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1.
J Immunother Cancer ; 10(10)2022 10.
Article in English | MEDLINE | ID: mdl-36261215

ABSTRACT

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.


Subject(s)
Human papillomavirus 16 , Papillomavirus Infections , Papillomavirus Vaccines , Uterine Cervical Neoplasms , Female , Humans , Immunodominant Epitopes , Inflammation/etiology , Ligands , Peptides , T-Lymphocytes , Toll-Like Receptor 2 , Uterine Cervical Neoplasms/virology , Papillomavirus Vaccines/adverse effects , Papillomavirus Infections/complications
2.
Proc Natl Acad Sci U S A ; 119(31): e2203167119, 2022 08 02.
Article in English | MEDLINE | ID: mdl-35881786

ABSTRACT

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.


Subject(s)
Glucuronidase , Glycoside Hydrolase Inhibitors , Neoplasm Metastasis , Animals , Cell Proliferation/drug effects , Glucuronidase/antagonists & inhibitors , Glycoside Hydrolase Inhibitors/pharmacology , Glycoside Hydrolase Inhibitors/therapeutic use , Heparan Sulfate Proteoglycans/metabolism , Heparitin Sulfate/metabolism , Humans , Mice , Neoplasm Metastasis/drug therapy
3.
Chem Sci ; 12(37): 12468-12475, 2021 Sep 29.
Article in English | MEDLINE | ID: mdl-34603678

ABSTRACT

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.

4.
Chemistry ; 27(40): 10461-10469, 2021 Jul 16.
Article in English | MEDLINE | ID: mdl-33991006

ABSTRACT

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.


Subject(s)
Staphylococcal Infections , Teichoic Acids , Cell Wall/metabolism , Glycosylation , Humans , Staphylococcus aureus/metabolism
5.
Chemistry ; 27(41): 10621-10627, 2021 Jul 21.
Article in English | MEDLINE | ID: mdl-33769608

ABSTRACT

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.


Subject(s)
ADP-Ribosylation , Serine , DNA Repair , Peptides , Protein Processing, Post-Translational , Serine/metabolism
6.
Chembiochem ; 22(2): 434-440, 2021 01 15.
Article in English | MEDLINE | ID: mdl-32864819

ABSTRACT

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.


Subject(s)
Antigens/metabolism , Mannosephosphates/metabolism , Peptides/metabolism , Toll-Like Receptors/metabolism , Antigens/chemistry , Humans , Ligands , Mannosephosphates/chemistry , Molecular Structure , Peptides/chemistry , Toll-Like Receptors/chemistry
7.
Chembiochem ; 22(7): 1215-1222, 2021 04 06.
Article in English | MEDLINE | ID: mdl-33180981

ABSTRACT

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 in 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.


Subject(s)
Antigens, Neoplasm/chemistry , Cancer Vaccines/immunology , Ligands , Toll-Like Receptor 2/chemistry , Vaccines, Synthetic/immunology , Antigens, Neoplasm/immunology , Cancer Vaccines/chemistry , Cell Line , Dendritic Cells/cytology , Dendritic Cells/metabolism , Drug Design , Humans , Interleukin-8/metabolism , Lipopeptides/chemical synthesis , Lipopeptides/chemistry , Lipopeptides/immunology , Lipoylation , Lymphocyte Activation , Toll-Like Receptor 2/metabolism , Vaccines, Synthetic/chemistry
8.
J Med Chem ; 63(20): 11691-11706, 2020 10 22.
Article in English | MEDLINE | ID: mdl-32960056

ABSTRACT

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.


Subject(s)
Cancer Vaccines/chemical synthesis , Glucosamine/analogs & derivatives , Lipid A/analogs & derivatives , Organophosphorus Compounds/chemistry , Ovalbumin/chemistry , Toll-Like Receptor 4/immunology , Adjuvants, Immunologic , Animals , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , Cytokines/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Glucosamine/chemistry , Glucosamine/immunology , Immunoglobulin G/blood , Ligands , Lymphocyte Activation/drug effects , Mice , Organophosphorus Compounds/immunology , Ovalbumin/immunology , Peptide Fragments/chemistry , Peptide Fragments/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Vaccines, Conjugate
9.
ACS Chem Biol ; 15(3): 728-739, 2020 03 20.
Article in English | MEDLINE | ID: mdl-32045202

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/chemical synthesis , Cancer Vaccines/chemical synthesis , Glycopeptides/chemistry , Lysine/chemistry , Mannose/chemistry , Vaccines, Conjugate/chemistry , Antigens, Neoplasm/metabolism , Antineoplastic Agents/metabolism , Cancer Vaccines/metabolism , Cell Culture Techniques , Cytokines/metabolism , Dendritic Cells , Epitopes/chemistry , Epitopes/metabolism , Fluorescent Dyes/chemistry , Humans , Optical Imaging , T-Lymphocytes , Toll-Like Receptor 7/metabolism , Vaccines, Conjugate/metabolism , Vaccines, Synthetic/chemistry , gp100 Melanoma Antigen/metabolism
10.
Front Chem ; 7: 650, 2019.
Article in English | MEDLINE | ID: mdl-31637232

ABSTRACT

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.

11.
Bioconjug Chem ; 30(4): 1150-1161, 2019 04 17.
Article in English | MEDLINE | ID: mdl-30865430

ABSTRACT

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.


Subject(s)
Dendritic Cells/immunology , Nod2 Signaling Adaptor Protein/immunology , Peptides/immunology , Toll-Like Receptor 2/immunology , Vaccines, Conjugate/immunology , Animals , Cytokines/biosynthesis , Dendritic Cells/cytology , HEK293 Cells , Humans , Mice , Mice, Transgenic , Monocytes/immunology , Vaccines, Conjugate/chemistry
12.
Biochemistry ; 58(6): 763-775, 2019 02 12.
Article in English | MEDLINE | ID: mdl-30513201

ABSTRACT

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.


Subject(s)
Amyloid/metabolism , Amyloidogenic Proteins/metabolism , B-Lymphocytes/metabolism , Myelin-Oligodendrocyte Glycoprotein/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Amyloid/immunology , Amyloid/toxicity , Amyloidogenic Proteins/chemical synthesis , Amyloidogenic Proteins/immunology , Amyloidogenic Proteins/toxicity , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , B-Lymphocytes/virology , Benzothiazoles/chemistry , Callithrix , Cell Line , Citrullination/immunology , Dendritic Cells/metabolism , Herpesvirus 4, Human , Humans , Mice, Inbred C57BL , Multiple Sclerosis, Chronic Progressive/immunology , Multiple Sclerosis, Chronic Progressive/metabolism , Multiple Sclerosis, Chronic Progressive/virology , Myelin-Oligodendrocyte Glycoprotein/chemical synthesis , Myelin-Oligodendrocyte Glycoprotein/immunology , Myelin-Oligodendrocyte Glycoprotein/toxicity , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Peptide Fragments/toxicity , Protein Aggregation, Pathological , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
13.
Chemistry ; 24(16): 4014-4018, 2018 Mar 15.
Article in English | MEDLINE | ID: mdl-29389054

ABSTRACT

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.


Subject(s)
Teichoic Acids/chemical synthesis , Alanine/metabolism , Cell Wall/chemistry , Glycosylation , Gram-Positive Bacteria/chemistry , Gram-Positive Bacteria/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/immunology , Molecular Structure , Solid-Phase Synthesis Techniques , Sugar Alcohols/chemistry , Teichoic Acids/chemistry , Teichoic Acids/immunology
14.
J Org Chem ; 82(24): 12992-13002, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29148768

ABSTRACT

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.

15.
Angew Chem Int Ed Engl ; 56(11): 2955-2959, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28170152

ABSTRACT

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 in 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.

16.
ACS Chem Biol ; 11(11): 3172-3178, 2016 11 18.
Article in English | MEDLINE | ID: mdl-27704768

ABSTRACT

Antigen recognition followed by the activation of cytotoxic T-cells (CTLs) is a key step in adaptive immunity, resulting in clearance of viruses and cancers. The repertoire of peptides that have the ability to bind to the major histocompatibility type-I (MHC-I) is enormous, but the approaches available for studying the diversity of the peptide repertoire on a cell are limited. Here, we explore the use of bioorthogonal chemistry to quantify specific peptide-MHC-I complexes (pMHC-I) on cells. We show that modifying epitope peptides with bioorthogonal groups in surface accessible positions allows wild-type-like MHC-I binding and bioorthogonal ligation using fluorogenic chromophores in combination with a Cu(I)-catalyzed Huisgen cycloaddition reaction. We expect that this approach will make a powerful addition to the antigen presentation toolkit as for the first time it allows quantification of antigenic peptides for which no detection tools exist.


Subject(s)
Epitopes/chemistry , Histocompatibility Antigens Class I/chemistry , Humans , T-Lymphocytes, Cytotoxic/immunology
17.
Oncotarget ; 7(41): 67087-67100, 2016 Oct 11.
Article in English | MEDLINE | ID: mdl-27564262

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/pharmacology , Cancer Vaccines/immunology , Oncogene Proteins, Viral/immunology , Repressor Proteins/immunology , T-Lymphocytes/immunology , Uterine Cervical Neoplasms/immunology , Female , Human papillomavirus 16 , Humans , Ligands , Lymph Nodes/immunology , Lymphocyte Activation , Oncogene Proteins, Viral/pharmacology , Repressor Proteins/pharmacology , Toll-Like Receptor 2/immunology
18.
Bioorg Med Chem Lett ; 26(15): 3641-5, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27289322

ABSTRACT

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.


Subject(s)
Cysteine/analogs & derivatives , Fluorescent Dyes/chemical synthesis , Lipoproteins/chemical synthesis , Cell Line , Cysteine/chemical synthesis , Cysteine/chemistry , Cysteine/pharmacokinetics , Dendritic Cells/drug effects , Dose-Response Relationship, Drug , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Humans , Interleukin-8/biosynthesis , Ligands , Lipoproteins/chemistry , Lipoproteins/pharmacokinetics , Molecular Structure , Solubility , Structure-Activity Relationship , Toll-Like Receptor 2/metabolism
19.
J Biol Phys ; 42(3): 299-315, 2016 06.
Article in English | MEDLINE | ID: mdl-26984615

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides/metabolism , Detergents/metabolism , Membranes, Artificial , Peptide Fragments/metabolism , Sodium Dodecyl Sulfate/metabolism , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Detergents/pharmacology , Dose-Response Relationship, Drug , Peptide Fragments/chemistry , Protein Binding , Rotation , Sodium Dodecyl Sulfate/pharmacology , Spin Labels
20.
ChemMedChem ; 11(2): 190-8, 2016 Jan 19.
Article in English | MEDLINE | ID: mdl-26059481

ABSTRACT

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment capable of triggering the innate immune system through interaction with the intracellular NOD2 receptor. To develop synthetic vaccine modalities composed of an antigenic entity (typically a small peptide) and a molecular adjuvant with well-defined activity, we previously assembled covalent MDP-antigen conjugates. Although these were found to be capable of stimulating the NOD2 receptor and were processed by dendritic cells (DCs) leading to effective antigen presentation, DC maturation--required for an apt immune response--could not be achieved with these conjugates. To improve the efficacy of these vaccine modalities, we equipped the MDP moiety with lipophilic tails, well-known modifications to enhance the immune-stimulatory activity of MDPs. Herein we report the design and synthesis of a lipophilic MDP-antigen conjugate and show that it is a promising vaccine modality capable of stimulating the NOD2 receptor, maturing DCs, and delivering antigen cargo into the MHC-I cross-presentation pathway.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/immunology , Antigens/chemistry , Antigens/immunology , Immunoconjugates/chemistry , Nod2 Signaling Adaptor Protein/immunology , Adjuvants, Immunologic/chemical synthesis , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dose-Response Relationship, Drug , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Structure-Activity Relationship
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