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1.
Chem Rec ; 21(11): 3313-3331, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34812564

ABSTRACT

Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood. The current state of the art suggests that the identity, abundancy and location of the glycans on the MUC backbone are all key parameters in the cellular and humoral response. This review shares lessons learned by us in over two decades of research in glycopeptide vaccines. By bridging synthetic chemistry and immunology, we discuss efforts in designing synthetic MUC1/4/16 vaccines and focus on the role of glycosylation patterns. We provide a brief introduction into the mechanisms of the immune system and aim to promote the development of cancer subunit vaccines.


Subject(s)
Cancer Vaccines , Glycopeptides , Mucins/immunology , Neoplasms/prevention & control , Cancer Vaccines/immunology , Glycosylation , Humans , Immunity , Neoplasms/immunology , Vaccines, Synthetic
2.
Mol Immunol ; 120: 74-82, 2020 04.
Article in English | MEDLINE | ID: mdl-32087569

ABSTRACT

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galß1-3(4)GlcNAcß. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.


Subject(s)
Antigens, Tumor-Associated, Carbohydrate/immunology , Amino Acid Sequence , Antibody Affinity , Antibody Specificity , Antigen-Antibody Reactions/immunology , Antigens, Tumor-Associated, Carbohydrate/chemistry , Carbohydrate Sequence , Epitopes/chemistry , Epitopes/immunology , Epitopes/isolation & purification , Humans , Immunity, Innate , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Jurkat Cells , Neoplasms/immunology
3.
Int J Med Sci ; 16(9): 1188-1198, 2019.
Article in English | MEDLINE | ID: mdl-31588183

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Breast Neoplasms/diagnosis , Breast Neoplasms/mortality , Mucin-1/metabolism , Animals , Biomarkers, Tumor/immunology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Deferoxamine/chemistry , Female , Humans , Immunohistochemistry/methods , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Mucin-1/immunology , Positron-Emission Tomography/methods , Prognosis , Radioisotopes/chemistry , Tissue Distribution , Zirconium/chemistry
4.
Cancer Immunol Res ; 7(1): 113-122, 2019 01.
Article in English | MEDLINE | ID: mdl-30413430

ABSTRACT

Preventive vaccination against tumor-associated endogenous antigens is considered to be an attractive strategy for the induction of a curative immune response concomitant with a long-lasting immunologic memory. The mucin MUC1 is a promising tumor antigen, as its tumor-associated form differs from the glycoprotein form expressed on healthy cells. Due to aberrant glycosylation in tumor cells, the specific peptide epitopes in its backbone are accessible and can be bound by antibodies induced by vaccination. Breast cancer patients develop per se only low levels of T cells and antibodies recognizing tumor-associated MUC1, and clinical trials with tumor-associated MUC1 yielded unsatisfactory therapeutic effects, indicating an urgent need to improve humoral immunity against this tumor entity. Herein, we demonstrate that preventive vaccination against tumor-associated human MUC1 results in a specific humoral immune response, a slowdown of tumor progression and an increase in survival of breast tumor-bearing mice. For preventive vaccination, we used a synthetic vaccine containing a tumor-associated glycopeptide structure of human MUC1 coupled to Tetanus Toxoid. The glycopeptide consists of a 22mer huMUC1 peptide with two immune dominant regions (PDTR and GSTA), glycosylated with the sialylated carbohydrate STN on serine-17. PyMT (polyomavirus middle T-antigen) and human MUC1 double-transgenic mice expressing human tumor-associated MUC1 on breast tumor tissue served as a preclinical breast cancer model.


Subject(s)
Cancer Vaccines/therapeutic use , Glycopeptides/immunology , Mammary Neoplasms, Experimental/therapy , Mucin-1/immunology , Tetanus Toxoid/immunology , Vaccines, Synthetic/therapeutic use , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Female , Humans , Immunoglobulin G/pharmacology , Mice, Transgenic , Middle Aged , Mucin-1/genetics , Triple Negative Breast Neoplasms/immunology
5.
Chembiochem ; 2018 Apr 06.
Article in English | MEDLINE | ID: mdl-29633523

ABSTRACT

The endothelial glycoprotein MUC1 is known to underlie alterations in cancer by means of aberrant glycosylation accompanied by changes in morphology. The heavily shortened glycans induce a collapse of the peptide backbone and enable accessibility of the latter to immune cells, rendering it a tumor-associated antigen. Synthetic vaccines based on MUC1 tandem repeat motifs, comprising tumor-associated 2,3-sialyl-T antigen, conjugated to the immunostimulating tetanus toxoid, are reported herein. Immunization with these vaccines in a simple water/oil emulsion produced a strong immune response in mice to which stimulation with complete Freund's adjuvant (CFA) was not superior. In both cases, high levels of IgG1 and IgG2a/b were induced in C57BL/6 mice. Additional glycosylation in the immunodominant PDTRP domain led to improved binding of the induced antisera to MCF-7 breast tumor cells, compared with that of the monoglycosylated peptide vaccine.

6.
ChemMedChem ; 13(1): 25-29, 2018 01 08.
Article in English | MEDLINE | ID: mdl-29193802

ABSTRACT

A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose-receptor-positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non-mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4+ T cells in the local lymph organs. Comparison of di- and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose-coupled vaccine and the non-mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells.


Subject(s)
Cancer Vaccines/immunology , Dendritic Cells/metabolism , Macrophages/metabolism , Mannose/chemistry , Mucin-1/immunology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cancer Vaccines/metabolism , Dendritic Cells/cytology , Dendritic Cells/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Lectins, C-Type/metabolism , Ligands , Lymph Nodes , MCF-7 Cells , Macrophages/cytology , Macrophages/immunology , Mannose Receptor , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred BALB C , Mucin-1/chemistry , Mucin-1/metabolism , Protein Binding , Receptors, Cell Surface/metabolism
7.
ChemMedChem ; 12(17): 1424-1428, 2017 09 07.
Article in English | MEDLINE | ID: mdl-28675699

ABSTRACT

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.


Subject(s)
Antigens, Tumor-Associated, Carbohydrate/therapeutic use , Breast Neoplasms/prevention & control , Cancer Vaccines/therapeutic use , Mucin-1/therapeutic use , Peptide Fragments/therapeutic use , Tetanus Toxoid/therapeutic use , Vaccines, Synthetic/therapeutic use , Animals , Antigens, Tumor-Associated, Carbohydrate/chemistry , Antigens, Tumor-Associated, Carbohydrate/immunology , Breast Neoplasms/immunology , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , Female , Humans , Immunization , MCF-7 Cells , Mice, Inbred C57BL , Mice, Transgenic , Mucin-1/chemistry , Peptide Fragments/chemistry , Tetanus Toxoid/chemistry , Tetanus Toxoid/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology
8.
ChemMedChem ; 12(10): 722-727, 2017 05 22.
Article in English | MEDLINE | ID: mdl-28440596

ABSTRACT

Fully synthetic MUC1 glycopeptide antitumor vaccines have a precisely specified structure and induce a targeted immune response without suppression of the immune response when using an immunogenic carrier protein. However, tumor-associated aberrantly glycosylated MUC1 glycopeptides are endogenous structures, "self-antigens", that exhibit only low immunogenicity. To overcome this obstacle, a fully synthetic MUC1 glycopeptide antitumor vaccine was combined with poly(inosinic acid:cytidylic acid), poly(I:C), as a structurally defined Toll-like receptor 3 (TLR3)-activating adjuvant. This vaccine preparation elicited extraordinary titers of IgG antibodies which strongly bound human breast cancer cells expressing tumor-associated MUC1. Beside the humoral response, the poly(I:C) glycopeptide vaccine induced a pro-inflammatory environment, very important to overcome the immune-suppressive mechanisms, and elicited a strong cellular immune response crucial for tumor elimination.


Subject(s)
Cancer Vaccines/immunology , Glycopeptides/immunology , Mucin-1/immunology , Poly I-C/immunology , Toll-Like Receptor 3/immunology , Vaccines, Synthetic/immunology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/pharmacology , Animals , Cancer Vaccines/genetics , Cancer Vaccines/pharmacology , Dendritic Cells , Humans , Mice , Poly I-C/chemistry , Vaccines, Synthetic/genetics , Vaccines, Synthetic/pharmacology
9.
Angew Chem Int Ed Engl ; 56(5): 1416-1421, 2017 01 24.
Article in English | MEDLINE | ID: mdl-28005299

ABSTRACT

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.


Subject(s)
Macrophages/metabolism , Oligosaccharides/metabolism , Selectins/metabolism , Cell Movement , Cells, Cultured , Flow Cytometry , Human Umbilical Vein Endothelial Cells , Humans , Inhibitory Concentration 50 , Ligands , Macrophages/cytology , Microscopy, Fluorescence, Multiphoton , Nanomedicine , Oligosaccharides/chemistry , Polymethacrylic Acids/chemistry , Selectins/chemistry , Sialyl Lewis X Antigen , Surface Plasmon Resonance , Tyramine/chemistry
10.
Chemistry ; 23(16): 3875-3884, 2017 Mar 17.
Article in English | MEDLINE | ID: mdl-27957769

ABSTRACT

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 core 2 ß-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched core 2 structures, which favor formation of linear core 1 or core 3 structures, and in particular, truncated tumor-associated antigen structures. The core 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 core 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 core 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.


Subject(s)
Cancer Vaccines/immunology , Glycopeptides/immunology , Mucin-1/immunology , Neoplasms/immunology , Polysaccharides/immunology , Vaccines, Synthetic/immunology , Animals , Antibody Formation , Cancer Vaccines/chemistry , Glycopeptides/chemistry , Humans , Immunity, Humoral , Mice , Mucin-1/chemistry , Neoplasms/therapy , Polysaccharides/chemistry , Protein Array Analysis , Vaccines, Synthetic/chemistry
11.
Proc Natl Acad Sci U S A ; 113(36): 10145-50, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27555590

ABSTRACT

T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.


Subject(s)
Casein Kinase II/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Animals , Casein Kinase II/deficiency , Casein Kinase II/genetics , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Forkhead Transcription Factors , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor , Humans , Interleukin-17 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments , Phosphorylation , Receptors, Interleukin , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , Severity of Illness Index , Signal Transduction , T-Lymphocytes, Regulatory/cytology , Th17 Cells/pathology
12.
Chembiochem ; 17(15): 1412-5, 2016 08 03.
Article in English | MEDLINE | ID: mdl-27188544

ABSTRACT

The Toll-like receptor 2 ligand Pam3 CysSer is of particular interest for the construction synthetic vaccines because of its ability to stimulate of the innate immune system. Such vaccines usually comprise Pam3 CysSer with the natural R-configuration at the glycerol 2-position. Pam3 CysSer peptide vaccines with natural configuration have been shown to be more efficient than the corresponding R/S diastereomers. In order to clarify whether the effect of the configuration of Pam3 Cys on the immune response also applies to glycopeptide vaccines, MUC1 glycopeptide-lipopeptide vaccines bearing either R- or R/S-configured Pam3 CysSerLys4 were compared for their immunological effects. In order to find out whether glycosylated MUC1 tandem repeat domains comprise not only B-cell epitopes but also T-cell epitopes, two-component vaccines containing the Pam3 CysSerLys4 lipopeptide and MUC1 glycopeptides with various glycosylation patterns were synthesized, and their immune reactions in mice were studied.


Subject(s)
Cancer Vaccines/chemistry , Lipoproteins/immunology , Mucin-1/immunology , Animals , Cancer Vaccines/chemical synthesis , Cancer Vaccines/pharmacology , Glycopeptides/immunology , Glycosylation , Humans , Immunity/drug effects , Lipoproteins/therapeutic use , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mucin-1/therapeutic use , Solid-Phase Synthesis Techniques , Stereoisomerism , Vaccines, Synthetic/chemistry
13.
Bioorg Med Chem ; 24(5): 1132-5, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26853835

ABSTRACT

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.


Subject(s)
Cancer Vaccines/immunology , Epitopes, T-Lymphocyte/immunology , Glycopeptides/immunology , Gold/chemistry , Metal Nanoparticles/chemistry , Mucin-1/immunology , Amino Acid Sequence , Animals , Cancer Vaccines/chemistry , Epitopes, T-Lymphocyte/chemistry , Genes, MHC Class II , Glycopeptides/chemistry , Humans , Immunization , MCF-7 Cells , Mice , Molecular Sequence Data , Mucin-1/chemistry , Neoplasms/immunology , Neoplasms/prevention & control
14.
Angew Chem Int Ed Engl ; 55(8): 2894-8, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26800384

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Breast Neoplasms/pathology , Breast/cytology , Cancer Vaccines/administration & dosage , Glycopeptides/immunology , Pancreatic Neoplasms/diagnosis , Female , Humans
15.
Chembiochem ; 16(6): 959-67, 2015 Apr 13.
Article in English | MEDLINE | ID: mdl-25755023

ABSTRACT

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.


Subject(s)
Antibodies, Neoplasm/immunology , Mucin-4/immunology , Pancreatic Neoplasms/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Cancer Vaccines/immunology , Cell Line, Tumor , Epitopes/immunology , Female , Humans , Immune Sera/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mucin-4/chemistry , Pancreatic Neoplasms/pathology , Tandem Repeat Sequences , Tetanus Toxoid/chemistry , Vaccination
16.
Adv Healthc Mater ; 4(4): 522-7, 2015 Mar 11.
Article in English | MEDLINE | ID: mdl-25327631

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/pharmacology , Cancer Vaccines/immunology , Glycopeptides/immunology , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Nanoparticles/chemistry , Oligodeoxyribonucleotides/chemistry , Amino Acid Sequence , Animals , Cations , Enzyme-Linked Immunosorbent Assay , Glycopeptides/chemistry , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/chemical synthesis , Mice, Inbred BALB C , Molecular Sequence Data , Nanoparticles/ultrastructure
17.
Angew Chem Int Ed Engl ; 53(51): 14245-9, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25318465

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/chemistry , Cancer Vaccines/chemical synthesis , Epitopes/chemistry , Glycopeptides/chemistry , Mucin-1/chemistry , T-Lymphocytes, Helper-Inducer/chemistry , Antigen-Antibody Reactions , Antigens, Neoplasm/immunology , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , Epitopes/immunology , Glycopeptides/immunology , Humans , Molecular Structure , Mucin-1/immunology , T-Lymphocytes, Helper-Inducer/immunology
18.
Chemistry ; 20(15): 4232-6, 2014 Apr 07.
Article in English | MEDLINE | ID: mdl-24623572

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Glycerol/chemistry , Glycopeptides/chemical synthesis , Polymers/chemistry , Animals , Antibodies/immunology , Antigen Presentation , Breast Neoplasms/drug therapy , Cancer Vaccines/therapeutic use , Click Chemistry , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Female , Glycopeptides/immunology , Humans , MCF-7 Cells , Mice , Mucin-1/chemistry , Mucin-1/metabolism , Tetanus Toxoid/chemistry , Tetanus Toxoid/metabolism
19.
Angew Chem Int Ed Engl ; 53(6): 1699-703, 2014 Feb 03.
Article in English | MEDLINE | ID: mdl-24449389

ABSTRACT

Multivalent synthetic vaccines were obtained by solid-phase synthesis of tumor-associated MUC1 glycopeptide antigens and their coupling to a Pam3 Cys lipopeptide through click reactions. These vaccines elicited immune responses in mice without the use of any external adjuvant. The vaccine containing four copies of a MUC1 sialyl-TN antigen showed a significant cluster effect. It induced in mice prevailing IgG2a antibodies, which bind to MCF-7 breast tumor cells and initiate the killing of these tumor cells by activation of the complement-dependent cytotoxicity complex.


Subject(s)
Cancer Vaccines/immunology , Glycopeptides/chemistry , Lipoproteins/chemistry , Mucin-1/chemistry , Amino Acid Sequence , Animals , Antibodies/immunology , Antibodies/pharmacology , Apoptosis/drug effects , Cancer Vaccines/chemical synthesis , Cancer Vaccines/chemistry , Click Chemistry , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Glycopeptides/chemical synthesis , Humans , Lipoproteins/chemical synthesis , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mucin-1/immunology , Mucin-1/metabolism , Rabbits
20.
Angew Chem Int Ed Engl ; 52(40): 10652-6, 2013 Sep 27.
Article in English | MEDLINE | ID: mdl-24038824

ABSTRACT

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.


Subject(s)
Cancer Vaccines/chemistry , Epitopes, T-Lymphocyte/chemistry , Glycopeptides/chemistry , Polymethacrylic Acids/chemistry , Amino Acid Sequence , Animals , Cancer Vaccines/immunology , Epitopes, T-Lymphocyte/immunology , Glycopeptides/immunology , Humans , MCF-7 Cells , Mice , Molecular Sequence Data , Mucin-1/chemistry , Mucin-1/immunology , Solubility , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology , Water/chemistry
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