Intradermal vaccination of HPV-16 E6 synthetic peptides conjugated to an optimized Toll-like receptor 2 ligand shows safety and potent T cell immunogenicity in patients with HPV-16 positive (pre-)malignant lesions.

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.

Lipid A analog CRX-527 conjugated to synthetic peptides enhances vaccination efficacy and tumor control.

Adjuvants play a determinant role in cancer vaccination by optimally activating APCs and shaping the T cell response. Bacterial-derived lipid A is one of the most potent immune-stimulators known, and is recognized via Toll-like receptor 4 (TLR4). In this study, we explore the use of the synthetic, non-toxic, lipid A analog CRX-527 as an adjuvant for peptide cancer vaccines. This well-defined adjuvant was covalently conjugated to antigenic peptides as a strategy to improve vaccine efficacy. We show that coupling of this TLR4 agonist to peptide antigens improves vaccine uptake by dendritic cells (DCs), maturation of DCs and T cell activation in vitro, and stimulates DC migration and functional T cell priming in vivo. This translates into enhanced tumor protection upon prophylactic and therapeutic vaccination via intradermal injection against B16-OVA melanoma and HPV-related TC1 tumors. These results highlight the potential of CRX-527 as an adjuvant for molecularly defined cancer vaccines, and support the design of adjuvant-peptide conjugates as a strategy to optimize vaccine formulation.

Developments in the Synthesis of Mycobacterial Phenolic Glycolipids.

The highly lipophilic outer barrier of mycobacteria, such as M. tuberculosis and M. leprae, is key to their virulence and intrinsic antibiotic resistance. Various components of this mycomembrane interact with the host immune system but many of these interactions remain ill-understood. This review covers several chemical syntheses of one of these components, mycobacterial phenolic glycolipids (PGLs), and outlines the interaction of these PGLs with the human immune system, as established using these well-defined pure compounds.

(Automated) Synthesis of Well-defined Staphylococcus Aureus Wall Teichoic Acid Fragments.

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.

Synthetic Phenolic Glycolipids for Application in Diagnostic Tests for Leprosy.

Point-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and prophylactic treatment strategies in leprosy control programs, thus positively contributing to clinical outcome and reducing transmission of this infectious disease. Levels of antibodies directed against the M. leprae-specific phenolic glycolipid I (PGL-I) closely correlate with an individual's bacterial load and a higher risk of developing leprosy. We describe herein the assembly of a set of PGL glycans carrying the characteristic phenol aglycon and featuring different methylation patterns. The PGL trisaccharides were applied to construct neoglycoproteins that were used to detect anti-PGL IgM antibodies in leprosy patients. ELISAs and quantitative lateral-flow assays based on up-converting nanoparticles (UCP-LFAs) showed that the generated PGL-I and PGL-II trisaccharide neoglycoconjugates can be applied for the detection of anti M. leprae IgM antibodies in POC tests.

Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens.

Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification. It is found that Langerin binding affinity increases with increasing number of mannosides. Hexavalent presentation of the mannosides resulted in binding affinities ranging from 3 to 12 µM. Trivalent presentation of the dimannosides and trimannosides led to Langerin affinity in the same range. The model melanoma gp100 antigenic peptide was subsequently equipped with a hexavalent cluster of the dimannosides and trimannosides as targeting moieties. Surprisingly, although the bifunctional conjugates were taken up in LCs in a Langerin-dependent manner, limited antigen presentation to cytotoxic T cells was observed. These results indicate that targeting glycan moieties on immunotherapeutic vaccines should not only be validated for target binding, but also on the continued effects on biology, such as antigen presentation to both CD8+ and CD4+ T cells.

Reagent controlled stereoselective synthesis of teichoic acid α-(1,2)-glucans.

The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools.

Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells.

Synthetic carbohydrate-based cell wall components from Staphylococcus aureus.

Glycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only in the last decades that glycoconjugate based vaccines have been effectively applied for controlling and preventing several infectious diseases, such as H. influenzae type b (Hib), N. meningitidis, S. pneumoniae or group B Streptococcus. Methicillin resistant S. aureus (MRSA) strains has been appointed by the WHO as one of those pathogens, for which new treatments are urgently needed. Herein we present an overview of the carbohydrate-based cell wall polymers associated with different S. aureus strains and the related affords to deliver well-defined fragments through synthetic chemistry.

Systematic Dual Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional Mannosylated Antigen.

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.

Formation of Immune Complexes with a Tetanus-Derived B Cell Epitope Boosts Human T Cell Responses to Covalently Linked Peptides in an Ex Vivo Blood Loop System.

Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin-derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer.

Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.

Synthesis and Macrodomain Binding of Mono-ADP-Ribosylated Peptides.

Mono-ADP-ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono-ADP-ribosylated proteins have been described. We report the synthesis of ADP-ribosylated peptides from the proteins histone H2B, RhoA and, HNP-1. An innovative procedure was applied that makes use of pre-phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP-ribosylated peptides, thus showing that the sequence surrounding ADP-ribosylated residues affects the substrate selectivity of macrodomains.

Synthesis and evaluation of fluorescent Pam3Cys peptide conjugates.

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.

Targeted Delivery of Fluorescent High-Mannose-Type Oligosaccharide Cathepsin Inhibitor Conjugates.

Three fluorescent cathepsin inhibitor glycoconjugates have been designed, synthesized, and evaluated in terms of their cell internalization and cathepsin inhibitory properties. The conjugates are composed of a peptide epoxysuccinate, capable of covalent and irreversible binding to cysteine proteases, coupled to a fluorescent BODIPY dye and functionalized with a mono-, tri-, or heptamannoside. Mannose-receptor-dependent uptake of the probes in live dendritic cells is shown to depend on the type of carbohydrate attached. Where uptake of the monomannoside is poor and mannose-receptor-independent, the intracellular labeling of cathepsins by the probes equipped with a tri- or heptamannoside conjugate appeared concentration- and mannose-receptor-dependent.

A multivalent ligand for the mannose-6-phosphate receptor for endolysosomal targeting of an activity-based probe.

The ubiquitously expressed mannose-6-phosphate receptors (MPRs) are a promising class of receptors for targeted compound delivery into the endolysosomal compartments of a variety of cell types. The development of a synthetic, multivalent, mannose-6-phosphate (M6P) glycopeptide-based MPR ligand is described. The conjugation of this ligand to fluorescent DCG-04, an activity-based probe for cysteine cathepsins, enabled fluorescent readout of its receptor-targeting properties. The resulting M6P-cluster-BODIPY-DCG-04 probe was shown to efficiently label cathepsins in cell lysates as well as in live cells. Furthermore, the introduction of the 6-O-phosphates leads to a completely altered uptake profile in COS and dendritic cells compared to a mannose-containing ligand. Competition with mannose-6-phosphate abolished all uptake of the probe in COS cells, and we conclude that the mannose-6-phosphate cluster targets the MPR and ensures the targeted delivery of cargo bound to the cluster into the endolysosomal pathway.

Exploring dual electrophiles in peptide-based proteasome inhibitors: carbonyls and epoxides.

Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though with considerable loss of activity.

Identification of a novel CD40 ligand for targeted imaging of inflammatory plaques by phage display.

The CD40/CD40L dyad is deemed to play a central role in several inflammatory processes, including atherosclerosis. As CD40 is overexpressed in atherosclerotic lesions, it constitutes a promising candidate for targeted imaging approaches. Here we describe the design of a novel, selective peptide ligand for CD40 by phage display. A synthetic peptide corresponding with the phage insert NP31 displayed nanomolar affinity for CD40. Affinity was further enhanced by mutimeric presentation of NP31. An essential 11-mer peptide motif was identified by truncation and alanine scan studies. Enriched phage selectively bound human CD40 and homed to inflammatory joints in a murine model of rheumatoid arthritis. NP31 ablated VEGF and IL-6 transcriptional activation and partially inhibited IL-6 production by CD40L-activated endothelial cells. Notably, NP31 did not only alter the biodistribution profile of a streptavidin scaffold but also markedly increased accumulation of the carrier in atherosclerotic aortic lesions of aged ApoE(-/-) mice in a CD40-dependent manner. This potent and selective peptide ligand has potential for targeted imaging and drug delivery approaches in CD40-dependent inflammatory disorders such as atherosclerosis.

Influence of O6 in mannosylations using benzylidene protected donors: stereoelectronic or conformational effects?

The stereoselective synthesis of ß-mannosides and the underlying reaction mechanism have been thoroughly studied, and especially the benzylidene-protected mannosides have gained a lot of attention since the corresponding mannosyl triflates often give excellent selectivity. The hypothesis for the enhanced stereoselectivity has been that the benzylidene locks the molecule in a less reactive conformation with the O6 trans to the ring oxygen (O5), which would stabilize the formed α-triflate and subsequent give ß-selectivity. In this work, the hypothesis is challenged by using the carbon analogue (C7) of the benzylidene-protected mannosyl donor, which is investigated in terms of diastereoselectivity and reactivity and by low-temperature NMR. In terms of diastereoselectivity, the C-7-analogue behaves similarly to the benzylidene-protected donor, but its low-temperature NMR reveals the formation of several reactive intermediate. One of the intermediates was found to be the ß-oxosulfonium ion. The reactivity of the donor was found to be in between that of the "torsional" disarmed and an armed donor.

Incorporation of non-natural amino acids improves cell permeability and potency of specific inhibitors of proteasome trypsin-like sites.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.