RESUMO
Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases. In the current work, three MUC1 antitumor vaccine candidates conjugated with different immune stimulants are evaluated immunologically. For assessment of the influence of the immune stimulant on antibody recognition, a comprehensive library of mucin 1 glycopeptides (>100 entries) is synthesized and employed in antibody microarray profiling; these range from small tumor-associated glycans (TN , STN , and T-antigen structures) to heavily extended O-glycan core structures (type-1 and type-2 elongated core 1-3 tri-, tetra-, and hexasaccharides) glycosylated in variable density at the five different sites of the MUC1 tandem repeat. This is one of the most extensive glycopeptide libraries ever made through total synthesis. On tumor cells, the coreâ 2 ß-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched coreâ 2 structures, which favor formation of linear coreâ 1 or coreâ 3 structures, and in particular, truncated tumor-associated antigen structures. The coreâ 2 structures are commonly found on healthy cells and the elucidation of antibody cross-reactivity to such epitopes may predict the tumor-selectivity and safety of synthetic vaccines. With the extended mucin core structures in hand, antibody cross-reactivity toward the branched coreâ 2 glycopeptide epitopes is explored. It is observed that the induced antibodies recognize MUC1 peptides with very high glycosylation site specificity. The nature of the antibody response is characteristically different for antibodies directed to glycosylation sites in either the immune-dominant PDTR or the GSTA domain. All antibody sera show high reactivity to the tumor-associated saccharide structures on MUC1. Extensive glycosylation with branched coreâ 2 structures, typically found on healthy cells, abolishes antibody recognition of the antisera and suggests that all vaccine conjugates preferentially induce a tumor-specific humoral immune response.
Assuntos
Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Mucina-1/imunologia , Neoplasias/imunologia , Polissacarídeos/imunologia , Vacinas Sintéticas/imunologia , Animais , Formação de Anticorpos , Vacinas Anticâncer/química , Glicopeptídeos/química , Humanos , Imunidade Humoral , Camundongos , Mucina-1/química , Neoplasias/terapia , Polissacarídeos/química , Análise Serial de Proteínas , Vacinas Sintéticas/químicaRESUMO
We report the preparation of gold nanoparticle (AuNP)-based vaccine candidates against the tumor-associated form of the mucin-1 (MUC1) glycoprotein. Chimeric peptides, consisting of a glycopeptide sequence derived from MUC1 and the T-cell epitope P30 sequence were immobilized on PEGylated AuNPs and the ability to induce selective antibodies in vivo was investigated. After immunization, mice showed significant MHC-II mediated immune responses and their antisera recognized human MCF-7 breast cancer cells. Nanoparticles designed according to this report may become key players in the development of anticancer vaccines.
Assuntos
Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Glicopeptídeos/imunologia , Ouro/química , Nanopartículas Metálicas/química , Mucina-1/imunologia , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/química , Epitopos de Linfócito T/química , Genes MHC da Classe II , Glicopeptídeos/química , Humanos , Imunização , Células MCF-7 , Camundongos , Dados de Sequência Molecular , Mucina-1/química , Neoplasias/imunologia , Neoplasias/prevenção & controleRESUMO
In studies within the realm of cancer immunotherapy, the synthesis of exactly specified tumor-associated glycopeptide antigens is shown to be a key strategy for obtaining a highly selective biological reagent, that is, a monoclonal antibody that completely differentiates between tumor and normal epithelial cells and specifically marks the tumor cells in pancreas tumors. Mucin MUC1, which is overexpressed in many prevalent cancers, was identified as a promising target for this strategy. Tumor-associated MUC1 differs significantly from that expressed by normal cells, in particular by altered glycosylation. Structurally defined tumor-associated MUC1 cannot be isolated from tumor cells. We synthesized MUC1-glycopeptide vaccines and analyzed their structure-activity relationships in immunizations; a monoclonal antibody that specifically distinguishes between human normal and tumor epithelial cells was thus generated.
Assuntos
Anticorpos Monoclonais/biossíntese , Neoplasias da Mama/patologia , Mama/citologia , Vacinas Anticâncer/administração & dosagem , Glicopeptídeos/imunologia , Neoplasias Pancreáticas/diagnóstico , Feminino , HumanosRESUMO
Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor-associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem-repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4-based vaccines induced very strong antigen-specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.
Assuntos
Anticorpos Antineoplásicos/imunologia , Mucina-4/imunologia , Neoplasias Pancreáticas/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Humanos , Soros Imunes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mucina-4/química , Neoplasias Pancreáticas/patologia , Sequências de Repetição em Tandem , Toxoide Tetânico/química , VacinaçãoRESUMO
Self-adjuvanting antitumor vaccines by multifunctional cationic nanohydrogels loaded with CpG. A conjugate consisting of tumor-associated MUC1-glycopeptide B-cell epitope and tetanus toxin T-cell epitope P2 is linked to cationic nanogels. Oligonucleotide CpG complexation enhances toll-like receptor (TLR) stimulated T-cell proliferation and rapid immune activation. This co-delivery promotes induction of specific MUC1-antibodies binding to human breast tumor cells without external adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Nanopartículas/química , Oligodesoxirribonucleotídeos/química , Sequência de Aminoácidos , Animais , Cátions , Ensaio de Imunoadsorção Enzimática , Glicopeptídeos/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Nanopartículas/ultraestruturaRESUMO
In a new concept of fully synthetic vaccines, the role of T-helper cells is emphasized. Here, a synthetic antitumor vaccine consisting of a diglycosylated tumor-associated MUC1 glycopeptide as the B-cell epitope was covalently cross-linked with three different T-helper-cell epitopes via squaric acid ligation of two linear (glyco)peptides. In mice this four-component vaccine administered without external immune-stimulating promoters elicit titers of MUC1-specific antibodies that were about eight times higher than those induced by a vaccine containing only one T-helper-cell epitope. The promising results indicate that multiple activation of different T-helper cells is useful for applications in which increased immunogenicity is required. In personalized medicine, in particular, this flexible construction of a vaccine can serve as a role model, for example, when T-helper-cell epitopes are needed that match human leukocyte antigens (HLA) in different patients.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/síntese química , Epitopos/química , Glicopeptídeos/química , Mucina-1/química , Linfócitos T Auxiliares-Indutores/química , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Glicopeptídeos/imunologia , Humanos , Estrutura Molecular , Mucina-1/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
For antitumor vaccines both the selected tumor-associated antigen, as well as the mode of its presentation, affect the immune response. According to the principle of multiple antigen presentation, a tumor-associated MUC1 glycopeptide combined with the immunostimulating T-cell epitope P2 from tetanus toxoid was coupled to a multi-functionalized hyperbranched polyglycerol by "click chemistry". This globular polymeric carrier has a flexible dendrimer-like structure, which allows optimal antigen presentation to the immune system. The resulting fully synthetic vaccine induced strong immune responses in mice and IgG antibodies recognizing human breast-cancer cells.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Glicerol/química , Glicopeptídeos/síntese química , Polímeros/química , Animais , Anticorpos/imunologia , Apresentação de Antígeno , Neoplasias da Mama/tratamento farmacológico , Vacinas Anticâncer/uso terapêutico , Química Click , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Glicopeptídeos/imunologia , Humanos , Células MCF-7 , Camundongos , Mucina-1/química , Mucina-1/metabolismo , Toxoide Tetânico/química , Toxoide Tetânico/metabolismoRESUMO
Highly decorated: Tumor-associated MUC1 glycopeptide and tetanus toxoid T-cell epitope P2 can be attached to water-soluble poly(N-(2-hydroxypropyl)methacrylamide) carriers by orthogonal ligation techniques. Fully synthetic vaccine A with additional nanostructure-promoting domains induced antibodies that exhibit high affinity to tumor cells.