Human MUC1 Transgenic Mice Research Articles

Prevention of Inflammation-Driven Colon Carcinogenesis in Human MUC1 Transgenic Mice by Vaccination with MUC1 DNA and Dendritic Cells

The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.

Evaluation of a novel monoclonal antibody against tumor-associated MUC1 for diagnosis and prognosis of breast cancer.

There is still a great unmet medical need concerning diagnosis and treatment of breast cancer which could be addressed by utilizing specific molecular targets. Tumor-associated MUC1 is expressed on over 90 % of all breast cancer entities and differs strongly from its physiological form on epithelial cells, therefore presenting a unique target for breast cancer diagnosis and antibody-mediated immune therapy. Utilizing an anti-tumor vaccine based on a synthetically prepared glycopeptide, we generated a monoclonal antibody (mAb) GGSK-1/30, selectively recognizing human tumor-associated MUC1. This antibody targets exclusively tumor-associated MUC1 in the absence of any binding to MUC1 on healthy epithelial cells thus enabling the generation of breast tumor-specific radiolabeled immune therapeutic tools.Methods: MAb GGSK-1/30 was used for immunohistochemical analysis of human breast cancer tissue. Its desferrioxamine (Df')-conjugate was synthesized and labelled with 89Zr. [89Zr]Zr-Df'-GGSK-1/30 was evaluated as a potential PET tracer. Binding and pharmacokinetic properties of [89Zr]Zr-Df'-GGSK-1/30 were analyzed in vitro using human and murine cell lines that express tumor-associated MUC1. Self-generated primary murine breast cancer cells expressing human tumor-associated MUC1 were transplanted subcutaneously in wild type and human MUC1-transgenic mice. The pharmacology of [89Zr]Zr-Df'-GGSK-1/30 was investigated using breast tumor-bearing mice in vivo by PET/MRT imaging as well as by ex vivo organ biodistribution analysis.Results: The mAb GGSK-1/30 stained specifically human breast tumor tissue and can be possibly used to predict the severity of disease progression based on the expression of the tumor-associated MUC1. For in vivo imaging, the Df'-conjugated mAb was radiolabeled with a radiochemical yield of 60 %, a radiochemical purity of 95 % and an apparent specific activity of 6.1 GBq/µmol. After 7 d, stabilities of 84 % in human serum and of 93 % in saline were observed. In vitro cell studies showed strong binding to human tumor-associated MUC1 expressing breast cancer cells. The breast tumor-bearing mice showed an in vivo tumor uptake of >50 %ID/g and clearly visible specific enrichment of the radioconjugate via PET/MRT.Principal conclusions: Tumor-associated MUC1 is a very important biomarker for breast cancer next to the traditional markers estrogen receptor (ER), progesterone receptor (PR) and HER/2-neu. The mAb GGSK-1/30 can be used for the diagnosis of over 90% of breast cancers, including triple negative breast cancer based on biopsy staining. Its radioimmunoconjugate represents a promising PET-tracer for breast cancer imaging selectively targeting breast cancer cells.

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.

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis

BackgroundMUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. MethodsWe employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. ResultsAs previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. ConclusionsAltogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung.

Immunization with a Synthetic Human MUC1 Glycopeptide Vaccine against Tumor-Associated MUC1 Breaks Tolerance in Human MUC1 Transgenic Mice.

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4+ T-, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor-associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines.

Abstract 4153: Abnormal expression of MUC1 mucin on colon epithelia stimulates production of pro-inflammatory cytokines promoting colitis-associated colon cancer in a murine model

Abstract MUC1 is a transmembrane glycoprotein aberrantly expressed in human adenocarcinoma as well as chronic inflammatory conditions. We have previously demonstrated that the tumor form of MUC1, in association with p65, upregulates the expression of pro-inflammatory cytokines in the tumor microenvironment, including IL-6 and TNF-alpha, by modulating their transcriptional activity in epithelial cancer cells. Here, we explored the mechanism underlying MUC1/p65-induced transcription of IL-6 and TNF-alpha in colon cancer cells and its significance for the microenviroment of colitis that is known to be the precursor to cancer. We used a mouse model of colitis-associated colon cancer where wild type (WT) and human MUC1 transgenic (MUC1.Tg) mice are given carcinogen azoxymethane (AOM), followed by three cycles of 1.2% dextran sodium sulfate (DSS) in drinking water to induce colitis. MUC1.Tg showed higher tumor incidence, decreased survival, increased body weight loss and shorter colon length. These features accelerated the severity of inflammation-induced colon cancer. Consistent with our previous in vitro data, expression of NF-kB family members was higher in MUC1.Tg mice compared to WT. We previously showed that in tumors, MUC1/p65 complex modulated the expression of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) and interaction with pro-inflammatory cytokine promoters. In order to understand the significance of MUC1/p65-modulated cytokines in progressive colitis that gives rise to colon cancer, we analyzed infiltration of inflammatory cells into the inflamed colon tissues. The number of infiltrating macrophages was much higher in AOM/DSS-treated MUC1.Tg mice compared to WT. ELISA assay and gene expression analyses demonstrated that the treatment with AOM/DSS in the presence of human MUC1 on the colonic epithelia resulted in a significant increase in M1 type macrophage-associated genes, including IL-6, TNF-alpha and iNOS, whereas M2 type macrophage markers such as Arginase 1, Ym1, and IL-10 were drastically reduced when compared to colon tissues of treated WT mice. Taken together, our findings reveal a pro-inflammatory role for MUC1 in colitis-related carcinogenesis. Our study also provides a mechanism by which MUC1 accelerates tumor initiation and progression. Citation Format: Sandra Cascio, Jacque Faylo, Jia Xue, Olivera Finn. Abnormal expression of MUC1 mucin on colon epithelia stimulates production of pro-inflammatory cytokines promoting colitis-associated colon cancer in a murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4153.

510. Treating Epithelial and Metastatic Malignancies with Glycoform-Specific Chimeric Antigen Receptors

Cancer-specific glycosylation deficiencies generating Tn or sTn O-linked glycans are found in >80% of the most lethal cancers (lung, prostate, breast, colon, ovary, pancreas). These hypoglycosylated cancer glycoantigens decrease cell-cell interactions and increase the mobility and metastatic potential of tumor cells. We developed a CAR specific to the Tn/sTn antigen of the oncogene Mucin-1 (MUC1), which is highly upregulated in many epithelial cancers and is heavily O- and N-glycosylated. Our CAR uses the scFv sequence derived from the 5E5 mAb and the endodomains of CD3zeta and 4-1BB that promote T cell activation and co-stimulation, respectively. We have shown that the 5E5 mAb and the 5E5 CAR are specific to the cancer-specific glycoform of MUC1 but not normal MUC1 through in vitro binding and cytokine assays and that the 5E5 CAR is non-reactive to normal human primary cells. We demonstrated that the 5E5 CAR T cells are responsive to ovarian, breast, lung, pancreatic, and leukemia cancers and developed NSG mouse xenograft models using Jurkat leukemia cell line and Hs766T metastatic pancreatic cell line that show rapid clearance of established tumors by 5E5 CAR T cells and prolonged survival of tumor-bearing mice. Restoring normal glycosylation in Jurkat leukemia cells abolishes the expression of the hypoglycosylated form of Muc1 and also prevents 5E5 CAR T cell cytotoxicity, further highlighting the specificity of the 5E5 CAR for the cancer-specific glycoform of MUC1. Infusion of human MUC1-transgenic mice with murine 5E5 CAR T cells showed an exquisite safety profile, while infusion of normal glycoform MUC1-specific murine CAR T cells (HMFG1 CAR, a previously described MUC1-specific CAR) demonstrate extreme safety concerns. These results demonstrate that targeting the cancer-specific glycoform of MUC1 provides a safe, yet potent cancer-specific immunotherapy for treating most epithelial malignancies and holds the potential to eradicate metastatic disease.

Abstract 2886: Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer

Abstract MUC1 is a transmembrane glycoprotein normally expressed at low levels in a fully glycosylated form on the apical surface of epithelial cells but it is found overexpressed and hypoglycosylated in human adenocarcinomas as well as sites of chronic epithelial inflammations. Aberrant glycosylation of MUC1 in cancer cells alters its normal function and affects the behavior of cancer cells. The MUC1 extracellular domain also participates in activation of the NF-kB family members such as phospho-IkBa and phospho-p65. Our data showed that the tumor form of MUC1, in association with p65, regulates the transcriptional activity of pro-inflammatory cytokines, including IL-6 and TNF-alpha, by binding to their promoter regions in both mouse and human cancer cells. Because proteomic analysis showed that MUC1 is able to interact directly with histone proteins, we hypothesized that MUC1 might influence the transcriptional activity of cytokines by inducing epigenetic modification. Here, we explored MUC1/p65-regulated transcription of IL-6 and TNF-alpha in colon cancer cells and discovered that MUC1/p65 complex modulated the expression and interaction of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) with pro-inflammatory cytokine promoters. Interestingly, the contribution of EzH2 in controlling the MUC1/p65-modulated cytokines expression was independent of its histone H3K27 methyltransferase activity but it shows novel properties in regulating chromatin remodeling that are currently under detailed investigation To investigate the in vivo significance of MUC1/p65-induced cytokines expression in the tumor microenvironment, we used human MUC1 transgenic mice that express MUC1 on the colonic epithelial cells, treated with azoxymethane to initiate and dextran sulfate sodium (AOM/DSS) to promote colorectal carcinogenesis. MUC1.Tg mice showed higher tumor incidence and decreased survival when compared with wild-type mice. Consistent with the in vitro data, NF-kB family members and EzH2 expression were higher in MUC1.Tg mice when compared to wild-type mice. In addition, the number of infiltrating macrophages strongly increased in AOM/DSS-treated colon tissues of MUC1.Tg mice. These are important findings that may explain the mechanism by which MUC1 and NF-kB regulate cancer progression. Citation Format: Sandra Cascio, Joshua Sciurba, Olivera Finn. Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2015-2886

Complex of MUC1, CIN85 and Cbl in Colon Cancer Progression and Metastasis.

We previously reported that CIN85, an 85 KDa protein known to be involved in tumor cell migration and metastasis through its interaction with Cbl, associates with MUC1 in tumor cells. MUC1/CIN85 complex also regulates migration and invasion of tumor cells in vitro. Here, we examined specifically human colon carcinoma tissue microarrays (TMA) by immunohistochemistry for the expression of MUC1 and CIN85 and their potential role in cancer progression and metastasis. We detected a significant increase in expression of both MUC1 and CIN85 associated with advanced tumor stage and lymph node metastasis. We further investigated if Cbl could also be present in the MUC1/CIN85 complex. Co-immunoprecipitation assay showed that Cbl co-localized both with CIN85 and with MUC1 in a human colon cancer cell line. To begin to investigate the in vivo relevance of MUC1 overexpression and association with CIN85 and Cbl in cancer development and progression, we used human MUC1 transgenic mice that express MUC1 on the colonic epithelial cells, treated with azoxymethane to initiate and dextran sulfate sodium (AOM/DSS) to promote colorectal carcinogenesis. MUC1.Tg mice showed higher tumor incidence and decreased survival when compared with wild-type mice. Consistent with the in vitro data, the association of MUC1, CIN85 and Cbl was detected in colon tissues of AOM/DSS-treated MUC1 transgenic mice. MUC1/CIN85/Cbl complex appears to contribute to promotion and progression of colon cancer and thus increased expression of MUC1, CIN85 and Cbl in early stage colon cancer might be predictive of poor prognosis.

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development

Antigen Choice Determines Vaccine-Induced Generation of Immunogenic versus Tolerogenic Dendritic Cells That Are Marked by Differential Expression of Pancreatic Enzymes

Dendritic cells (DC) elicit immunity to pathogens and tumors while simultaneously preserving tolerance to self. Efficacious cancer vaccines have been a challenge because they are based on tumor Ags, some of which are self-Ags and thus subject to self-tolerance. One such Ag is the tumor-associated mucin MUC1. Preclinical testing of MUC1 vaccines revealed existence of peripheral tolerance to MUC1 that compromises their efficacy. To identify mechanisms that act early postvaccination and might predict vaccine outcome, we immunized human MUC1 transgenic mice (MUC1.Tg) i.v. with a MUC1 peptide vaccine against which they generate weak immunity and wild-type (WT) mice that respond strongly to the same peptide. We analyzed differences in splenic DC phenotype and function between the two mouse strains at 24 and 72 h postvaccination and also performed unbiased total gene expression analysis of the spleen. Compared to WT, MUC1.Tg spleens had significantly fewer DC, and they exhibited significantly lower expression of costimulatory molecules, decreased motility, and preferential priming of Ag-specific Foxp3(+) regulatory T cells. This tolerogenic DC phenotype and function was marked by a new putative biomarker revealed by the microarray: a cohort of pancreatic enzymes (trypsin, carboxypeptidase, elastase, and others) not previously reported in DC. These enzymes were strongly upregulated in the splenic DC from vaccinated WT mice and suppressed in the splenic DC of vaccinated MUC1.Tg mice. Suppression of the enzymes was dependent on regulatory T cells and on signaling through the IL-10R and correlated with global downregulation of DC immunostimulatory phenotype and function.

DC enzymes as early biomarkers of an effective vaccine (46.7)

Abstract Previous work by our lab and others has shown that human MUC1 transgenic mice (MUC1.tg) are hyporesponsive to vaccination with some forms of MUC1 and fail to mount a spontaneous immune response against MUC1 positive tumors. An interesting correlate of hyporesponsiveness to MUC1 vaccination is that as early as 24 hours post vaccination, splenic dendritic cells (DC) in wild type (WT) mice upregulate expression of a group of proteolytic enzymes previously characterized as functioning primarily in pancreatic catabolism (e.g. Trypsin-1, Trypsin-4, Carboxypeptidase B1). This upregulation does not occur in the MUC1.tg animals. Using an in vitro co-culture system and in vivo antibody depletion, we show that regulation of enzyme expression in DC is dependent on their interaction with CD4 T cells such that CD4 effector T cells (Teff) up-regulate these enzymes, whereas the presence of activated CD4 regulatory T cells (Treg) actively suppress this activity in an IL-10 dependent manner. TLR agonists do not upregulate enzyme expression, suggesting differences are due to alternative DC activation pathways following their interaction with T cells. We propose that one or more of these enzymes, which appear to be co-regulated, could serve as early markers of, and most likely also as contributors to, DC activation by helper T cells. In contrast, lack of their expression marks insufficient activation or active suppression by Tregs.

Intracellular signaling “fingerprint” in CD4 T cells in response to vaccination (165.14)

Abstract Early biomarkers of vaccine efficacy need to be established. We hypothesize that intracellular signaling within CD4 T cells as early as 24 and 48 hours post vaccination could yield a “fingerprint” that is predictive of the ultimate effectiveness of the elicited immune response. In our model, there are reduced responses to MUC1 peptide (MUC1p) vaccination in T cells in human MUC1 transgenic mice (MUC1.Tg) mice compared to wildtype (WT) mice. We are examining phosphorylation downstream of TCR, cell survival, proliferation, and apoptosis signaling cascades post vaccination with either DC loaded with MUC1p (DC:MUC1p) or soluble MUC1p in conjunction with poly:ICLC (sMUC1p:ICLC). Adoptively transferred TCR transgenic CD4 T cells specific for MUC1p displayed no statistically significant differences in signaling during priming in WT vs MUC1.Tg mice at 1, 6, 12 and 24 hrs post vaccination with DC:MUC1p. However, there were trends towards decreased phosphorylation in STAT1, STAT4, and increased pLCK in WT mice. We are currently examining signaling differences in the endogenous memory response in MUC1.Tg and WT mice 1 and 24 hrs post secondary boost of either DC:MUC1p or sMUC1p:ICLC vaccines to determine if differences exist upon secondary exposure and if signaling can be altered. Vaccinated MUC1.Tg and WT mice have been challenged with B16.MUC1 to correlate early T cell signaling with control of tumor burden and intracellular signaling occurring in the tumor microenvironment.

Prevention of colitis-associated colon cancer using a vaccine to target abnormal expression of the MUC1 tumor antigen

Association between chronic inflammation and cancer development is exemplified by inflammatory bowel disease (IBD) where patients with chronic uncontrolled colitis have a significantly increased risk of developing colitis-associated colorectal cancer (CACC). CACC appears to progresses through the inflammation-dysplasia-carcinoma sequence. This highlights the need to identify targets and interventions that reduce inflammation and prevent development of dysplasia in the context of IBD. Using the dextran sulfate sodium (DSS) mouse model of chronic colitis and CACC, we show that an important target of intervention in human disease would be the epithelial cell molecule MUC1 that is aberrantly expressed on inflamed colonocytes and promotes inflammation and progression to CACC. We show that a MUC1 vaccine can ameliorate chronic colitis and prevent development of dysplasia in the colon and thus extend survival in human MUC1 transgenic mice. This study supports the potential of prophylactic vaccines to target antigens that become aberrantly expressed in chronic inflammation (e.g., IBD) and continue to be expressed on the associated cancers (e.g., colon cancer), to prevent and/or treat both diseases.

Therapeutic MUC1-Based Cancer Vaccine Expressed in Flagella-Efficacy in an Aggressive Model of Breast Cancer

MUC1, a tumor-associated antigen overexpressed in many carcinomas, represents a candidate of choice for cancer immunotherapy. Flagella-based MUC1 vaccines were tested in therapeutic setting in two aggressive breast cancer models, comprising the implantation of the 4T1-MUC1 cell line in either Balb/c, or Human MUC1 transgenic mice in which spontaneous metastases occurs. Recombinant flagella carrying only 7 amino acid of MUC1 elicited therapeutic activity, affecting both the growth of established growing tumors and the number of metastases. Higher therapeutic activity was achieved with an additional recombinant flagella designed with the SYFPEITHI algorithm. The vaccines triggered a Th1 response against MUC1 with no evident autoimmune response towards healthy MUC1-expressing tissues. Recombinant flagella carrying a 25-residue fragment of MUC1, induced the most effective response, as evidenced by a significant reduction of both the size and growth rate of the tumor as well as by the lower number of metastases, and expanding life span of vaccinated mice.

Activating and tolerizing signaling “fingerprint” in T cells responding to DC/tumor MUC1 vaccine (156.23)

Abstract Early biomarkers of efficacy of vaccines are lacking. We hypothesize that intracellular signaling in T cells following administration of the vaccine yields a “fingerprint” predictive of an effective or a tolerogenic immune response and could be used as a biomarker. Our lab has explored immune responses to human tumor MUC1 peptide vaccine in wildtype (WT) versus human MUC1 transgenic mice (MUC1.Tg) and found that they were significantly lower in MUC1Tg. To “fingerprint” signaling differences between the effective immune response in WT versus the lower (tolerogenic) response in MUC1.Tg mice, we vaccinated both with DC loaded with MUC1 peptide, harvested spleens (n=3) at 1,6,12, and 24 hours post vaccination and analyzed phosphorylation downstream of TCR and costimulatory molecules, as well as activation of cell survival, proliferation, and apoptosis signaling cascades. We saw early increases in pSTAT1, pSTAT4, and p38MAPK with a trend towards higher phosphorylation in WT mice that did not reach statistical significance. There was also a trend towards higher phosphorylation in WT mice in pLCK and pAKT downstream of the TCR and costimulatory molecules. Interestingly, at certain timepoints we observed increases in signaling in MUC1.Tg mice, which may serve as compensatory mechanisms that we will further investigate. We are challenging these two vaccinated groups of mice with MUC1+ tumor in an attempt to correlate early T cell activation with later protection from tumor growth.

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.

Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas

A bigenic MUC1.Tg/MIN mouse model was developed by crossing Apc/(MIN/+) (MIN) mice with human MUC1 transgenic mice to evaluate MUC1 antigen-specific immunotherapy of intestinal adenomas. The MUC1.Tg/MIN mice developed adenomas at a rate comparable to that of MIN mice and had similar levels of serum MUC1 antigen. A MUC1-based vaccine consisting of MHC class I-restricted MUC1 peptides, a MHC class II-restricted pan-helper peptide, unmethylated CpG oligodeoxynucleotide and GM-CSF caused flattening of adenomas and significantly reduced the number of large adenomas. Immunization was successful in generating a MUC1-directed immune response evidenced by increased MUC1 peptide-specific anti-tumor cytotoxicity and IFN-gamma secretion by lymphocytes.

Cutting Edge: Transgenic Expression of Human MUC1 in IL-10−/− Mice Accelerates Inflammatory Bowel Disease and Progression to Colon Cancer

Epithelial cell MUC1 is aberrantly expressed on human epithelial adenocarcinomas where it functions as a regulator of immune responses and an oncogene. Normally expressed at low levels in healthy colonic epithelium, MUC1 was reported to be overexpressed in human inflammatory bowel disease (IBD) and thus may be expected to play an important role in regulating chronic inflammation and its progression to colitis-associated colon cancer. Studies in the immunobiology and pathology of IBD and colitis-associated colon cancer have been done in various mouse models but none could properly address the role of MUC1 due to low homology between the mouse and the human molecule. We report that IL-10(-/-) mice, a widely accepted mouse model of IBD, crossed to human MUC1-transgenic mice, develop MUC1(+) IBD characterized by an earlier age of onset, higher inflammation scores, and a much higher incidence and number of colon cancers compared with IL-10(-/-) mice.

Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcalpha1-O-Ser/Thr (Tn) and NeuAcalpha2-6GalNAcalpha1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96-107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures.