MUC1 Antibodies Research Articles

Preliminary Study on the Modification of Quantum Dots/Geothermal Silica Nanocomposites with Breast Cancer Antibody (MUC-1)

In this research, a silica-based nanosensor is synthesized using the sol-gel method and subsequently modified with quantum dots and MUC-1 antibody to detect MCF7, a cell line commonly found in breast cancer. Synthesis of silica nanoparticles was conducted through the sol-gel process using NaOH and HCl. The characterization using surface area analysis shows that geothermal silica based nanoparticles exhibit specific size of 31.2 nm with specific surface area of 192.37 m2/g. The nanoparticles were then modified using Cd-based Carboxyl Quantum dots to give fluorescence properties, obtaining SiNP@QD. Characterization was performed using UV-Vis and fluorescence spectroscopy to understand the photostability of nanoparticles in PBS buffer in various concentrations. The fluorescent nanoparticles were then immobilized with MUC-1 antibody and the successful conjugation was confirmed with FTIR showing peaks at 1548 and 1637 cm−1 corresponding to the amide I and amide II stretching vibration, respectively. The MUC-1 antibody modified silica nanoparticles is potential to be applied as nanosensor for the optical detection of MCF-7 cell line as one of breast cancer biomarkers.

Antitumor effect of a novel humanized MUC1 antibody-drug conjugate on triple-negative breast cancer

Breast cancer is the most common malignant cancer in women. Triple-negative breast cancer (TNBC) has a poorer prognosis than other subtypes and is challenging to treat. MUC1 is a therapeutic target in breast and pancreatic cancer. We developed a novel humanized antibody that specifically binds MUC1 expressed in breast cancer cells and conjugated a humanized MUC1 (HzMUC1) antibody to monomethyl auristatin (MMAE). HzMUC1-MMAE showed an anti-proliferative effect on HER2 positive trastuzumab-resistant breast cancer. Immunoprecipitation indicated that HzMUC1 recognized native MUC1 in TNBC cells. Confocal microscopy showed that HzMUC1 bound MUC1 on the surface of TNBC cells, and the conjugates exhibited the same binding ability to HCC70 as unconjugated HzMUC1 by cell-based ELISA. Treatment of TNBC cells with HzMUC1-MMAE reduced growth of MUC1-positive cells and induced G2/M cell cycle arrest and apoptosis. In a mouse model of breast cancer, HzMUC1-MMAE significantly reduced the growth of tumors established by subcutaneous injection of HCC70 TNBC cells. Therefore, HzMUC1-ADC has therapeutic potential for TNBC.

Therapeutic effect of a MUC1-specific monoclonal antibody-drug conjugates against pancreatic cancer model

BackgroundPancreatic cancer is one of the most aggressive malignancies without effective targeted therapies. MUC1 has emerged as a potential common target for cancer therapy because it is overexpressed in a variety of different cancers including the majority of pancreatic cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1 have been reported. Recently, we generated a humanized MUC1 antibody (HzMUC1) specific to the interaction region between MUC1-N and MUC1-C. In this study, we generated the antibody drug conjugate (ADC) by conjugating HzMUC1 with monomethyl auristatin (MMAE), and examined the efficacy of HzMUC1-MMAE against the MUC1-positive pancreatic cancer in vitro and in vivo.MethodsWestern blot and immunoprecipitation were used to detect MUC1 in pancreatic cancer cells. MUC1 localization in pancreatic cancer cells was determined by confocal microscopy. HzMUC1 was conjugated with the monomethyl auristatin (MMAE), generating the HzMUC1-MMAE ADC. Colony formation assay and flow cytometry were used to assess the effects of the HzMUC1-MMAE cell viability, cell cycle progression and apoptosis. Capan-2 and CFPAC-1 xenograft model were used to test the efficacy of HzMUC1-MMAE against pancreatic cancer.ResultsHzMUC1 antibody binds to MUC1 on the cell surface of pancreatic cancer cells. HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in pancreatic cancer cells. Importantly, HzMUC1-MMAE significantly reduced the growth of pancreatic xenograft tumors by inhibiting cell proliferation and enhancing cell death.ConclusionOur results indicate that HzMUC1-ADC is a promising novel targeted therapy for pancreatic cancer. HzMUC1-ADC should also be an effective drug for the treatment of different MUC1-positive cancers.

Expresión de la mucina asociada a membrana MUC-1/ y su relación con Ki67 en el carcinoma mucoepidermoide de glándulas salivales

Background: Salivary gland tumors are rare and with different malignant potential. Immunohistochemistry is useful, especially Ki67, a marker of cell proliferation, for its prognostic information. On the other side, the mucin MUC-1 is overexpressed and loses its exclusively apical membranous localization and is aberrantly glycosylated in mucoepidermoid carcinoma of salivary glands. In the present work, we investigated the quantitative expression of tumor proliferative index and localization and overexpression of MUC-1 in the mucoepidermoid carcinoma of the human salivary glands, not studied is these organs, to determine their relationship with the histological grade and prognosis. Methods: H/E staining and immunohistochemistry with MUC-1 and Ki67 antibodies was performed on 10 cases. Results: The markings of the mucoepidermoid carcinoma depended on the histological grade; in low- grade tumors with MUC-1 only 10 to 25% of the cells were labeled, in contrast to high- grade tumors, with 75 to 90% labeled cells. They also differed in cell proliferation; in the low-grade tumors was 15% and in high-grade tumors it was greater than 30%. Conclusion: MUC-1 overexpression is significantly higher in high-grade mucoepidermoid carcinoma, as is Ki67 marking, results that are associated with tumor prognosis.

Longitudinal Modulation of Loco-Regional Immunity in Ovarian Cancer Patients Receiving Intraperitoneal Chemotherapy.

The immune tumor microenvironment (TME) of epithelial ovarian cancer (EOC) carries both effector and suppressive functions. To define immune correlates of chemotherapy-induced tumor involution, we performed longitudinal evaluation of biomarker expression on serial biological specimens collected during intraperitoneal (IP) platinum-based chemotherapy. Serial biological samples were collected at several time points during IP chemotherapy. RNA from IP fluid cells and tumor tissue was analyzed via NanoString. Meso Scale Discovery (MSD) multiplex assay and ELISA for MUC1 antibodies were performed on plasma and IP fluid. Differentially expressed genes in IP fluid demonstrate an upregulation of B cell function and activation of Th2 immune response along with dampening of Th1 immunity during chemotherapy. MSD analysis of IP fluid and gene expression analysis of tumor tissue revealed activation of Th2 immunity and the complement system. Anti-MUC1 antibodies were detected in IP fluid samples. IP fluid analysis in a secondary cohort also identified chemotherapy-induced B cell function genes. This study shows that serial IP fluid sampling is an effective method to capture changes in the immune TME during chemotherapy and reveals treatment induced changes in B cell function and Th2 immunity.

Tumour-associated Mucin1 correlates with the procoagulant properties of cancer cells of epithelial origin

BackgroundCancer-associated thrombosis (CAT) is caused, at least in part, by procoagulant factors produced by the tumour itself. Although MUC1 is an established biomarker for the diagnosis, immunotherapy, and prognosis of cancer, it is unclear whether it contributes to the procoagulant phenotype of cancer cells. MethodsMUC1 knockdown breast cancer MCF-7 cells were used to investigate the influence of overexpression of MUC1 on procoagulant parameters. In addition, the effect of treating normal human epithelial cells with extracellular vesicles from several human breast and pancreatic cancer cell lines, which overexpress MUC1, was determined. The impact of a pharmacological anti-MUC1 antibody on cancer cells was also analysed. ResultsThe level of a range of procoagulant proteins was observed to correlate with the MUC1 level of human breast and pancreatic cancer cell lines. MUC1 downregulation in MCF-7 cells led to a reduction in the procoagulant parameters particularly thrombin activity. The levels of selected tumorigenic markers, procoagulant proteins and miRNAs associated with tumorigenicity and thromboembolism were also modulated by treatment of normal cells with tumour cell derived extracellular vesicles in correlation with that of the extracellular vesicles donor cells. Moreover, the procoagulant properties were also reduced by an anti-MUC1 antibody in these cancer cells. ConclusionsA range of procoagulant proteins found in human breast and pancreatic cancer cells were shown to exhibit a positive correlation with the level of MUC1 and thereby potentially contribute to the pathogenesis of CAT. The reduction of the procoagulant activity by MUC1 antibody could be an additional beneficial effect of its therapeutic efficacy. These findings also suggest that the level of tumour associated MUC1 could be of use as a risk factor for CAT.

Comparative study on the antitumor effects of gemcitabine polybutylcyanoacrylate nanoparticles coupled with anti-human MUC1 and CA199 monoclonal antibodies on pancreatic cancer in vitro and in vivo

Background and Study AimsThis study was designed to compare the antitumor effects of anti-human MUC1 monoclonal antibody with those of anti-human CA199 monoclonal antibody coupled with drug-loaded polybutylcyanoacrylate nanoparticles on human pancreatic cancer cell lines and pancreatic cancer-bearing model animals and to screen more efficient targeting molecules. Patients and MethodsGemcitabine-loaded nanospheres were prepared by emulsion polymerization (GEM-PBCA-NP), and then, anti-MUC1 monoclonal antibody was coupled with GEM-PBCA-NP (MUC1-GEM-PBCA-NP), and anti-human CA199 monoclonal antibody was coupled with GEM-PBCA-NP (CA199-GEM-PBCA-NP), using the chemical crosslinking method. The cell-killing rates were detected using MTT assay. The changes in the tumor cell cycle and apoptosis after treatment were detected using flow cytometry. Then, the subcutaneous planting method was adopted to establish an animal model of pancreatic cancer: two nanometer microspheres were injected into the body of nude mice via the tail vein; the tumor suppression effect was detected after treatment; then, the groups were compared. ResultsIn vitro, the cell-killing rate of each experimental group was significantly different from that of the control group (P < 05). The MUC1-GEM-PBCA-NP group had a significantly higher cell-killing rate than the other groups (P < 05). The apoptosis rate of the MUC1-GEM-PBCA-NP treatment group was significantly higher than that of other groups (P < 05). In vivo, the tumor inhibition rate of the MUC1-GEM-PBCA-NP treatment group was 72.69% ± 4.29%, which was significantly higher than those of other groups (P < 0.05). The tumor inhibition rate of the CA199-GEM-PBCA-NP treatment group was 56.58% ± 5.11%, which was significantly higher than those of other control groups (P < 0.05). At the end of treatment, the average tumor mass of the MUC1-GEM-PBCA-NP treatment group was 433.55 ± 12.49 mg, which was significantly lower than those of other groups (P < 0.05). ConclusionCompared with CA199-GEM-PBCA-NP, MUC1-GEM-PBCA-NP is more effective in vitro and in vivo. MUC1 could be a target molecule in treating pancreatic cancer.

A novel humanized MUC1 antibody-drug conjugate for the treatment of trastuzumab-resistant breast cancer.

Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic.

Expression of Tumour-Associated MUC1 Is a Poor Prognostic Marker in Breast Cancer in Kumasi, Ghana.

Background Immunohistochemical assessment of breast cancer and stratification into the basic molecular subtypes afford a much deeper insight into the biology of breast cancer, while presenting with opportunities to exploit personalized, targeted treatment. Traditionally, the oestrogen, progesterone, and epidermal growth factor receptors are assessed. MUC1, a transmembrane mucin, has been demonstrated a potential prognostic and metastatic marker in breast cancer. However, there have been a limited number of studies addressing the predictive and prognostic features of MUC1 in African breast cancer. This study aims at addressing the expression profiles of MUC1 and other biomarkers in Ghanaian breast cancer and determines its predictive and prognostic characteristics, in relation to other clinicopathological features. Methods Haematoxylin and eosin (H&E) slides of breast cancer cases were reviewed and 203 suitable cases were selected for tissue microarray (TMA) construction and immunohistochemistry. Anti-ER, PR, HER2, Ki-67, and MUC1 antibodies were used. Results from the immunostaining were analysed using SPSS version 23. Results About 59% of cases expressed MUC1. Majority of cases in the study showed a lack of expression of all three traditional markers (29% expressed ER, 10.9% PR, and 20.7% HER2). Ki-67 index were 62.1% (low), 16.5% (moderate), and 21.4% (high). MUC1 expressions among the molecular classes were luminal A (60.7%), luminal B (68.8%), HER2 overexpression (87.5%), and triple negative (56.6%). There were significant associations between MUC1 and HER2 overexpression (p=0.01) and triple negative (p < 0.01). Conclusion The high proportion of breast cancer cases expressing MUC1, as well as its association with the two most aggressive molecular classes, indicate a substantial role in the biology of breast cancer in our cohort, and it is an indication of poor prognosis.

Magnetic-Based Enrichment of Rare Cells from High Concentrated Blood Samples.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

A Physiologically-Based Pharmacokinetic Model for Targeting Calcitriol-Conjugated Quantum Dots to Inflammatory Breast Cancer Cells.

Quantum dots (QDs) conjugated with 1,25 dihydroxyvitamin D3 (calcitriol) and Mucin‐1 (MUC‐1) antibodies (SM3) have been found to target inflammatory breast cancer (IBC) tumors and reduce proliferation, migration, and differentiation of these tumors in mice. A physiologically‐based pharmacokinetic model has been constructed and optimized to match experimental data for multiple QDs: control QDs, QDs conjugated with calcitriol, and QDs conjugated with both calcitriol and SM3 MUC1 antibodies. The model predicts continuous QD concentration for key tissues in mice distinguished by IBC stage (healthy, early‐stage, and late‐stage). Experimental and clinical efforts in QD treatment of IBC can be augmented by in silico simulations that predict the short‐term and long‐term behavior of QD treatment regimens.

Subtyping of intraductal papillary mucinous neoplasms - pitfalls of MUC1 immunohistochemistry.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions of pancreatic ductal adenocarcinoma (PDAC). Current edition of WHO Classification of Tumors of the Digestive System recognizes four different subtypes (gastric, intestinal, pancreatobiliary, and oncocytic) and recommends analysis of mucin expression (MUC1, MUC2, MUC5AC, MUC6) as well as evaluation of architectural and cell differentiation patterns for correct classification. However, there is no consensus on MUC1 expression of IPMN-lesions in the literature. Current recommendations are based on studies where antibodies against the core MUC1 protein or sialylated MUC1 (tumor associated MUC1), not the fully glycosylated MUC1 were used. We have recently reported that MUC1 is strongly expressed in both gastric and intestinal types IPMN specimens from the cystic wall, obtained by endoscopic ultrasound guided microbiopsy procedure. We have used a commercial MUC1 antibody, validated and recommended for diagnostic use, which recognizes fully glycosylated MUC1. Based on the above, we propose a revision of the WHO Classification, specifying that antibodies against tumor associated MUC1 should be used for IPMN subtyping.

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition

Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles

BackgroundPancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target.MethodsPoly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles.ResultsIn vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs.ConclusionsThe study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.

360 (PB-155) - The effect of monoclonal MUC1 antibody loaded on pegilated carbon nanotubes on MCF7 cell line

360 (PB-155) - The effect of monoclonal MUC1 antibody loaded on pegilated carbon nanotubes on MCF7 cell line

Immunogenicity of chimeric MUC1-HER2 vaccine against breast cancer in mice.

Objective(s):Breast cancer is one of the most common cancers in the world and is on the increase. MUC1 and HER2 as tumor-associated antigens (TAAs) are abnormally expressed to some extent in 75–80% of breast cancers. In our present research, a novel chimeric MUC1-HER2 (HM) protein was designed and used to study whether an immune response can be generated against these TAAs. In vitro analysis of the HER2-MUC1 construct confirmed the co-expression of MUC1 and HER2.Materials and Methods:BALB/c mice were immunized with this novel chimeric protein. The humoral immune response was assessed by enzyme-linked immunosorbent assay (ELISA). Then, BALB/c mice were injected subcutaneously 2×105 4T1-MUC1-HER2 tumor cells. Subsequently, tumor size and tumor necrosis measurements, MTT, cytokines assay and survival test were performed.Results:The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer.Conclusion:The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer.

Clinicopathological Study of Carcinoma of the Ampulla of Vater with Special Reference to MUC1, MUC2 and MUC5AC Expression.

The ampulla of Vater consists of papilla, common channel, distal common bile duct and the distal main pancreatic duct. Ampullary carcinomas arise from two different types of mucosa, reflecting their broad histomorphological spectrum. Adenocarcinomas originating in the ampulla of Vater are classified as having either 'Intestinal' or 'Pancreatobiliary' type of differentiation. Pancreatobiliary type has consistently shown a worse prognosis. Various types of mucins are distributed in normal tissues and gastrointestinal tumours. MUC1, MUC2 and MUC5AC are known to be the most important amongst all. To study clinicopathological features of different types of carcinoma of ampulla with MUC1, MUC2 and MUC5AC expression. We included 20 cases of ampullary adenocarcinoma in this study, diagnosed after radical surgery. The histopathological slides were reviewed and the tumours were classified into intestinal type, pancreatobilliary and other types. Representative block of tumours was subjected to immunohistochemical (IHC) study with MUC1, MUC2 and MUC5AC antibodies. Results were subsequently analysed. Pancreaticobiliary type of ampullary carcinoma cases predominated (50%) over intestinal type (25%). Carcinomas of "other types" included one case each of mucinous carcinoma, adenosquamous carcinoma and undifferentiated carcinoma and well differentiated neuroendocrine tumours. Pancreatobiliary type of carcinomas was associated with higher tumour stage and grade and lymph node metastasis. All the pancreatobiliary type expressed MUC1 and all intestinal type carcinomas expressed MUC2. MUC 5AC expression was seen predominantly in pancreatobiliary type of ampullary carcinomas. Mucin markers are associated with two different subtypes of ampullary carcinomas which have different prognosis. Immunohistochemical study of mucin expression can define the different types of carcinomas of ampulla of Vater with better objective criteria.

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases.

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases.

Synthesis of a MUC1 Mucin Cyclic Dimer Peptide and Its Antibody Binding Properties as Revealed by STD-NMR

In a previous study we showed that the shortened MUC1 mucin peptide GVTSAPD could bind monoclonal antibody (mAb). We proceeded on to make a cyclic peptide of the same sequence to see if it would be more effective in binding antibody. We were able to synthesize and isolate two different cyclic mucin peptides: 1) a monomer cyclic peptide with sequence GVTSAPD which we did not study due to difficulties in achieving homogeneity, and 2) a dimer cyclic peptide with sequence GVTSAPDGVTSAPD that was successfully isolated and studied. We describe here the results of the dimer cyclic peptide-antibody interactions obtained by Saturation Transfer Difference NMR (STDNMR). The results indicated that the protons of all residues experienced STD effects, notably being more pronounced at Pro, Val, Ala and Asp compared to the linear peptide GVTSAPD. The Pro residue exhibited STD peaks for all its side chain protons with stronger intensity at ProHγ while Ala, Val and Thr are localized to methyl groups. KEYWORDS: Muc1 Antibody Recognition Epitope, STD NMR, Mucin Peptide-antibody Interactions, Cyclodimer Peptide.

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody.

The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.