Publications by authors named "Sylvie Bay"

35 Publications

Engineering Synthetic Lipopeptide Antigen for Specific Detection of subsp. Infection.

Front Vet Sci 2021 23;8:637841. Epub 2021 Apr 23.

INRAE, Université de Tours, ISP, Nouzilly, France.

Unlike other MAC members, subsp. (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as or the more closely related species subsp. , the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.
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http://dx.doi.org/10.3389/fvets.2021.637841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103206PMC
April 2021

Interferon-γ Response of subsp. Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

Front Vet Sci 2021 26;8:645251. Epub 2021 Mar 26.

Animal and Plant Health Agency, Addlestone, United Kingdom.

Despite its potential for early diagnosis of subsp. (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or subsp. (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Complex-specific antibody and PPDj-induced IFN-γ responses were monitored . Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.
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http://dx.doi.org/10.3389/fvets.2021.645251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034290PMC
March 2021

The fully synthetic glycopeptide MAG-Tn3 therapeutic vaccine induces tumor-specific cytotoxic antibodies in breast cancer patients.

Cancer Immunol Immunother 2020 May 7;69(5):703-716. Epub 2020 Feb 7.

Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, 28 rue du Dr Roux, 75015, Paris, France.

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT CD4 T-cell epitope. This promiscuous CD4 T-cell epitope can bind to a wide range of HLA-DRB molecules and is thus expected to activate CD4 T-cell responses in a large part of the human population. The MAG-Tn3 vaccine was formulated with the GSK-proprietary immunostimulant AS15, composed of CpG7909, MPL, and QS21, which has been shown to stimulate both innate and humoral responses, in addition to being well tolerated. Here, seven patients with localized breast cancer with a high-risk of relapse were immunized with the MAG-Tn3 vaccine formulated with AS15. The first results of phase I clinical trial demonstrated that all vaccinated patients developed high levels of Tn-specific antibodies. Moreover, these antibodies specifically recognized Tn-expressing human tumor cells and killed them through a complement-dependent cytotoxicity mechanism. Overall, this study establishes, for the first time, the capacity of a fully synthetic glycopeptide cancer vaccine to induce specific immune responses in humans.
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http://dx.doi.org/10.1007/s00262-020-02503-0DOI Listing
May 2020

Cleaved CD31 as a target for in vivo molecular imaging of inflammation.

Sci Rep 2019 12 20;9(1):19560. Epub 2019 Dec 20.

Nuclear Medicine Department, X. Bichat Hospital, APHP and DHU FIRE, F-75018, Paris, France.

There is a need for new targets to specifically localize inflammatory foci, usable in a wide range of organs. Here, we hypothesized that the cleaved molecular form of CD31 is a suitable target for molecular imaging of inflammation. We evaluated a bioconjugate of D-P8RI, a synthetic peptide that binds all cells with cleaved CD31, in an experimental rat model of sterile acute inflammation. Male Wistar rats were injected with turpentine oil into the gastrocnemius muscle two days before Tc-HYNIC-D-P8RI (or its analogue with L-Proline) SPECT/CT or [F]FDG PET/MRI. Biodistribution, stability study, histology, imaging and autoradiography of Tc-HYNIC-D-P8RI were further performed. Biodistribution studies revealed rapid elimination of Tc-HYNIC-D-P8RI through renal excretion with almost no uptake from most organs and excellent in vitro and in vivo stability were observed. SPECT/CT imaging showed a significant higher Tc-HYNIC-D-P8RI uptake compared with its analogue with L-Proline (negative control) and no significant difference compared with [F]FDG (positive control). Moreover, autoradiography and histology revealed a co-localization between Tc-HYNIC-D-P8RI uptake and inflammatory cell infiltration. Tc-HYNIC-D-P8RI constitutes a new tool for the detection and localization of inflammatory sites. Our work suggests that targeting cleaved CD31 is an attractive strategy for the specific in vivo imaging of inflammatory processes.
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http://dx.doi.org/10.1038/s41598-019-56163-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925130PMC
December 2019

An IgG-induced neutrophil activation pathway contributes to human drug-induced anaphylaxis.

Sci Transl Med 2019 07;11(500)

Unit of Antibodies in Therapy and Pathology, Institut Pasteur, UMR 1222 INSERM, 75015 Paris, France.

Anaphylaxis is a systemic acute hypersensitivity reaction that is considered to depend on allergen-specific immunoglobulin E (IgE) antibodies and histamine release by mast cells and basophils. Nevertheless, allergen-specific IgG antibodies have been proposed to contribute when the allergen is an abundant circulating large molecule, e.g., after infusions of therapeutic antibodies or dextran. Data from animal models demonstrate a pathway involving platelet-activating factor (PAF) release by monocytes/macrophages and neutrophils activated via their Fc gamma receptors (FcγRs). We hypothesized that such a pathway may also apply to small drugs and could be responsible for non-IgE-mediated anaphylaxis and influence anaphylaxis severity in humans. We prospectively conducted a multicentric study of 86 patients with suspected anaphylaxis to neuromuscular-blocking agents (NMBAs) during general anesthesia and 86 matched controls. We found that concentrations of anti-NMBA IgG and markers of FcγR activation, PAF release, and neutrophil activation correlated with anaphylaxis severity. Neutrophils underwent degranulation and NETosis early after anaphylaxis onset, and plasma-purified anti-NMBA IgG triggered neutrophil activation ex vivo in the presence of NMBA. Neutrophil activation could also be observed in patients lacking evidence of classical IgE-dependent anaphylaxis. This study supports the existence of an IgG-neutrophil pathway in human NMBA-induced anaphylaxis, which may aggravate anaphylaxis in combination with the IgE pathway or underlie anaphylaxis in the absence of specific IgE. These results reconcile clinical and experimental data on the role of antibody classes in anaphylaxis and could inform diagnostic approaches to NMBA-induced acute hypersensitivity reactions.
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http://dx.doi.org/10.1126/scitranslmed.aat1479DOI Listing
July 2019

Selection of similar single domain antibodies from two immune VHH libraries obtained from two alpacas by using different selection methods.

Immunol Lett 2017 08 6;188:89-95. Epub 2017 Jul 6.

Institut Pasteur, CITECH, Plateforme d'Ingénierie des Anticorps, Paris, France. Electronic address:

The two most used methods to select camelid single-domain antibody-fragments (VHHs) are: displaying their repertoires on the surface of filamentous bacteriophages (phage display) or linking them to ribosomes (ribosome display). In this study, we compared specific VHHs isolated from two different immune libraries coming from two different alpacas by using these two selection methods. Three anti-GFAP (glial fibrillary acidic protein) VHHs were derived from an immune library obtained by ribosome display after immunization of one alpaca with purified GFAP, a protein expressed by astroglial cells. In parallel, three other anti-GFAP VHHs were derived from an immune library by phage display after immunization of another alpaca with a human brain tissue extract containing GFAP. All the VHHs were closely related and one VHH was found to be strictly identical in both studies. This highlights the selection pressure exerted by the camelid immune system to shape the paratope of an antibody against a defined antigen.
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http://dx.doi.org/10.1016/j.imlet.2017.07.001DOI Listing
August 2017

Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease.

MAbs 2017 Aug/Sep;9(6):1016-1027. Epub 2017 Jun 28.

a Institut Pasteur, Unité de Chimie des Biomolécules, Département Biologie Structurale et Chimie , Paris , France.

Today, molecular imaging of neurodegenerative diseases is mainly based on small molecule probes. Alternatively, antibodies are versatile tools that may be developed as new imaging agents. Indeed, they can be readily obtained to specifically target any antigen of interest and their scaffold can be functionalized. One of the critical issues involved in translating antibody-based probes to the clinic is the design and synthesis of perfectly-defined conjugates. Camelid single-domain antibody-fragments (VHHs) are very small and stable antibodies that are able to diffuse in tissues and potentially cross the blood brain barrier (BBB). Here, we selected a VHH (R3VQ) specifically targeting one of the main lesions of Alzheimer's disease (AD), namely the amyloid-beta (Aß) deposits. It was used as a scaffold for the design of imaging probes for magnetic resonance imaging (MRI) and labeled with the contrastophore gadolinium using either a random or site-specific approach. In contrast to the random strategy, the site-specific conjugation to a single reduced cysteine in the C-terminal part of the R3VQ generates a well-defined bioconjugate in a high yield process. This new imaging probe is able to cross the BBB and label Aß deposits after intravenous injection. Also, it displays improved r1 and r2 relaxivities, up to 30 times higher than a widely used clinical contrast agent, and it allows MRI detection of amyloid deposits in post mortem brain tissue of a mouse model of AD. The ability to produce chemically-defined VHH conjugates that cross the BBB opens the way for future development of tailored imaging probes targeting intracerebral antigens.
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http://dx.doi.org/10.1080/19420862.2017.1342914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540085PMC
June 2017

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mol Microbiol 2017 Aug 15;105(4):525-539. Epub 2017 Jun 15.

Infectiologie et Santé Publique, INRA, Université de Tours, UMR1282, Nouzilly, F-37380, France.

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.
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http://dx.doi.org/10.1111/mmi.13717DOI Listing
August 2017

Rearranged Biosynthetic Gene Cluster and Synthesis of Hassallidin E in Planktothrix serta PCC 8927.

ACS Chem Biol 2017 07 17;12(7):1796-1804. Epub 2017 May 17.

Institut Pasteur , Collection of Cyanobacteria, Paris, France.

Cyanobacteria produce a wide range of natural products with antifungal bioactivity. The cyclic glycosylated lipopeptides of the hassallidin family have potent antifungal activity and display a great degree of chemical diversity. Here, we report the discovery of a hassallidin biosynthetic gene cluster from the filamentous cyanobacterium Planktothrix serta PCC 8927. The hassallidin gene cluster showed heavy rearrangement and marks of genomic plasticity. Nucleotide bias, differences in GC content, and phylogenetic incongruence suggested the acquisition of the hassallidin biosynthetic gene cluster in Planktothrix serta PCC 8927 by horizontal gene transfer. Chemical analyses by liquid chromatography and mass spectrometry demonstrated that this strain produced hassallidin E, a new glycosylated hassallidin variant. Hassallidin E was the only structural variant produced by Planktothrix serta PCC 8927 in all tested conditions. Further evaluated on human pathogenic fungi, hassallidin E showed an antifungal bioactivity. Hassallidin production levels correlated with nitrogen availability, in the only nitrogen-fixing Planktothrix described so far. Our results provide insights into the distribution and chemical diversity of cyanobacterial antifungal compounds as well as raise questions on their ecological relevance.
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http://dx.doi.org/10.1021/acschembio.7b00093DOI Listing
July 2017

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs.

Glycobiology 2017 01 22;27(2):140-153. Epub 2016 Nov 22.

Laboratory of Tumor Immunology and Glycobiology, Institut Pasteur de Montevideo, Mataojo 2020 (C.P. 11400), Montevideo, Uruguay

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.
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http://dx.doi.org/10.1093/glycob/cww111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224595PMC
January 2017

Large-scale synthesis and structural analysis of a synthetic glycopeptide dendrimer as an anti-cancer vaccine candidate.

Org Biomol Chem 2016 Dec;15(1):114-123

Institut Pasteur, Unité Chimie des Biomolécules, 75724 Paris Cedex 15, France. and CNRS UMR 3523, 75724 Paris Cedex 15, France.

Herein, we report a new process that enables the gram-scale production of a fully synthetic anti-cancer vaccine for human use. This therapeutic vaccine candidate, named MAG-Tn3, is a high-molecular-weight tetrameric glycopeptide encompassing carbohydrate tumor-associated Tn antigen clusters and peptidic CD4 T-cell epitopes. The synthetic process involves (i) the stepwise solid-phase assembly of protected amino acids, including the high value-added Tn building blocks with only 1.5 equivalents, (ii) a single isolated intermediate, and (iii) the simultaneous deprotection of 36 hindered protective groups. The resulting MAG-Tn3 was unambiguously characterized using a combination of techniques, including a structural analysis by nuclear magnetic resonance spectroscopy. The four peptidic chains are flexible in solution, with a more constrained but extended conformation at the Tn3 antigen motif. Finally, we demonstrate that, when injected into HLA-DR1-expressing transgenic mice, this vaccine induces Tn-specific antibodies that mediate the killing of human Tn-positive tumor cells. These studies led to a clinical batch of the MAG-Tn3, currently investigated in breast cancer patients (phase I clinical trial). The current study demonstrates the feasibility of the multigram-scale synthesis of a highly pure complex glycopeptide, and it opens new avenues for the use of synthetic glycopeptides as drugs in humans.
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http://dx.doi.org/10.1039/c6ob01931eDOI Listing
December 2016

Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets.

J Control Release 2016 12 6;243:1-10. Epub 2016 Oct 6.

Institut Pasteur, CITECH, Plateforme d'Ingénierie des Anticorps, 75724 Paris Cedex 15, France. Electronic address:

Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology.
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http://dx.doi.org/10.1016/j.jconrel.2016.09.019DOI Listing
December 2016

Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D.

PLoS One 2016 23;11(6):e0157962. Epub 2016 Jun 23.

Department of Biomedical Sciences, University of Sassari, 07100, Sassari, Italy.

Aims/hypothesis: Although numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria.

Methods: Plasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c133-141, MAP3865c125-133, MAP2404c70-85 and MAP1,4αgbp157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis.

Results: Responses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%.

Conclusions: MAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends support to the involvement of MAP in T1D.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919038PMC
July 2017

The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

Cancer Immunol Immunother 2016 Mar 4;65(3):315-25. Epub 2016 Feb 4.

Unité de Régulation Immunitaire et Vaccinologie, Equipe Labellisée Ligue Contre le Cancer, Institut Pasteur, 25 rue du Docteur Roux, 75015, Paris, France.

Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.
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http://dx.doi.org/10.1007/s00262-016-1802-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779142PMC
March 2016

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

Res Vet Sci 2015 Oct 1;102:118-21. Epub 2015 Aug 1.

UMR1282, Infectiologie et Santé Publique (ISP-311), INRA Centre Val de Loire, F-37380 Nouzilly, France. Electronic address:

After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.
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http://dx.doi.org/10.1016/j.rvsc.2015.07.017DOI Listing
October 2015

Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

Int J Oncol 2013 Sep 28;43(3):775-84. Epub 2013 Jun 28.

Department of Immunobiology, School of Medicine, UdelaR, 11800 Montevideo, Uruguay.

There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.
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http://dx.doi.org/10.3892/ijo.2013.2000DOI Listing
September 2013

MGL signaling augments TLR2-mediated responses for enhanced IL-10 and TNF-α secretion.

J Leukoc Biol 2013 Aug 6;94(2):315-23. Epub 2013 Jun 6.

VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam, the Netherlands.

DCs orchestrate immune responses to infectious pathogens and disturbances in tissue integrity. Equipped with C-type lectins, DCs can respond to environmental changes in glycosylation. Many C-type lectins are capable of modulating TLR activation, thereby facilitating tailor-made immune reactions. Here, we investigated the signaling properties of the C-type lectin MGL and show that MGL engagement by agonistic antibodies or carbohydrate ligands couples to TLR signal transduction for increased IL-10 and TNF-α secretion by human monocyte-derived DCs. MGL triggering especially synergized with TLR2-induced pathways, leading to elevated IL-10 mRNA levels and enhanced TNF-α mRNA stability. In addition, MGL signaling promoted phosphorylation of the MAPK ERK and the transcription factor CREB. Whereas specific inhibitors of p90RSK blocked the MGL-induced cytokine secretion, AP-1 was not involved. Strikingly, NF-κB was only crucial for the IL-10 response and dispensable for TNF-α production. Together, our results demonstrate that MGL activation of the ERK-p90RSK-CREB axis converges with TLR2-induced pathways, thereby fine-tuning the DC maturation phenotype.
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http://dx.doi.org/10.1189/jlb.1012520DOI Listing
August 2013

Specific IgG response against Mycobacterium avium paratuberculosis in children and adults with Crohn's disease.

PLoS One 2013 2;8(5):e62780. Epub 2013 May 2.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR989, Paris, France.

Background And Aims: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P).

Methods: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease.

Results: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease.

Conclusions: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062780PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642204PMC
December 2013

Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer.

Cancer Immunol Immunother 2013 Jun 21;62(6):1107-22. Epub 2013 Apr 21.

Departamento de Anatomía Patológica y Citología del Hospital de la Mujer, Centro Hospitalario Pereira Rossell, Montevideo, Uruguay.

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.
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http://dx.doi.org/10.1007/s00262-013-1425-7DOI Listing
June 2013

A Tn antigen binding lectin from Myrsine coriacea displays toxicity in human cancer cell lines.

J Nat Med 2013 Apr 30;67(2):247-54. Epub 2012 May 30.

Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, 11800, Montevideo, Uruguay.

The Tn antigen (GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. In the present study we characterize the biochemical and functional properties of the Myrsine coriacea lectin (McL). We show that McL is an unusual high molecular weight highly glycosylated protein, which displays a strong Tn binding activity. The lectin exhibits in vitro inhibition of proliferation in the six cancer cell lines evaluated, in a dose-dependent manner (the strongest activity being against HT-29 and HeLa cells), whereas it does not exhibit toxicity against normal lymphocytes. McL could be exploited in the design of potential new tools for the diagnosis or treatment of cancer.
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http://dx.doi.org/10.1007/s11418-012-0671-xDOI Listing
April 2013

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses.

J Biol Chem 2011 Mar 30;286(10):7797-7811. Epub 2010 Dec 30.

From the Institut Pasteur, Department of Immunology, Immune Regulation and Vaccinology Unit, 75015 Paris,; INSERM, U1041, Paris,. Electronic address:

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.
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http://dx.doi.org/10.1074/jbc.M110.209742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048667PMC
March 2011

Glycosidic Tn-based vaccines targeting dermal dendritic cells favor germinal center B-cell development and potent antibody response in the absence of adjuvant.

Blood 2010 Nov 18;116(18):3526-36. Epub 2010 Aug 18.

Immune Regulation and Vaccinology Unit, Department of Immunology, Institut Pasteur, 25-28 Rue du Dr. Roux, Paris Cedex 15, France.

In vivo targeting of C-type lectin receptors is an effective strategy for increasing antigen uptake and presentation by dendritic cells (DCs). To induce efficient immune response, glycosylated tumor-associated Tn antigens were used to target DCs through binding to macrophage galactose-type lectin (MGL). The capacity of Tn-glycosylated antigens-and the multiple antigenic glycopeptide Tn3 therapeutic candidate vaccine-to target mouse and human MGL(+) DCs are demonstrated, especially regarding dermal DCs. In mice, MGL(+) CD103(-) dermal DCs efficiently captured and processed glycosylated Tn antigen in vivo, inducing a potent major histocompatibility complex (MHC) class II-restricted T-cell response. Intradermal immunization with Tn-glycopeptides induced high levels of Th2 cytokines-even in the presence of unmethylated cytosine-phosphate-guanosine-and was associated with increased expansion of the germinal center B-cell population. Therefore, MGL acts as an efficient endocytic antigen receptor on dermal DCs in vivo, able to prime Tn-specific T- and B-cell responses. Moreover, even in the absence of adjuvant, immunization with this glycosidic Tn-based vaccine induced high levels of anti-Tn antibody responses, recognizing human tumor cells. In vivo DC-targeting strategies, based on Tn-MGL interactions, constitute a promising strategy for enhancing antigen presentation and inducing potent antibody response.
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http://dx.doi.org/10.1182/blood-2010-04-279133DOI Listing
November 2010

The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis.

Mol Microbiol 2008 Dec 17;70(6):1342-57. Epub 2008 Oct 17.

Biology of Gram-Positive Pathogens, Department of Microbiology, CNRS URA 2172, Paris, France.

DegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU-dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU(D55N)) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU(D55N) mutant. We also show that the small-molecular-weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesDeltaptaDeltaackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes.
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http://dx.doi.org/10.1111/j.1365-2958.2008.06496.xDOI Listing
December 2008

Cinnamic acid, an autoinducer of its own biosynthesis, is processed via Hca enzymes in Photorhabdus luminescens.

Appl Environ Microbiol 2008 Mar 1;74(6):1717-25. Epub 2008 Feb 1.

Unité de Génétique des Membranes Bactériennes, CNRS URA 2172, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.

Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.
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http://dx.doi.org/10.1128/AEM.02589-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268303PMC
March 2008

Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium.

Vaccine 2008 Jan 20;26(2):257-68. Epub 2007 Nov 20.

UR1282, Infectiologie Animale, Santé Publique, INRA centre de Tours, F-37380 Nouzilly, France.

Background: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced.

Methods: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals.

Conclusions: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis.
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http://dx.doi.org/10.1016/j.vaccine.2007.10.059DOI Listing
January 2008

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides.

J Am Soc Mass Spectrom 2007 Aug 25;18(8):1405-13. Epub 2007 Apr 25.

Laboratoire des Mécanismes Réactionnels, Ecole Polytechnique, CNRS, Palaiseau, France.

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups.
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http://dx.doi.org/10.1016/j.jasms.2007.04.008DOI Listing
August 2007

Carbohydrate antigens: synthesis aspects and immunological applications in cancer.

Mini Rev Med Chem 2006 Dec;6(12):1357-73

CNRS, URA2128, Unité de Chimie Organique, Paris, France.

Tumor Associated Carbohydrate Antigens (TACAs) constitute powerful tools as tumor markers and as targets for anticancer immunotherapy. In this review, methods of production of glycopeptide-based vaccines, as well as results of preclinical and clinical studies in cancer patients are discussed.
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http://dx.doi.org/10.2174/138955706778992996DOI Listing
December 2006

Efficient monitoring of enzymatic conjugation reaction by surface-enhanced laser desorption/ionization time of flight mass spectrometry for process optimization.

Bioconjug Chem 2006 Mar-Apr;17(2):559-64

Unité de Chimie Organique URA CNRS 2128, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris, Cedex 15, France.

Efficient analysis of bioconjugation reactions is one the most challenging task for optimizing and eventually achieving the reproducible production of large amount of conjugates. In particular, the complexity of some reaction mixtures precludes the use of most of the existing methods, because of the presence of large amounts of contaminants. As an alternative method, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for monitoring an in vitro enzymatic transglycosylation of N-acetylgalactosamine (GalNAc) residues to a recombinant mucin protein MUC6. For this reaction, catalyzed by the uridine 5'-diphospho-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), we used either a recombinant ppGalNAc-T1 or a mixture of ppGalNAc-Ts contained in MCF7 tumor cell extracts. In the present study, we show that SELDI-TOF MS offers unique advantages over the traditional methodologies. It is a rapid, accurate, sensitive, reproducible, and very convenient analytical method for monitoring the course of a bioconjugation, even in heterogeneous samples such as cell extracts. SELDI-TOF MS proved very useful for optimizing the reaction parameters of the transglycosylation and for achieving the large scale preparation of Tn antigen-glycosylated mucins for antitumor immunotherapy applications.
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http://dx.doi.org/10.1021/bc0502661DOI Listing
July 2006

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination.

Glycobiology 2006 May 31;16(5):390-401. Epub 2006 Jan 31.

Unité de Chimie Organique URA CNRS 2128, Institut Pasteur, Paris, France.

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.
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http://dx.doi.org/10.1093/glycob/cwj082DOI Listing
May 2006