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.

Synthesis and immunological evaluation of self-adjuvanting MUC1-macrophage activating lipopeptide 2 conjugate vaccine candidates.

We describe herein the synthesis and immunological evaluation of self-adjuvanting mucin 1 (MUC1)-macrophage activating lipopeptide 2 (MALP2) (glyco)peptide vaccine candidates. Vaccine constructs were shown to induce high titres of class-switched IgG antibodies in C57BL/6 mice after four immunisations despite the lack of a helper T cell epitope.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD8(+) CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1(+), CD133(+)) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation.

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α-N-acetylgalactosamine (GalNAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal-regulated kinase 1,2 (ERK1,2) and nuclear factor-κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen-presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen-specific CD8(+) T-cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.

Tumor burden influences cytotoxic T cell development in metastatic breast cancer patients--a phase I/II study.

The influence of tumor burden on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Four previously treated metastatic breast cancer patients, two with macroscopic disease and two with no evidence of disease, in complete remission (CR), were enrolled. Each apheretic peripheral blood mononuclear cell (PBMC) sample was stimulated twice with MUC-1 before infusion back into the patients. CTL responses against MCF-7 cell line and cytokine production were measured before infusion. Patients received two monthly CTL infusions and were monitored for toxicity, tumor response as well as tumor marker levels. The CTL generated from patients with high tumor burdens had less cytokine production and lower cytotoxicity of MCF-7 than the CTL of patients in CR. The differences between the two groups were observed after the two MUC1 in vitro stimulations of the cells obtained in first apheresis. This difference increased after the two MUC1 stimulations of the cells obtained in the second apheresis. The cytotoxicity function was sustained from the first infusion to the second apheresis only for the patients in CR. This suggests that tumor burden had an inverse effect on the function of the generated CTL.

Progression of pancreatic adenocarcinoma is significantly impeded with a combination of vaccine and COX-2 inhibition.

With a 5-year survival rate of <5%, pancreatic cancer is one of the most rapidly fatal malignancies. Current protocols for the treatment of pancreas cancer are not as effective as we desire. In this study, we show that a novel Mucin-1 (MUC1)-based vaccine in combination with a cyclooxygenase-2 inhibitor (celecoxib), and low-dose chemotherapy (gemcitabine) was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive pancreatic ductal adenocarcinomas. The study was conducted in an appropriate triple transgenic model of spontaneous pancreatic cancer induced by the KRAS(G12D) mutation and that expresses human MUC1 as a self molecule. The combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor. The mechanism for the increased immune response was attributed to the down-regulation of circulating prostaglandin E(2) and indoleamine 2, 3,-dioxygenase enzymatic activity, as well as decreased levels of T regulatory and myeloid suppressor cells within the tumor microenvironment. The preclinical data provide the rationale to design clinical trials with a combination of MUC1-based vaccine, celecoxib, and gemcitabine for the treatment of pancreatic cancer.

Pre-clinical safety evaluation of heat shock protein 65-MUC1 peptide fusion protein.

With a goal of developing a medication for the treatment of MUC1 expressing human cancers, a recombinant heat shock protein 65-MUC1 fusion protein (HSP65-MUC1) between BCG derived heat shock protein 65 (HSP65) and MUC1 derived peptide (MUC1) was developed. To move the HSP65-MUC1 into a phase I clinical trial, a comprehensive non-clinical safety evaluation was conducted. The evaluation comprised of single-dose toxicity and repeat-dose toxicity studies both in mice and rhesus monkeys. The data from the study indicates that the treatment with HSP65-MUC1 is not associated with obvious toxicity in the tested animals. The changes in clinical chemistry and hematology in both the mice and monkeys were considered to be mild because there were no indications of overt toxicity after administering HSP65-MUC1. The data provided here contributed to the approval of initiating a phase I clinical trial with HSP65-MUC1 for the treatment of patients with MUC1-positive breast cancer in China.

MUC1 gene-derived glycoprotein assays for monitoring breast cancer (CA 15-3, CA 27.29, BR): are they measuring the same antigen?

CONTEXT: There are 2 general types of assays measuring MUC1 gene-derived glycoprotein: assays for cancer antigen (CA) 15-3, which are sandwich assays, and assays for CA 27.29, which are competitive assays. These 2 types of assays measure slightly different parts of this tandem-repeat molecule. Across-method assay differences hinder the exchange of patient test values among integrated health care networks and among countries. OBJECTIVE: This report evaluates the method differences among these assays to determine if the differences between these assays are mainly related to variations in calibration or differences in analyte specificity. DESIGN: Data from 22 College of American Pathologists survey challenges were analyzed to compare 10 commercial assay methods for these 2 related analytes. In addition, data from 58 patient samples were analyzed to compare 3 of these assays. RESULTS: The linear correlation coefficients comparing the within-method medians of these proficiency test distributions were very high (>0.99) for all of the methods; however, the regression slopes varied from 0.836 to 1.095. The regression slopes for the patient specimens varied similarly, but the correlation coefficients were lower. CONCLUSIONS: This study indicates that many of the test value differences for these measurements are due to differences in assay calibration rather than differences in the specificity of the assay measurement systems. Survey test data potentially could be used to help harmonize these assay differences.

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers.

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.

Dendritic cell-tumor cell fusion and staphylococcal enterotoxin B treatment in a pancreatic tumor model.

BACKGROUND: Surgical resection of pancreatic tumors removes gross disease but not metastases. Adjuvant therapy such as chemotherapy and radiation treatment is of little value in metastatic pancreatic cancer. The hypothesis of this investigation is that specific and effective immunotherapeutic vaccine (dendritic/tumor cell fusion) will activate cytotoxic T lymphocytes (CTLs), leading to the eradication of spontaneous pancreatic cancer. METHODS: We have developed a double transgenic mouse model (MET) that forms spontaneous pancreatic tumors and expresses the human MUC1 antigen. Seven-week-old MET mice (n = 8) were treated every 3 weeks with the vaccine. In addition, these mice received 50 microg of superantigen staphylococcal enterotoxin B (SEB), a known T cell stimulant, prior to the first vaccination. A second treatment group received SEB alone (n = 8) and controls received no treatment (n = 9). MUC1-specific CTLs were measured by chromium release assay. At 10 weeks of age and at necropsy, MUC1 serum levels were measured using a MUC1-specific ELISA. RESULTS: Mice were known to harbor microscopic foci of cancer at birth. Survival was enhanced in vaccine as well as SEB-treated mice (75% CI +/- 0.42) compared to controls (11% CI +/- 0.28) and both groups of treated mice exhibited mature CTLs without in vitro stimulation. MUC1 serum levels of the vaccine group were 50% less than that of control (P < 0.04) at 10 weeks. MUC1 serum levels directly correlated with tumor weight at necropsy (r = 0.86). CONCLUSIONS: This is the first evidence that MUC1-specific CTLs can be stimulated to enhance survival in a spontaneous tumor model.