences in immune regulation by other CD4 T cell subsets (Th17, Treg) with consequences for MUC1 vaccination in preclinical models. PA Dept of Health, CMRF.

Erratum: Experimental Study on New Immunotherapy of Oral Cancer by Using Monoclonal Anti-MUC1 Antibody[Journal of Japanese Society for Oral Mucous Membrane vol.14 No.2 39-45

Erratum: Experimental Study on New Immunotherapy of Oral Cancer by Using Monoclonal Anti-MUC1 Antibody[Journal of Japanese Society for Oral Mucous Membrane vol.14 No.2 39-45

Surface Mucin-1 does not play a role in dendritic cell migration

Mucin-1 (MUC1) is a transmembrane glycoprotein that is upregulated upon maturation of dendritic cells (DC) in vitro or in vivo. One of the proposed functions of surface expressed MUC1 is its involvement in migration of cells. We hypothesized that MUC1 is involved in DC migration since mature DC (mDC) are highly migratory cells and MUC1 is upregulated on the surface of DC upon maturation. In this study we cultured DC using two maturation cocktails, one cocktail containing IL-4, GM-CSF, TNFα, PGE2, IL-1β and IL-6 (TP1,6-DC) and the other IL-13, GM-CSF, Ribomunyl and IFN-γ (RI-DC). Both maturation cocktails render DC with a similar surface phenotype including CCR7 expression, but only the former induces a migratory capacity of DC to a CCL19 gradient. To analyze the role of surface-expression of MUC1 on TP1,6-DC, that are capable of migration, expression of MUC1 was prevented by adding an anti-MUC1 antibody (Ab) during the maturation process. Compared with matured DC in the absence of the Ab, no difference was observed in chemokine-induced migratory behaviour between the MUC1+ and MUC1− DC populations in a standard Transwell chemotaxis assay, nor in organotypic cultures. Our data clearly demonstrate that surface MUC1 on DC does not influence intrinsic cell-motility, nor is it involved in cell–cell and cell–matrix dependent migration.

Separation, purification and overexpression of anti-MUC1 nanobody from VHH phagemid library

One of the most important goals of antibody engineering was to reduce the size of antibodies in order to reduce side effects, resulting in the production of, single chain variable fragments (Scvf) and single domain antibodies (SdAbs). Natural SdAbs were discovered in camelidae and llamas in 1993 by chance. They are part of the humoral response of camelidae (Camelidae dromedaries and Camelidae bacterianus) and llamas (Llama glama) consisting of the variable heavy chain (VH) of the whole antibody. They are called VHH in order to avoid the confusion with the ordinary VH in human beings. Their small size in the nanometer range gained them the nick name of nanobodies. They are easily separated from immunized and non-immunized phagemid libraries and are more stable in high temperatures (90°C), compared to humanized or chimeric antibodies. These unique features make them a good potential to be used as targeting or diagnostic tools, especially in targeting tumor markers. Mucin 1 (MUC1) a member of the mucin family, is known to be one of those tumor associated antigen, being over-expressed in breast, lung, colon, pancreas, stomach, prostate and ovary carcinomas. In this study we tried to separate and purify an anti-MUC1 coding antibody from a camelid phage library and overexpress it in prokaryotic settings in order to be used as a targeting agent for gene delivery to breast cancer cell line.

Effect of the antibody immunotherapy by the anti-MUC1 monoclonal antibody to the oral squamous cell carcinoma

Effect of the antibody immunotherapy by the anti-MUC1 monoclonal antibody to the oral squamous cell carcinoma

Breast tumor targeting with 99mTc-HYNIC-PR81 complex as a new biologic radiopharmaceutical

Human epithelial mucin, MUC1, is commonly overexpressed in adenocarcinoma that includes more than 80% of breast cancers. The PR81 is a murine anti-MUC1 monoclonal antibody (MAb) that was prepared against the human breast cancer. We developed an indirect method for labeling of this antibody with (99m)Tc in order to use the new preparation in immunoscintigraphy studies of BALB/c mice bearing breast tumors. The (99m)Tc-PR81 complex was prepared using the HYNIC as a chelator and tricine as a coligand. The labeling efficiency determined by instant thin-layer chromatography (ITLC) was 89.2%+/-4.7%, and radiocolloides measured by cellulose nitrate electrophoresis were 3.4%+/-0.9%. The in vitro stability of labeled product was determined at room temperature by ITLC and in human serum by gel filtration chromatography - 88.3%+/-4.6% and 79.8%+/-5.7% over 24 h, respectively. The integrity of labeled MAb was checked by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, and no significant fragmentation was seen. The results of cell binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies performed in female BALB/c mice with breast tumor xenografts at 4, 16 and 24 h after the (99m)Tc-HYNIC-PR81 injection demonstrated a specific localization of the compound at the site of tumors and minimum accumulation in non target organs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 4, 8, 12, 16, 20, 24, 28, 32 and 36 h after the complex injection. The tumors were visualized with high sensitivity after 8 h. The findings showed that the new radiopharmaceutical is a promising candidate for radioimmunoscintigraphy of the human breast cancer.

Initiation of Mammalian O-Mannosylation in Vivo Is Independent of a Consensus Sequence and Controlled by Peptide Regions within and Upstream of the α-Dystroglycan Mucin Domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

Upregulated expression of complement inhibitory proteins on bladder cancer cells and anti‐MUC1 antibody immune selection

Membrane complement inhibitors (CD46, CD55 and CD59) are upregulated in some human cancers indicating that they play a role in immune evasion. We investigated complement inhibitor expression in bladder cancer and examined the hypothesis that selective pressure of an antibody response (anti-MUC1) results in the upregulated expression of complement inhibitors on tumor cells. Paired samples of tumor and normal tissue from 22 bladder cancer patients were analyzed for expression of MUC1, CD46, CD55 and CD59, and matched serum samples analyzed for anti-MUC1 IgM and IgG levels. Relationships between anti-MUC1 antibody levels and complement inhibitor expression were investigated. MUC1 mRNA was upregulated in 86% of tumor samples. CD46 was upregulated in 77%, CD55 in 55% and CD59 in 59% of tumors. Low titer anti-MUC1 IgM was detected in normal human sera, but was elevated in 41% of the bladder cancer patients. Anti-MUC1 IgG was virtually absent from normal sera, but present in 32% of the cancer patients. There was a direct relationship between anti-MUC1 antibody titer and expression level of complement inhibitors. Analysis of the correlation of each antibody with the expression of each complement inhibitor by Spearman's rank test revealed a strong correlation between both anti-MUC1 IgM and IgG levels and increased expression of CD46 and CD55, and combined anti-MUC1 IgM/IgG levels correlated with increased expression of all 3 complement inhibitors. In conclusion, the data demonstrate upregulated complement inhibitor expression and the presence of an anti-MUC1 antibody response in bladder cancer patients and support the hypothesis of antibody-mediated immune selection.

Overexpression and altered glycosylation of MUC1 in malignant mesothelioma

Current interest in the MUC1/EMA mucin relates to its role in malignancy, and its potential as a therapeutic target. MUC1/EMA expression has been observed in the majority of epithelioid mesotheliomas. However, little is known of the characteristics of MUC1/EMA in mesothelioma. Herein, we studied the cell surface and soluble expression of the MUC1/EMA glycoprotein, and determined the mRNA and genomic expression profiles in mesothelioma. We found that the anti-MUC1 antibody, E29, was the most diagnostically useful of seven antibody clones examined with a sensitivity of 84% (16 out of 19 cases) and no false positive results. MUC1 mRNA expression was significantly higher in mesothelioma samples than in benign mesothelial cells. No amplification of the MUC1 gene was observed by FISH. Seven of 9 mesothelioma samples expressed MUC1-secreted mRNA isoform in addition to the archetypal MUC1/transmembrane form. CA15.3 (soluble MUC1) levels were significantly higher in the serum of mesothelioma patients than in healthy controls but were not significantly different to levels in patients with benign asbestos-related disease. CA15-3 in effusions could differentiate malignant from benign effusions but were not specific for mesothelioma. Thus, as in other cancers, alterations in MUC1 biology occur in mesothelioma and these results suggest that specific MUC1 characteristics may be useful for mesothelioma diagnosis and should also be investigated as a potential therapeutic target.

Gemcitabine (gem) can enhance immunotherapy (IT) of pancreatic cancer – importance of schedule

With a 5‐year survival rate of only 4%, non‐resectable pancreatic cancer is in urgent need of novel approaches to supplement current therapies. This study assessed parameters for combining gem and IT with the anti‐MUC1 antibody, AR20.5. Upon injection, AR20.5 forms immune complexes with circulating MUC1, leading to enhanced T cell immunity. To examine the effects of gem on immune cells and antigen processing, three parallel approaches were conducted: a) an in vitro co‐culture system of immature DC, tumor and T cells; b) a time course study of gem (60 mg/kg) in Panc02 tumor bearing mice; and c) a combination chemo‐IT study in MUC1‐transgenic mice with established Panc02.MUC1 tumors. In vitro, gem did not affect T cell or DC survival at concentrations ≤5 μg/mL. Interestingly, gem enhanced maturation of DC and T cell proliferation to non‐specific stimuli (anti‐CD3/CD28) as well as to the co‐cultured tumor cells. The in vivo time course study confirmed minor effects of gem on T and B cell survival with rapid recovery. Gem increased DC maturation in the spleen and tumor as well as DC migration to the tumor by Day 5. Further, gem significantly reduced T regs in the draining LN (Day 7) and tumor (Day 10). Reduction in myeloid derived suppressor cells was transient (Day 5–7) in spleen and tumor. Tumor control studies showed synergistic effects for combination gem‐IT over single agents when IT was given 1 week delayed relative to gem.

Effect of the Antibody Immunotherapy by the Anti-MUC1 Monoclonal Antibody to the Oral Squamous Cell Carcinoma in Vitro

To conserve both form and function in the oral area, effective and selective drugs against oral cancer will be required. We focused on MUC1, a transmembrane glycoprotein, that is considered tumor-associated antigens (TAAs) for cancer therapy. Recently, studies were done to evaluate the patterns of MUC1 expression in oral squamous cell carcinomas (OSCCs) and it was found that higher MUC1 expression correlates with tumor invasion and metastasis. Using oral squamous cell carcinoma cell lines, we demonstrate here that tumor-specific targeting of MUC1 with the specific monoclonal antibody C595 has functional consequences with regard to complement deposition on MUC1-expressing oral cancer cell lines. Anti-MUC1 monoclonal antibody (mAb) also induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) to OSCC cells, and these effects were strongly correlated with MUC1 expression. Thus, these results indicate that anti-MUC1 mAb could provide a useful tool against OSCCs, and may provide insight into the development of low side-effect targeting therapy for this malignant disease.

Expression of MUC1 mucin in full-term pregnancy human placenta

MUC1 mucin is a component of glycocalyx in human endometrium and may play an important role in generation of "receptive window" at embryo implantation. Considering that MUC1 expression in human placenta is changed during pregnancy, and that MUC1 structure and function are not completely known in this organ, we have undertaken isolation of this mucin and detection of glycan epitopes, since they are crucial for its properties. Samples of human placenta were homogenized and MUC1 was extracted in different conditions with the use of ionic or non-ionic detergents. Identification of this glycoprotein was performed by Western and lectin blotting, after its purification on Sepharose 4B column. The best extraction of MUC1 glycoprotein was achieved with a non-ionic detergent, Triton X-100. Reactions with anti-MUC1 antibody showed a few glycoforms with molecular weights between 116 and 205 kDa, with the most visible glycoform approximating 205 kDa. Reactions with lectins enabled detection of carbohydrate antigens, such as T and Tn with sialic acid linked by alpha2, 3 and to a lesser extent by alpha2, 6 bond. MUC1 mucin is present in several glycoforms on the maternal side of human placenta after term delivery. They contain short glycan structures, similar to some tumor carbohydrate antigens.

PAM4-Reactive MUC1 Is a Biomarker for Early Pancreatic Adenocarcinoma

The anti-MUC1 monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens were assessed by immunohistology for expression of the PAM4-reactive, non-variable number of tandem repeats (VNTR), MUC1 epitope, and the VNTR epitope bound by the MA5 MAb. The PAM4-reactive MUC1 epitope was not detected in normal pancreas but was expressed in 87% (48 of 55) of invasive pancreatic adenocarcinomas, including early stage 1 disease: PAM4 labeled 94% (44 of 47) of the earliest PanIN lesions, PanIN-1A and 1B, along with 91% (10 of 11) of PanIN-2, 40% (2 of 5) of PanIN-3, and 86% (31 of 36) of intraductal papillary mucinous neoplasia lesions. A mostly diffuse pattern of labeling was observed. A second, unrelated, anti-MUC1 MAb, MA5, showed considerably less sensitivity with early PanIN-1 lesions; only 61% (25 of 41) were positive and the labeling did not differentiate normal pancreas from PanINs. The results suggest that expression of the PAM4-reactive antigen may represent an early event in the development of invasive pancreatic adenocarcinoma, and is unrelated to the VNTR peptide core epitopes of MUC1. Detection of this biomarker using immunohistology, in vitro immunoassays, and in vivo antibody-based imaging may provide new opportunities for the early detection and improved diagnosis of pancreatic cancer.

Epitope Mapping of PR81 anti-MUC1 Monoclonal Antibody Following PEPSCAN and Phage Display Techniques

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.