RESUMEN
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.
Asunto(s)
Glucuronidasa , Inhibidores de Glicósido Hidrolasas , Metástasis de la Neoplasia , Animales , Proliferación Celular/efectos de los fármacos , Glucuronidasa/antagonistas & inhibidores , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/uso terapéutico , Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Ratones , Metástasis de la Neoplasia/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Antígenos/metabolismo , Manosafosfatos/metabolismo , Péptidos/metabolismo , Receptores Toll-Like/metabolismo , Antígenos/química , Humanos , Ligandos , Manosafosfatos/química , Estructura Molecular , Péptidos/química , Receptores Toll-Like/químicaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/química , Vacunas contra el Cáncer/inmunología , Ligandos , Receptor Toll-Like 2/química , Vacunas Sintéticas/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/química , Línea Celular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Diseño de Fármacos , Humanos , Interleucina-8/metabolismo , Lipopéptidos/síntesis química , Lipopéptidos/química , Lipopéptidos/inmunología , Lipoilación , Activación de Linfocitos , Receptor Toll-Like 2/metabolismo , Vacunas Sintéticas/químicaRESUMEN
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.
Asunto(s)
ADP-Ribosilación , Serina , Reparación del ADN , Péptidos , Procesamiento Proteico-Postraduccional , Serina/metabolismoRESUMEN
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.
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Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Linfocitos B/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Amiloide/inmunología , Amiloide/toxicidad , Proteínas Amiloidogénicas/síntesis química , Proteínas Amiloidogénicas/inmunología , Proteínas Amiloidogénicas/toxicidad , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/virología , Benzotiazoles/química , Callithrix , Línea Celular , Citrulinación/inmunología , Células Dendríticas/metabolismo , Herpesvirus Humano 4 , Humanos , Ratones Endogámicos C57BL , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Crónica Progresiva/virología , Glicoproteína Mielina-Oligodendrócito/síntesis química , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Agregación Patológica de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Péptidos/inmunología , Receptor Toll-Like 2/inmunología , Vacunas Conjugadas/inmunología , Animales , Citocinas/biosíntesis , Células Dendríticas/citología , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Monocitos/inmunología , Vacunas Conjugadas/químicaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Detergentes/metabolismo , Membranas Artificiales , Fragmentos de Péptidos/metabolismo , Dodecil Sulfato de Sodio/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Fragmentos de Péptidos/química , Unión Proteica , Rotación , Dodecil Sulfato de Sodio/farmacología , Marcadores de SpinRESUMEN
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.
Asunto(s)
Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/síntesis química , Daño del ADN , Reparación del ADN , Espectroscopía de Resonancia Magnética , Fosforilación , Poli Adenosina Difosfato Ribosa/química , Técnicas de Síntesis en Fase SólidaRESUMEN
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.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos/química , Antígenos/inmunología , Azidas/química , Reactividad Cruzada/inmunología , Células Dendríticas/química , Células Dendríticas/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Papillomavirus Humano 16 , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Neoplasias del Cuello Uterino , Femenino , Humanos , Epítopos Inmunodominantes , Inflamación/etiología , Ligandos , Péptidos , Linfocitos T , Receptor Toll-Like 2 , Neoplasias del Cuello Uterino/virología , Vacunas contra Papillomavirus/efectos adversos , Infecciones por Papillomavirus/complicacionesRESUMEN
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.
RESUMEN
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.
Asunto(s)
Vacunas contra el Cáncer/síntesis química , Glucosamina/análogos & derivados , Lípido A/análogos & derivados , Compuestos Organofosforados/química , Ovalbúmina/química , Receptor Toll-Like 4/inmunología , Adyuvantes Inmunológicos , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Glucosamina/química , Glucosamina/inmunología , Inmunoglobulina G/sangre , Ligandos , Activación de Linfocitos/efectos de los fármacos , Ratones , Compuestos Organofosforados/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Vacunas ConjugadasRESUMEN
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.
Asunto(s)
Antineoplásicos/síntesis química , Vacunas contra el Cáncer/síntesis química , Glicopéptidos/química , Lisina/química , Manosa/química , Vacunas Conjugadas/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Vacunas contra el Cáncer/metabolismo , Técnicas de Cultivo de Célula , Citocinas/metabolismo , Células Dendríticas , Epítopos/química , Epítopos/metabolismo , Colorantes Fluorescentes/química , Humanos , Imagen Óptica , Linfocitos T , Receptor Toll-Like 7/metabolismo , Vacunas Conjugadas/metabolismo , Vacunas Sintéticas/química , Antígeno gp100 del Melanoma/metabolismoRESUMEN
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.
RESUMEN
Novel transition metal catalysts based on oligonucleotides can be easily obtained by functionalization of 5-iodouridine with phosphine ligands, resulting in good asymmetric induction in palladium catalyzed allylic nucleophilic substitution.
Asunto(s)
ADN Catalítico/síntesis química , Idoxuridina/análogos & derivados , Oligonucleótidos/síntesis química , Paladio/química , Fosfinas/análisis , Catálisis , Cristalografía , ADN Catalítico/química , Idoxuridina/química , Ligandos , Oligonucleótidos/química , Elementos de Transición/químicaRESUMEN
Solid-phase methodology for synthesis of adenosine diphosphate ribose oligomers (ADPr-oligomers) of defined length is described using an advanced 2-ribosyl adenosine phosphoramidite building block that is prepared in solution via an expeditious and high-yielding method. The methodology is based on phosphitylation of a phosphomonoester as the key condensation step in the solid-phase protocol. As ADP-ribosylation appears to be an important yet poorly understood post-translational modification of proteins, the presented methodology for rapid synthesis of ADPr-fragments of exactly defined length should be of interest for researchers in the field of structural biology and cell biology. © 2016 by John Wiley & Sons, Inc.
RESUMEN
The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16+ cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4+ and CD8+ T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16+ cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas contra el Cáncer/inmunología , Proteínas Oncogénicas Virales/inmunología , Proteínas Represoras/inmunología , Linfocitos T/inmunología , Neoplasias del Cuello Uterino/inmunología , Femenino , Papillomavirus Humano 16 , Humanos , Ligandos , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Proteínas Oncogénicas Virales/farmacología , Proteínas Represoras/farmacología , Receptor Toll-Like 2/inmunologíaRESUMEN
Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8(+) T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4(+) as well as CD8(+) T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8(+) and CD4(+) T-cell priming capacity compared with free SLPs injected together with a free TLR2-L. Vaccination with TLR2-L SLP conjugates leads to efficient induction of antitumor immunity in mice challenged with aggressive transplantable melanoma or lymphoma. Our data indicate that TLR2-L SLP conjugates are suitable to promote integrated antigen-specific CD8(+) and CD4(+) T-cell responses required for the antitumor effects. Collectively, these data show that TLR2-L SLP conjugates are promising synthetic vaccine candidates for active immunotherapy against cancer.
Asunto(s)
Vacunas contra el Cáncer , Lipopéptidos/inmunología , Receptor Toll-Like 2/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Línea Celular Tumoral , Células Dendríticas/inmunología , Ligandos , Linfoma/patología , Linfoma/terapia , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Toll-Like 2/agonistas , Carga TumoralRESUMEN
The synthesis of two ribonucleoprotein fragments of unprecedented complexity is reported. These hybrid biomolecules are prepared combining the use of an automated solid phase peptide and oligonucleotide synthesizer on a single solid support.