MUC1-specific Antibodies Research Articles

An efficient and safe MUC1-dendritic cell-derived exosome conjugate vaccine elicits potent cellular and humoral immunity and tumor inhibition in vivo

Antitumor vaccines are a promising strategy for preventing or treating cancers by eliciting antitumor immune responses and inducing protective immunity against specific antigens expressed on tumor cells. Vaccine formulations that enhance the humoral and cellular immune responses of vaccine candidates would be highly beneficial but are still limited. Here we developed an antitumor vaccine candidate by conjugating a MUC1 glycopeptide antigen to dendritic cell-derived exosomes (Dex). In vivo, the MUC1-Dex construct induced high MUC1-specific IgG antibody titers with strong binding affinities for MUC1-positive tumor cells and promoted cytokine secretion. Moreover, CD8+ T cells from immunized mice exhibited strong cytotoxicity against MUC1-positive tumor cells. Importantly, in both preventative and therapeutic tumor-bearing mouse models, the construct inhibited tumor growth and prolonged survival. Collectively, these results demonstrate that Dex is a promising vaccine carrier that can be used as adjuvant to enhance the immunological efficacy of tumor vaccines. Statement of significance•We prepared an efficient and biosafe antitumor vaccine candidate by conjugating Dex with MUC1 glycopeptide using a two-step biorthogonal crosslinking strategy.•MUC1-Dex enhanced the expression of costimulatory molecules CD80 and CD86 on DCs to promote their maturation whilst simultaneously upregulating MHC I and MHC II expression to increase antigen presentation by DCs.•MUC1-Dex conjugates stimulated the production of MUC1-specific IgG antibodies in vivo, and the antibodies bound strongly to MUC1-expressing tumor cells.•The constructed vaccines induced a strong antibody response and antigen specific CTLs to kill the tumor and delayed tumor development in a therapeutic and prophylactic tumor bearing mouse model.

The Adjuvant of α-Galactosylceramide Presented by Gold Nanoparticles Enhances Antitumor Immune Responses of MUC1 Antigen-Based Tumor Vaccines.

BackgroundTherapeutic tumor vaccines are one of the most promising strategies and have attracted great attention in cancer treatment. However, most of them have shown unsatisfactory immunogenicity, there are still few available vaccines for clinical use. Therefore, there is an urgent demand to develop novel strategies to improve the immune efficacy of antitumor vaccines.PurposeThis study aimed to develop novel adjuvants and carriers to enhance the immune effect of MUC1 glycopeptide antigen-based antitumor vaccines.MethodsAn antitumor vaccine was developed, in which MUC1 glycopeptide was used as tumor-associated antigen, α-GalCer served as an immune adjuvant and AuNPs was a multivalent carrier.ResultsImmunological evaluation results indicated that the constructed vaccines enabled a significant antibody response. FACS analysis and immunofluorescence assay showed that the induced antisera exhibited a specific binding with MUC1 positive MCF-7 cells. Moreover, the induced antibody can mediate CDC to kill MCF-7 cells. Besides stimulating B cells to produce MUC1-specific antibodies, the prepared vaccines also induced MUC1-specific CTLs in vitro. Furthermore, the vaccines significantly delayed tumor development in tumor-bearing mice model.ConclusionThese results showed that the construction of vaccines by presenting α-GalCer adjuvant and an antigen on gold nanoparticles offers a potential strategy to improve the antitumor response in cancer immunotherapy.

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.

Circulating Myeloid Derived Suppressor Cells (MDSC) That Accumulate in Premalignancy Share Phenotypic and Functional Characteristics With MDSC in Cancer.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate in circulation of cancer patients and at tumor sites where they suppress anti-tumor immunity. We previously reported that in a colon cancer prevention trial of a MUC1 vaccine tested in individuals at increased risk for colon cancer, those who did not mount immune response to the vaccine had higher pre-vaccination levels of circulating MDSC compared to those who did. We also reported that individuals with pancreatic premalignancy, Intraductal Papillary Mucinous Neoplasm (IPMN), had increased circulating levels of MDSC that inversely correlated with spontaneous antibody responses against the pancreatic tumor associated antigen MUC1, abnormally expressed on IPMN. Accumulation of MDSC in cancer and their immunosuppressive role had been well established but their presence in premalignancy was unexpected. In this study we compared MDSC in premalignancy with those in cancer with the hypothesis that there might be differences in the composition of various MDSC subpopulations and their immunosuppressive functions due to different lengths of exposure to disease and/or different tissue microenvironments. In cohorts of patients with premalignant polyps, colon cancer, premalignant IPMN, and pancreatic cancer, we confirmed higher levels of MDSC in premalignancy compared to healthy controls, higher levels of MDSC in cancer compared to premalignancy, but no difference in their subpopulation composition or immunosuppressive capacity. We show that levels of MDSC in premalignancy correlate negatively in vivo with spontaneous MUC1-specific antibody responses and in vitro with polyclonal T cell proliferation and IFN-γ secretion.

Synthesis of a Self-Adjuvanting MUC1 Vaccine via Diselenide-Selenoester Ligation-Deselenization.

Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphipathic nature of the molecules, which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology, the diselenide-selenoester ligation (DSL), to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glyco- and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam2Cys. This construct was shown to elicit MUC1-specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the helper T cell epitope PADRE both boosted the antibody response and resulted in elevated cytokine production.

ICOSL-augmented adenoviral-based vaccination induces a bipolar Th17/Th1 T cell response against unglycosylated MUC1 antigen

Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10 months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo, establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine.

Immunogenicity of a Tripartite Cell Penetrating Peptide Containing a MUC1 Variable Number of Tandem Repeat (VNTR) and A T Helper Epitope.

Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses.

CpG ODN1826 as a Promising Mucin1-Maltose-Binding Protein Vaccine Adjuvant Induced DC Maturation and Enhanced Antitumor Immunity.

Mucin 1 (MUC1), being an oncogene, is an attractive target in tumor immunotherapy. Maltose binding protein (MBP) is a potent built-in adjuvant to enhance protein immunogenicity. Thus, a recombinant MUC1 and MBP antitumor vaccine (M-M) was constructed in our laboratory. To enhance the antitumor immune activity of M-M, CpG oligodeoxynucleotides 1826 (CpG 1826), a toll-like receptor-9 agonist, was examined in this study as an adjuvant. The combination of M-M and CpG 1826 significantly inhibited MUC1-expressing B16 cell growth and prolonged the survival of tumor-bearing mice. It induced MUC1-specific antibodies and Th1 immune responses, as well as the Cytotoxic T Lymphocytes (CTL) cytotoxicity in vivo. Further studies showed that it promoted the maturation and activation of the dendritic cell (DC) and skewed towards Th1 phenotype in vitro. Thus, our study revealed that CpG 1826 is an efficient adjuvant, laying a foundation for further M-M clinical research.

Combination of mAb-AR20.5, anti-PD-L1 and PolyICLC inhibits tumor progression and prolongs survival of MUC1.Tg mice challenged with pancreatic tumors.

A substantial body of evidence suggests the existence of MUC1-specific antibodies and cytotoxic T cell activities in pancreatic cancer patients. However, tumor-induced immunosuppression renders these responses ineffective. The current study explores a novel therapeutic combination wherein tumor-bearing hosts can be immunologically primed with their own antigen, through opsonization with a tumor antigen-targeted antibody, mAb-AR20.5. We evaluated the efficacy of immunization with this antibody in combination with PolyICLC and anti-PD-L1. The therapeutic combination of mAb-AR20.5 + anti-PD-L1 + PolyICLC induced rejection of human MUC1 expressing tumors and provided a long-lasting, MUC1-specific cellular immune response, which could be adoptively transferred and shown to provide protection against tumor challenge in human MUC1 transgenic (MUC.Tg) mice. Furthermore, antibody depletion studies revealed that CD8 cells were effectors for the MUC1-specific immune response generated by the mAb-AR20.5 + anti-PD-L1 + PolyICLC combination. Multichromatic flow cytometry data analysis demonstrated a significant increase over time in circulating, activated CD8 T cells, CD3+CD4-CD8-(DN) T cells, and mature dendritic cells in mAb-AR20.5 + anti-PD-L1 + PolyICLC combination-treated, tumor-bearing mice, as compared to saline-treated control counterparts. Our study provides a proof of principle that an effective and long-lasting anti-tumor cellular immunity can be achieved in pancreatic tumor-bearing hosts against their own antigen (MUC1), which can be further potentiated using a vaccine adjuvant and an immune checkpoint inhibitor.

High Affinity Binding of a MUC1 Specific Antibody to a Glycopeptide Mediated by Conformational Selection

MUC1 is a transmembrane glycoprotein and is a target for antibody‐mediated immunotherapy. In cancer cells, MUC1 is upregulated, and undergoes abnormal glycosylation resulting in truncated carbohydrate chains being added to the protein. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. The peptide sequence, and the cancer specific truncated glycans are both available for antibody recognition. There is significant interest in determining which is the more suitable target for antibody immunotherapy. We investigated the role of tumor specific glycosylation in antigen recognition by the therapeutic antibody AR20.5. Using microscale thermophoresis we explored the affinity of AR20.5 to cancer specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than against the naked peptide. Given these results we postulated that A20.5 must specifically bind the carbohydrate as well as the peptide. Using X‐ray crystallography we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts supports a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide that is recognized by the antibody. High affinity binding of AR20.5 to the MUC1 glycopeptide is not driven by specific antibody‐antigen contacts, but rather glycosylation alters the conformational equilibrium of the antigen which allows the antibody to select the correct coformation. This study demonstrates a novel mechanism of antibody‐antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.Support or Funding InformationThis work was supported through start‐up funds from the College of Science and Math at CSUF, and by a research grant from Quest Pharmatech Inc. to CLB. MR was supported by rGrants from ASI at CSUF and FSSRA awards from the College of Science and Math at CSUF.

Pre-clinical toxicity and immunogenicity evaluation of a MUC1–MBP/BCG anti-tumor vaccine

Mucin 1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas and is an attractive target in tumor immunotherapy. Our previous study showed that the MUC1–MBP/BCG anti-tumor vaccine induced a MUC1-specific Th1-dominant immune response, simulated MUC1-specific cytotoxic T lymphocyte killing activity, and could significantly inhibit MUC1-expression B16 cells' growth in mice. To help move the vaccine into a Phase I clinical trial, in the current study, a pre-clinical toxicity and immunogenicity evaluation of the vaccine was conducted. The evaluation was comprised of a single-dose acute toxicity study in mice, repeat-dose chronic toxicity and immunogenicity studies in rats, and pilot toxicity and immunogenicity studies in cynomolgus monkeys. The results showed that treatment with the MUC1–MBP/BCG anti-tumor vaccine did not cause any organ toxicity, except for arthritis or local nodules induced by BCG in several rats. Furthermore, the vaccine significantly increased the levels of IFN-γ in rats, indicating that Th1 cells were activated. In addition, the results showed that the MUC1–MBP/BCG anti-tumor vaccine induced a MUC1-specific IgG antibody response both in rats and cynomolgus monkeys. Collectively, these data are beneficial to move the MUC1–MBP/BCG anti-tumor vaccine into a Phase I clinical trial.

Abstract POSTER-THER-1434: Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host

Abstract Purpose: Immunotherapy with monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance antitumor responses has recently showed clinical promise in advanced solid tumors. However, most of the preliminary evidence on therapeutic efficacy comes from melanoma, prostate and lung cancer. Further studies on other tumor types (including ovarian cancer), and on additional, potentially more effective regimens that take into consideration the administration route and baseline immune status of the host, are currently needed. Approach: We recently generated a triple transgenic MUC1+/-KrasG12D/+PtenloxP/loxP (MKP) mouse model that carries a conditional, oncogenic Kras mutation, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors that overexpress MUC1 tumor-associated antigen. Using an orthotopic MKP ovarian tumor, we generated a new ovarian cancer cell line (2F8). MUC1.Tg mice injected IP with 8x10E5 of completely syngeneic 2F8 cells develop IP tumors. Mice wild-type for MUC1 were similarly challenged with 2F8 cells, thus creating an isogenic system, in which the hosts are syngeneic to all other tumor antigens, except for human MUC1. Results: At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Anti-PDL1 was administered IP every 2 weeks (200 mg/dose) for a total of 3 doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to a late tumor stage. The anti-PD-L1 treated MUC1.Tg mice showed substantial T cell infiltration within the tumor and significantly increased survival (p= 0.001) compared to isotype control- treated mice. In contrast, wild type mice with isogenic 2F8 tumors developed spontaneous, high titer anti-MUC1 antibodies and did not respond to anti-PD-L1 therapy. However, earlier treatment (at day 11, instead of day 21) post tumor challenge, higher frequency of IP injections (weekly) and higher overall dose (seven doses, 200 μg/dose) increased treatment efficacy in these mice and led to increased survival. Anti-PDL1 therapy did not change the amplitude of MUC1 specific antibodies in responding mice. In addition to the above quantitative and qualitative changes in systemic and intratumoral T cells, broad range profiling using microarrays are ongoing and will reveal the hallmarks of response in anti-PD-L1 treated mice. Conclusions: These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer and can increases survival despite (1) treatment initiation at late stage, (2) low dose/low frequency of administration and (3) low PD-L1 expression on tumors. By employing an isogenic animal model that favors anti-tumor humoral immunity, we show here that tumor-bearing hosts with antibody-driven immune imbalance at time of treatment initiation are non-responsive and may require a more dose intensive regimen. This is in line with our previous findings showing inverse correlation between survival and pre-existing MUC-1 specific antibody levels in ovarian cancer patients receiving IP Interleukin -2 (IL-2). Our studies have high translational potential and support IP administration of PD-L1 blockers in ovarian cancer patients, while taking into consideration pre-existent, anti-tumor antibodies. Citation Format: Jyothi Mony, Lixin Zhang, Tejas Tirodkar, Esther Elishaev, Joan Brozick, Robert P. Edwards, Anda M. Vlad. Intraperitoneal anti-PD-L1 increases survival in a novel ovarian cancer model and its in vivo efficacy is influenced by baseline anti-tumor immunity of the host [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1434.

Mucin 1-specific B cell immune responses and their impact on overall survival in breast cancer patients

Considering the diverse functions of B cells, responses to tumor-associated antigens (TAA) have been thought to be the main source of B cell-mediated antitumor immunity. Polymorphic epithelial mucin (MUC1) is considered one of the most specific TAA in patients with breast cancer. The present study aims to dissect the level and subclasses of naturally occurring anti-MUC1 antibodies in regard to tumor biologic parameters, clinical characteristics and overall survival. In 288 primary, non-metastatic breast cancer patients, pretreatment serum levels of anti-MUC1 immunoglobulin G (IgG) and its subclasses G1–4 as well as immunoglobulin M (IgM) were analyzed via ELISA. With respect to overall survival (Kaplan–Meier analysis), tumor biologic parameters as hormone receptor status, human epidermal growth factor receptor 2 (Her2), Ki-67 expression and tumor grading have been correlated as well as clinical characteristics as nodal involvement, tumor stage and patients' age at the time of diagnosis. Median follow-up time was 148 mo (IQR: 73.1–158.5 mo). A significant increase in IgG antibody titers was correlated highly significantly with an improved overall survival of patients. In multivariate analysis, total IgG proved to be an independent prognostic marker for overall survival (p = 0.002). IgG subclass analysis did not reveal any correlation of IgG1, IgG3 and IgG4 levels with overall survival, while increased immunoglobulin G2 (IgG2) values, although statistically not significant, tended to correlate with prolonged patient survival. MUC1-specific IgM antibodies were shown not to be predictive of overall survival. Altogether, humoral immune responses appear to play a crucial part in the tumor immunity of breast cancer patients. The present data confirms the positive impact of tumor-specific IgG on prolonged overall survival in breast cancer patients. MUC1-antibody testing might be a useful tool to identify high-risk patients who may need adjuvant therapy and potentially might benefit from MUC1-directed immunotherapy.

Autoantibodies against the signal peptide domain of MUC1 in patients with multiple myeloma: Implications for disease diagnosis and prognosis

Naturally generated autoantibodies to tumor-associated antigens such as MUC1 can assist in cancer diagnosis and prognosis. While previous studies have concentrated on the tandem repeat array domain of MUC1, here we focused on MUC1's signal peptide domain. We used ELISA assays with MUC1-specific epitopes and antibodies to quantify soluble MUC1 antigen and anti-MUC1 autoantibodies against the tandem repeat array and signal peptide domains in 15 naïve donors and 27 multiple myeloma cancer patients. We showed a significant increase in up to 24-fold (P<0.004) only in the levels of anti-MUC1 signal peptide autoantibodies in the sera of multiple myeloma patients vs. naïve donors. This increase stemmed chiefly from the preferred immunogenicity of the signal peptide. Moreover, a significant positive correlation (R(2)=0.5361, P<0.048, Pearson correlation) was shown between the levels of soluble MUC1 and anti-MUC1 signal peptide autoantibodies in multiple myeloma patients with progressive disease while under therapy. This is an initial report on the existence of autoantibodies to a signal peptide domain in general and to the MUC1 signal peptide domain in particular in cancer patients. The autoantibodies had MUC1 rather than signal peptide specificity. The specific nature of the antigen leading to generation of these autoantibodies is still unclear because it is unlikely that the target antigen is a major histo-compatibility complex-peptide complex and we could not trace soluble MUC1 signal peptide fragments in naïve donors and multiple myeloma patients. Further validation of these findings may improve diagnostic and prognostic capabilities for MUC1-positive multiple myeloma patients and potentially, patients with other MUC1-positive cancers, as well.

Recombinant plant‐expressed tumour‐associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.

Recognition of pleural mesothelioma by mucin-1(950-958)/human leukocyte antigen A*0201-specific CD8+ T-cells

Recent clinical investigations have demonstrated that T-cell-based immunotherapy of malignant pleural mesothelioma (MPM) could represent an alternative to the other therapeutic strategies. However, its development suffers from the lack of identified tumour antigenic targets. Mucin (MUC)1, which is expressed and recognised by cytotoxic T-cells in numerous cancer types, has not been investigated as a potential immune target in MPM. Thus, the objective of this study was to analyse MUC1 expression by MPM cells and to determine whether this antigen can be the target of cytotoxic CD8+ T-cells (cytotoxic T-lymphocytes (CTLs)). We first evaluated the expression and glycosylation of MUC1 by MPM cell lines using different MUC1-specific monoclonal antibodies. We then obtained a CTL clone specific for a MUC1 peptide (residues 950-958) presented by human leukocyte antigen (HLA)-A*0201 and studied its interferon-γ and cytotoxic response to MPM cell lines. We found that all MPM cell lines expressed MUC1 protein at the cell surface with different glycosylation profiles. We also observed that HLA-A*0201+ MPM cell lines are recognised and lysed by a HLA-A*0201/MUC1(950-958)-specific CTL clone independently of the MUC1 glycosylation profile. Thus, MUC1 expression and antigen presentation by MPM cells may represent an attractive target for immunotherapeutic treatment of MPM despite its hyperglycosylated profile.

Exposure to abnormal self antigens during non-malignant events may provide immunological defense against tumors (165.15)

Abstract Antibodies and T cells specific for several tumor associated antigens exist not only in cancer patients but also in healthy individuals. We hypothesize that non-malignant events such as viral infections elicit abnormal expression of self antigens on infected cells and specific immune memory for those antigens. Abnormal expression of the same antigens on tumor cells triggers specific immune responses and provides anti-tumor immunosurveillance. We are testing this hypothesis by examining changes in expression of known tumor associated antigens MUC1 and cyclin B1 during influenza virus infection of MUC1 transgenic mice. We found increased MUC1 and cyclin B1 expression in acutely infected lung tissue collected three days post infection. Furthermore, MUC1 became hypoglycosylated, the form found on cancer cells. Importantly, we found increased MUC1-specific and cyclin B1-specific antibodies in serum from mice post-infection compared to pre-infection. These antigens are likely to be representative of a much larger number of normal cell molecules that undergo changes in expression and induce an immune response important for cancer immunosurveillance. To address this, post-infection serum was blotted against tumor lysate separated by SDS-PAGE and compared to serum from uninfected mice. We found a marked increase in reactivity with several tumor proteins. In future experiments we will correlate immunity to specific antigens with cancer immunosurveillance.

Genetic profiling of circulating tumor cells in patients with advanced colorectal cancer.

e14113 Background: We have developed a new preanalytical enrichment method for circulating carcinoma cells (CTC) based on EpCAM and MUC1 specific antibodies coupled to immunomagnetic beads. Molecular detection and tumor cell characterization was performed with a multimarker panel by real-time RT-PCR. Here we present first results of a universal marker panel for local advanced and/or metastatic colorectal cancer. Methods: The high affinity antibodies BM7 (MUC-1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment from 10 ml peripheral EDTA-blood of patients with documented metastatic and/or local advanced disease. Separated cells were lysed and used for mRNA isolation and c-DNA synthesis. Real-time quantitative RT-PCR approaches with SYBR assays (Eurogentec) and FAM-labeled TaqMan probes selected with the UniversalProbeLibrary system (Roche AG, Basel, CH) were developed for the epithelial markers cytokeratin 19 and 20 (CK19/20), EpCAM, CEA, Survivin, metastasis associated in colon cancer (MACC) and HIF-1alpha. Results: Sensitivity of the the multimarker panel was validated in calibration tests with 2 and 10 cultured carcinoma cells (embedded tumor cell calibrators, ETC) and the specificity of the panel was confirmed by examination of blood from healthy donors. Positivity rate of ETC controlled real-time RT-PCR on the basis of the multimarker panel was 70% (32 of 46 patients) with local advanced and/or metastatic disease. The marker with the highest prevalence was EpCAM (52%) followed by Survivin (32%), MUC-1 (24%), CK19 (24%), HIF-1 (31%) MACC (15%) and CEA 14%. Conclusions: We have used embedded tumor cells (ETC) as internal calibrators for accurate process control and normalization of the immunobead quantitative RT-PCR technique. The newly introduced surrogate marker panel is linked to cellular characteristics as apoptosis, invasion, angiogenesis and stem cell phenotype. Therefore it may help to give additional information on prognosis and therapy response. It can be used in patient follow-up to detect early metastasis. It also might lead to both tailored therapy regimes and revised indications for adjuvant treatment, independent of lymph node involvement. No significant financial relationships to disclose.

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.

TRANSLATIONAL IMPACT

Endometriosis results from the mislocalization of cells that usually form the uterine lining, to ectopic locations. The condition can be asymptomatic, but for 10% of women in their reproductive years, it is associated with pelvic pain, fatigue, and irregular or heavy periods. Whether or not it produces symptoms, endometriosis can impair fertility and increase the likelihood of miscarriage and stillbirth in women who become pregnant. Mislocalized cells often form ectopic lesions, also called implants or nodules, on organs within the pelvic cavity, including the ovaries, intestines and bladder. Surgery and drugs are used to reduce the extent of lesions, to promote fertility and to relieve pain, but endometriosis is often recurring and can precipitate ovarian epithelial tumors.Endometriosis is mediated partly by the immune system but very few endometriosis antigens have been identified. The cell surface glycoprotein, mucin 1 or MUC1, is a known ovarian tumor-associated antigen that is present in ectopic lesions of ovarian endometriosis. However, the role of MUC1 expression in endometriosis and its potential as a biomarker for ovarian cancer are unknown.Here, the authors use double transgenic MUC1Kras mice, with inducible ovarian endometriosis, to study changes in MUC1 expression during progression to endometriosis. The mice develop benign, MUC1-positive ovarian lesions that closely resemble human endometriosis. MUC1 expression in the affected ovaries increases significantly as lesions develop, stimulating the production of MUC1-specific antibodies. High IgM and IgG antibody titers appear early in the disease process and are maintained. During the later stages of disease, MUC1Kras mice, like women with the disease, show an expansion of immunosuppressive Foxp3+ CD4 regulatory T cells in the draining lymph nodes. The authors suggest that developing tolerance to MUC1 during endometriosis may inhibit immune competence and enable cancer progression.The authors describe and characterize an animal model of human endometriosis. The pattern of MUC1 expression and its impact on the immune system of the host should facilitate the development of novel immune-based approaches. MUC1Kras mice could also further clarify the role of MUC1 in endometriosis and provide an in vivo platform for testing MUC1 vaccines to treat endometriosis and prevent ovarian cancer.