Mucin 1 Transgenic Mice Research Articles

Enhancing T Cell and Antibody Response in Mucin-1 Transgenic Mice through Co-Delivery of Tumor-Associated Mucin-1 Antigen and TLR Agonists in C3-Liposomes.

Mucin-1 (MUC1) is a highly relevant antigen for cancer vaccination due to its overexpression and hypo-glycosylation in a high percentage of carcinomas. To enhance the immune response to MUC1, our group has developed C3-liposomes that encapsulate the MUC1 antigen along with immunostimulatory compounds for direct delivery to antigen-presenting cells (APCs). C3-liposomes bind complement C3, which interacts with C3-receptors on APCs, resulting in liposomal uptake and the delivery of tumor antigens to APCs in a manner that mimics pathogenic uptake. In this study, MUC1 and Toll-like receptor (TLR) agonists were encapsulated in C3-liposomes to provoke an immune response in transgenic mice tolerant to MUC1. The immune response to the C3-bound MUC1 liposomal vaccine was assessed by ELISA, ELISpot, and flow cytometry. Co-administering TLR 7/8 agonists with MUC1 encapsulated in C3-liposomes resulted in a significant antibody response compared to non-encapsulated MUC1. This antibody response was significantly higher in females than in males. The co-encapsulation of three TLR agonists with MUC1 in C3-liposomes significantly increased antibody responses and eliminated sex-based differences. Furthermore, this immunization strategy resulted in a significantly increased T cell-response compared to other treatment groups. In conclusion, the co-delivery of MUC1 and TLR agonists via C3-liposomes greatly enhances the immune response to MUC1, highlighting its potential for antigen-specific cancer immunotherapy.

Delivering Two Tumour Antigens Survivin and Mucin-1 on Virus-Like Particles Enhances Anti-Tumour Immune Responses.

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

Abstract 4571: Development of glycoconjugate based anticancer immunotherapy

Abstract Tumor associated carbohydrate antigens (TACAs) are attractive targets for the development of anticancer immunotherapy. Mucin-1 (MUC1) is a representative member of the TACA family. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anti-cancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen respectively have been synthesized. Bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize the immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding with MUC1 expressing melanoma B16-MUC1 cells and effectively killed these cells in vitro. Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that MUC1-Tf based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potentials as anti-cancer vaccines. Citation Format: Xuefei Huang. Development of glycoconjugate based anticancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4571.

Synthesis and Immunological Evaluation of Disaccharide Bearing MUC-1 Glycopeptide Conjugates with Virus-like Particles.

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.

Abstract P4-06-20: Delivering tumour antigens survivin and mucin-1 on virus-like particles for breast cancer immunotherapy

Abstract Breast cancer is the most frequently diagnosed cancer in women worldwide. Although there are a variety of treatment options available, breast cancer is a difficult disease to treat and many patients experience recurrence following treatment. We have previously shown that tumour antigens delivered on virus-like particles (VLP) induce a targeted anti-cancer immune response. In this study we investigated whether combining the two tumour antigens survivin and mucin 1 as vaccine targets can induce a superior anti-cancer immune response for breast cancer immunotherapy. VLP were designed to recombinantly express the murine survivin epitope. Following expression of Survivin-VLP, aberrantly glycosylated mucin 1 (MUC1) peptide was conjugated onto the Survivin-VLP using intracellular cleavable bis-arylhydrazone linking strategy. Western Blot analysis and electron microscopy confirmed Survivin-VLP expression and UV absorption confirmed conjugation of the MUC1 peptide to the Survivin-VLP. C57mg.MUC1 breast cancer cells were injected into the mammary fat pad of C57Bl/6 and MUC1 transgenic mice. Once tumours were palpable, mice were vaccinated with the Survivin-MUC1-VLP and controls of DPBS, VLP without antigen or VLP delivering either survivin or MUC1 antigen. Tumour growth and mouse survival were monitored for 80 days. Mice vaccinated with Survivin-MU1-VLP delivering both antigens showed enhanced survival (D78) compared to mice vaccinated with VLP delivering only one of the antigens (D66) and controls (D53). Delivery of two tumour antigens induced an enhanced anti-tumour immune response compared to delivery of single tumour antigens. The induction of multiple immune responses against different tumour antigens may apply additional evolutionary pressure upon tumours, prolonging the ability for tumours to develop resistance, or escape through the proliferation of a resistant subpopulation. The use of VLP for the delivery of multiple antigens and adjuvants represents a promising approach to improve cancer immunotherapy for breast cancer. Citation Format: Kramer K, Braeden D, Young VL, Walker GF, Ward VK, Young SL. Delivering tumour antigens survivin and mucin-1 on virus-like particles for breast cancer immunotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-20.

Protective Epitope Discovery and Design of MUC1-based Vaccine for Effective Tumor Protections in Immunotolerant Mice.

Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qβ carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qβ-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qβ-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.

Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model.

Monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance anti-tumor responses show clinical promise in advanced solid tumors. Most of the preliminary evidence on therapeutic efficacy of immune checkpoint blockers comes from studies in melanoma, lung and renal cancer. To test the in vivo potential of programmed death-ligand 1 (PD-L1) blockade in ovarian cancer, we recently generated a new transplantable tumor model using human mucin 1 (MUC1)-expressing 2F8 cells. The MUC1 transgenic (MUC1.Tg) mice develop large number of intraperitoneal (IP) tumors following IP injection of 8 × 10(5) syngeneic 2F8 cells. The tumors are aggressive and display little T cell infiltration. Anti-PD-L1 antibody was administered IP every 2 weeks (200 μg/dose) for a total of three doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to late tumor stage. The anti-PD-L1 treatment led to substantial T cell infiltration within the tumor and significantly increased survival (p = 0.001) compared to isotype control-treated mice. When the same therapy was administered to wild-type mice challenged with 2F8 tumors, no survival benefit was observed, despite the presence of high titer anti-MUC1 antibodies. However, earlier treatment (day 11) and higher frequency of IP injections restored the T cell responses and led to prolonged survival. Splenocyte profiling via Nanostring using probes for 511 immune genes revealed a treatment-induced immune gene signature consistent with increased T cell-mediated immunity. These findings strongly support further preclinical and clinical strategies exploring PD-L1 blockade in ovarian cancer.

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

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

Abstract 1237: Mechanism of action of L-BLP25 in preclinical pancreatic tumor models.

Abstract Background. L-BLP25 is an investigational antigen-specific cancer immunotherapeutic agent targeting human mucin 1 (MUC1). A single low dose of cyclophosphamide (CPA) is known to be immunostimulatory and may enhance the anti-tumor effects of L-BLP25 when given 3 days before treatment initiation. Here we report the immune and anti-tumor efficacy of L-BLP25 with CPA pretreatment in subcutaneous (s.c.) and orthotopic models of murine pancreatic cancer. These models allow for reproduction of the clinical scenario, where 8 weekly doses of L-BLP25 are administered. Methods. Murine pancreatic tumor cells Panc02 transfected with human MUC1 were injected for comparison s.c. or orthotopically into human MUC1 transgenic mice. Mice received either vehicle control (PBS), CPA (100 mg/kg), L-BLP25 (100 μg), or L-BLP25 (100 μg) + CPA (100 mg/kg). CPA was administered intravenously (i.v.) 3 days prior to initiation of 8 weekly intradermal (i.d.) or s.c. L-BLP25 injections. Tumor volume was monitored and splenic cell populations were quantified by FACS. In the s.c. tumor model, the contribution of CD4+ and CD8+ T cell subsets to the inhibition of tumor growth was studied. In the orthotopic model, antigen-specific IFN-γ secretion was assessed via ex vivo T cell re-stimulation with antigen alone. Results. In the s.c. model of pancreatic cancer, tumor volume was reduced compared to vehicle at day 49 after i.d. administration of L-BLP25 (41.4%, p<0.02) and L-BLP25 + CPA (55.5%, p<0.02). In multiple studies, tumor volume was consistently lower in mice treated with L-BLP25 + CPA vs. L-BLP25 alone. L-BLP25 + CPA s.c. treatment was effective at day 49 with 44% greater inhibition of tumor growth and more pronounced extended survival compared to i.d. treatment, supporting the route of administration in the clinic. Various splenic immune cell populations were increased by s.c. L-BLP25 + CPA treatment compared to vehicle and L-BLP25 alone, including the CD8+ effector memory/Treg ratio. Mice treated with s.c. L-BLP25 + CPA after CD8+ T cell depletion had a greater tumor volume compared to T cell-sufficient mice, while CD4+ T cell depletion had no effect. In the orthotopic model of pancreatic cancer, median survival was improved with s.c. L-BLP25 + CPA treatment vs. vehicle (p<0.05) and vs. L-BLP25 alone (non-significant). When stimulated ex vivo with the BP25 peptide, the frequency of IFN-γ producing CD4+ T cells isolated from the spleens of mice treated with L-BLP25 + CPA was significantly higher than in all other treatment groups (p<0.001). CD8+ T cells were found to produce no IFN-γ after in vitro re-stimulation. Conclusions. When administered after a single, low-dose pre-treatment of CPA, L-BLP25 immunotherapy exhibits anti-tumor activity, immune activation and prolonged survival in murine pancreatic cancer models. T cell depletion data indicate a CD8+ T cell-mediated anti-tumor response, whilst IFN-γ production by CD4+ helper T cells may contribute to prolonged survival. Citation Format: Kenneth Hance, Bo Marelli, Jin Qi, Guozhong Qin, Huakui Yu, Hong Wang, Xiaomei Xu, Robert Tighe, Beatrice Brunkhorst, Yan Lan, Robert Hofmeister, Helen Sabzevari, Michael Wolf. Mechanism of action of L-BLP25 in preclinical pancreatic tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2013-1237

Abstract 3644: Pancreatic tumor cells that develop within Muc1 knock-out mice generate less immunosuppressive MDSCs in vitro

Abstract Epithelial Mucin 1 (MUC1) is a well characterized tumor associated antigen found to be over expressed and aberrantly glycosylated in human pancreatic adenocarcinomas. In recent years, Myeloid- derived suppressor cells (MDSCs), major regulators of the immune response, have been implicated in the progression of cancer. We have recently shown that Muc1 null mice have a higher propensity of MDSCs and are unable to reject allogeneic tumors as effectively as their wild-type counterparts. Here, we studied the effects of tumor derived soluble factors (TDSFs) from pancreatic cancer cell lines that over expressed human MUC1 (KCM) or were null for Muc1 (KCKO) on the generation of MDSCs from bone marrow (BM) cells in vitro. The two cell lines were derived from a spontaneous mouse model of pancreatic ductal adenocarcinoma (PDA) that was bred to the Muc1-null (KCKO) or the human MUC1 transgenic (KCM) mice. BM cells were cultured in complete DMEM supplemented with 10ng/ml GM-CSF and 10ng/ml IL-4 + 30% v/v conditioned media (TCCM) from KCKO or KCM cell lines for 5 days. On day 3, floating cells were removed and fresh media with cytokines and TCCM was added. On day 5, cells were stained for MDSCs and analyzed by flow cytometry. Statistical analysis was performed using GraphPad software and p values were calculated using One-way ANOVA. We observed a significant increase in the monocytic (CD11b+Ly6C+Ly6G-) and the granulocytic (CD11b+Ly6C+LyG+) MDSC subpopulations when naïve BM cells were cultured in the presence of TCCM from KCKO versus KCM cells (p<0.05). Upon further analysis of certain myeloid and granulocytic maturation, activation and immune modulatory markers on the CD11b+Gr1+ MDSCs, we found an up-regulation of CD11c and CD115 molecules on the CD11b+Gr1+ MDSCs generated in the presence of KCKO TCCM but not in the presence of KCM TCCM (p<0.01). This indicated the presence of dendritic cell and macrophage phenotypic markers which arise from the maturation of the monocytic subpopulation. Indeed, we confirmed that the up-regulation of the CD11c and CD115 markers were only found on the monocytic but not on the granulocytic subpopulation when BM cells were treated with KCKO TCCM compared to KCM TCCM (p<0.01). Since the monocytic subpopulation possess most of the immune suppressive activity, we hypothesized that KCKO TCCM induces a more mature monocytic subpopulation of MDSC that may not be immunosuppressive. Thus, when KCKO and KCM cells were injected in vivo in a asyngeneic normal C57BL/6 mice, the KCM cells grew significantly faster than the KCKO cells which were not only slow growing but remained as a stable disease. This could be partially explained by the induction of a less suppressive MDSC phenotype. These findings could lead to therapies that manipulate the immune regulatory cell population maturing into a less immune suppressive state in a tumor setting allowing the body to mount an effective immune response against cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3644. doi:10.1158/1538-7445.AM2011-3644

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

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

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses

DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly- l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 μg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.

Intradermal vaccination of MUC1 transgenic mice with MUC1/IL-18 plasmid DNA suppresses experimental pulmonary metastases

MUC1 (mucin 1) is a transmembrane glycoprotein normally expressed on epithelia of the pancreas, breast, prostate, colon, and lung. However, this self-antigen is over-expressed and aberrantly glycosylated in adenocarcinomas, thereby making it a potential target for immunotherapy. Toward this goal, DNA plasmids encoding human MUC1 (pMUC1) and mouse interleukin-18 (pmuIL-18) were developed, and previous work demonstrated pMUC1/pmuIL18 vaccination protected MUC1 transgenic mice (MUC1.Tg) from subcutaneous tumor challenge. This report shows that pMUC1/pmuIL-18 is effective in preventing and treating pulmonary metastases in MUC1.Tg mice. Vaccination with pMUC1 or pmuIL-18 alone was insufficient to elicit measurable anti-tumor effects. However, co-administration of pMUC1 with pmuIL-18 reduced the incidence of lung tumors and prolonged survival. Furthermore, pMUC1/pmuIL-18 immunization protected mice from challenge with MUC1 + tumors, but not from MUC1 − tumors, indicating that the anti-tumor effect is antigen-specific. More importantly, pMUC1/pmuIL-18 was effective in treating established tumors. Finally, in vivo antibody-mediated lymphocyte depletion and neutralization of interferon γ (IFNγ) revealed that CD8 + T cells and IFNγ mediate the anti-tumor immunity. Collectively, these results demonstrate that pMUC1/pmuIL-18 breaks tolerance to MUC1, and induces antigen-specific immunity with protective and therapeutic benefit. This suggests that pMUC1/pmuIL-18 DNA vaccination may provide clinical benefit for patients with MUC1 + tumors.

A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice

MUC1 (mucin 1) is a tumor-associated antigen that is overexpressed in many adenocarcinomas. Active immunotherapy targeting tumors expressing MUC1 could have great treatment value. MUC1 DNA vaccines were evaluated in MUC1 transgenic (MUC1.Tg) mice challenged with MC38/MUC1 + tumor cells. Vaccination with MUC1 plasmid DNA (pMUC1) alone was insufficient to induce tumor protection. However, co-administration of pMUC1 with a plasmid encoding murine interleukin-18 (pmuIL-18) resulted in significant tumor protection and survival after tumor challenge. Protection was durable in the absence of additional vaccination, as demonstrated by continued protection of vaccinated mice following tumor rechallenge. Mice surviving challenges with MC38/MUC1 + cells showed significant protection after challenge with MUC1 − MC38 tumor cells, suggesting that these mice had developed immune responses to epitopes shared between the tumor cell lines. Antibody-mediated depletion of lymphocyte subsets demonstrated that protection was due largely to CD4 + T cells. This work demonstrates that a naked DNA vaccine can break tolerance to MUC1 and induce an immune response capable of mediating both significant protection from tumor challenge and increased survival.

Mucin-1 is expressed on dendritic cells, both in vitro and in vivo

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.

Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells.

The tumour-associated antigen mucin 1 (MUC1) is a multifunctional protein involved in protection of mucous membranes, signal transduction, and modulation of the immune system. More than 70% of cancers overexpress MUC1, making MUC1 a potential target for immunotherapy. In the present study, MUC1 transgenic mice were crossed with syngeneic strains that express the polyomavirus middle-T oncogene (PyMT) driven by the mouse mammary tumour virus promoter long-terminal repeat (MMTV-LTR). The resultant breed (MMT mice) developed spontaneous MUC1-expressing mammary carcinomas with 100% penetrance at 8-15 weeks of age. As found in human breast cancer, the mammary carcinoma in MMT mice arose in multiple stages. Immunization with fusions of dendritic cells and MUC1-positive tumour cells (FC/MUC1) induced MUC1-specific immune responses that blocked or delayed the development of spontaneous breast carcinomas. In contrast, there was no delay of tumour development in MMT mice immunized with irradiated MC38/MUC1 tumour cells. The efficacy of fusion cells was closely correlated with the timing of initial immunization. Immunization with FC/MUC1 initiated in MMT mice at < 1, 1-2 and 2-3 months of age rendered 33, 5 and 0% of mice free of tumour, respectively, up to 6 months. Whereas mice immunized in the later stage of tumour development succumbed to their disease, immunization resulted in control of tumour progression and prolongation of life. These results indicate that immunization with FC/MUC1 can generate an anti-MUC1 response that is sufficient to delay the development of spontaneous mammary carcinomas and control tumour progression in MMT mice.