RESUMO
Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.
Assuntos
Glucuronidase , Inibidores de Glicosídeo Hidrolases , Metástase Neoplásica , Animais , Proliferação de Células/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Metástase Neoplásica/tratamento farmacológicoRESUMO
Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.
Assuntos
Antígenos/metabolismo , Manosefosfatos/metabolismo , Peptídeos/metabolismo , Receptores Toll-Like/metabolismo , Antígenos/química , Humanos , Ligantes , Manosefosfatos/química , Estrutura Molecular , Peptídeos/química , Receptores Toll-Like/químicaRESUMO
Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity inâ vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/imunologia , Ligantes , Receptor 2 Toll-Like/química , Vacinas Sintéticas/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Desenho de Fármacos , Humanos , Interleucina-8/metabolismo , Lipopeptídeos/síntese química , Lipopeptídeos/química , Lipopeptídeos/imunologia , Lipoilação , Ativação Linfocitária , Receptor 2 Toll-Like/metabolismo , Vacinas Sintéticas/químicaRESUMO
Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.
Assuntos
Infecções Estafilocócicas , Ácidos Teicoicos , Parede Celular/metabolismo , Glicosilação , Humanos , Staphylococcus aureus/metabolismoRESUMO
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase.
Assuntos
ADP-Ribosilação , Serina , Reparo do DNA , Peptídeos , Processamento de Proteína Pós-Traducional , Serina/metabolismoRESUMO
Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34-56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35-55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.
Assuntos
Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Linfócitos B/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Amiloide/imunologia , Amiloide/toxicidade , Proteínas Amiloidogênicas/síntese química , Proteínas Amiloidogênicas/imunologia , Proteínas Amiloidogênicas/toxicidade , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Benzotiazóis/química , Callithrix , Linhagem Celular , Citrulinação/imunologia , Células Dendríticas/metabolismo , Herpesvirus Humano 4 , Humanos , Camundongos Endogâmicos C57BL , Esclerose Múltipla Crônica Progressiva/imunologia , Esclerose Múltipla Crônica Progressiva/metabolismo , Esclerose Múltipla Crônica Progressiva/virologia , Glicoproteína Mielina-Oligodendrócito/síntese química , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Agregação Patológica de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.
Assuntos
Células Dendríticas/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Peptídeos/imunologia , Receptor 2 Toll-Like/imunologia , Vacinas Conjugadas/imunologia , Animais , Citocinas/biossíntese , Células Dendríticas/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Monócitos/imunologia , Vacinas Conjugadas/químicaRESUMO
Teichoic acids (TAs) are key components of the Gram-positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well-defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid-phase synthesis approach for the rapid generation of well-defined glycosylated, glycerol-based TA oligomers. Building on the use of a "universal" linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA-microarray, which is used to reveal, for the first time, the binding preferences of anti-LTA (lipoteichoic acids) antibodies at the molecular level.
Assuntos
Ácidos Teicoicos/síntese química , Alanina/metabolismo , Parede Celular/química , Glicosilação , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Estrutura Molecular , Técnicas de Síntese em Fase Sólida , Álcoois Açúcares/química , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologiaRESUMO
The development of effective protecting group chemistry is an important driving force behind the progress in the synthesis of complex oligosaccharides. Automated solid-phase synthesis is an attractive technique for the rapid assembly of oligosaccharides, built up of repetitive elements. The fact that (harsh) reagents are used in excess in multiple reaction cycles makes this technique extra demanding on the protecting groups used. Here, the synthesis of a set of oligorhamnan fragments is reported using the cyanopivaloyl (PivCN) ester to ensure effective neighboring group participation during the glycosylation events. The PivCN group combines the favorable characteristics of the parent pivaloyl (Piv) ester, stability, minimal migratory aptitude, minimal orthoester formation, while it can be cleaved under mild conditions. We show that the remote CN group in the PivCN renders the PivCN carbonyl more electropositive and thus susceptible to nucleophilic cleavage. This feature is built upon in the automated solid-phase assembly of the oligorhamnan fragments. Where the use of a Piv-protected building block failed because of incomplete cleavage, PivCN-protected synthons performed well and allowed the generation of oligorhamnans, up to 16 monosaccharides in length.
RESUMO
A new class of phosphanylmethylphosphonate reagents has been developed to enable the controlled synthesis of methylene bisphosphonate mono- and diesters. Condensation of such reagents with an alcohol of choice through azole-mediated phosphoramidite chemistry followed by inâ situ oxidation provides orthogonally protected methylene bisphosphonate tetraesters. Global deprotection of the tetraester leads to terminal methylene bisphosphonates. Alternatively, selective deprotection at the terminal phosphonate followed by a condensation between the acquired methylene bisphosphonate triester and a second alcohol leads to methylene bisphosphonates diesters.
RESUMO
Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.
Assuntos
Cisteína/análogos & derivados , Corantes Fluorescentes/síntese química , Lipoproteínas/síntese química , Linhagem Celular , Cisteína/síntese química , Cisteína/química , Cisteína/farmacocinética , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Humanos , Interleucina-8/biossíntese , Ligantes , Lipoproteínas/química , Lipoproteínas/farmacocinética , Estrutura Molecular , Solubilidade , Relação Estrutura-Atividade , Receptor 2 Toll-Like/metabolismoRESUMO
The amyloid ß (A ß) peptide is important in the context of Alzheimer's disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the hydrophobic part of A ß suggests a possible membrane activity of the peptide. We employed spin-label EPR to investigate the aggregation process of A ß1-40 in the presence of the sodium dodecyl sulfate (SDS) detergent as a membrane-mimicking agent. In this work, the effect of SDS on A ß is studied using two positions of spin label, the N-terminus and position 26. By comparing the two label positions, the effect of local mobility of the spin label is eliminated, revealing A ß aggregation in the SDS concentration regime below the critical micelle concentration (CMC). We demonstrate that, at low SDS concentrations, the N-terminus of A ß participates in the solubilization, most likely by being located at the particle-water interface. At higher SDS concentrations, an SDS-solubilized state that is a precursor to the one A ß/micelle state above the CMC of SDS prevails. We propose that A ß is membrane active and that aggregates include SDS. This study reveals the unique potential of EPR in studying A ß aggregation in the presence of detergent.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Detergentes/metabolismo , Membranas Artificiais , Fragmentos de Peptídeos/metabolismo , Dodecilsulfato de Sódio/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Fragmentos de Peptídeos/química , Ligação Proteica , Rotação , Dodecilsulfato de Sódio/farmacologia , Marcadores de SpinRESUMO
The post-translational modification of proteins that is known as adenosine diphosphate ribosylation (ADPr) regulates a wide variety of important biological processes, such as DNA-damage repair and cellular metabolism. This modification is also involved in carcinogenesis and the process of aging. Therefore, a better understanding of the function of ADP-ribosylation is crucial for the development of novel therapeutics. To facilitate the elucidation of the biology of ADPr, the availability of well-defined fragments of poly(ADP-ribose) is essential. Herein we report a solid-phase synthetic approach for the preparation of ADP-ribose oligomers of exactly defined length. The methodology is exemplified by the first reported synthesis of an ADP-ribose dimer and trimer.
Assuntos
Adenosina Difosfato Ribose/química , Poli Adenosina Difosfato Ribose/síntese química , Dano ao DNA , Reparo do DNA , Espectroscopia de Ressonância Magnética , Fosforilação , Poli Adenosina Difosfato Ribose/química , Técnicas de Síntese em Fase SólidaRESUMO
The activation of CD8(+) T-cells requires the uptake of exogenous polypeptide antigens and proteolytic processing of these antigens to octamer or nonamer peptides, which are loaded on MHC-I complexes and presented to the T-cell. By using an azide as a bioorthogonal protecting group rather than as a ligation handle, masked antigens were generated-antigens that are not recognized by their cognate T-cell unless they are deprotected on the cell using a Staudinger reduction.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos/química , Antígenos/imunologia , Azidas/química , Apresentação Cruzada/imunologia , Células Dendríticas/química , Células Dendríticas/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Propriedades de SuperfícieRESUMO
The covalent attachment of an innate immune system stimulating agent to an antigen can provide active vaccine modalities capable of eliciting a potent immune response against the incorporated antigen. Here we describe the design, automated synthesis and immunological evaluation of a set of four muramyl dipeptide-peptide antigen conjugates. Muramyl dipeptide (MDP) represents a well-known ligand for the intracellular NOD2 receptor and our study shows that covalently linking an MDP-moiety to an antigenic peptide can lead to a construct that is capable of stimulating the NOD2 receptor if the ligand is attached at the anomeric center of the muramic acid. The constructs can be processed by dendritic cells (DCs) and the conjugation does not adversely affect the presentation of the incorporated SIINFEKL epitope on MHC-I molecules. However, stimulation of the NOD2 receptor in DCs was not sufficient to provide a strong immunostimulatory signal.
RESUMO
BACKGROUND: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP. METHODS: A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-)malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 µg per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays. RESULTS: Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial. CONCLUSIONS: Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity. TRIAL REGISTRATION NUMBERS: NCT02821494 and 2014-000658-12.
Assuntos
Papillomavirus Humano 16 , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Epitopos Imunodominantes , Inflamação/etiologia , Ligantes , Peptídeos , Linfócitos T , Receptor 2 Toll-Like , Neoplasias do Colo do Útero/virologia , Vacinas contra Papillomavirus/efeitos adversos , Infecções por Papillomavirus/complicaçõesRESUMO
ADP-ribosylation is a pivotal post-translational modification that mediates various important cellular processes producing negatively charged biopolymer, poly (ADP-ribose), the functions of which need further elucidation. Toward this end, the availability of well-defined ADP-ribose (ADPr) oligomers in sufficient quantities is a necessity. In this work, we demonstrate the chemical synthesis of linear ADPr oligomers of defined, increasing length using a modified solid phase synthesis method. An advanced phosphoramidite building block temporarily protected with the base sensitive Fm-group was designed and implemented in the repeating pyrophosphate formation via a P(v)-P(iii) coupling procedure on Tentagel solid support. Linear ADPr oligomers up to a pentamer were successfully synthesized and their affinity for the poly-(ADP-ribose)-binding macrodomain of the human oncogenic helicase and chromatin remodeling enzyme ALC1 was determined. Our data reveal a length-dependent binding manner of the nucleic acid, with larger ADPr oligomers exhibiting higher binding enthalpies for ALC1, illustrating how the activity of this molecular machine is gated by PAR.
RESUMO
Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.
Assuntos
Vacinas Anticâncer/síntese química , Glucosamina/análogos & derivados , Lipídeo A/análogos & derivados , Compostos Organofosforados/química , Ovalbumina/química , Receptor 4 Toll-Like/imunologia , Adjuvantes Imunológicos , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glucosamina/química , Glucosamina/imunologia , Imunoglobulina G/sangue , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Compostos Organofosforados/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas ConjugadasRESUMO
Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of DCs is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the induction of antigen-specific T cells. The natural O-mannosidic linkage is susceptible to enzymatic degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and streamline the synthesis of mannosylated peptide antigens, we here describe the development of an acid-stable C-mannosyl lysine, which allows for the inline introduction of mannosides during solid-phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization capacities and binding affinities as the O-mannosyl analogs. Moreover, the antigen conjugates were capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and providing enhanced gp100 presentation to CD8+ and CD4+ T cells, similar to their O-mannosyl counterparts. Our results demonstrate that the C-mannose lysine is a valuable building block for the generation of anticancer peptide-conjugate vaccine modalities.
Assuntos
Antineoplásicos/síntese química , Vacinas Anticâncer/síntese química , Glicopeptídeos/química , Lisina/química , Manose/química , Vacinas Conjugadas/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Vacinas Anticâncer/metabolismo , Técnicas de Cultura de Células , Citocinas/metabolismo , Células Dendríticas , Epitopos/química , Epitopos/metabolismo , Corantes Fluorescentes/química , Humanos , Imagem Óptica , Linfócitos T , Receptor 7 Toll-Like/metabolismo , Vacinas Conjugadas/metabolismo , Vacinas Sintéticas/química , Antígeno gp100 de Melanoma/metabolismoRESUMO
Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known "high mannose" structures, that we presented in a systematically increasing number of copies (n = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs.